Compounds, combinations, and methods of preparation and uses therefor

Abstract

Disclosed are combinations, including, for example, combinations comprising zingerone and at least one additional compound, as well as compositions comprising these combinations. Disclosed also are methods for preparing and using these combinations, and methods for preparing and using these compositions. Specifically disclosed are beneficial compositions, including pharmaceutical compositions, dietary compositions, and methods employing these.

Description

COMPOUNDS, COMBINATIONS, AND METHODS OF PREPARATION AND USES THEREFOR RELATED APPLICATION

[0001] This application claims the benefit of US provisional patent application number 63 / 556,369, filed on 21 February 2024, the entire contents of which are hereby incorporated by reference. FIELD

[0002] The present disclosure relates to combinations, including, for example, compositions comprising zingerone and one or more additional compounds. Specifically noted are beneficial compositions, including pharmaceutical compositions and dietary compositions, and uses for these compositions. BACKGROUND

[0003] Ginger (Zingiber officinale) is a flowering plant whose rhizome is widely used as a spice and in traditional medicine. If consumed in reasonable quantities, ginger has few negative side effects. It is on the FDA's "generally recognized as safe" list.

[0004] The characteristic fragrance and flavour of ginger result from volatile oils that compose 1-3% of the weight of fresh ginger, primarily consisting of zingerone, shogaols, and gingerols with 6-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]- 5-hydroxy-3-decanone) as the major pungent compound.6-gingerol

[0005] Zingerone has a lower pungency and a spicy-sweet aroma. Fresh ginger contains minimal zingerone, but it can be produced by cooking or drying of the ginger root. This causes dehydration of gingerol through the loss of a water molecule to produce zingerone and hexanal. Zingerone is also called vanillylacetone.zingerone

[0006] Shogaols are more pungent and have higher antioxidant activity and are not found in raw ginger, but are formed from gingerols during heating, storage or via acidity. Shogaol is a dehydrated form of gingerol.shogaol

[0007] Given the current emphasis on compositions that include natural components and mixtures of natural components, there is a need for new compositions, including plant-based compositions, and particularly those with immune-modulating and anti-inflammatory activity. The present application aims to meet one or more of these needs or to at least provide the public with a useful alternative. SUMMARY

[0008] In one aspect, the present disclosure encompasses a combination comprising: compound I and compound II, these compounds being of formula I and formula II, respectively:, wherein:R1is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3;R2 is -H, or when taken together with R4 and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3 is -H or when taken together with R2 forms =O; and R4is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, andwherein: R10 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R11is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R12 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R13is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds.

[0009] In particular aspects:

[0010] Compound I and compound II are different compounds.

[0011] Compound I is selected from the group consisting of:acetyl zingerone zingeronezingerol ethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone4-hydroxy-3-methoxycinnamic acid ethyl esterhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acidconiferyl aldehyde eugenol4-hydroxy-3-methoxybenzoic acid and any salts, solvates, hydrates, and protected forms thereof.

[0012] Compound I is selected from the group consisting of: zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

[0013] Compound I is selected from the group consisting of: zingerone and acetyl zingerone, and any salts, solvates, hydrates, and protected forms thereof.

[0014] Compound II is selected from the group consisting of:ferulic acidhydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatevanilpyruvic acid methyl-3-methoxy-4-hydroxystyryl ketone3,4,5-trihydroxycinnamic acid sinapic acidcinnamic acid p-coumaric acidand any salts, solvates, hydrates, and protected forms thereof.

[0015] Compound II is selected from the group consisting of: ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

[0016] Compound I or compound II is a synthetic compound.

[0017] Compound I or compound II is a naturally occurring compound.

[0018] Compound I or compound II is provided as a botanical extract.

[0019] The botanical extract consists essentially of a compound I or compound II.

[0020] The botanical extract consists essentially of zingerone.

[0021] The botanical extract is produced by a method disclosed herein.

[0022] Compound I and compound II are formulated as one or more pharmaceutical compositions.

[0023] Compound I and compound II are formulated as one or more dietary compositions.

[0024] Compound I and compound II are formulated as one or more foods, beverages, additives, and dietary supplements.

[0025] The combination is for use in treating or preventing inflammation in a subject.

[0026] Compound I and compound II are administered to the subject: (i) simultaneously; (ii) sequentially; (iii) separately; or (iv) as a co-formulation.

[0027] In one other aspect, the present disclosure encompasses a kit comprising compound I and compound II of a preceding aspect.

[0028] The kit comprises at least one of the following: one or more containers, one or more buffers, one or more excipients, or instructions for use.

[0029] Compound I and compound II are provided in separate containers.

[0030] Compound I and compound II are provided in the same container.

[0031] Compound I and compound II are formulated as one or more pharmaceutical compositions.

[0032] Compound I and compound II are formulated as one or more dietary compositions.

[0033] Compound I and compound II are provided as different formulations for administration.

[0034] Compound I and compound II are provided as the same formulations for administration.

[0035] Compound I and compound II are provided as a co-formulation for administration.

[0036] Compound I and / or compound II is formulated as a solid, semi-solid, or liquid.

[0037] Compound I and / or compound II is formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule.

[0038] Compound I and / or compound II is formulated to be provided in a sachet.

[0039] The kit is for use in treating or preventing inflammation in a subject.

[0040] Compound I and compound II are administered to the subject: (i) simultaneously; (ii) sequentially; (iii) separately; or (iv) as a co-formulation.

[0041] In one other aspect, the present disclosure encompasses a composition comprising compound I and compound II of a preceding aspect.

[0042] In various aspects:

[0043] The composition is formulated as a pharmaceutical composition.

[0044] The composition is formulated as a dietary composition.

[0045] The composition is formulated as a food, a beverage, an additive, or a dietary supplement.

[0046] The composition is formulated as a solid, semi-solid, or liquid.

[0047] The composition is formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule.

[0048] The composition is provided in a sachet.

[0049] The composition further comprises one or more anti-inflammatory agents.

[0050] The composition further comprises one or more of: an analgesic compound, antipyretic compound, and psychotropic compound.

[0051] The composition further comprises one or more of: a cannabinoid compound. mushroom compound, non-steroid anti-inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound.

[0052] The composition further comprises one or more of: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

[0053] The composition is formulated as a dosage form comprising about 10 mg to about 3000 mg of compound I or compound II.

[0054] The composition is formulated as a dosage form comprising about 10 mg to about 1500 mg of compound I or compound II.

[0055] The composition is formulated as a dosage form comprising about 10 mg to about 1000 mg of compound I or compound II.

[0056] The composition is formulated as a dosage form comprising about 10 mg to about 500 mg of compound I or compound II.

[0057] The composition is formulated as a dosage form comprising about 10 mg to about 300 mg of compound I or compound II.

[0058] The composition is formulated as a dosage form comprising about 10 mg to about 150 mg of compound I or compound II.

[0059] The composition is formulated as a dosage form comprising about 10 mg to about 100 mg of compound I or compound II.

[0060] The composition is formulated as a dosage form comprising about 10 mg to about 75 mg of compound I or compound II.

[0061] The composition is formulated as a dosage form comprising about 10 mg to about 50 mg of compound I or compound II.

[0062] The composition is for use in treating or preventing inflammation in a subject.

[0063] In one other aspect, the present disclosure encompasses a method of treating or preventing inflammation comprising administering to a subject compound I and compound II of a preceding aspect.

[0064] In various aspects:

[0065] The inflammation requires modulation.

[0066] The inflammation is acute or chronic inflammation.

[0067] The inflammation is an inflammatory disorder.

[0068] The inflammation is inflammation of one or more of: an immune disorder; an arthritic disorder; an infection; a cardiac, circulatory, or pulmonary disorder; a neurological disorder; and a neoplastic disorder.

[0069] The inflammation is an inflammation affecting one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen, lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

[0070] The inflammation is inflammation of one or more of: Alzheimer’s disease, early stage Alzheimer’s disease, ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerated colitis), Crohn's disease, dementia, early stage dementia, depression, diabetes, fibromyalgia, gout, infection (e.g., microbial infection), immune mediated inflammatory disease, inflammatory bowel disease (IBD), interstitial cystitis, multiple sclerosis (MS), polymyalgia psoriasis, scleroderma, and Sjögren’s syndrome, and systemic lupus erythematosus (SLE; lupus).

[0071] The inflammation is inflammation of one or more of: rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, Sjögren’s syndrome arthritis.

[0072] The inflammation is inflammation of one or more of: atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disorder, and cytokine storm syndrome.

[0073] The inflammation is inflammation of one or more of: breast cancer, leukaemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0074] The inflammation is inflammation of one or more of: a blister, dermatitis, eczema, hive, lesion, papule, plaque, psoriasis, rash, rosacea, ulcer, and wound.

[0075] In other aspects:

[0076] Compound I and compound II are administered as different formulations.

[0077] Compound I and compound II are administered as the same formulations.

[0078] Compound I and compound II are administered as a co-formulation.

[0079] Compound I and compound II are administered simultaneously.

[0080] Compound I and compound II are administered sequentially.

[0081] Compound I and compound II are administered separately.

[0082] Compound I and compound II are co-administered with one or more anti- inflammatory agents.

[0083] Compound I and compound II are co-administered with one or more of: an analgesic compound, antipyretic compound, and psychotropic compound.

[0084] Compound I and compound II are co-administered with one or more of: a cannabinoid compound. mushroom compound, non-steroid anti-inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound.

[0085] Compound I and compound II are co-administered with one or more of: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone,methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

[0086] In other aspects:

[0087] The subject is administered a dose of about 10 mg to about 3000 mg of compound I or compound II.

[0088] The subject is administered a dose of about 10 mg to about 1500 mg of compound I or compound II.

[0089] The subject is administered a dose of about 10 mg to about 1000 mg of compound I or compound II.

[0090] The subject is administered a dose of about 10 mg to about 500 mg of compound I or compound II.

[0091] The subject is administered a dose of about 10 mg to about 300 mg of compound I or compound II.

[0092] The subject is administered a dose of about 10 mg to about 150 mg of compound I or compound II.

[0093] The subject is administered a dose of about 10 mg to about 100 mg of compound I or compound II.

[0094] The subject is administered a dose of about 10 mg to about 75 mg of compound I or compound II.

[0095] The subject is administered a dose of about 10 mg to about 50 mg of compound I or compound II.

[0096] In one other aspect, the present disclosure encompasses use of compound I and compound II of a preceding aspect for preparing a combination therapy for treating or preventing inflammation in a subject.

[0097] In various aspects:

[0098] The inflammation requires modulation.

[0099] The inflammation is acute or chronic inflammation.

[0100] The inflammation is an inflammatory disorder.

[0101] The inflammation is inflammation of one or more of: an immune disorder; an arthritic disorder; an infection; a cardiac, circulatory, or pulmonary disorder; a neurological disorder; and a neoplastic disorder.

[0102] The inflammation is an inflammation affecting one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen, lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

[0103] The inflammation is inflammation of one or more of: Alzheimer’s disease, early stage Alzheimer’s disease, ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerated colitis), Crohn's disease, dementia, early stage dementia, depression, diabetes, fibromyalgia, gout, infection (e.g., microbial infection), immune mediated inflammatory disease, inflammatory bowel disease (IBD), interstitial cystitis, multiple sclerosis (MS), polymyalgia psoriasis, scleroderma, and Sjögren’s syndrome, and systemic lupus erythematosus (SLE; lupus).

[0104] The inflammation is inflammation of one or more of: rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, Sjögren’s syndrome arthritis.

[0105] The inflammation is inflammation of one or more of: atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disorder, and cytokine storm syndrome.

[0106] The inflammation is inflammation of one or more of: breast cancer, leukaemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0107] The inflammation is inflammation of one or more of: a blister, dermatitis, eczema, hive, lesion, papule, plaque, psoriasis, rash, rosacea, ulcer, and wound.

[0108] In other aspects:

[0109] Compound I and compound II are formulated in different forms for administration.

[0110] Compound I and compound II are formulated in the same forms for administration.

[0111] Compound I and compound II are co-formulated for administration.

[0112] Compound I and compound II are formulated for simultaneous administration.

[0113] Compound I and compound II are formulated for sequential administration.

[0114] Compound I and compound II are formulated for separate administration.

[0115] Compound I and compound II are formulated for co-administration with one or more anti-inflammatory agents.

[0116] Compound I and compound II are formulated for co-administration with one or more of: an analgesic compound, antipyretic compound, and psychotropic compound.

[0117] Compound I and compound II are formulated for co-administration with one or more of: a cannabinoid compound. mushroom compound, non-steroid anti- inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound.

[0118] Compound I and compound II are formulated for co-administration with one or more of: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

[0119] In other aspects:

[0120] An effective dosage for the therapy is about 10 mg to about 3000 mg of compound I or compound II.

[0121] An effective dosage for the therapy is about 10 mg to about 1500 mg of compound I or compound II.

[0122] An effective dosage for the therapy is about 10 mg to about 1000 mg of compound I or compound II.

[0123] An effective dosage for the therapy is about 10 mg to about 500 mg of compound I or compound II.

[0124] An effective dosage for the therapy is about 10 mg to about 300 mg of compound I or compound II.

[0125] An effective dosage for the therapy is about 10 mg to about 150 mg of compound I or compound II.

[0126] An effective dosage for the therapy is about 10 mg to about 100 mg of compound I or compound II.

[0127] An effective dosage for the therapy is about 10 mg to about 75 mg of compound I or compound II.

[0128] An effective dosage for the therapy is about 10 mg to about 50 mg of compound I or compound II.

[0129] In one aspect, the botanical extract of a preceding aspect is produced by a method comprising: (i) subjecting ginger root to an alkaline treatment in alkaline solution; or (ii) subjecting juice obtained from ginger root to an alkaline treatment in an alkaline solution; or (iii) subjecting juice and marc obtained from ginger root to alkaline treatment in alkaline solution.

[0130] In specific aspects:

[0131] The ginger root is fresh.

[0132] The ginger root is dried.

[0133] The ginger root is dried at about 40 to about 70 degrees Celsius, or at about 40 to about 60 degrees Celsius, or at about 55 to about 65 degrees Celsius, or at about 60 degrees Celsius.

[0134] The juice is obtained by macerating and / or pressing the ginger root.

[0135] The marc is obtained by juicing, macerating, and / or pressing the ginger root.

[0136] The ginger root is diced and subjected to the alkaline treatment.

[0137] The ginger root is diced, dried, and subjected to the alkaline treatment.

[0138] The alkaline treatment is carried out at about 40 to about 70 degrees Celsius.

[0139] The alkaline treatment is carried out at about 50 to about 60 degrees Celsius.

[0140] The alkaline treatment is carried out at about 55 to about 65 degrees Celsius.

[0141] The alkaline treatment is carried out at about 60 degrees Celsius.

[0142] The alkaline treatment is carried out for about 1-72 hours.

[0143] The alkaline treatment is carried out for about 1-48 hours.

[0144] The alkaline treatment is carried out for about 1-24 hours.

[0145] The alkaline treatment is carried out for about 1-30 hours, or about 1-20 hours, or about 1-10 hours, or about 1-5 hours.

[0146] The alkaline treatment is carried out for about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours.

[0147] The alkaline treatment is carried out for about 2 hours.

[0148] The alkaline treatment is carried out for about 1 hour.

[0149] Potassium hydroxide (KOH) is used.

[0150] A liquid form of potassium hydroxide (KOH) is used.

[0151] About 0.1% to about 6% KOH (v / v) is used. About 0.5% to about 5.5% KOH (v / v) is used. About 1% to about 6% KOH (v / v) is used. About 1.5% to about 5.5% KOH (v / v) is used. About 2% to about 4% KOH (v / v) is used. About 1.5% KOH (v / v) to about 3.5% KOH (v / v) is used.

[0152] Calcium hydroxide Ca(OH)2is used.

[0153] About 0.5% to about 4% Ca(OH)2 (v / v) is used. About 1.5% to about 3.5% Ca(OH)2 (v / v) is used. About 2% to about 3% Ca(OH)2 (v / v) is used.

[0154] After alkaline treatment, the alkaline solution is neutralised.

[0155] The alkaline solution is neutralised with citric acid.

[0156] The alkaline solution is cooled during neutralisation to alleviate excess heat.

[0157] The alkaline solution is neutralised to obtain a pH of about 6.5 to about 7.5.

[0158] The neutralised alkaline solution is freeze dried.

[0159] The neutralised solution is heat dried.

[0160] The neutralised alkaline solution is subjected to extraction of the zingerone.

[0161] The neutralised solution is dried and optionally subjected to extraction of the zingerone.

[0162] The drying is at about 50 degrees Celsius to at about 70 degrees Celsius.

[0163] The drying is at about 55 degrees Celsius to about 65 degrees Celsius.

[0164] The drying is at about 60 degrees Celsius.

[0165] The drying is for at least 24 hours.

[0166] The drying is for about 24 hours to about 28 hours.

[0167] The dried material is optionally milled.

[0168] The zingerone is optionally further extracted by one or more alcohol extraction steps.

[0169] The zingerone is optionally further extracted by one or more ethanol extraction steps.

[0170] The ethanol extraction is carried at about 35 degrees Celsius to at about 65 degrees Celsius.

[0171] The ethanol extraction is carried at about 45 degrees Celsius to about 55 degrees Celsius.

[0172] The ethanol extraction is carried at about 50 degrees Celsius.

[0173] The ethanol extraction is carried out for at least 7 days.

[0174] The ethanol extraction is carried out for 24 hours or less.

[0175] The ethanol extraction is carried out for at least 4 hours.

[0176] The ethanol extraction is carried out for about 4 to about 8 hours.

[0177] The ethanolic extract is optionally dried.

[0178] The zingerone is extracted using supercritical fluid extraction.

[0179] The zingerone is extracted by a supercritical fluid extraction followed by an alcohol extraction step.

[0180] The method produces a product, this being a composition that comprises zingerone.

[0181] The composition is free from or substantially free from aldehydes.

[0182] The composition is a botanical extract.

[0183] The composition is an ethanolic extract.

[0184] The composition is a powder.

[0185] In one aspect, the botanical extract of a preceding aspect is produced by a method comprising: subjecting ginger root extract to an alkaline treatment.

[0186] The ginger root extract is obtained by supercritical fluid extraction of the ginger root.

[0187] The ginger root extract is obtained by alcohol extraction of the ginger root.

[0188] The ginger root extract is obtained by juicing the ginger root.

[0189] The ginger root extract is obtained by juicing the ginger root to obtain a juice and a marc.

[0190] The juicing includes macerating and / or pressing the ginger root.

[0191] The alkaline treatment is carried out at about 30 to about 70 degrees Celsius.

[0192] The alkaline treatment is carried out at about 50 about 60 degrees Celsius.

[0193] The alkaline treatment is carried out at about 55 to about 65 degrees Celsius.

[0194] The alkaline treatment is carried out at about 60 degrees Celsius.

[0195] The alkaline treatment is carried out for about 1-72 hours.

[0196] The alkaline treatment is carried out for about 1-48 hours.

[0197] The alkaline treatment is carried out for about 1-24 hours.

[0198] The alkaline treatment is carried out for about 1-30 hours, or about 1-20 hours, or about 1-10 hours, or about 1-5 hours.

[0199] The alkaline treatment is carried out for about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours.

[0200] The alkaline treatment is carried out for about 2 hours.

[0201] The alkaline treatment is carried out for about 1 hour.

[0202] Potassium hydroxide (KOH) is used.

[0203] A liquid form of potassium hydroxide (KOH) is used.

[0204] About 0.1% to about 6% KOH (v / v) is used. About 0.5% to about 5.5% KOH (v / v) is used. About 1% to about 6% KOH (v / v) is used. About 1.5% to about 5.5% KOH (v / v) is used. About 2% to about 4% KOH (v / v) is used. About 1.5% KOH (v / v) to about 3.5% KOH (v / v) is used.

[0205] Calcium hydroxide Ca(OH)2is used.

[0206] About 0.5% to about 4% Ca(OH)2 (v / v) is used. About 1.5% to about 3.5% Ca(OH)2(v / v) is used. About 2% to about 3% Ca(OH)2(v / v) is used.

[0207] After alkaline treatment, the alkaline solution is neutralised.

[0208] The alkaline solution is neutralised to obtain a pH of about 6.5 to about 7.5 or about 7.0 to about 7.3.

[0209] After neutralisation of the alkaline solution, the neutralised material is dried.

[0210] The dried material is optionally milled.

[0211] The dried material is optionally further extracted.

[0212] The zingerone is optionally further extracted by one or more alcohol extraction steps.

[0213] The zingerone is optionally further extracted by one or more ethanol extraction steps.

[0214] The ethanol extraction is carried out for at least 7 days.

[0215] The ethanol extraction is carried out for 24 hours or less.

[0216] The ethanol extraction is carried out for at least 4 hours.

[0217] The ethanol extraction is carried out for about 4 to about 8 hours.

[0218] The ethanolic extract is optionally dried.

[0219] The zingerone is optionally further extracted using supercritical fluid extraction.

[0220] The zingerone is optionally further extracted by a supercritical fluid extraction followed by an alcohol extraction step.

[0221] The method produces a product, this being a composition that comprises zingerone.

[0222] The composition is free from or substantially free from aldehydes.

[0223] The composition is a botanical extract.

[0224] The composition is an ethanolic extract.

[0225] The composition is a powder.

[0226] In one aspect, the botanical extract of a preceding aspect is produced by a method comprising: (i) subjecting ginger root to an alkaline treatment in alkaline solution; or (ii) subjecting juice obtained from ginger root, and optionally marc obtained from ginger root, to an alkaline treatment in an alkaline solution, wherein the alkaline solution comprises about 1% to about 6% KOH (v / v), wherein the alkaline treatment is carried out for about 1 to about 2 hours, and wherein following alkaline treatment, the alkaline solution is neutralised to a pH of about 6.5 to about 7.5.

[0227] The method produces a product, this being a composition that comprises zingerone.

[0228] The composition is free from or substantially free from aldehydes.

[0229] The composition is a botanical extract.

[0230] The composition is an ethanolic extract.

[0231] The composition is a powder.

[0232] The foregoing brief summary broadly describes the features and technical advantages of certain embodiments of this disclosure. Further technical advantages will be described in the detailed description and examples that follows.

[0233] Novel features that are believed to be characteristic will be better understood from the detailed description when considered in connection with any accompanying figures and examples. However, the figures and examples provided herein are intended to help illustrate what is disclosed or assist with developing an understanding what is disclosed, and are not intended to limit the scope of this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0234] Figure 1. Photograph depicting fresh ginger root.

[0235] Figure 2. HPLC UV chromatogram traces (280 nm) for alkaline treated ginger.

[0236] Figure 3. Schematic showing processing comparison.

[0237] Figure 4A: Photograph depicting juicing machine and raw ginger prior to juicing.

[0238] Figure 4B: Photograph depicting juicing of raw ginger in progress.

[0239] Figure 5A: Ginger juice treated with KOH (0.5%) and analysed by HPLC. Peak areas shown for zingerone (Z) and gingerol (G).

[0240] Figure 5B: Ginger juice treated with KOH (1%) and analysed by HPLC. Peak areas shown for zingerone (Z) and gingerol (G).

[0241] Figure 5C: Ginger juice treated with KOH (2%) and analysed by HPLC. Peak areas shown for zingerone (Z) and gingerol (G).

[0242] Figure 6A: Ginger marc produced by pressing.

[0243] Figure 6B: Ginger juice produced by pressing.

[0244] Figure 7A: Schematic showing ethanolic extraction process and evaporation.

[0245] Figure 7B: Flow chart for a large scale ginger juicing method.

[0246] Figure 7C: Flow chart for a large scale zingerone extraction method with ginger juice as starting material.

[0247] Figure 7D: Flow chart for a large scale zingerone extraction method with ginger marc as starting material.

[0248] Figure 7E-7F: Stability of zingerone extract obtained by large scale production. Testing was carried out over two months. Figure 7E shows Zingerone content at 5°C and 40°C. Figure 7F shows pH levels at 5°C and 40°C.

[0249] Figure 8A: GCMS TIC analysis for ethanolic extract.

[0250] Figure 8B: Comparison of ethanolic extract with hexanal standard. Shown are 2-7 minute regions of chromatogram.

[0251] Figure 9: Dose response curve for cytotoxicity assay. Disclosed botanical extract assessed.

[0252] Figure 10: Dose response curve for nitric oxide assay. Disclosed botanical extract assessed.

[0253] Figure 11: Dose response curve for IL-6 assay. Disclosed botanical extract assessed.

[0254] Figure 12: Dose response curve for cytotoxicity, NO, and IL-6 assays. Disclosed botanical extract assessed.

[0255] Figure 13: Cell viability of RAW264.7 cells was measured with the WST- 1 assay. Comparative study for disclosed botanical extract and commercially sourced zingerone.

[0256] Figure 14: Interleukin (IL)-6 produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). Comparative study for disclosed botanical extract and commercially sourced zingerone.

[0257] Figure 15: Interleukin (IL)-10 produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). Comparative study for disclosed botanical extract and commercially sourced zingerone.

[0258] Figure 16: Tumour necrosis factor (TNF)-α produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). Comparative study for disclosed botanical extract and commercially sourced zingerone.

[0259] Figures 17A-17B: Both testing methods showed efficacy of dexamethasone as a positive control. Figure 17A shows results for Method 1 analysis. Figure 17B shows results for Method 2 analysis.

[0260] Figures 18A-18B: Both testing methods performed similarly for MTT scoring. Figure 18A shows results for Method 1 analysis. Figure 18B shows results for Method 2 analysis.

[0261] Figures 19A-19D: Disclosed botanical extract showed a cytotoxic effect at 150, 100, 75 and 50 µM. Data is presented as the mean of three biological replicates ± SEM. Non-linear regression was computed using GraphPad Prism 9.0. Figure 19A shows results for synthetic zingerone. Figure 19B shows results for acetyl zingerone. Figure 19C shows results for ferulic acid. Figure 19D shows results for disclosed botanical extract.

[0262] Figure 20: Disclosed botanical extract produces a dose dependent inhibition of IL-6 production from stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0263] Figure 21: Treatment with the disclosed botanical extract at 25 µM produces significantly less IL-6 compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 µM. Data is presented as the mean of three biological replicates ± SEM. Repeated Measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P < 0.05, ***: P < 0.001

[0264] Figure 22: Treatment with the disclosed botanical extract at 25 µM significantly lowered IL-6 levels compared to vehicle control. Data is presented as the mean of three biological replicates ± SEM. Repeated Measures Two-Way ANOVA with a Sidak correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P<0.05, **: P<0.01.

[0265] Figures 23A-23D: No observable effect on IL-6 production from unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM. Figure 23A shows results for synthetic zingerone. Figure 23B shows results for acetyl zingerone. Figure 23C shows results for ferulic acid. Figure 23D shows results for disclosed botanical extract.

[0266] Figure 24: Disclosed botanical extract reduces TNF (TNF-α) production by stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0267] Figure 25: Treatment with the disclosed botanical extract at 25 µM produces significantly less IL-6 compared to treatment with acetyl zingerone at 150 µM.Data is presented as the mean of three biological replicates ± SEM. Repeated Measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P < 0.05.

[0268] Figure 26: Reduction of TNF production by stimulated RAW264.7 cells. Ferulic acid at 150 µM produces a significant reduction of TNF compared to vehicle. Data is presented as the mean of three biological replicates ± SEM. Repeated Measures Two- Way ANOVA with a Sidak correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P<0.05, **: P<0.01.

[0269] Figures 27A-27D: No observable effect on TNF production from unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM. Figure 27A shows results for synthetic zingerone. Figure 27B shows results for acetyl zingerone. Figure 27C shows results for ferulic acid. Figure 27D shows results for disclosed botanical extract.

[0270] Figure 28: Disclosed botanical extract reduces NO levels in stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0271] Figure 29: Treatment with the disclosed botanical extract at 25 µM produces significantly lower NO levels compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 µM. Data is presented as the mean of three biological replicates ± SEM. Repeated Measures One-Way ANOVA with a Turkey correction for multiple comparisons was computed in GraphPad Prism9.0.

[0272] Figure 30: Treatment with synthetic zingerone at 150 µM produces a significantly higher levels of NO compared to treatment with vehicle. Data is presented as the mean of three biological replicates ± SEM. Repeated Measures Two-Way ANOVA with a Sidak correction for multiple comparisons was computed in GraphPad Prism 9.0.

[0273] Figure 31: Cotreatment of zingerone and ferulic acid did not substantially reduce the MTT score of LPS-Stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0274] Figure 32: Cotreatment of acetyl zingerone and ferulic acid did not substantially reduce the MTT score of LPS-Stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0275] Figure 33: Cotreatment of zingerone and ferulic acid did not substantially reduce the MTT score of unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0276] Figure 34: Cotreatment of acetyl zingerone and ferulic acid did not substantially reduce the MTT score of unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0277] Figure 35: Cotreatment of zingerone with ferulic acid inhibited IL-6 production in LPS stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0278] Figures 36A-36D: Cotreatment of zingerone with ferulic acid inhibited IL- 6 production in LPS-stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM. Repeated measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P<0.05, **: P<0.01. Figure 36A shows results for individual treatments and cotreatments with zingerone at 75 µM and ferulic acid at 75 µM. Figure 36B shows results for individual treatments and cotreatments with zingerone at 75 µM and ferulic acid at 150 µM. Figure 36C shows results for individual treatments and cotreatments with zingerone at 150 µM and ferulic acid at 75 µM. Figure 36D shows results for individual treatments and cotreatments with zingerone at 150 µM and ferulic acid at 150 µM.

[0279] Figure 37: Cotreatment of acetyl zingerone with ferulic acid inhibited IL- 6 production from LPS stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0280] Figures 38A-38D: Cotreatment of acetyl zingerone with ferulic acid inhibited IL-6 production in LPS-stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM. Repeated measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. Figure 38A shows results for individual treatments and cotreatments with acetyl zingerone at 75 µM and ferulic acid at 75 µM. Figure 38B shows results for individual treatments and cotreatments with acetyl zingerone at 75µM and ferulic acid at 150 µM. Figure 38C shows results for individual treatments and cotreatments with acetyl zingerone at 150 µM and ferulic acid at 75 µM. Figure 38D shows results for individual treatments and cotreatments with acetyl zingerone at 150 µM and ferulic acid at 150 µM.

[0281] Figures 39A-39C: No observable effect seen in treatment of unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM. Figure 39A shows results for individual treatments with zingerone, acetyl zingerone, and ferulic acid. Figure 39B shows results for cotreatments with zingerone and ferulic acid. Figure 39C shows results for cotreatments with acetyl zingerone and ferulic acid.

[0282] Figure 40: Cotreatment with zingerone and ferulic acid outperformed individual treatments in inhibiting TNF production from LPS stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0283] Figures 41A-41D: Cotreatment with zingerone and ferulic acid showed a significantly greater inhibition of TNF production when compared to individual treatments. Data is presented as the mean of three biological replicates ± SEM. Repeated measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P<0.05, **: P<0.01.

[0284] Figure 42: Cotreatment with acetyl zingerone and ferulic acid outperformed individual treatments in inhibiting TNF production from LPS stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ± SEM.

[0285] Figures 43A-43D: Cotreatment with acetyl zingerone and ferulic acid showed a significantly greater inhibition of TNF production when compared to individual treatments. Data is presented as the mean of three biological replicates ± SEM. Repeated measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P< 0.05, **: P<0.01.

[0286] Figure 44: TNF levels in unstimulated conditions were substantially lower relative to stimulated conditions. Data is presented as the mean of three independent replicates ± SEM.

[0287] Figure 45: Cotreatment with zingerone and ferulic acid blocked elevation of NO in RAW264.7 cells as compared to treatment with zingerone alone. Data is presented as the mean of three biological replicates ± SEM.

[0288] Figure 46: Cotreatment with acetyl zingerone and ferulic resulted in lower NO levels as compared to acetyl zingerone treatment alone. Data is presented as the mean of three biological replicates ± SEM.

[0289] Figure 47: Change in body weight from baseline. Mice were immunised subcutaneously in the hind legs on day 0 with type 2 chicken collagen (100 ^g / mouse) in complete Freund’s adjuvant (containing 500 ^g / mouse Mycobacterium tuberculosis) and weighed daily. Starting from 3 days after immunisation, mice were treated orally with the botanical extract (BE) at 1.9 (low), 3.88 (med), or 5.69 mg / mouse (high) in sweetened condensed milk (SCM) daily. Vehicle treated mice were administered SCM alone. (a) The % change in body weight from baseline (day 0) is shown as the mean ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. (b) The % change in body weight from day 1 is shown as the mean ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. **** p<0.0001 by two-way ANOVA.

[0290] Figure 48: Plasma IgM levels at day 7 and 14. Mice were immunised with collagen and treated as indicated for Figure 47. Plasma was collected from mice on day 7 post immunisation by tail bleed (light grey circles) and on day 14 by cardiac puncture (black circles). IgM was assessed by ELISA. Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. The dashed line shows the mean value of the vehicle-treated group at day 7 while the dotted line is the mean value from healthy animals. P values were calculated by 1-way ANOVA with Fisher’s least significant differences post test.

[0291] Figure 49: Collagen-specific IgG at day 14. Mice were immunised with collagen and treated as indicated for Figure 47. Plasma was collected from mice on day 14 by cardiac puncture. Collagen-specific IgG was assessed by commercial ELISA (Chondrex). Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. The dashed line shows the mean value of the vehicle-treated group at day 14. Minimal collagen-specific IgG was detected in healthy animals (open circles). P values were calculated by Kruskal-Wallis with an uncorrected Dunn’s post test.

[0292] Figure 50: Plasma TNF levels at day 14. Mice were immunised with collagen and treated as indicated for Figure 47. Plasma was collected from mice on day 14 by cardiac puncture. TNF was assessed by ELISA and shown as the mean ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. The dashed line shows the mean value of the vehicle-treated group at day 14. P values were calculated by 1-way ANOVA with Fisher’s least significant differences post test.

[0293] Figure 51: Change in body weight from baseline. Mice were immunised as indicated for Figure 47. Starting from 3 days after immunisation, mice were treated daily by oral gavage with zingerone (25 mg / kg), ferulic acid (25 mg / kg), or zingerone (12.5 mg / kg)+ferulic acid (12.5 mg / kg) in 0.4% methylcellulose / 8% ethanol. Vehicle treated mice were administered the same volume of 0.4% methylcellulose / 8% ethanol alone. (a) The % change in body weight from baseline (day 0) is shown as the mean ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. (b) Comparison of zingerone (12.5 mg / kg)+ferulic acid (12.5 mg / kg) treatment to dexamethasone (5 mg / kg in 0.4% methylcellulose / 8% ethanol; daily by oral gavage). The % change in body weight from day 0 is shown as the mean ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. **** p<0.0001 by two-way ANOVA.

[0294] Figure 52: Collagen-specific IgG at day 14. Mice were immunised with collagen as indicated for Figure 47 and treated as indicated for Figure 51. Plasma was collected from mice on day 14 by cardiac puncture. Collagen-specific IgG was assessed by commercial ELISA (Chondrex). Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. Minimal collagen-specific IgG was detected in healthy animals. P values were calculated by Kruskal-Wallis with an uncorrected Dunn’s post test. **p<0.01 and *p<0.05.

[0295] Figure 53: Plasma IgM levels at day 7. Mice were immunised with collagen as indicated for Figure 47 and treated as indicated for Figure 51. Plasma was collected from mice on day 7 by tail bleed. IgM was assessed by ELISA. Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. P values were calculated by 1-way ANOVA with Fisher’s least significant differences post test. **p<0.01

[0296] Figure 54: Plasma IL-6 levels at day 7 (a) and day 14 (b). Mice were immunised with collagen as indicated for Figure 47 and treated as indicated for Figure 51. Plasma was collected from mice on day 7 by tail bleed and day 14 by cardiac puncture. IL- 6 was assessed by cytometric bead array (CBA). Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. P values were calculated by 1-way ANOVA with Fisher’s least significant differences post test. *p<0.05

[0297] Figure 55: Plasma IL-17A levels at day 7 (a) and day 14 (b). Mice were immunised with collagen as indicated for Figure 47 and treated as indicated for Figure 51.Plasma was collected from mice on day 7 by tail bleed and day 14 by cardiac puncture. IL- 17A was assessed by CBA. Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. P values were calculated by Kruskal-Wallis with an uncorrected Dunn’s post test. **p<0.01 and *p<0.05

[0298] Figure 56: Plasma IL-1^ levels at day 7 (a) and day 14 (b). Mice were immunised with collagen as indicated for Figure 47 and treated as indicated for Figure 51. Plasma was collected from mice on day 7 by tail bleed and day 14 by cardiac puncture. IL- 1^ was assessed by CBA. Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. P values were calculated by Kruskal-Wallis with an uncorrected Dunn’s post test.

[0299] Figure 57: Plasma monocyte chemoattractant protein (MCP)-1 levels at day 7 (a) and day 14 (b). Mice were immunised with collagen as indicated for Figure 47 and treated as indicated for Figure 51. Plasma was collected from mice on day 7 by tail bleed and day 14 by cardiac puncture. MCP-1 was assessed by CBA. Shown are the means ^ SEM of values from individual mice (n=8 / group) from 2 independent experiments. Comparison between zingerone alone and zingerone+ferulic acid P values were calculated by Mann-Whitney.

[0300] Figure 58: Summary of key disease-associated parameters indicating that the combination of zingerone+ferulic acid has a distinct pattern of benefits compared to either zingerone or ferulic acid alone. Shown is a heatmap using the mean values illustrated in Figures 52 and 54-57. The highest mean (white) was considered 100% effect and the lowest mean (black) considered 0% effect. DETAILED DESCRIPTION

[0301] The following description sets forth numerous exemplary configurations, parameters, and the like. It should be recognised, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments.

[0302] All references, including patents and patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Nor does discussion of any reference constitute an admissionthat such reference forms part of the common general knowledge in the art, in New Zealand or in any other country. Definitions

[0303] Where a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Thus, each range that is specified (e.g., 1 to 10) includes all possible combinations of numerical values between the lowest value and the highest value enumerated (e.g., 1, 1.1, 2, 3, 3.3, 4, 5.5, 6, 7, 8.9, 9 and 10) and also any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.9), and, therefore, all sub- ranges of all ranges expressly disclosed herein are hereby expressly disclosed. The numeric values provided in parentheses here are only examples of what is specifically intended and all possible combinations of numerical value between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure in a similar manner.

[0304] In each instance herein, in descriptions, embodiments, and examples of the present disclosure, the terms “comprising”, “including”, etc., are to be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as to opposed to an exclusive sense, that is to say in the sense of “including but not limited to”.

[0305] As used herein “and / or” means additionally or alternatively.

[0306] In the present description, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” can be taken to mean one element or more than one element.

[0307] Throughout this description, the term “about” is used to indicate that a value includes the standard deviation of error for the method being employed to determine the value, for example, levels of compounds or dosage levels, as described in detail herein. In particular, the term “about” encompasses up to a 10% deviation (positive and negative) in the stated value or range.

[0308] The term “comprising”, as used herein, may refer to the presence of a compound in a composition. As exemplifications, the compound may be at least 1%, at least 2%, at least 4%, at least 5%, at least 10%, at least 12%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90 by weight of the composition (% w / w). Alternatively, the compound may be at least 1%, at least 2%, at least 4%, at least 5%, at least 10%, at least 12%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by volume of the composition (%v / v or %w / v).

[0309] The term “consisting essentially of”, as used herein, may refer to the presence of a compound in a product. The product may be, for example, a composition as described herein, or may be, for example, a product produced by a method as described herein. As exemplifications, for solids, the compound may be at least 90% by weight of the product, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97, at least 98%, at least 99%, by weight of the product (% w / w). For liquids or semi-solids, the compound may be at least 90% by volume of the product, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% by volume of the product (%v / v or % w / v).

[0310] The term “substantially free” in relation to aldehydes refers to a product having negligible aldehyde levels. The product may be, for example, a composition as described herein, or may be, for example, a product produced by a method as described herein. As exemplifications, aldehyde levels may be less than 20 ppm, less than 15 ppm, less than 10 ppm, less than 7.5 ppm, less than 5 ppm, less than 2 ppm, less than 1.5 ppm, less than 1 ppm, less than 0.75 ppm, less than 0.5 ppm, less than 0.2 ppm, less than 0.1 ppm, less than 0.05 ppm, less than 0.005 ppm, or less than 0.0005 ppm.

[0311] The term “alkaline treatment” as used herein means the exposure of a sample (e.g., ginger, ginger juice, ginger marc, or any combination thereof) to an aqueous solution containing alkali having a pH greater than 7. Included, without limitation, are solutions comprising, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide and any combination thereof. It is to be appreciated that the alkaline treatment can occur at a range of temperatures as described herein and that the alkaline solution may be heated prior to or during exposure to the sample comprising ginger or anextract from ginger. The alkaline solution will have a chemically effective amount of alkali present to convert at least some gingerol present in the sample to zingerone. Particular methodologies are described in detail herein.

[0312] Generally speaking, an “extract” of this disclosure will refer to a botanical extract (also referred to as a “botanical drug”). More specifically, an “extract” refers to a composition where one or more liquid, solid, or chemical constituents of a plant or plant part has been isolated or concentrated. For example, a liquid, solid, or semi-solid extract may be obtained. An extract may be obtained by one or more of: juicing, pressing, macerating, mashing, milling, or other standard processes. Solvent-based extraction is also included. Solid extracts are specifically noted, for example, powders obtained from drying or evaporation. As specific exemplifications, an extract may be prepared as a dry form, or may be prepared in the form of a solution. A “zingerone extract” refers to an extract comprising zingerone, as prepared / produced from ginger root (i.e., ginger rhizome, which can also be referred to as “ginger”). Particular extracts and their production methods are described in detail herein.

[0313] The term “composition” as used herein encompasses a product comprising one or more active components (e.g., combinations of compounds as set out herein), and one or more suitable excipients comprising other ingredients. These may be physiologically acceptable excipients. Encompassed is any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the active components. In particular aspects, the composition may comprise any suitable solvate or salt of each compound. As specific examples, an extract of this disclosure can be prepared as a composition suitable for administration to a subject, or suitable for formulation for administration to a subject. Various exemplary compositions are described in detail herein.

[0314] A “pharmaceutical composition” refers to a composition administered to a subject, for example, to treat or prevent inflammation. A “dietary composition” refers to a composition to be ingested by a subject, for example, to alleviate or prevent inflammation.

[0315] The term “salt” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminium, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc,and the like. Noted are ammonium, calcium, magnesium, potassium, and sodium salts. Salts in the solid form may exist in more than one crystal structure and may also be in the form of hydrates. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylene-diamine, diethylamine, 2- diethylaminoethanol, 2-dimethylamino-ethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When a compound is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, ethanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Noted are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, fumaric, and tartaric acids.

[0316] The term “solvate” refers to an aggregate that consists of a solute ion or molecule with one or more solvent molecules. Solvates include hydrates, that is, aggregates of a compound of interest with water.

[0317] As used herein, “administration of” or “administering” refers to the providing at least one active component or a composition formulated to contain at least one active component to a subject. The administration can be by any suitable route and via any suitable formulation. In some cases, it may be useful to use different routes of administration and / or different formulations in the same subject. For example, one or more oral formulations may be used, or one or more topical formulations may be used, or one or more oral formulation may be used in conjunction with one or more topical formulations. Non-limiting exemplifications of routes of administration and formulations for administration are provided herein.

[0318] An “anti-inflammatory agent” refers to a constituent that mitigates one or more symptoms of inflammation. Included amongst these are pharmaceutical agents, phytochemical agents, plant components, plant extracts, and essential oils, as well astisanes and other infusions, along with various other constituents, to assist in the reduction of inflammation. Such may be utilised in combination with the composition(s) and compound(s) this disclosure, and may be used to assist in regulating an immune response.

[0319] “Co-administration” or “co-administering” refers to the combined use of active components, for example, for therapy or for cosmetic enhancement, and includes the administration of co-formulations (i.e., combination formulations), as well as the simultaneous, sequential, or separate administration of distinct formulations.

[0320] Similarly, “in conjunction” refers to the combined use of one or more active components and a device / procedure. This can include use of the active component(s) preceding use of the device / procedure, simultaneously with the device / procedure, and / or following use of the device / procedure.

[0321] As used herein, “simultaneous” administration means that an active component and at least one other additional active component are administered to a subject together or essentially together. This may be, for example, administration by the same route or by different routes, and may be, for example, administration in a single dose (e.g., via co- formulations).

[0322] As used herein “separate” administration means that an active component and at least one other active component are administered to a subject by different means. This may be, for example, administration by two different routes (i.e., via different formulations). As an exemplification, this may occur where one active component is administered by infusion and at least one other active component is administered orally during the infusion or before or after the infusion.

[0323] As used herein “sequential” administration means that an active component and at least one other active component are administered at different points in time. As an exemplification, this may occur where one active component is administered via one route (e.g., orally) before or after administration of another active component via another route (e.g., infusion). Alternatively, this administration may be by the same route (i.e., via same formulations). For example, this may occur by oral administration of one active component followed by oral administration of another active component.

[0324] The term “inflammation” includes any degree of inflamed tissue in a subject that persists for any period of time. Particular exemplifications include acuteinflammation or chronic inflammation. The inflammation may be associated with pain. The inflammation may be that of the joint, skin, lung, heart, circulatory system, digestive tract, genitourinary tract, etc. These and other types of inflammation are encompassed herein.

[0325] A “symptom” of inflammation includes one or more of: pain, heat, redness, swelling, and loss of function. Also noted as markers of inflammation are the production of proinflammatory cytokines (e.g., IL-6, TNF-α) and / or proinflammatory small molecules (e.g., NO), as well as the activation and / or accumulation of immune cells.

[0326] As used herein, a “subject” may be a human or non-human animal, particularly a mammal, including cattle, sheep, goats, pigs, horses, and other livestock, including, as well, dogs, cats, and other domesticated pets. In particular aspects, the subject is a human being.

[0327] “Preventing” as used herein refers to halting or delaying the onset or progression of inflammation or a disorder involving inflammation. A preventative measure may result in the stoppage or delay of development of the inflammation or the disorder, or its symptom(s), a prevention of progression of the inflammation or the disorder, or its symptom(s), or a lessening of the developed inflammation or disorder, or its symptom(s), if such happen to arise. A preventative measure may also act in supporting, maintaining, and / or protecting of a bodily system. It should be understood that the term “treating or preventing” does not exclude the possibility of obtaining both treatment and prevention of the disorder. A “therapeutic” effect or “therapeutic” method may include treatment, or prevention, or both.

[0328] “Treating” as used herein refers to ameliorating or resolving inflammation or a disorder involving inflammation. A treatment will result in the reduction, e.g., amelioration or resolution, of the inflammation or the disorder, or one or more symptoms of the inflammation or the disorder. Resolution in the context of a treatment includes partial or complete reversal of the inflammation or the disorder, or its symptom(s). Partial or complete healing is encompassed by this, e.g., an improvement in one or more relevant health parameters. A treatment can include a lessening in the expression of the inflammation or the disorder, or its symptom(s). A treatment may also suppress existing inflammation / inflammatory disorder or its symptom(s), or put existinginflammation / inflammatory disorder or its symptom(s) into remission. In the context of “treating”, the healing of wounds and rashes is specifically noted.

[0329] “Alleviation” refers to ameliorating inflammation or a disorder involving inflammation. An alleviation will result in the reduction, e.g., amelioration, of the inflammation or the disorder, or one or more symptoms of the inflammation or the disorder. Healing is specifically encompassed, e.g., an improvement in one or more relevant health parameters. An alleviation includes a lessening in the expression of the inflammation or the disorder, or its symptom(s). An alleviation may also be an action in suppressing existing inflammation / inflammatory disorder or its symptom(s). Alleviation of the inflammation of wounds and rashes is specifically noted.

[0330] The term “effective amount” refers to a sufficient quantity of the active component(s), in a suitable composition, and in a suitable dosage form to treat or prevent a noted disorder, or at least one symptom thereof. The “effective amount” will vary depending on the component(s) used, the type of therapy, and the species, age, weight, heath, etc, of the subject to be treated.

[0331] “Combination” refers to combined use of two or more components (e.g., two or more active components). Usage may be by co-formulated components (i.e., combination formulations), or by simultaneous, sequential, or separate use of components (e.g., via different formulations, same formulations, or co-formulations). These and other specific combinations are encompassed in this disclosure. Compounds and combinations

[0332] The applicant has determined that combinations of compound I (e.g., zingerone or acetyl zingerone) and compound II (e.g., ferulic acid) provide significantly increased anti-inflammatory activity. Therefore, the present disclosure relates generally to these particular combinations, and compositions comprising these combinations, and methods of preparation and use of such.

[0333] In one aspect, the combination of this disclosure comprises compound I and compound II, these compounds being of formula I and formula II, respectively:, wherein: R1 is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3; R2is -H, or when taken together with R4and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3is -H or when taken together with R2forms =O; and R4 is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, andwherein: R10 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R11 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R12 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R13 is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds.

[0334] The combination may consist essentially of compound I and compound II.

[0335] As exemplifications, compound I may be selected from the group consisting of:acetyl zingerone zingeronezingerol ethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone 4-hydroxy-3-methoxycinnamic acid ethyl ester6-gingerolhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acidconiferyl aldehyde eugenol4-hydroxy-3-methoxybenzoic acid and any salts, solvates, hydrates, and protected forms thereof.

[0336] Of particular note are zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

[0337] As exemplifications, compound II may be selected from the group consisting of:ferulic acid hydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatevanilpyruvic acid methyl-3-methoxy-4-hydroxystyryl ketone3,4,5-trihydroxycinnamic acid sinapic acidcinnamic acid p-coumaric acid and any salts, solvates, hydrates, and protected forms thereof.

[0338] Of particular note are ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

[0339] In various aspects, the compounds of this disclosure may be synthetic or naturally occurring. The disclosed compounds may be provided as a botanical extract. The botanical extract may comprise of one or more of the disclosed compounds. For example, the botanical extract may comprise zingerone, as described herein. In addition, the botanical extract may be produced by methodology as described herein. It is expected that, in a specific combination, compound I and compound II will be different compounds.

[0340] The compounds of this disclosure, including compound I and compound II, may be utilised as any pharmaceutically acceptable salts. Examples include acid addition salts of strong mineral acids such as HCl and HBr salts and addition salts of strong organic acids such as a methanesulfonic acid salt. Further examples of salts include sulphates and acetates such as trifluoroacetate or trichloroacetate. Other examples are provided herein. Salts a solid form may exist in more than one crystal structure and may also be in the form of hydrates.

[0341] The compounds of this disclosure may be formulated as prodrugs. For example, amino groups may be protected with a group which can be cleaved in vivo, to liberate the biologically active compound. In particular, the prodrug may be an amine prodrug. Examples of amine prodrugs include sulphomethyl, HSO3-FMOC, and salts thereof, as well as others. See, e.g., Bergen et al., Antimicrob Agents and Chemotherapy, 2006, 50, 1953; Schechter et al., J.Med Chem 2002, 45(19) 4264; Krise and Oliyai in Biotechnology: Pharmaceutical Aspects, 2007, 5(2), 101-131. The compounds of this disclosure may be formulated as solvates. Examples of solvates include hydrates.

[0342] The compounds of this disclosure may be prepared such that one or more atom is replaced by a naturally occurring or non-naturally occurring isotope. In one aspect,the isotope may be a stable isotope. For example, a compound may include deuterium. As exemplifications, H may be in any isotopic form, including1H,2H (D), and3H (T); C may be in any isotopic form, including12C,13C, and14C; O may be in any isotopic form, including16O and18O; and the like.

[0343] The compounds of this disclosure may be provided as one or more particular forms, for example, geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans- forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a-and b-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half chair-forms; and any combination thereof.

[0344] Specifically noted are isomers or isomeric forms, which include all of the above forms, except tautomeric forms. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are readily obtained by utilising standard methods, or by adapting the methods taught herein.

[0345] The compounds disclosed herein may be utilised in protected forms. Thus, one or more functional groups within compound may be provided with a protecting group to prevent their unintended reaction, for example during synthesis or storage. For example, it may be convenient or desirable to prepare, purify, and / or handle a compound in a chemically protected form. By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, e.g., Green and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley and Sons, 1999.

[0346] The compounds of this disclosure may be utilised in a substantially purified form and / or in a form substantially free from contaminants. For example, the substantially purified form may be at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90% by weight, e.g., at least 95% byweight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight. In certain aspects, the contaminants may represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1 % by weight.

[0347] In certain aspects, the compounds of this disclosure may be provided with a kit. The kit may include compound I premixed with or separate from compound II. The kit may provide the disclosed compounds as pharmaceutical compositions or dietary compositions. The kit may include compound I in the same formulations for administration as compound II or in different formulations for administration as compound II. The kit may include compound I and compound II co-formulated for administration. For example, The kit may include compound I and / or compound II formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule. The kit may include compound I and / or compound II formulated to be provided in a sachet. The disclosed compounds (e.g., formulated as composition(s)) may be provided in one or more containers in the kit. Additional components may also be provided with the kit, for example, one or more buffers or one or more excipients, intended for use with one or more of the disclosed compounds. Optionally, instructions may be provided with the kit, as well as any other item, such as any number of containers, labels, or medical tools, including bottles, pads, syringes, tubes, etc. The instructions for the administration of a pharmaceutical composition may include information as to dosage, dosing schedule, routes of administration, amongst other information. The instructions for utilisation of a dietary composition may include information as to recommended use. The kit may comprise a description of selecting a subject suitable for treatment based on identifying whether that subject has inflammation or an inflammatory disorder, or a symptom of such, or is at risk of having such. The kit may include one or more reagents determining levels of the compound(s) in the subject. In this way, the subject may be assessed for the response to any therapies being utilised. Obtaining compounds

[0348] The compounds of this disclosure may be obtained by various methods. The compounds may be synthetic or naturally occurring. The compounds may be provided by commercial suppliers. The compounds may be provided in botanical extracts. Forexample, a botanical extract may comprise compound I (e.g., zingerone) or compound II (e.g., ferulic acid). Alternatively, a botanical extract may comprise compound I (e.g., zingerone) or compound II (e.g., ferulic acid).

[0349] In certain aspects, zingerone may be obtained by subjecting the ginger root to a particular treatment and obtaining an extract comprising zingerone. For example, alkaline treatment of ginger root may be utilised in accordance with WO 2023 / 018338, which is hereby incorporated by reference herein. Exemplary methods are also provided herein. Other preparation methods include: biotransformation of dehydrozingerone to zingerone, e.g., utilising filamentous fungi (see, for example, Svetaz et al., 2014, Int J Mol Sci, 15(12):22042-22058), chemical synthesis of zingerone, e.g., starting from 3-(4- hydroxy-3-methoxyphenyl)-N-methoxy-N-methylpropanamide (see, for example, Sanders and Seidel, 1992, J Agr Food Chem, 40(2):263-265), retro aldol condensation reactions utilising gingerol (see, for example, Mangal et al., 2022, Front Chem 10:https: / / doi.org / 10.3389 / fchem.2022.902719), amongst others.

[0350] In specific aspects, ferulic acid may be obtained from one or more of: commelinid plants (e.g., grasses, rice, wheat, oats, palms), vegetables (e.g., spinach, beets), flowers, fruits, leaves, beans, seeds of coffee, artichoke, peanuts, and other nuts (see, e.g., Antonopoulou et al., 2021, Front Nutr.8: 777576). If ferulic acid is free esterified and not bound on the cell wall matrix, a direct extraction step can be applied in order to recover the valuable compound. Efficient extraction with green solvents has been reported, such aqueous ethanol solution (e.g., Sun and Wang, 2008, Journal of the Chinese Institute of Chemical Engineers, 39:653-6) or hot water (e.g., Pazo-Cepeda et al., 2021, Food Biosci, 44:101374). Physical activation, such as ultrasound or microwave, may enhance extraction yields. If ferulic acid is encased in the cell wall matrix, pretreatment methods are usually applied, such as alkaline hydrolysis or enzymatic hydrolysis. For example, ferulic acid can be released enzymatically using feruloyl esterases (e.g., Buranov and Mazza, 2009, Food Chemistry, 115(4): 1542-1548). After pre-treatment, ferulic acid may then be purified from enzymatic solution by the use of activated charcoal (e.g., Ou et al., 2007, Journal of Food Engineering, 78(4):1298-1304), ethanol washing (e.g., Sibhatu et al., 2021, Lwt, 135:110009), resins (e.g., Dupoiron et al., 2017, Industr Crops Prod, 105:148-55), magnetic nanoparticles (e.g., Qu et al., 2017, Sep Sci Technol, 52:1022-30), membrane filtration (e.g., Couteau and Mathaly, 1997, Ind Crops Prod, 6:237-52; Domingos et al.,2020, Sep Purif Technol, 242:116755), and chromatographic methods, such counter- current high performance liquid chromatography (e.g., Tang et al., 2021, J Chromatogr A, 1636:461772).

[0351] Commercially available sources may also be utilised. In regard to zingerone, suppliers include, for example, Vigon International, BOC Sciences, ECHEMI, ChemDirect, Sigma-Aldrich, and others. Commercial suppliers for ferulic acid include, for example, Vingon International, Shaanxi Guanjie Technology / Guanjie Biotech, Feng Zhi Jin, and others.

[0352] In one particular aspect, this disclosure provides a method of producing zingerone. The inventors have found that zingerone compositions prepared from ginger root in accordance with the disclosed methods have significant anti-inflammatory activity, which exceed the activity of commercially available zingerone compositions. Therefore, the present disclosure provides zingerone compositions prepared from ginger root, and methods of preparation of such.

[0353] In one aspect, the zingerone may be obtained from ginger root by subjecting the ginger root to an alkaline treatment. The alkaline treatment may include incubation in an alkaline solution as described herein. As starting material, the ginger root may be fresh ginger root. For example, to assist with preparation, it may be helpful to optimise the period of time that the ginger root is retained in the soil prior to harvesting. In this way, the ginger root that is utilised will be fresh and will retain the advantageous characteristics of fresh ginger root.

[0354] The disclosed preparation methods produce a highly efficacious botanical extract. As exemplifications, to optimise freshness, the ginger root may be harvested less than 48 hours before processing, less than 24 hours before processing, or less than 12 hours before processing, or less than 6 hours before processing, or less than 3 hours before processing. For example, fresh ginger may have a moisture content of about 80% to about 95%, about 81% to about 95%, or about 82% to about 95%, or about 83% to about 95%, or about 85% to about 95% on a wet basis.

[0355] Alternatively, the ginger root may be dried prior to treatment. For example, The ginger root may be dried at about 40° to about 70°C, or at about 55° to about 65°C, or at about 60°C. Drying may be carried out for about 1-72 hours, or about 1-48 hours, orabout 1-24 hours, or about 1-20 hours, or about 1-18 hours, or about 1-10 hours, or about 1-5 hours.

[0356] In certain aspects, the ginger root selected for use in the disclosed methods may have a minim level of gingerol, e.g., 6-gingerol. For example, the ginger root (e.g., fresh ginger root) may have about 0.3 to about 10 mg / g, or about 0.3 to about 9 mg / g, or about 0.3 to about 8 mg / g, or about 0.3 to about 7 mg / g, or about 0.3 to about 6 mg / g, or about 0.4 to about 5 mg / g of 6-gingerol. As further exemplifications, the ginger root may have at least 1 mg / g, at least 2 mg / g, at least 3 mg / g, at least 4 mg / g, or at least 5 mg / g of 6-gingerol. Thus, in certain circumstances, it may be advantageous to test levels of gingerol, e.g., 6-gingerol, in the starting material before commencing a method as disclosed herein.

[0357] As one aspect, the method comprises subjecting juice and / or marc from the ginger root to an alkaline treatment. The ginger juice and / or ginger marc may be obtained by macerating and / or pressing. The macerating may comprise homogenising with a blender, food processor, or similar machinery. For pressing, machine or hand presses may be utilised. Screw pressing is specifically noted. The solid material remaining after juicing (ginger marc) may be re-juiced to obtain ginger juice. This may be repeated as needed. The various juice samples and marc samples may be combined before alkaline treatment, e.g., juice sample A + juice sample B, or marc sample A + marc sample B, or juice sample A, B + marc sample A, B.

[0358] Optionally, the ginger marc may be subjected to hot water treatment to obtain a diluted juice. For example, water may be added to the marc at a ratio of about 6 to about 1 (~6:1), or about 5 to about 1 (~5:1), or about 4 about 1 (~4:1), or about 3 to about 1 (~3:1) by weight. The water may be, for example, at about 40°C to about 80°C, or at about 50°C to about 70°C, or at about 55°C to about 65°C, or at about 60°C. The incubation time in the water may be about 5 minutes to about 60 minutes, or about 10 minutes to about 30 minutes, or about 15 minutes to about 20 minutes, or about 15 minutes. The diluted juice samples may then be subjected to alkaline treatment. The diluted juice samples may be combined with other juice samples before alkaline treatment.

[0359] In one aspect, potassium hydroxide (KOH) may be used in the alkaline treatment. For example, a solid form of KOH may be used, e.g., KOH pellets. As an exemplification, the solid form of KOH may be at about 100% starting concentration, orat about 90% to about 100% starting concentration. Alternatively, a liquid form of KOH may be used in the alkaline solution. As an exemplification, the liquid form of KOH may be about 50% starting concentration, or about 40% to about 60%, or about 45% to about 55%. The concentration of KOH used in the treatment mixture (e.g., final concentration) may be, for example, about 0.1% to about 6%, or about 0.5% to about 5.5%, or about 1% to about 6%, or about 1.5% to about 5.5%, or about 2% to about 4%, or about 1.5% to about 3.5%, or about 2% (v / v). As an alternative to this, calcium hydroxide Ca(OH)2may be used in the alkaline treatment. For example, a liquid form of Ca(OH)2may be used in the alkaline solution. The concentration of Ca(OH)2 used in the treatment mixture may be, for example, about 0.5% to about 4%, about 1.5% to about 3.5%, about 1% to about 2%, or about 3.0% (v / v). Liquid forms may include, for example, stock solutions of about 25% to about 65%, or about 30% to about 60%, or about 35% to about 55%, or about 45% to about 55%, or about 50%.

[0360] In certain aspects, the alkaline treatment may achieve a pH level for the treatment solution of about pH 9 to about pH 14, or about pH 9.5 to about pH 13.5, or about pH 10 to about pH 13.5, or about pH 10.5 to about pH 13.5, or about pH 11.5 to about pH 13.5, or about pH 12.5 to about pH 13.5, or a pH of at least 13. The alkaline treatment may be carried out for a sufficient time and at a sufficiently elevated temperature to obtain desired levels of zingerone. For example, the alkaline treatment may be carried out for about 1-72 hours, or about 1-48 hours, or about 1-24 hours. Further examples include treatment for about 1 to about 30 hours, or about 1 to about 20 hours, or about 1 to about 10 hours, or about 1 to about 15 hours, or about 1 to about 7 hours, or about 1 to about 6 hours, or about 1 to about 5 hours, or about 1 to about 4 hours, or about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours, or at least 1 hour, or about 2 hours, or about 1 hour. As particular examples, the alkaline treatment may be carried out at about 40° to about 70°C, or about 50° to about 60°C, or about 55° to about 65°C, or about 60°C. It will be understood that lower temperatures can allow for longer treatment periods. For example, alkaline treatments performed at room temperature can carried out for about 3 days to about 9 days, or for about 5 days to about 9 days, or for about 5 days to about 7 days.

[0361] Following alkaline treatment, the treatment mixture may be further processed, for example, by one or more of: neutralisation, extraction, and drying. Forneutralisation, citric acid or other acid composition may be utilised. As exemplifications, neutralisation may achieve a pH of about 6.4 to about 7.4, or about 6.5 to about 7.5, or about 6.6 to about 7.6, or about 6.9 to about 7.4, or about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. For example, about 10 to about 700 g / L citric acid may be utilised, or about 10 to about 600 g / L, or about 10 to about 500 g / L, or about 10 to about 400 g / L, or about 10 to about 300 g / L, or about 10 to about 200 g / L, or about 10 to about 100 g / L, or about 10 to about 50 g / L, or about 10 to about 40 g / L, or about 10 to about 30 g / L, or about 10 to about 20 g / L, or about 15 to about 16 g / L citric acid may be utilised.

[0362] For extraction, zingerone extraction may be achieved by one or more alcohol extractions, e.g., one or more ethanol extractions. As exemplifications, an alcohol extraction, e.g., ethanol extraction, may be carried out for about 1-72 hours, or about 1-48 hours, or about 1-24 hours, or about 6-24 hours, or about 8-24 hours, or about 12-24 hours, or about 18-24 hours. In one specific aspect, alcohol extraction may be carried out for 24 hours or less, for example, for at least 4 hours, or for about 4 to about 12 hours, or for about 4 to about 8 hours. The alcohol extraction may be carried out, for example, at about 35°C to about 65°C, or at about 45°C to about 55°C, or at least 50°C, or at about 50°C.

[0363] One or more drying steps may be used before and / or after extraction (e.g., alcohol extraction. For example, freeze drying or heat drying may be utilised. In particular, the amount of ethanol in the extract composition may be reduced by air drying, rotary evaporation, lyophilisation, or other techniques. With certain drying methods (e.g., rotary drying), the temperature and pressure can be increased to remove residual liquid(s). For example, drying can be carried out under vacuum, for example, at 50 mBar or less, 45 mBar or less, or 40 mBar or less. As further examples, drying can be carried out at about 35°C to about 50°C, or at about 35°C to about 45°C, or at least 30°C, or at least 35°C, or at least 40°C, or at about 40°C. Where the material is dried after neutralisation but before alcohol extraction, drying may be carried out, e.g., for at least 18 hours, or at least 24 hours, or at least 28 hours, or at least 30 hours, or about 18 to about 28 hours, or about 24 to about 28 hours. The drying may be carried out, for example, at about 45°C to about 75°C, or at about 55°C to about 65°C, or no more than 60°C, or at about 60°C. Other alternatives for drying are described herein.

[0364] As part of the initial processing, the ginger root may be washed or sterilised. The plant component (e.g., fruit or seed) may be passed through an assembly having one or more roller brushes for removing any adhering foreign matter. Conventional washing techniques may then be employed. For example, it is possible to use a series of spray nozzles to wash the components. Wash additives aiding cleansing or reducing the microbial count on the plant components may be employed according to local regulations and requirements. For example, the plant components may be washed by a chlorine wash and / or an ozone impregnated water wash followed by a fresh water rinse.

[0365] As noted, it may be desirable for a liquid or semi-solid zingerone composition to be prepared from ginger root. As described herein, zingerone components may be extracted by chemical means (e.g., solvent-based extraction). Solvent-based extraction may utilise one or more of: water, methanol, ethanol, or 2-propanol, extraction. Supercritical fluid extraction, for example CO2extraction may also be used to extract zingerone. Also suitable are emulsions, pastes, suspensions, and syrups. For example, in certain aspects, it may be desirable to use a paste from the ginger root or from the ginger root component (e.g., zingerone or zingerone extract). As an exemplification, the ginger root may be heated for several hours, strained, and reduced to a thick, concentrated form. Upon thickening, the paste can be spread on a flat sheet, or transferred to a packaging, for example, a bag, tube, jar, bottle, or other container. The paste may be transferred aseptically. It may be desired to prepare the paste from mature plant components. The paste may be a smooth preparation.

[0366] In specific aspects, the present disclosure encompasses mechanical means (e.g., juicing means such as maceration and / or pressing) for extracting zingerone from ginger root. In one embodiment, a pressing assembly may be adapted to perform a pulping or comminution process. Such process can be relatively mild and gentle (soft pulping) compared to conventional fruit pulping techniques. With soft pulping, no significant disintegration or lysis of cells is utilised. The press belts may be multiple loops rotated about a series of pulleys. The distance separating the press belts may decrease in the direction of travel of the plant component. In this way, increased force may be exerted upon the plant component as it travels along the length of the pressing assembly. In a particular aspect, a pressing assembly or mechanical press may be used to obtain juice from the ginger root, as described herein. Alternatively, or in addition to this, mechanicalmaceration may be used to obtain juice. For example, commercial juicing equipment may be utilised.

[0367] The ginger root component (e.g., zingerone or zingerone extract) may be processed by a freezing step. This may be followed by or used in conjunction with a drying or evaporation step. In an alternative embodiment, the component is dried or evaporated, and then processed to a powder without an intervening freezing step. Methods involving air drying or heat-assisted drying (e.g., oven drying) may be used. Drying may be obtained, for example, by one or more of: sun or solar drying, hot air drying, batch drying, rotary drying, tunnel drying, belt drying, fluidised bed drying, impingement drying, puff drying, drum drying, spray drying, vacuum drying, freeze drying, or osmotic drying. Exemplary temperatures for drying include about 50°C-70°C, about 55°C-65°C, or at least 50°C, at least 55°C, at least 60°, or at least 65°C. Evaporation may be obtained, for example, by one or more of: pan evaporation, batch evaporation, tube evaporation, rising film evaporation, falling film evaporation, rising-falling film evaporation, or agitated film evaporation. Combinations of various drying and evaporation methods may be used. For example, filtering followed by freeze drying may be used.

[0368] If freezing is used, it may be desirable to freeze the ginger root component (e.g., zingerone or zingerone extract) as soon as possible after it is produced to maintain freshness. However, freezing may be carried out within 24 or 48 hours, as needed. Standard freezing methodologies may be utilised. Blast freezing is particularly desirable for use with the present disclosure. The component may be frozen in standard sized pales, which are used to collect the frozen product after processing. The component, for example, can be stored frozen (e.g., at -18°C) until it is required. Optionally, the component may then be freeze dried, i.e., lyophilised. Freeze drying techniques are widely used. The freeze-drying cycle may be up to 48 hours. In particular aspects, the process may be carried out to such that water formation is avoided, and the moisture content is minimised during processing. It will be understood that freeze drying / lyophilising does not exclude the use of higher temperatures (i.e., higher than freezing temperatures). For example, higher temperatures may be used for removing residual moisture during the secondary drying phase for lyophilisation / freeze drying procedures.

[0369] The resulting dried or evaporated component from ginger root (e.g., zingerone or zingerone extract) may then be milled into a powder, which can then beutilised as appropriate. Standard milling methods may be utilised. Standard mesh sizes may be used to produce the powder, for example, US 20, US 23, US 30, US 35, US 40, US 45, or US 50 mesh sizes may be used. The sieve size for the powder may range from 1.0 to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or may be about 1.0 mm, about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47 mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm.

[0370] The composition (e.g., containing compound I and / or compound II) may be prepared as a pharmaceutical composition. The composition can also be prepared as a dietary composition, for example, a functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement). In various aspects, the composition may be prepared in liquid or solid form, or semi-solid form. Various formulations are encompassed in this disclosure. In certain aspects, it may be desirable to formulate the composition into a powder. The powder may be provided in free flowing form or as a solid cake. The composition may be provided as a powder for forming a suspension, powder for forming a solution, bulk granules, or bulk powder. The powder may be prepared as tablets or capsules, or other formulations, as described in detail herein.

[0371] It will be understood that, for any liquid or semi-solid product obtained from ginger root, the liquid / semi-solid may be used in this form or may be dried or evaporated to obtain a powder form for use as a pharmaceutical composition or dietary composition, as described herein. In the same way, it will be understood that, for any solid product obtained from ginger root, the solid may be used as such (e.g., with milling, sieving, or other processing), or may be re-suspended to obtain a liquid or semi-solid form for use as a pharmaceutical composition or dietary composition, as described herein. Compositions

[0372] The applicant has found that the utilisation of compound I (e.g., zingerone or acetyl zingerone) in combination with compound II (e.g., ferulic acid) provides significantly improved anti-inflammatory and immunomodulating activities that are useful for reducing cellular inflammation markers, reducing tissue damage, antioxidant action, as well as for treating or preventing inflammation and inflammatory disorders in a subject.

[0373] Accordingly, compositions (e.g., pharmaceutical compositions, dietary compositions) that allow the combined use of compound I and compound II are particularly advantageous in addressing inflammation and various inflammatory disorders. Combineduse may be achieved through co-formulations or through formulations being used together. For example, compound I and compound II may be used together as different formulations (e.g., for oral and infusion administration) or may be used together as the same formulations (e.g., both for oral administration).

[0374] In particular aspects, the compositions may be formulated to allow for simultaneous, separate, or sequential administration of compound I and compound II. As examples, compound I and compound II can both be formulated for oral administration (i.e., same formulations) and given simultaneously or sequentially to a subject. Compound I and compound II can also be co-formulated for oral administration and given to a subject. Alternatively, compound I can be formulated for oral administration, while compound II can be formulated for infusion (i.e., different formulations) and given simultaneously or sequentially to a subject. As a further example, compound I can be formulated for infusion, while compound II can be formulated for oral administration (i.e., different formulations) and given simultaneously or sequentially to a subject. It will be understood that various forms for administration can be combined and utilised in accordance with the present disclosure.

[0375] The present disclosure encompasses various formulations, including pharmaceutical compositions and dietary compositions. As non-limiting examples, the percentage of the compound in the composition may be about 0.01% to about 30%, or about 1% to about 30%, or about 1% to about 15%, or about 1% to about 10%, or about 1% to about 9%, or about 1% to about 8%, or about 0.1% to about 7%, or about 0.1% to about 6%, or about 0.1% to about 5%, or about 0.1% to about 4%, or about 0.1% to about 4%, or about 0.1% to about 3%, or a percentage of at least about 1%, at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, or at least about 50%, or a percentage of about 6.25%, about 12.5%, or about 25%, these percentages being representative of v / v values for a liquid composition, or w / w values for a solid composition, or w / v values for a liquid or semi-solid composition. In any of the various forms disclosed herein (e.g., liquid, solid, semi-solid, etc), the composition may be free from or substantially free from aldehydes.

[0376] As exemplifications, a solid may include about 0.5 to about 300 mg / g compound I or compound II, or about 1 to about 150 mg / g compound I or compound II, orabout 1 to about 100 mg / g compound I or compound II, or about 1 to about 80 mg / g compound I or compound II, or about 1 to about 60 mg / g compound I or compound II, or about 1 to about 50 mg / g compound I or compound II, or about 1 to about 40 mg / g compound I or compound II, or about 1 to about 20 mg / g compound I or compound II, or about 1 to about 10 mg / g compound I or compound II, or about 12 mg / g compound I or compound II, or about 10 to about 60 mg / g compound I or compound II, or about 10 to about 50 mg / g compound I or compound II, or about 10 to about 40 mg / g compound I or compound II, or about 10 to about 30 mg / g compound I or compound II, or about 10 to about 20 mg / g compound I or compound II, or about 10 to about 15 mg / g compound I or compound II, or at least about 50 mg / g, or at least about 40 mg / g, or at least about 20 mg / g, or at least about 10 mg / g compound I or compound II (w / w).

[0377] Similarly, as further exemplifications, a liquid or semi-solid composition may include about 0.5 to about 300 mg / ml compound I or compound II, or about 1 to about 150 mg / ml compound I or compound II, or about 1 to about 100 mg / ml compound I or compound II, or about 1 to about 80 mg / ml compound I or compound II, or about 1 to about 60 mg / ml compound I or compound II, or about 1 to about 50 mg / ml compound I or compound II, or about 1 to about 40 mg / ml compound I or compound II, or about 1 to about 20 mg / ml compound I or compound II, or about 1 to about 10 mg / ml compound I or compound II, or about 12 mg / ml compound I or compound II, or about 10 to about 60 mg / ml compound I or compound II, or about 10 to about 50 mg / ml compound I or compound II, or about 10 to about 40 mg / ml compound I or compound II, or about 10 to about 30 mg / ml compound I or compound II, or about 10 to about 20 mg / ml compound I or compound II, or about 10 to about 15 mg / ml compound I or compound II, or at least about 50 mg / ml, or at least about 40 mg / ml, or at least about 20 mg / ml, or at least about 10 mg / ml compound I or compound II (w / v).

[0378] In various aspects, topical compositions may be prepared, for example: for use on hands (e.g., hand creams), pre-operative tissue (e.g., surgical preparations for skin), mucous membranes (e.g., treatments for bladder, urethral, or vaginal inflammation, or cleansing of these cavities prior to medical procedures), wounds or burns (e.g., ointments, bandages, or dressings), mouth or throat (e.g., mouthwashes or lozenges), or eye (e.g., eye drops or ointments).

[0379] As non-limiting examples, topical compositions may include one or more of: diluents (e.g., ethanol or other alcohol), emollients (e.g., PEG-45, palm kernel glycerides, or isopropyl myristate), humectants (e.g., glycerine or methylpropanediol), carriers (e.g., one or more oils), occlusive agents (e.g., mineral oil or dimethicone) other conditioning agents (e.g., behentrimonium methosulfate or polyquaternium-7), and surfactants (e.g., mild surfactants (e.g., amphoacetate, isethionate, sulfosuccinate, in particular, sodium lauroamphoacetate, sodium cocoyl isethionate, disodium oleoamido sulfosuccinate, sodium lauryl sulfate, sodium C14-16 olefin sulfonate). Exemplary oils include olive oil, coconut oil (e.g., coconut-derived MCT oil), palm oil (e.g., palm kernel- derived MCT oil), any other MCT oil (medium-chain triglyceride oil), and any combination thereof. Other possible carriers include lecithin (e.g., liquid form) and propylene glycol. Any combination of the carriers set out herein is also noted.

[0380] In yet other aspects, the compositions may be prepared for various routes of administration, including oral formulas. Also included are compositions prepared for other routes of enteral or parenteral administration. Enteral formulations include but are not limited to: oral, rectal, sublingual, sublabial, and buccal preparations. Parenteral formulations include but are not limited to: nasal, intraocular, vaginal, intralesional, transdermal, and transmucosal preparations. Standard methods are available for formulating pharmaceutical compositions. See, e.g., Remington: Essentials of Pharmaceutics, 2013, Pharmaceutical Press, London.

[0381] In particular aspects, the compositions of this disclosure may be prepared as a powder, or in any other suitable dosage form. Topical formulations may be prepared, for example, as aerosols, balms, creams, dressings, drops, emulsions, films, foams, gels, jellies, liquids, lotions, masks, oils, ointments, pastes, powders, salves, soaps, sprays, suspensions, solutions, tinctures, and vapours. Further topical formulations include bandages, dressings, patches, pads, sponges, strips, tapes, and others noted herein.

[0382] As described herein, the compositions may be formulated as a semi-solid or liquid composition, for example, for oral administration (e.g., taken directly by mouth or via inclusion in capsules or in other forms), or for enteral or parenteral administration (e.g., taken by injection, feeding tube, or in other forms). Alternatively, the composition may be formulated as a powder to be encapsulated, tableted, or added to or incorporated in other products.

[0383] Oral formulations may be prepared, for example, as draughts, drops, elixirs, emulsions, liquids, linctuses, solutions, sprays, suspensions, syrups, tonics, or, as films, gels, gummies, jellies, lozenges, nuggets, pastes, purees, pomaces, powders, pills, or strips. In other aspects, oral formulations may be prepared as tablets or as capsules, for example, with liquid, semi-solid, or solid contents. Oral formulations may be provided in sachet form, for example, a powder sachet, or a gel or jelly sachet. Included also are oral formulations comprising thin strips, or comprising solids in a capsule to mix with food or drink. The oral formulation may be provided as shooters or shots (to be consumed by mouth), for example, liquid shots, gel, or jelly shots, paste shots, or powder shots.

[0384] Particularly encompassed are delayed release formulas, extended release formulas, as well as formulas for rapid disintegration. Capsules, for example gel capsules, are specifically encompassed, as well as sachets and chewable tablets. Additionally, included are combination formulas, which include the powder of the present disclosure mixed with other beneficial agents, e.g., one or more anti-inflammatory agents. Other formulas are also possible, as described herein.

[0385] The dissolution time for an oral formulation can be modified for a rapid effect or for sustained release. Oral formulations may also contain a mixture of slow and fast release particles to produce rapid and sustained absorption in the same dose. Special coatings can be used with oral formulations such as tablets and capsules to impart resistance to stomach acids. Oral formulations can also be coated with sugar, varnish, or wax to improve taste.

[0386] Thus, tablets may be prepared as rapid dissolve tablets and capsules may be prepared as extended release capsules. The tablets may be scored tablets, chewable tablets, effervescent tablets, orally disintegrating tablets, or tablets for forming a suspension. The capsules may be gel capsules, for example, and may include solid, semi- solid, or liquid contents. This includes gel capsules made by single piece gel encapsulation and two piece gel encapsulation. Hard shell capsules and soft shell capsules are specially noted. Non-gelatine capsules are also noted, as well as caplets.

[0387] It will be understood that certain formulations will be suitable for topical or other applications. Particular formulations of interest are: eye formulas (e.g., drops, ointments), ear formulas (e.g., drops, ointments), nasal or airway formulas (e.g., drops, sprays, insufflation compositions, inhalation compositions, nebulisation compositions),skin formulas (e.g., soaps, sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, patches, tapes, bandages, dressings, sponges, vapours) throat or mouth formulas (e.g., drops, lozenges, mouthwashes, toothpastes, sprays, pastes, gels, jellies, gummies), mucous membrane formulas (e.g., sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, dressings, sponges).

[0388] Solid, semi-solid, and liquid compositions can combine compound I and / or compound II with one more other compounds to ensure a stable and active composition. For example, an oral formulation, such as a tablet or capsule, may include: about 5 to about 50% w / w compound I or compound II; up to about 80% w / w of one or more fillers, lubricants, glidants, or binders; and up to about 10% w / w of compound I or compound II to ensure easy disintegration, disaggregation, and dissolution of the tablet in the stomach or the intestine. In one particular exemplification, a botanical extract of this disclosure (e.g., ethanolic tincture) may be mixed with one or more carrier substances such as glycerol, glyceryl esters, hydrogenated oils, polyethylene glycols, poloxamers, etc, and included in a capsule (e.g., gel capsule).

[0389] Thus, the composition may contain various excipients, for example, one or more: solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, thickening agents, viscosity enhancers / reducers, emollients, surfactants, chelating agents, adjuvants, anti-adherents, anti-caking agents, binders, coatings, disintegrants, lubricants, glidants, flow agents, sorbents, flavours, flavour masking agents, colours, sweeteners, or preservatives.

[0390] As exemplifications, the composition may include less than 1% of a preservative, for example, about 0.005% to about 0.5%, or about 0.05% to about 0.15%, or may include about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, or about 0.2% of a preservative, these percentages being representative of w / v values or w / w values. Useful preservatives include but are not limited to sorbic acid, sodium sorbate, potassium sorbate, citric acid, ascorbic acid, malic acid, tartaric acid, propionic acid, and benzoic acid, for example, in the form of its sodium salt, e.g., sodium benzoate.

[0391] Other useful excipients include but are not limited to: stearin, magnesium stearate, and stearic acid; saccharides and their derivatives, e.g., disaccharides: sucrose, lactose; polysaccharides and their derivatives, e.g., starches, cellulose or modifiedcellulose such as microcrystalline cellulose and cellulose ethers such as hydroxypropyl cellulose; sugar alcohols such as isomalt, xylitol, sorbitol and maltitol; proteins such as gelatin; polysaccharides such as pectin; gums and other thickeners, e.g., acacia gum, gellan gum, guar gum, locust bean gum, xanthan gum, agar, arrowroot carrageenan, gelatine, glycerine, kudzu, lecithin, starch; synthetic polymers such as polyvinylpyrrolidone, polyethylene glycol; fatty acids, plant based surfactants; e.g., sunflower lecithin, waxes, shellac, plastics, and plant fibres, e.g., corn protein zein; hydroxypropyl methylcellulose; cross linked polymers, e.g., cross linked polyvinylpyrrolidone (crospovidone), and cross linked sodium carboxymethyl cellulose (croscarmellose sodium); sodium starch glycolate; silicon dioxide, fumed silica, talc, and magnesium carbonate. Any combination of excipients may be utilised.

[0392] A wide array of delivery systems may be utilised, for example, nanoparticle delivery systems such as polymeric nanoparticles (e.g., PEG, PLGA, PLA, chitosan, etc), lipid-based nanoparticles (e.g., liposomes, micellar nanoparticles, phytosomes etc), nanocrystals / nanoshells, and inorganic nanoparticles (e.g., metal nanoparticles, dendrimers, etc). For example, phytosomes, which may include lecithin, may be used to increase absorption of the zingerone or zingerone extract, both topically and orally.

[0393] Liquid compositions may be stored, for example, in vials, bags, ampoules, cartridges, or prefilled syringes. The composition may also be transferred from a vial to a larger container and mixed with other materials. Dried or evaporated compositions may be stored, for example, in vials, cartridges, dual chamber syringes, or prefilled mixing systems. Before administration, a dry-form composition may be reconstituted as a liquid before being administered.

[0394] Exemplary unit dosages of the composition include: about 0.1 mg to about 1000 mg compound I or compound II, about 1 mg to about 500 mg compound I or compound II, about 1 mg to about 250 mg compound I or compound II, about 1 mg to about 200 mg compound I or compound II, about 1 mg to about 100 mg compound I or compound II, about 1 mg to about 50 mg compound I or compound II, or about 1 mg to about 25 mg compound I or compound II. The dosage may be formulated for administration once per week, twice per week, three times per week, every other day, once per day, twice per day, or three times per day, or more as needed. The dosage may beadjusted for paediatric, geriatric, overweight, underweight, or other patients, where required. Dosage modification can be made in accordance with standard methods. It is to be appreciated therefore that a wide range of unit dose forms may be envisioned and prepared. Methods of use

[0395] As noted herein, compound I (e.g., zingerone or acetyl zingerone) utilised in combination with compound II (e.g., ferulic acid) find specific applications in treating or preventing inflammation and various health conditions associated with inflammation. For example, the combined utilisation of these compounds may be employed in reducing proinflammatory cytokine or proinflammatory small molecule levels in a subject. In particular aspects, the proinflammatory cytokine may be an interleukin cytokine such as IL-6. The proinflammatory cytokine may be a tumour necrosis factor such as TNF. The proinflammatory small molecule may be nitric oxide.

[0396] As indicated above, combined utilisation can be achieved be through co- formulations or by distinct formulations that are used in combination. For example, the compositions may be formulated to allow for simultaneous, separate, or sequential administration of compound I and compound II. The compositions of the present disclosure may be prepared as one or more pharmaceutical forms. In addition, or as an alternative to this, the compositions can be prepared as one or more dietary forms, for example, functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement).

[0397] The composition of this disclosure may comprise (or may consist essentially of) one or more of the disclosed compounds. As examples, compound I and / or compound II may be present in the composition in one or more synthetic forms or one or more naturally occurring forms. Compound I and / or compound II may be present in the composition as one or more botanical extracts. In certain aspects, the composition may comprise (or may consist essentially of) zingerone or a zingerone extract, for example, as produced by the methods set out herein.

[0398] In various aspects, the disclosed compositions may be used to target one or more inflammatory disorders. The inflammation is an inflammation affecting one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen,lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

[0399] Disorders of interest include but are not limited to: immune system disorders (e.g., autoimmune disorders) such as Alzheimer’s disease (e.g., early stage Alzheimer’s disease), ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerated colitis), Crohn's disease, dementia (e.g., early stage dementia), depression, disease, diabetes, fibromyalgia, gout, immune mediated inflammatory disease, infection (e.g., microbial infection), inflammatory bowel disease (IBD), interstitial cystitis, multiple sclerosis (MS), polymyalgia psoriasis, scleroderma, and Sjögren’s syndrome, and systemic lupus erythematosus (SLE; lupus); arthritic disorders such as rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, Sjögren’s syndrome arthritis; cardiac, circulatory, and pulmonary disorders such as atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, and coronavirus (e.g., Covid-19) respiratory disorders, cytokine storm syndrome; and neoplastic disorders including various cancers and tumours, such as breast cancer, leukaemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0400] Other disorders of interest include musculoskeletal disorders (e.g., disorders of the bones, cartilage, digits, joints, limbs, muscles, tendons, etc), and back disorders (e.g., conditions of the spine or soft tissue of the back). Also noted are inflammatory disorders associated with infections, e.g., gingival inflammation. Noted specifically are anti-inflammatory therapies for the skin. As non-limiting examples, the disclosed compositions may be used for one or more of: blisters, dermatitis, eczema, hives, lesions, papules, plaques, psoriasis, rashes, rosacea, ulcers, and wounds. Wounds may be acute or chronic (e.g., non-healing or recurring wounds). Surgical wounds and scarring are specifically noted.

[0401] In particular aspects, the disclosed compositions (e.g., containing compound I and / or compound II) may be used together with one or more anti-inflammatory agents. For example, the composition may be prepared as a combined formulation with one or more anti-inflammatory agents. Alternatively, the one or more anti-inflammatoryagents may be utilised in distinct formulations. Where distinct formulations are used, it is possible to coordinate use with the disclosed composition(s), for example, via simultaneous or sequential administration. In addition, a composition as described herein may be used in conjunction with various medical or non-medical procedures. Use of the disclosed composition may be carried out prior to, during, or after the procedure(s), or any combination thereof.

[0402] As examples, anti-inflammatory agents may include one or more constituents obtained from plants, for example, one or more plant compounds, concoctions, extractions, and / or oils. These include constituents from: manuka (e.g., L. scoparium), houhere (e.g., Hoheria angustifolia, Hoheria glabrata, Hoheria lyallii, Hoheria populnea, Hoheria sexstylosa), horopito (e.g., Pseudowintera colorata), kawakawa (e.g., Piper excelsum), koromiko (e.g., Hebe stricta, Hebe salicifolia, or Hebe elliptica), poroporo (e.g., Solanum aviculare), pukatea (e.g., Laurelia novae-zelandiae), and others. Extracts from manuka (e.g., manuka oil) and kawakawa (e.g., leaf extraction) are specifically noted. Also noted are constituents from Psilocybe spp., such as P. azurescens, P. semilanceata, and P. cyanescens, as well as those from Cannabis spp., such as C. sativa.

[0403] Also included are essential oils, such as those from basil, bergamot, chamomile (e.g., Roman chamomile), clary sage, clove, copaiba, eucalyptus, fennel, frankincense, helichrysum, hops, lavender, marjoram (e.g., sweet marjoram), patchouli, peppermint, rose, rosemary, tea tree, thyme, and turmeric. Further included are honey (e.g., manuka honey), arnica (e.g., arnica oil, cream, or gel), activated charcoal, capsaicin, sesame, yarrow (e.g., for various skin formulas), and comfrey (e.g., for ointments or creams).

[0404] As further examples, anti-inflammatory agents may include one or more drug compounds. Included amongst these are analgesic agents, antipyretic agents, and psychotropic agents. Exemplifications include acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, and others. NSAID compounds and salicylate compounds are specifically noted. Noted as well are opioid compounds (e.g., KOR inhibitors) and steroid compounds (e.g., corticosteroids). Exemplifications include butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, andeptazocinem. Further exemplifications include betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone. Cannabinoid compounds and mushroom compounds are also noted. Exemplifications include cannabidiol, cannabigerol, and tetrahydrocannabinol. Further exemplifications include psilocybin and psilocin.

[0405] Various anti-inflammatory agents may be adapted and utilised in accordance with this disclosure. Any combination of anti-inflammatory agents can be utilised. Any medical devices and procedures may also be used in conjunction with the disclosed compounds and compositions.

[0406] As described herein, compositions of the present disclosure are useful as anti-inflammatory formulations. In particular aspects, compositions can be used in methods to reduce or delay inflammation in certain tissue. This tissue includes: a joint, skin, ear, eye, nose, mouth, gums, throat, digestive, cardiac, circulatory, pulmonary, vaginal, and urinary tract tissues, and others noted herein. The compositions may be applied, for example, to burns, to lessen the chance of inflammation, or to the skin before surgery, to combat inflammation on the skin around the operation site. The compositions may be used as hand cleansers (e.g., soaps or hand sanitisers), to be applied with or without water. The compositions may be used for minor skin irritations, cuts, or grazes. The compositions may be used as mouthwashes or gargles, for example, to combat inflammation from mouth sores or gingival inflammation. Compositions may also be utilised as lozenges and throat sprays, for example, to relieve a sore throat. Eye drops or ointments may be used to combat inflammation of the eye, including the eye lid.

[0407] The compositions of the present disclosure also find use as formulations, which can be used in methods for treating or preventing inflammations and inflammatory disorders, as described herein. The inflammation may affect one or more physiological components, including one or more parts of: the articular system, circulatory system, respiratory system, digestive system, renal system, excretory system, reproductive system, integumentary system, nervous system, lymphatic system, endocrine system, muscular system, skeletal system, and sensory system.

[0408] Various routes of administration may be used for the compositions, including parenteral (e.g., topical) and enteral (e.g., oral) administration, as described herein. Enteral administration may be by duodenal tubing or gastric tubing, includingnasogastric tubing, or other standard means. Oral administration may be by tablets, capsules, sachets, drops, elixirs, linctuses, solutions, emulsions, suspensions, draughts, purees, pastes, pomaces, syrups, gels, jellies, gummies, tonics, or various other means. Topical administration may be by drops, sprays, ointments, soaps, pads, sponges, dressings, bandages, or various other means. Standard modes of administration may be utilised by a skilled person. The compositions disclosed herein are not limited to a particular form for administration.

[0409] As exemplary dosages, one or more compositions may be administered to provide a dose of about 1 to about 3000 mg compound I or compound II, or about 100 to about 3000 mg compound I or compound II, or about 100 to about 2500 mg compound I or compound II, or about 100 to about 2000 mg compound I or compound II, or about 1 to about 1800 mg compound I or compound II, or about 100 to about 1600 mg compound I or compound II, or about 100 to about 1400 mg compound I or compound II, or about 100 to about 1200 mg compound I or compound II, or about 100 to about 1000 mg compound I or compound II. Additional exemplifications include about 10 to about 300 mg dose of compound I or compound II, or about 10 to about 200 mg dose of compound I or compound II, or about 10 to about 150 mg dose of compound I or compound II, or about 10 to about 100 mg dose of compound I or compound II, or about 10 to about 80 mg dose of compound I or compound II, or about 10 to about 60 mg dose of compound I or compound II, or about 10 to about 55 mg dose of compound I or compound II, or about 10 to about 50 mg dose of compound I or compound II, or about 10 to about 40 mg dose of compound I or compound II. These ranges of dosages are particularly useful for one or more compositions that are dried and milled to a powder.

[0410] The European Food Safety Authority (EFSA) has classified zingerone safe for animal consumption (e.g., rats) with No Observed Adverse Effects (NOAEL) based on a dose of up to 128 mg / kg / day (EFSA, 2016, 14(8):4557). Exemplary dosages may be determined, for example, for an average 70 kg human subject. Such exemplary dosages may include, for example, about 1 mg / kg to about 50 mg / kg, or about 5 mg / kg to about 50 mg / kg, or about 5 mg / kg to about 45 mg / kg, or about 5 mg / kg to about 40 mg / kg, or about 5 mg / kg to about 45 mg / kg, or about 5 mg / kg to about 35 mg / kg, or about 5 mg / kg to about 30 mg / kg, or about 5 mg / kg to about 25 mg / kg, or about 5 mg / kg to about 20 mg / kg, or about 5 mg / kg to about 15 mg / kg for compound I or compound II.

[0411] Additional exemplary dosages may include, for example, about 0.1 mg / kg to about 20 mg / kg, or about 0.1 mg / kg to about 15 mg / kg, or about 0.1 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 8 mg / kg, or about 0.5 mg / kg to about 6 mg / kg, or about 0.1 mg / kg to about 4 mg / kg, or about 0.1 mg / kg to about 2 mg / kg, or about 0.1 mg / kg to about 1 mg / kg, for compound I or compound II. Further exemplary dosages may include, for example, about 5 mg / kg, about 6 mg / kg, about 8 mg / k, about 8.8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 12 mg / kg, about 12.5 mg / kg, about 14 mg / kg, about 16 mg / kg, about 17 mg / kg, about 17.5 mg / kg, about 18 mg / kg, about 20 mg / kg, about 22 mg / kg, about 24 mg / kg, about 26 mg / kg, about 26.6 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, or about 30 mg / kg, or any dosage range therein, for example, about 5 mg / kg to about 30 mg / kg, or about 8 mg / kg to about 28 mg / kg, or about 12 mg / kg to about 28 mg / kg, for compound I or compound II.

[0412] The dosages as indicated herein may be administered once per week, every other day, once per day, twice per day, three times per day, or less or more, as needed. Administration may be made with food, or before a meal. The appropriate dosage and dosage form will be readily determined by skilled persons. EXAMPLES

[0413] The examples described herein are provided for the purpose of illustrating specific embodiments and are not intended to limit this disclosure in any way. Example 1: Preparation of Zingerone

[0414] An initial sample of fresh ginger (400 gm) as shown in Figure 1 was sourced from New Zealand and cleaned of dirt and soil. The cleaned ginger was then diced or chopped finely and subjected to alkaline treatment (800 g of 0.5% potassium hydroxide in distilled water). The resulting mixture was stirred and left in an oven at 60°C for 22 hours. It is to be appreciated that the resulting mixture could also be placed in a water bath and maintained at around 60°C for the desired time period.

[0415] The pH of the mixture was then adjusted to pH 7 by addition of concentrated citric acid and the treated plant material was spread out on a metal tray and dried in an oven at 60°C for 20 hours. The resulting dried material weighed 35 grams resulting in a dry yield of 8.75%. The dried material was then scraped into a flask and covered with 95% ethanol for extraction (210 ml). The flask was shaken and placed in an oven at 40°C for 16 hours. The extract (Extract 1) was filtered off using a glass funnel witha glass wool plug. The remaining plant material was extracted again with 95% ethanol (Extract 2) and then twice more with 50% ethanol (Extracts 3 and 4).

[0416] The extracts were analysed directly for zingerone content. A 294 mg chopped sample of the fresh ginger was also extracted with 2 ml of ethanol and this extract was also analysed. The results are shown in Table 1. Table 1. Extract composition Extract Volume Weight (g) Total Total *Dried (mL) zingerone 6-gingerol Weight (g) (mg) (mg) 1 142 119 67 94 4.04 2 140 116 14 21 1.18 3 112 93 8 15 4 104 86 3 9 Totals 92 139 * The dry weight of Extract 1 and Extract 2 was estimated by drying a 5 ml portion of each extract. Results

[0417] The 400 g fresh ginger root supplied was shown to contain 260 mg of 6- gingerol (i.e. 0.65 mg / g). This would mean the theoretical maximum yield of zingerone would be in the order of 171 mg from the treated material (loss of weight due to lower molecular weight of zingerone vs 6-gingerol). About 50% of the 6-gingerol was unconverted. It is envisioned that further studies could be used to increase the alkaline conversion of 6-gingerol to zingerone.

[0418] The ethanolic extraction was done using the minimal volume needed to cover the treated plant material. This meant that Extract 1, the most concentrated one, had a zingerone content of 0.47 mg / mL. It is envisioned that this concentration could, in principle, be increased using either a multi-steeping process or by evaporation of some of the ethanol. Reducing the volume of ethanol by 80% would yield a solution with 2.35 mg zingerone / mL.

[0419] It is envisioned that drying the extract fully would give a concentration of approximately 16 mg / g of solid extract. Higher concentrations would be expected if starting with a higher initial content of 6-gingerol and / or with more complete conversion. A theoretical 25 mg dose, for example, could then potentially be achieved by formulationof the dried extract directly into an oil or glycerol carrier to give the required zingerone dose in one or two 500 mg capsules.

[0420] The majority of the zingerone was extracted in the first extraction. There are situations where double extraction would be beneficial. The third and fourth extraction increased the totals by modest amounts. Overall, ethanol extraction was seen to be highly effective, and very efficient and inexpensive as a preparative method. Example 2: Preparation of Zingerone

[0421] Overview: These studies show that an aqueous alkaline treatment followed by freeze-drying of fresh ginger greatly improved conversion, reaching a ~1% zingerone content in the dry ginger. The treated ginger was then extracted with supercritical CO2and CO2 + ethanol co-solvent with a combined extraction yield of 3% and an average concentration of zingerone in the extracted oleoresin of approximately 12%. In addition, it was found that drying of fresh ginger at a moderate temperature (60°C) followed by supercritical CO2extraction resulted in an extraction yield of 4.6%. The extracted oleoresin was then alkaline treated and the final product contained approximately 15% zingerone.

[0422] Drying: Samples of the fresh ginger material imported from Fiji were sliced into 2-5 mm slices and placed on a single layer on perforated oven trays. Drying was performed in a forced convection. Drying was carried out at a moderate temperature (60°C) with the goal of eliminating moisture without conversion of gingerol. This process was considered finished when the moisture content of the ginger reached 7%. The dried ginger obtained was stored refrigerated until it was used in the extraction trials.

[0423] Catalysed conversion: Small scale, preliminary trials were performed by treating around 1.6 g of fresh chopped Fijian sourced ginger with aqueous solutions of 0.5% KOH (pH 14), 1% Ca(OH)2 (pH 11.6) and 1% sodium carbonate (pH 10.5). The volume:weight ratio of reagent added:ginger was around 3:1. The samples were then shaken and placed in a fumehood at room temperature, or ovens at 37°C or 60°C, overnight, before being analysed. It is to be appreciated that suitable water baths could be used to keep the samples at the desired temperature for the desired period of time.

[0424] Once the alkaline treatment had been selected, 5.2 kg of fresh ginger was minced using a vertical cutter mixer (RobotCoupe R45). The ginger was then mixed with a 3:1 liquid:solid ratio (volume: weight) of KOH 0.5% (~0.1 N), resulting in a pH ~ 12.5. The mixture was manually stirred and placed in an oven at 60°C for 24 hours. After thistime, the mixture was neutralised by adding concentrated citric acid (625 g / L) to a pH ~ 7.2. The neutralised mixture was then freeze-dried, and the ginger obtained in this process was stored refrigerated until it was used in the extraction trials.

[0425] Catalysed conversion results: As noted, different alkalis were tested: 0.5% potassium hydroxide or KOH (pH 14), 1% calcium hydroxide or Ca(OH)2 (pH 11.6), and 1% sodium carbonate or Na2CO3 (pH 10.5). The content of zingerone and 6-gingerol was quantified for these experiments (Table 2), and it was concluded that a treatment with KOH at 60°C was the most efficient and gave the highest zingerone concentration. Table 2 shows the amounts of 6-gingerol mg / g and zingerone (mg / g) obtained for the respective conditions, with results expressed on a wet basis. HPLC traces are shown in Figure 2. It can be seen from the HPLC traces that Ca(OH)2at 1% and KOH at 0.5% perform similarly. At 60°C for KOH 0.5% the ratio of zingerone:6-gingerol is 6.5. At 60°C for Ca(OH)21.0% the ratio of zingerone:6-gingerol is 4.0. Table 2. Peak areas at 280 nm for zingerone and 6-gingerol in treated samples Alkali Temperature 6-gingerol (mg / g) Zingerone (mg / g) KOH Room temperature 1.4 0.2 KOH 37°C 1.0 0.3 KOH 60°C 0.4 0.7 Na2CO3 Room temperature 1.4 0.1

[0426] This treatment was applied to a larger sample of fresh ginger (5.2 kg) and the resulting treated ginger was then neutralised and freeze-dried. The yield of the freeze- dried ginger was 17%, i.e., 17 g of treated, freeze-dried ginger per 100 g of minced raw ginger. The zingerone and 6-gingerol content of the freeze-dried ginger was measured at 10.2 and 3.3 mg / g, respectively (dry basis). See Table 2-1. The zingerone content was at least 10 times higher than that of the oven dried ginger. Table 2-1: Composition of treated and freeze-dried ginger Wet basis Wet basis Dry basis Dry basis Treatment Moisture 6-Gingerol Zingerone 6-Gingerol Zingerone (mg / g) (mg / g) (mg / g) (mg / g)KOH 0.5% 4.5% 3.2 9.8 3.3 10.2 at 60°C + freeze-dried

[0427] Extraction: Supercritical extraction trials were carried out using the alkaline treated ginger. The alkaline treated, freeze-dried ginger was lightly crushed by hand and placed in a 2 L extraction vessel with sintered filter discs at both ends, filling the vessel completely. The extraction was carried out as described above until a sharp decrease in the extraction rate was observed, corresponding with a CO2:feed ratio of 13:1. At this point, the ethanol co-solvent pump was started, and ethanol was added to the CO2 stream with a ratio of approximately 10 wt% (i.e.10 g ethanol per 100 g CO2). The ethanol pump was stopped after a 2:1 ethanol:feed had been introduced (2 g ethanol per g of feed). CO2was then circulated to flush out any ethanol remaining in the bed. When the extraction was finished, the plant was depressurized, and the residual marc was allowed to degas overnight before being unloaded. The ethanol present in the extract was removed by rotary evaporation under vacuum. Extraction parameters are listed in Table 3. Table 3. Summary for extraction parameters Extraction 1 Extraction 2 Feed material Untreated ginger Alkaline treated ginger (dried at 60°C) (freeze-dried) Solvent phase CO2CO2=> CO2+ ethanol Extraction pressure 300 bar 300 bar Extraction temperature 40°C 40°C Separator pressure 55 bar 55 bar Separator temperature 40°C 40°C Feed mass 358.6 g 322.41 g Average CO2 flow rate 2.8 kg / h 2.8 kg / h Average ethanol flow rate 4.5 g / min Ethanol % 10% Ethanol to feed ratio 2:1 CO2 to feed ratio 37.5:1 55:1

[0428] Analysis: Samples were prepared for analysis by addition of methanol after neutralisation as needed. Extracts were dissolved directly in methanol. Analysis was by HPLC with an acetonitrile / 0.1% formic acid gradient. Detection was performed at 280 nm, and the column used was a Phenomenex Kinetex C18 (150 X 2.1 mm). Zingerone eluted at around 2 minutes and 6-gingerol at 5.2 minutes. Quantification of zingerone and6-gingerol was obtained from a standard curve prepared using analytical standards of these compounds.

[0429] Extraction results: As noted, the alkaline treated and subsequently freeze- dried ginger was extracted with CO2followed by CO2+ethanol co-solvent. No free water was observed in the CO2extract, and the ethanol obtained in the CO2+ethanol extract was removed by rotary evaporation under vacuum. The extracts had a sweet, caramelized fragrance. The yield obtained with CO2was 1.5%. Addition of 10% ethanol co-solvent allowed for extraction of an additional 1.5%. The composition of the different fractions is shown in Table 4 and Table 5. See, also, Figure 3. Table 4. Composition of extracts obtained from alkali treated ginger 6-Gingerol Zingerone (mg / g) (mg / g) untreated CO2extract 290 0 treated CO2extract 103 153 treated CO2 extract + ethanol 52 91 treated Marc 1.2 5.4 Table 5. Zingerone (Z) mass balance for the extraction of alkali treated gingerFeed CO2 extractCO2extract +MarcMassEtOH extract balance g Z mg / g Z mg g Z mg / g Z mg g Z mg / g Z mg g Z mg / g Z mg 322 9.8 3143 4.8 153 735 4.7 91 428 312 5.4 1687 90.6%

[0430] It can be seen from the results that for the alkali treated sample, the CO2extract contained 153 mg / g of zingerone (15.3%) and 103 mg / g (10.3%) 6-gingerol, with a zingerone / gingerol ratio of approximately 1.5. The CO2+ethanol extract contained 91 mg / g of zingerone and 52 mg / g of gingerol (zingerone / gingerol ratio of approximately 1.75). The marc or residual ginger post extraction was also analysed, and found to contain 5.4 mg / g of zingerone. When the mass of feed, extracts and marc is taken into account (Table 4), the zingerone mass balance can be calculated at 90.6%. This indicates that 90.6% of the zingerone present initially in the feed is accounted for in the extracts and marc. The difference could be caused by degradation during extraction or in the ethanol removal step. The zingerone extraction yield (i.e., grams of zingerone extracted per 100 g of zingerone in the feed) was only 37% when calculated based on the extracts. However, 54% of the initial zingerone present in the feed remains unextracted in the marc, so the zingeroneextraction yield can also be calculated as 46% when based on the marc results. This accounts for the “missing” zingerone. Since the proportion of unextracted zingerone is significant, the extraction process could be further improved to reduce this. The extraction yield of gingerol is higher than that of zingerone (82% in the untreated sample and 71% in the treated sample), since it is more soluble in CO2.

[0431] In another experiment, a CO2 extract obtained from untreated ginger root was subjected to an alkaline treatment to study the conversion of gingerol into zingerone. Overnight treatment with both 0.1 N and 1 N KOH at 60°C worked well, with a resulting concentration of zingerone in the treated ginger of around 15%. The resulting material appeared to be much cleaner than the alkaline treated crude ginger, so is an interesting alternative process that could provide an even more cost effective extraction process overall as the conversion process is on a smaller volume of material. In fact, for the same amount of starting fresh ginger (100 kg), and based on the results obtained in this work, the extraction of untreated ginger followed by alkaline treatment of the extract would yield almost twice as much zingerone in the final product than the alternative process (see comparison in the table below). However, even in this case, the overall zingerone yield of the process is ~0.1% (0.1 kg zingerone per 100 kg fresh ginger). It is expected that further optimisation is possible.

[0432] In another experiment a sample of CO2 extract of untreated ginger was mixed with KOH and converted to a zingerone rich extract. The post-treatment neutralisation step involved the separation of the zingerone as a zingerone enriched resin from the aqueous reaction mixture.3 grams of oleoresin was taking in duplicate and 9 ml of 1 N KOH was added to the samples in a plastic vial. The samples were then shaken and left overnight in a 60 degrees oven. The treated samples were then neutralised by the addition of 10 ml of 1 N HCl. This was a small excess of acid to ensure that all of the KOH was neutralised. The addition was performed in two steps with mixing after each step. The samples were centrifuged at 2000 rpm to separate the water from the oleoresin. After centrifugation the bulk of the water was then removed by pipette. The resultant resin was then removed. Absolute alcohol was added to one of the resin samples (5 ml ethanol in approximately 3 grams resin) to produce a tincture (Sample 1). The other sample was kept in resin form (Sample 2). Sample 1 and Sample 2 were analysed for zingerone content.The concentration of zingerone in the tincture (Sample 1) was calculated at 23 mg / g of tincture while that of the treated resin (Sample 2) was 52 mg / g.

[0433] These values for both Samples 1 and 2 were lower than those obtained with earlier treatments for which 150 mg / g zingerone content was estimated. In this work the resin Sample 2 had been separated from the water and analysed. Subsequently the separated water was also analysed and estimated to contain another 50-55 mg (approximately 25%) of zingerone for each 3 g batch. Zingerone has been reported as having quite limited water solubility so this result was quite unexpected. Because it appears that a significant portion of the zingerone is staying in the water and not separating out with the resin, this method is contrasted to direct addition of alcohol to the crude neutralised product.

[0434] Because the water is able to extract some of the zingerone from the resin it may also be possible to produce a high yield of zingerone after treatment by drying down the total neutralised alkaline treatment product. Further process optimisation is to be undertaken to improve yields given the finding that after alkaline treatment zingerone appears to be more soluble in water than previously reported. Example 3: Further reaction methods and comparisons

[0435] Overview: Zingerone is not naturally present in ginger; it is a conversion product from gingerol through a treatment process. In these studies, a sample of alkaline treated and dried ginger was received from Samoa and extracted with ethanol using two different sets of extraction conditions: extraction at 40°C for 20 h (Extract A) and at room temperature for 7 days (Extract B). The resulting zingerone concentration of the extracts was found to be 41.4 mg / g for Extract A and 43.8 mg / g for Extract B.

[0436] Extraction A: The treated ginger produced by SROS (Scientific Research Organisation of Samoa) was supplied in two separate plastic bags. The contents of both bags were combined, frozen at -80°C and subsequently milled using a knife mill (Wiley) with a 2 mm mesh attached. The milled material (782.3 g) was then placed in a round bottom flask along with food grade ethanol with a ratio of approximately 5:1 by weight. The flask was then placed in a water bath at 40°C and 5 rpm stirring overnight (total extraction time 20 hours). After this time, the mixture was filtered under vacuum and the ethanol was removed by rotary evaporation under vacuum to produce 43.8 g of highly viscous, dark brown resin with a characteristic ginger aroma. Extraction yield was 5.6%.10 g of this resin (Extract A) were taken out and stored refrigerated and under nitrogen flush for possible future bioassays.

[0437] Extraction B: The ginger was frozen and milled as described above. The milled material (785.9 g) was placed in a bucket along with food grade ethanol with a ratio of approximately 5:1 by weight. The ginger was macerated in ethanol at room temperature (22-29°C) over 7 days. Samples were taken on days 1, 3 and 7. After 7 days, the mixture was filtered under vacuum and the ethanol was removed by rotary evaporation under vacuum to produce 49.3 g of highly viscous, dark brown resin with a characteristic ginger aroma (Figure 1), very similar to the one obtained in Extraction A. 10 g of this resin (Extract B) were taken out and stored refrigerated and under nitrogen flush for possible future bioassays. Extraction yield was 6.3%.

[0438] Zingerone and aldehyde analysis: Quantification of zingerone was carried out by HPLC in the starting material (i.e., treated ginger) and the final two resins, as well as in the samples on days 1, 3 and 7 for Extraction B (note that the liquid sample from day 7 is equivalent to the final resin sample). The HPLC quantification method included methanol addition and grinding the sample before analysis. Zingerone content for all fractions is shown in Table 6. Zingerone mass balance and yield are shown in Table 7. Table 6. Comparing extraction conditions – zingerone content for different fractions Treated Day 1 Day 3 Day 7 Final resin ginger sample sample sample Zingerone extract A 3.1 mg / g 41.4 mg / g Zingerone extract B 3.1 mg / g 0.38 mg / ml 0.44 mg / ml 0.4 mg / ml 43.8 mg / g Table 7. Zingerone mass balance and yield FeedZingerone Zingerone Extract Zingerone Zingerone Zingerone Extraction (g) in feed in feed (g) in extract in extract yield Yield (mg / g) (g) (mg / g) (g) Ext A. 782.3 3.1 2.4 43.8 41.4 1.8 75% 5.6% Ext B. 785.9 3.1 2.4 49.3 43.8 2.2 89% 6.3%

[0439] As indicated above, Extract A was obtained by treatment at 40 degrees, duration 20 hrs, while Extract B was obtained by treatment at room temperature, duration 7 days. The results for Tables 6 and 7 show that higher levels of zingerone were obtained by longer treatment at room temperature, although high yields were also obtained by a temperature increase to 40°C.

[0440] To determine zingerone content in the starting material, this was first extracted into a suitable solvent. In one process, this extraction was carried out with ethanol resulting in a lower zingerone content (1.44 mg / g). In a second process, this was carried out using methanol and grinding the ginger along with the solvent in a mortar and pestle. This resulted in a higher zingerone content (3.1 mg / g). For reference, the zingerone content reported by the Samoan lab for this material was 1.86 mg / g.

[0441] Based on this, the zingerone yield (i.e., the amount of zingerone in the extract relative to the zingerone in the feed) was estimated at 75% for Extraction A and 89% for Extraction B. The 6-gingerol peak seen in the HPLC analysis when determining zingerone content was consistently observed at around 1 / 8th the peak area of zingerone. This suggests that extraction had little effect on the zingerone to gingerol ratio.

[0442] A sample of both final resins was taken up in ethanol and examined by GCMS for aldehyde analysis. A very low level of hexanal was seen (too low to quantify). Hexanal is a side product of the reaction to form zingerone but is somewhat volatile. The identity of the hexanal peak was confirmed by library matching of MS data and a separate injection of a hexanal standard. Discussion

[0443] Each of the studies described herein was effective in producing zingerone. A comparison of the results from Examples 2 and 3 is provided in Table 8. Table 8. Comparison of zingerone yields Zingerone Difference to fresh ginger Fresh ginger 0.1 mg / g Baseline Dried ginger (180°C, 10 hr) 0.7 mg / g 7-fold higher KOH 0.5% at 60°C 10.2 mg / g 102-fold higher + freeze-dried (Example 2) Disclosed processing method 43.8 mg / g 438-fold higher Samoan ginger (Example 3) Disclosed processing method 310 mg / g expected 3100-fold higher Fijian ginger (Example 2) Disclosed processing method 88.9 mg / g 889-fold higher Fijian ginger (Example 6) Disclosed processing method 177.8 mg / g expected 1778-fold higher Fiji ginger (Example 5)

[0444] Similar values for fresh and dried ginger were obtained by Li et al. See Li et al., 2016, “Chemical characterization and antioxidant activities comparison in fresh, dried, stir-frying and carbonized ginger” J Chromatogr B Analyt. Technol. Biomed. Life Sci.1011: 223-32. Example 2, as described above, advances well beyond a standard retro- aldol reaction. Example 3, as described directly above, provides further advancements, utilising temperature and pH adjustments, and extraction. Further advancements are provided in Examples 5, 6, and 12, as described herein.

[0445] Regarding the Samoan ginger, it is noted that the ginger was not harvested at the requested time (9 months in the ground), and this affected the level of gingerol present in the ginger and subsequently the zingerone contend in the end product. Therefore, it is expected that further gains can be obtained. Regarding the Fijian ginger, this was substantially higher in zingerone compared to the Samoan ginger (10.2 : 1.44 = 7.08 x higher). This means that the total yield can be extrapolated for the Fijian ginger as 310 mg / g, if the experimental conditions of Example 3 were to be applied. That is: 43.8 mg / g (amount obtained from Samoan ginger in Example 3) x 7.08 (higher starting content in Fijian ginger) = 310 mg / g.

[0446] Table 5 in Example 2 shows the output of the pH treated ginger followed by CO2 extraction. It was found that 322 grams of fresh ginger provides 153 mg / g zingerone. In comparison to this, Example 3 utilises 785.9 grams (2.4 times more compared to amount of product used in Example 2) and provides 43.8 mg / g zingerone. However, this lower yield can be explained by the lower levels in the starting material from Samoa (1.44 mg / g zingerone). Example 4: Processing methods using juicing and alkaline treatment

[0447] Summary: Ginger root was mechanically juiced and the levels of 6- gingerol were determined for juice and remnant solids. The majority of the 6-gingerol was present in the juice. Treatment of the juice with alkali showed that effectively all of the 6- gingerol was converted to zingerone in 5-6 hrs at 60°C.

[0448] Overview for juicing: Fresh ginger (500 to 1000 g) was pre-treated by blending / macerating and pressing the ginger. The liquid fraction was retained, and the marc was further washed with warm water (4 parts water to 1 part ginger) at a temperature of 55-60°C for a period of 10-15 minutes. This was done in a covered vessel. This was then pressed again. Each fraction was analysed for 6-gingerol content (a total of 5analyses), namely: 1) Fresh ginger sample immediately prior to processing; 2) Liquid fraction following initial blending / maceration; 3) Ginger marc following initial blending / maceration; 4) 2nd liquid fraction collected following further washing of initial marc; 5) Final ginger marc following washing then pressing as above. A moisture content reading was made on each lot of ginger marc, after final pressing (i.e. samples 3) and 5) above).

[0449] Overview for alkaline treatment: An alkaline treatment was performed on the liquid fractions to establish the conversion to zingerone. Samples were taken at a range of timepoints and analysed for zingerone content. The samples were subjected to small scale treatments with KOH (5% as previously). Treatment was carried out using 1 ml samples at room temperature, 30°C and 60°C samples at 1, 2, 3 and 5 hours. Additionally, one sample was treated at 60°C for 24 hours. Analysis of up to 15 samples was carried out.

[0450] Juicing method: Two samples of fresh ginger root were obtained, one locally (organic, from Evithé in Petone) and a second received from Phil Rasmussen in Auckland. Both samples appeared to be plumper / juicier than normal supermarket ginger root. The moisture content was determined by slicing up approximately 10 g of each root, freezing with liquid air and then freeze drying. The 6-gingerol content was determined extraction of root with methanol. For this, around 5 g of each sample was cut into 4-5 pieces and crushed using a small kitchen garlic press. The crushed root and juice were extracted with methanol (2 x 15 ml) at 60°C for 20 minutes. This was followed by HPLC analysis with detection at 280 nm. The results are shown in Table 9, below. Table 9. Moisture content of gingers Sample % solid after freeze drying 6-gingerol content (mg / g) Auckland sample 16 1.46 Petone sample 5.3 1.58

[0451] The Auckland sample was chosen for the juicing work. For this, 635 g ginger root was processed using a home juicer. This contained a rotating screw drive with mesh juice filter and adjustable solids nozzle (see Figures 4A-4B). The juicer removed 516.3 g (81%) of liquid (juice 1, J1) with 101.5 g of solid (marc 1, M1) collected. Some of the dry solid was removed for analysis and 90.5 g extracted with 360 ml of hot tap water. This was allowed to sit for 15 minutes before running this material through the juicer. From this step, 350 g of juice (juice 2, J2) was recovered along with 70.2 g of solid (marc 1, M2).

[0452] The two liquids were refrigerated overnight. Both were cloudy with settled solids. These samples were shaken up prior to analysis or treatment. The juice samples were analysed for 6-gingerol content by mixing a sample with ethanol (1:1), centrifuging and direct injection of the supernatant. The solid content of the two pressed solids M1 and M2 were 37% and 26.3% respectively.

[0453] The results are shown in Table 10, below. The gingerol values were multiplied by weight to give the total amounts of gingerol in each material. The juice was found to contain 81.6% of the measured gingerol. The total gingerol calculated for the feed was lower than the recovered amount suggesting a partial extraction of gingerol from the root. The percentage gingerol was based on the total measured gingerol for the marc and juices (rather than the feed measurement). Table 10. Weights and gingerol content Sample weight (mg) 6-gingerol (mg / g) total 6-gingerol (mg) % 6-gingerol Feed 635 1.46 928 Juice 1 516.3 1.58 814 75 Juice 2 350 0.2 72 6.6 Marc 1 101.5 1.95 198 16.3 Marc 2 90.5 1.816 146 13.4 Sum of Juice 1, 2 + Marc 2 1085

[0454] Alkaline treatment: This work was performed on the first juice (J1) recovered from the juicing process (above). Samples of juice (after shaking to suspend all solids) were allowed to react with KOH at RT, 30 or 60°C for 1, 2, 3, 5, and also at 60°C for 24 hours. Three concentrations of KOH were also trialled, 0.5, 1.0 and 2.0 %. A solution of 2 N KOH was prepared (5.6 g KOH in 50 ml water). To generate 0.5%, 1% and 2% KOH concentrations in each sample, 0.25, 0.5 or 1 ml of the 2 N KOH was added to 5.5 ml of juice and shaken. The samples were then placed at RT (lab), 30°C (water bath) or 60°C (drying oven). Sampling for HPLC analysis was done by taking 200 µl from each sample, adding 200 µl of 1 N HCl, and then 500 µl of ethanol. After centrifugation the sample was directly injected into the HPLC. The peak areas of zingerone and gingerol were compared (see below).

[0455] These results demonstrated that treatment with 2% KOH was able to achieve complete conversion to zingerone within 5 hours. See Figure 5C. Incubation at 60°C was particularly favourable. See Figure 5C. The results are presented as peaks areasfor zingerone (Z) and gingerol (G). It was noted that the KOH treated samples had solid present which settled in the tube. For analysis, the tubes were shaken and a sample was taken with a wide bore tip to avoid plugging. Example 5: Additional processing methods using juicing and alkaline treatment

[0456] Overview: The aim is to produce an extract from ginger having the 6- gingerol converted to zingerone by an alkaline-catalysed reversed aldol reaction. The current process seeks to reduce treatment time and water use. This process was trialled at a scale of roughly 40 kg before further production at a 200 kg scale will be undertaken.

[0457] In brief, fresh ginger was received and processed by treating it in alkali followed by freeze-drying to produce a treated ginger powder. This powder was extracted with ethanol at room temperature for 3 days, with samples taken at 24, 48 and 72 hours to assess the progress of the extraction. The ethanol extraction is detailed in Example 6.

[0458] Methodology: Alkaline pre-treatment and drying of imported fresh ginger were carried out. For this, fresh ginger (36.67 kg) was pressed in a Vincent Corporation CP-4 screw press to make two streams: a ginger juice and a marc. Screw Press settings were a VSD speed of 50%, and a cone air pressure of 2 bar. The marc from the initial press was pressed a second time to remove any remaining juice.

[0459] The two juices were combined. The juice was heated to 60°C and KOH was added to a final concentration of 2% w / w. The alkalised juice was held at 60°C for 5 hours to convert gingerol to zingerone. The juice was neutralised to a pH of 7.2 by addition of anhydrous citric acid. The juice, now containing zingerone, was freeze-dried and ground.

[0460] Samples were taken of: the fresh ginger (ZINGO); the two marcs (GMARC and GMARC2); the juice before KOH additions (GKOH0); the juice after 2, 3, and 5 hours of treatment (GKOH2, GKOH3, GKOH5); the final dried extract (GPE).

[0461] In addition, a sample of GMARC2 was extracted with hot water as follows. Water was added to GMARC2 at a ratio of 5:1 w:w. The mixture was heated to 60°C and held at this temperature for 15 minutes. The extract was separated from the solids by screw pressing with the same settings as above. Samples were taken of: the extract (GMARC2 HWEX); the marc (HW MARC). Each sample was analysed for total solids (LOD, 16 hrs 100°C) and gingerol or zingerone content by HPLC.

[0462] Sample analysis: Analysis was performed using HPLC with UV detection at 280 nm. Sample preparation was as follows: (1) liquid samples such as juice were diluted 1:1 with ethanol and centrifuged. In-process liquid samples with alkali present were diluted with 1 N HCl and ethanol 1:1:1 (2) solid samples such as raw ginger or ginger marc were extracted by double extraction with ethanol (ultrasonication, heat at 60°C for 20 minutes, vortexed and centrifuged) and combining supernatants. Solid extracts (from approximately 5 g) were generally made up to 50 mL for analysis. The raw fresh ginger was roughly chopped and then blended with ethanol using an ULTRA-TURRAX® type mixer. Quantification was performed by comparison to a standard curve prepared using zingerone. A molecular weight correction was made for gingerol.

[0463] Pressing: 36.67 kg of raw Fijian ginger was received and pressed. Pressing was effective, producing a large volume of light green juice, and a fibrous marc.28.92 kg of juice was recovered from first press.6.6 kg of marc was then pressed a second time to recover an additional 2.18 kg of juice. Total yield of juice was 31.1 kg, equivalent to 86% of the incoming raw ginger by mass. The final marc mass recovered was 4.05 kg. There was typically 1-2 kg of holdup in the screw press at the end of a run. This gave rise to a minor difference noted between the feed mass and the combined mass of marc and juice.

[0464] Hot water extraction: 3.62 kg of GMARC2 and 18.1 kg of water were heated to 60°C and then separated by screw pressing after 15 minutes of extraction at 60°C. Next, 21.18 kg of mixture was pressed. Noting that approximately 500 g of water was lost as evaporation during the extraction. From this, 17.62 kg of extract was recovered and subsampled for analysis. In addition, 2.69 kg of marc was recovered.

[0465] Alkaline treatment: For this, 1.236 kg of 50% KOH solution was added to the 31.1 kg of ginger juice, to reach a target KOH concentration of 2%. The pH after KOH addition was 12.18. On addition of KOH the colour of the juice changed for a light green to a reddish brown. The juice was held at 60°C for 5 hours, and then neutralised by the addition of 500 g anhydrous citric acid. The pH after citric acid addition was 7.23.

[0466] Freeze drying: The treated juice was transferred to freeze dryer trays and frozen overnight before transferring to the Cuddon FD80 freeze dryer. A total of 28.51 kg of juice was loaded onto trays and dried. Approximately 3 kg of juice was lost prior to freezing and drying as a result of manual handling. After drying, 2.86 kg of dried extract was collected- a total of 10% of the mass of juice dried. This was ground and subsampled.After grinding and subsampling and handling losses a total of 2.19 kg was packed into foil bags for storage until further processing. Approximately 1.6 kg of this was sent for ethanol extraction (see Example 6).

[0467] Mass balance summaries are shown below. Juicing Inputs fresh ginger 100 kg Outputs ginger juice 85.79 kg ginger marc 14.21 kg Alkaline treatment Inputs ginger juice 85.79 kg 50% KOH solution 3.37 kg anhydrous citric acid 1.36 kg Outputs treated ginger juice 90.53 kg Freeze drying Inputs treated ginger juice 90.53 kg Outputs dried ginger extract 9.08 kg

[0468] Gingerol and zingerone levels were measured as described above. The results are shown in Table 11, below. Table 11. Gingerol and zingerone levels Sample 6-Gingerol (mg / g) Zingerone (mg / g) Basis ZING0 (feed) 0.522 N / A Wet GMARC 1 1.08 N / A Wet GMARC 2 1.99 N / A Wet MARC HW 0.22 N / A Wet GKOH-0 6.90 0.00 Dry GKOH-2 0.93 5.76 Dry GKOH-3 0.90 5.74 Dry GKOH-5 0.79 5.32 Dry GPE N / A 5.70 Dry

[0469] This table shows the gingerol content of different fractions during pre- treatment and the treatment process. From these measurements, there was a total of 19.1 g of 6-gingerol in the 36.67 kg of raw ginger feed. The dried ginger extract (GPE) had a zingerone content of 5.7 mg / g. Therefore, the total zingerone in the 2.86 kg of dried powder (before losses and milling losses), was 16.30 g. The concentration of gingerol in the juicebefore conversion (GKOH-0) was 6.9 mg / g on a dry basis. When converted back to a wet basis, using a solids concentration of 6.67% (before the addition of KOH and anhydrous citric acid increased the total solids content to approximately 10%), the total gingerol in the juice was 14.32 g. The GKOH-2, -3, -5 values for 6-gingerol were to be confirmed.

[0470] The mass balances between the gingerol in the juice, and the zingerone in the final powdered extract are not precisely aligned. This may be due to variations in measurements. The marc numbers appear to be somewhat elevated, in view of the total mass balance calculations. When the marc was extracted with hot water, the extract had a 6-gingerol content of 0.22 mg / g, corresponding to a total of 5.7 g of 6-gingerol in this extract, roughly 25% of the 6-gingerol in the feed. For water extraction, this required a total of 18 kg of water. This in turn would increase the mass of KOH and citric acid, and the drying loads by an additional 58%. Thus, in certain circumstances, it may be desirable to omit water extraction.

[0471] Notably, these experiments showed that almost total recovery of 6-gingerol (in the form of zingerone) in the final product was achieved. The samples taken during the conversion reactions - GKOH2, GKOH3, and GKOH5 - show that the conversion from 6- gingerol to zingerone occurred during the first two hours of treatment, with no significant increase in zingerone levels in samples taken after 2 hours of treatment. Given that the conversion appears to be complete after 2 hours, this incubation time (or even shorter incubation times) will be sufficient.

[0472] It was concluded that the processing method, including pressing followed by KOH treatment in the juice phase, was an effective production method. This is an advancement well beyond standard alkaline-catalysed reversed aldol reactions. Further experiments will employ more than 200 kg ginger as starting material. 6-Gingerol levels in this batch were 1.1 mg / g and this was expected to reflect in the corresponding zingerone levels. Example 6: Ethanol extraction process and analysis

[0473] Overview: Fresh ginger was received and processed by treating it in alkali followed by freeze-drying to produce a treated ginger powder (see Example 5, above). This powder was extracted with ethanol at room temperature for 3 days, with samples taken at 24, 48 and 72 hours to assess the progress of the extraction.

[0474] Methodology: The treated ginger produced as above (Example 5) was stored refrigerated until used. Approximately half of the received ginger was extracted with XNS food grade ethanol at room temperature. The treated ginger, along with ethanol (using a ginger:ethanol ratio of 1:5 by weight), was placed in a 10 L glass vessel equipped with an overhead stainless-steel stirrer. The mixture was stirred for 72 hours, with a stirring speed sufficient as to not allow sedimentation of solids at the bottom.

[0475] After 24 hours had passed, the stirrer was turned off and the solids were allowed to settle for 10 minutes before taking a 50 mL sample from the top. After another 24 hours had passed, a second sample was taken out using the same procedure. After a total 72 hours had passed, stirring was stopped and the mixture was filtered under vacuum using filter paper. Samples were taken of the cake and a final tincture sample, representing 72 hours, was taken from the filtrate. The remaining filtrate was labelled ZINGOEE. A ~300 mL sample of ZINGOEE was taken and stored refrigerated in a glass bottle. In a further step, all ZINGOEE was evaporated to create a total resin volume of 43.5 g at a value of 88.9 mg / g zingerone.

[0476] The remaining ethanolic tincture was evaporated under vacuum using a Buchi R220SE rotary evaporator operating at 50 mbar and 40°C until a volume reduction of approximately 31-fold was achieved. The resulting concentrated extract (ZINGOCE) was then analysed and once the zingerone content was confirmed, a small sample of this concentrated extract was used to produce a standardised tincture containing ~12 mg zingerone per gram by diluting it with food grade ethanol. Two separate samples of this standardised tincture were sent to SCU (Australia) for analysis, and a third sample was retained onsite for zingerone analysis.

[0477] Results: The process and results are set out in Figure 7A. For these experiments, 801.5 g of the received treated ginger was used in the ethanol extraction, along with 4007.3 g of food grade ethanol. After 72 hours at room temperature (16-20°C) under stirring, the mixture was filtered and 3556.7 g of a clear brown, aromatic ethanol tincture was obtained [ZINGOEE], as well as 1033.4 g of cake (i.e., spent ginger solids). Approximately 140 g of ethanol was lost due to evaporation during the extraction. The total weight of ethanolic tincture produced, including the samples taken at 24 and 48 hours, was 3631.7 g.

[0478] Quantification of zingerone was carried out by HPLC in the starting material (i.e., treated ginger, GPE) and the samples after 24, 48 and 72 hour ethanol extraction at room temperature. The spent ginger solids (i.e., the cake from the filtration process) was also analysed. Zingerone content for all fractions is shown in Table 12. HPLC results showed little difference between the three extraction times, indicating that 24 hours is sufficient for extraction. Table 12. Zingerone content (mg / g) for samples Treated ginger 24 h sample 48 h sample 72 h sample Spent ginger solids [GPE] [ZINGOEE] [CAKE] 5.7 1.07 1.08 1.1 0.6

[0479] The filtrate [ZINGOEE] had a zingerone concentration of 1.1 mg / g, i.e., 3.88 g zingerone are present in the liquid, or 85% of the starting zingerone, indicating reasonable recovery. The cake had a zingerone concentration of 0.61 mg / g, i.e., 0.63 g of zingerone in the cake. However, it was noted that the cake had certain amount of ethanolic solution still present. In future processing, the cake could be washed with clean ethanol to flush out as much extract as possible.

[0480] The zingerone mass balance for the ethanol extraction process is 98.5%. This is based on 3.88 g in the extract, plus 0.63 g in the cake, divided by 4.58 g in the feed. It was determined that room temperature extraction provided advantageous recovery of zingerone. It is possible that increasing the extraction temperature could lead to higher zingerone recovery, but room temperature extraction is clearly effective.

[0481] After taking ~300 mL sample of ZINGOEE, the remaining extract (3249 g) was evaporated under vacuum to produce 104.9 g of concentrated extract [ZINGOCE] with a zingerone content of 33 mg / g (3.46 g of zingerone). This concentrated extract had a total solids content of 37.1% (measured as loss on drying at 110°C), indicating that the final oleoresin weight that could be achieved if all ethanol were removed would be 38.9 g. If this number is extrapolated to the total amount of ZINGOEE produced, the resulting extraction yield for the process is around 5.4%. A standardised tincture containing 12 mg / g of zingerone was prepared by combining 25.5 g of ZINGOCE with 44.5 g of food grade ethanol. Samples of this tincture were sent for further testing.

[0482] In addition, further analysis showed that no aldehydes were present in the final product. See Figures 8A-8B. For these assessments, a sample of an ethanolic extractof treated ginger was analysed using GCMS for the presence of aldehydes. If present, this would be expected as (predominantly) hexanal derived from 6-gingerol. It is expected that the powder preparations (e.g., pre-extraction powders, as in Examples 4 and 5, above) will also lack aldehydes. Next steps will include evaporation of the ethanolic tincture to produce a thick ethanolic paste.

[0483] Conclusions: The proposed method which included screw pressing followed by KOH treatment in the juice phase, neutralisation with citric acid, and then freeze drying, proved to be highly effective, with almost total recovery of 6-gingerol in the juice phase achieved, as well as total conversion to zingerone after 5 hours of treatment with 2% KOH at 60°C. The pre-processed dried powder currently contained 5.32 mg / g zingerone, which is at least two times more efficient than previous manufacturing techniques.

[0484] The ethanol extraction at room temperature for 24 hours is an optional step achieving at least 85% recovery of zingerone. The recovery could be further increased by washing the solids with fresh ethanol after extraction. In these methods, the ethanolic extract [ZINGOEE] was evaporated to achieve a significant volume reduction, followed by reconstitution of the concentrated extract [ZINGOCE] with fresh ethanol in order to produce a standardised tincture containing the target dose of 12 mg / g zingerone.

[0485] For the extraction process, the overall Z mass balance (out / in) was 98.5%. The zingerone content in the filtrate (ZINGOEE) was 1.1 mg / g, and the zingerone content in the concentrated extract (ZINGOCE) was 33 mg / g. The method produced 104.9 g of ZINGOCE at 33 mg / g, leaving 3.46 g zingerone. This number is lower than the 3.88 g in ZINGOCE because ~400 g of ZINGOCE was removed before evaporation (for testing, plus ~300 mL retention sample). Taking this into account, the calculations fit very well. The overall zingerone mass balance before evaporation was calculated as 98.5 % (= (0.63 + 3.88) / 4.58).

[0486] The results obtained in this work indicate that 100 kg of fresh ginger with a 6-gingerol content of 0.5 mg / g would yield 3.6 kg of standardised tincture with a zingerone content of 12 mg / g. From this, it can be taken that higher 6-gingerol level should produce a higher yield of zingerone.Example 6A: Large scale production process

[0487] Overview: A large scale production process has been carried out. This allows up to 700 ± 50 kg raw ginger rhizome per run as outlined below.

[0488] Step 1: Initial juicing of ginger rhizome.

[0489] Introduction: A total of 1360 kg raw ginger was delivered to Phytex to Synergy Food group manufacturing plant in Brookvale, Sydney. Material was required to be stored under refrigerated conditions (4oC ± 3oC) when stored overnight. In processing, material was transferred back to refrigerated conditions to the extent possible to reduce the risk of degradation. Ginger juice was collected in 200 L blue poly drums for transport back to Phytex facility. The solid marc was collected in a separate 200 L blue poly drum (see, e.g., Figure 7B). Equipment and juicing areas were cleaned and maintenance / calibration was performed on equipment. Clean tags or cleaning logs were used as needed.

[0490] Run A details: Ginger rhizomes were divided into two equal ~700 kg runs. The second 700 kg batch was stored under refrigeration until use in the following week. The first 700 kg batch was passed through the belt press juicer (Voran EBP500). All juice obtained from the juicer was collected into 200 L blue HDPE drums (provided by Phytex) on a wooden pallet. All solid / marc obtained from the juicer was placed into a separate 200 L blue HDPE drum (provided by Phytex). The solid marc was passed through the belt press juicer (Voran EBP500) a second time to remove further liquid from the solid marc. The liquid from the second press was combined with the original 200 L blue HDPE drums containing the first-pressed liquid.

[0491] Run B details: The following week, juicing was commenced for the second 700 kg batch. The second 700 kg batch was passed through the belt press juicer (Voran EBP500). All juice obtained from the juicer was placed into 200 L blue HDPE drums (provided by Phytex) on a wooden pallet. All solid / marc obtained from the juicer was collected into a separate 200 L blue HDPE drum (provided by Phytex). The solid marc was passed through the belt press juicer (Voran EBP500) a second time to remove further liquid from the solid marc. The liquid from the second press was combined with the original 200 L blue HDPE drums containing the first-pressed liquid.

[0492] The total amounts of ginger juice and solid marc were transferred for in- process sampling and readiness for Ultrafiltration (UF). The mass balance and percentage yield were calculated and recorded. At the end of Step 1, approximately 600 L ± 50 Lginger juice and 200 kg + 50 kg ginger marc / solid was obtained from each batch of 700 kg + 50 kg ginger rhizome.

[0493] Step 2 (i): Alkaline treatment of ginger juice and ginger marc. See, e.g., flowchart in Figure 7C.

[0494] Introduction: Run A and Run B were performed in two equal treatments with KOH. The extraction vessel had maximum capacity of 400 L, so each run (A or B) was performed in two parts (run A1 / A2 and run B1 / B2). Equipment maintenance / calibration and cleaning status was checked before proceeding with treatment. Clean tags or cleaning logs were used as needed.

[0495] Processing of ginger juice (300 L per run): The volume of ginger juice obtained in the multiple 200 L HDPE drums was measured. Net weights were recorded for all HDPE drums. The ginger juice / settleable solids remaining in each 200 L HDPE drum were mixed using a 1 m plastic mixing paddle to ensure solids were resuspended. A clamp was attached to the decanting lid (lid with 25 mm poly valve) to each 200 L HDPE drum. Using the drum lifter all solid fractions were poured into the extraction vessel to combine with the UF concentrate. A wall mounted stirrer purple propeller was inserted into the extraction vessel with 2 x 100 mm stainless steel propellers attached. Propeller heights were set to the appropriate height with to mix the juice volume. The wall mounted stirrer was connected on utilities board and stirring was commenced in a clockwise direction at 50 Hz. The settleable sludge from the bottom of each drum was scooped out and transferred to the extraction vessel (to be dissolved by the heat and alkaline treatment). Each drum was hosed with I L water to remove all residual ginger juice, and this was added to the extraction vessel.

[0496] Adding potassium hydroxide to ginger juice: Stirring being commenced, KOH was added at 2.0% w / w. This was based on the volume of total juice obtained from ginger rhizome (raw material). Before KOH addition, the volume for the ginger juice and the initial pH level were recorded. A blue HDPE drum was tared on the 150 kg scale. The total amount of ginger juice was weighed for inclusion in the alkaline treatment. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffer solutions, and calibration was checked with a pH 2.0 buffer solution. In the extraction vessel, 50% KOH was added to the ginger juice extract at the rate of: wt. KOH (kg) = wt. ginger juice (kg) x 0.03 kg KOH (50%). Addition at this rate increased the pH to 13.0 + 1.0. All pH reading adjustmentswere incorporated with automatic temperature compensation (Mettler Toledo pH meter). When the pH range was reached, stirring was continued for a further 15 minutes (minimum). The final pH and volume of added KOH was recorded.

[0497] Alkaline treatment procedure for ginger juice: A stainless steel steam coil was inserted into the extraction vessel and connected to the steam inlet on the utilities wall using the dedicated steam hose. The steam valve was turned on at the wall to allow steam to heat the coil. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffer solutions, and calibration was checked with a pH 2.0 buffer solution. The ginger juice (pH greater than 13) was heated to 60oC + 2oC with stirring for 60 minutes. The steam flow was adjusted to ensure the alkaline liquid remained with the temperature range (60oC + 2oC). A sample was taken from the stirred alkaline ginger juice (20 mL) to monitor conversion of 6-gingerol to zingerone. The volume of the alkaline ginger juice was recorded.

[0498] Neutralisation procedure for ginger juice: The alkaline ginger juice was stirred for another 5 minutes before neutralising the solution with citric acid >98.0%. The SS coil within the extraction vessel was connected with cooling water and the cooling water was circulated. The addition of citric acid (>98%) to the alkaline juice represented a strong acid-base reaction, hence being exothermic and therefore generating heat. The cooling water was applied to alleviate excess heat from the solution. Prior to use, the citric acid was prepared. This was based on a raw material starting weight of 1340 kg. Citric acid granules (18.0 kg) were weighed out into a 20 L purple polythene bucket. Extra citric acid aqueous solution (50% w / v) was prepared by dissolving 1.0 kg citric acid granules into 1.0 L purified water in a 20 L purple polythene bucket. This was dissolved with a stainless steel hand stirrer. Whilst alkaline ginger juice was stirring in the extraction vessel, citric acid granules were added. This was done by adding 10 kg and waiting for the granules to dissolve. The pH of the solution was monitored during this process. The slow addition of citric acid was continued using 500 ml scoops at a time and waiting 30 seconds before the adding the next scoop. Once all of the citric acid was added and dissolved (18.0 kg), a pH reading was obtained. Then the 50% citric acid aqueous solution was added until a pH range of 7.0-7.3 was obtained. When this desired range was reached, stirring was continued for a further 15 minutes. All pH reading adjustments were incorporated with automatic temperature compensation (Mettler Toledo pH meter). The resulting solution was then noted as “zingerone liquid”. The final pH, volume of added citric acid (>98%), and volumeof zingerone liquid was recorded. A sample was taken from the zingerone liquid (20 mL) to monitor conversion of 6-gingerol to zingerone.

[0499] Step 2 (ii): Alkaline treatment of ginger marc. See, e.g., flowchart in Figure 7D.

[0500] Processing ginger marc / solids (50 kg per run): The wall mounted stirrer was inserted into the extraction vessel with three large 150 mm propellers attached. Stirring was commenced at 50 Hz. From the 200 L HDPE drum, 50 kg total solid marc was added to the extraction vessel. It was ensured that the solid was continually drawn into the liquid and the solution was moving freely with the stirrer. If the solution became too viscous, an addition 20 L of water was added.

[0501] Adding potassium hydroxide to ginger marc / solids: KOH was added at 2% w / w. This was based on based on weight of the volume of total juice extract obtained from ginger rhizome raw material. A blue HDPE drum was tared on the 150 kg scale. The total amount of marc was weighed for inclusion in the alkaline treatment. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffer solutions, and calibration was checked with a pH 2.0 buffer solution. In the extraction vessel, 50% KOH was added to the ginger juice extract at the rate of: wt. KOH (kg) = wt. ginger marc (kg) x 0.03 kg KOH (50%). The calculations accounted for the weight of ginger marc (50 kg) and water added (20 kg). Addition at this rate increased the pH to 13.0 + 1.0. When the pH range was reached, stirring was continued for a further 15 minutes (minimum). The final pH and volume of added KOH was recorded.

[0502] Alkaline treatment procedure for ginger marc / solids: The SS steam coil was s inserted into the extraction vessel and connected to the steam inlet on the utilities wall using the dedicated steam hose. The steam valve was turned on at the wall to allow steam to heat the coil. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffer solutions, and calibration was checked with a pH 2.0 buffer solution. The ginger marc (pH greater than 13) was heated to 60oC ± 2oC with stirring for 60 minutes. The steam flow was adjusted to ensure the alkaline liquid remained with the temperature range (60oC + 2oC). A sample was taken from the stirred alkaline ginger marc (20 mL) to monitor conversion of 6-gingerol to zingerone. The volume of the alkaline ginger marc was recorded.

[0503] Neutralisation of ginger marc / solids: The alkaline ginger marc was stirred for another 5 minutes before neutralising the solution with citric acid >98.0%. The SS coil within the extraction vessel was connected with cooling water and the cooling water was circulated. This was applied to alleviate excess heat from the exothermic reaction (acid + base). Prior to use, the citric acid was prepared as noted above. Whilst alkaline ginger marc was stirring in the extraction vessel, citric acid granules were added. This was done by adding 5 kg and waiting for the granules to dissolve. The pH of the solution was monitored during this process. The slow addition of citric acid was continued using 500 ml scoops at a time and waiting 30 seconds before the adding the next scoop. Once all of the citric acid was added and dissolved (18 kg, total), a pH reading was obtained. Then the 50% citric acid aqueous solution was added until a pH range of 7.0-7.3 was obtained. When this desired range was reached, stirring was continued for a further 15 minutes. All pH reading adjustments were incorporated with automatic temperature compensation (Mettler Toledo pH meter). The resulting solution was then noted as “zingerone marc”. The final pH, volume of added citric acid (>98%), and volume of zingerone marc was recorded. A sample was taken from the zingerone marc (20 mL) to monitor conversion of 6-gingerol to zingerone. The mass balance and percentage yield were calculated.

[0504] Step 3: Production of zingerone powder by drying and milling.

[0505] Oven drying: Equipment maintenance / calibration and cleaning status were confirmed. The drying over was set up with calibrated COMARK digital logger with stainless steel probe. Time was recorded for each drum of zingerone concentrate to be taken from the cold room to prepare for drying. The Brix% and theoretical zingerone content were noted. The oven was loaded with all trays having Teflon mats for drying. The drum was mixed using a polypropylene paddle before using a 4 L jug to remove a volume of liquid. This was subsequently transferred to a 2 L jug to allow placement into each tray. Next, 3 L was poured onto the Teflon mat for each tray. The trays were loaded onto each shelf to ensure a gap between the trays (not less than 30 mm) and allow for airflow and to allow breaking up of dried materials. The oven temperature was set at 60°C. After 24 hours, each tray was inspected and scraped to release the softer portions off the Teflon mat as needed. The underside of the product was positioned on the top of the flipped tray. The product was gently broken up using the plastic spatula to facilitate drying. The trays were loaded back into the oven and dried for a further 4 hours (28 hours total). When sufficientdried (at 24 or 28 hours), the product was crushed manually with a stainless steel spatula and collected in 100 micron poly bag. Each bag was weighed and recorded and placed into a 100 L square mobile tub. The square mobile tub was weighed and recorded. The product was then ready for milling.

[0506] Milling: Prior to milling, the stainless steel hammer mill was checked as clean and fit with a clean 1 mm mesh screen (“fine zingerone screen”). The lid was fitted with a 3 mm rubber mat seal and the thumb screw was firmly tightened to lock the lid in place. The hammer mill was run for 10 seconds to ensure correct rotation and clearances. A 100 micron poly bag was fitted to the outlet of the mill chamber and tightened with an adjustable clamp. The overhead dust extractor was activated. Slowly, the coarse dry zingerone material was transferred into the feed chute using the poly scoop. Two scoops at a time were emptied into the chute and the inlet flap was opened every few seconds. All the material in the chute was passed through the stainless steel screen and collected in a poly bag collector. All bags were weighed and calculations were made to determine mass balance and percentage yield. A representative sample (20 g) was removed from each bag and assessed for percentage moisture determination. All 20 kg bags were placed into an additional 100-micron poly bag. The remaining air void was evacuated before sealing with a double seal using an Impulse heat sealer. The bags were stored in the cold room. Quality analysis was performed for the final product (zingerone powder). See Table 12A, below. Quality indicators were found to be excellent. Table 12A. Zingerone extract (powder) quality assessment Content of zingerone (HPLC) 0.2 to 0.4% Pesticide residues Less than limit of reporting Aflatoxins Less than limit of reporting Total aerobic microbial count (cfu / g) 180 cfu / ml Total yeast and mould count (cfu / g) < 10 cfu / ml E. coli and salmonella Absent Elemental impurities Cu = 5.4 mg / kg Zn = 31 mg / kg Less than limit of reporting for As, Cd, Cr, Hg, Ni, Pb Acid insoluble ash 1% Water soluble ash 35.8% Total ash 37.1% Gingerol content ≤ 0.1% Water determination 3.8%

[0507] Step 4: Ethanol extraction of zingerone powder.

[0508] Introduction: Up to 75 kg dry powder was able to be extracted in a single extraction step as outlined below. Equipment maintenance / calibration and cleaning status (including extraction vessel) was confirmed. The 40 mm stainless steel strainer was attached to the outlet in the bottom of the extraction vessel. This was hand tightened into the bottom 25 mm thread to strain out the solids after extraction stirring. The dedicated stainless steel heating coil was fitted inside the extraction vessel for connection to steam later in this step. The glass condenser coil was attached to the lid of the extraction tank and the inlet / outlet was connected to the cooling water inlet / outlets on the wall. The cooling water was circulated through the glass condenser coil.

[0509] Single extraction: For the extraction, 95% ethanol was added to the clean extraction vessel with the lid fitted. The following addition rate was utilised: vol 95% ethanol = amt. zingerone powder (kg) x 5 kg. The purple WMS shaft was inserted through the bearing gland in the lid of the extraction vessel. Stirring was commenced at 200-300 rpm to facilitate complete mixing of the ethanol solution. Slowly, 75 kg of dry zingerone powder (from Step 3) was added to the extraction vessel through the 100 mm access port on the lid of the vessel. Stirring was continued during this addition. Once all the powder was added, the access port was closed and the lid was sealed. All initial temperatures were recorded before heating. Then, the steam supply was connected to the stainless steel coil in the extraction vessel. Whilst stirring, the temperature of the ethanolic solution was monitored and maintained at a minimum of 50°C for at least 4 hours. The stirrer was turned off and the solution was allowed to stand for 20 minutes. The final temperature was recorded prior to filtration of the ethanolic extract. A sample was removed from the ethanolic extract (20 mL) and tested for zingerone.

[0510] Filtering of residual solid from the ethanolic extract: The contents of the ethanolic extract were drained into two clean 200 L HDPE drums. Stirring was continued while draining. The ethanolic extract was allowed to sit for 30 minutes, to allow any solids to settle. The hopper plate filter was set up with a Z1 pad (5 micron). This was fitted and compacted with a stainless steel plunger tool. The upper layer of the ethanolic extract was decanted through the hopper plate filter using the drum lifter. The decanted solution was poured thorough a 55 micron pre-filter and collected into buckets. The filtered ethanolic extract was collected from the hopper plate filter into two clean blue HDPE drums. When the hopper plate filter was filled with solid components, a full vacuum was applied to theunit for 10 minutes, until all the liquid was collected. The hopper plate filter was emptied after every vacuum filter and set aside in clean poly buckets. This was repeated until all of the ethanolic extract had been filtered through the Z1 pad. The volume of 95% ethanol wash was recorded. A sample was removed from the combined filtered ethanolic extract (20 mL) and tested for zingerone. The two drums with filtered ethanolic extract were stored in the cold room.

[0511] Rotary evaporation of the ethanolic extract: Three 15 L buckets of filtered ethanolic extract were removed from the cold room and allowed to equilibrate to room temperature. A Heidolph round bottom flask was prepared, dried, and weighed. The Heidolph 20 L rotary evaporator was set up with 1 L fresh 95% ethanol added to the RBF. The cooling water inlet / outlet lines was connected to appropriate valves on the wall and the valve was adjusted to alter flow rate as required. The rotary evaporator was connected to the Heidolph vacuum pump. A white 4 mm HDPE tube, submerged in 95% ethanol, was connected to the inlet tap on the rotary evaporator. Circulation of cooling water was commenced through the glass condenser. Residual water was removed during the evaporation stage by running at a minimum 50 mBar and 40°C. Accordingly, the vacuum was set and slow rotation of the round bottom flask was commenced (approximately 60 rpm). As each bucket was emptied, the inlet tube was transferred to the next bucket until all three buckets (45 L total) had been bled into the rotary evaporator and collected in the condenser flask. Observations were made for pressure, colour, etc.

[0512] Standardisation of ethanolic extract: For each rotary evaporator run (45 L filtered ethanolic extract in three buckets), the volume was condensed down to the 4 L volume line marked on the outside of the RBF. This line represented roughly the standard mark that 45 L Ethanol Extract is condensed down to 4 L (12.5% mg / ml). At that point, a sample of the concentrated tincture (10 mL) was removed and tested for zingerone. If the concentration of zingerone was greater than 12.5 mg / ml, 1 L filtered combined ethanolic extract from Step 4 was added and evaporation was continued until the correct concentration was achieved. If the concentration of zingerone was less than 12.5 mg / ml, evaporation was continued until the correct concentration was achieved. Twelve amber 4 L glass bottles were rinsed with fresh 95% ethanol and left to drain for 30 minutes. When standardised to 12.5 mg / ml, the RBF was removed from the rotary evaporator and placed into a 15 L bucket. The standardised zingerone tincture was decanted into the rinsed anddrained bottles, leaving 30 mm from the bottom thread on the bottle. The filled bottles were capped with poly lids having Teflon seals and hand tightened. Parafilm was wrapped around the bottle neck and lid. Sealed bottles were placed into 100 micron poly bags and double heat sealed. These were placed in a box and the box was put into the cold room. Quality analysis was performed for the final product (ethanolic extract). See Table 12B, below. Quality indicators were found to be excellent. Stability of the final product was assessed and confirmed after 2 months of storage under accelerated conditions (40˚C ±2˚C / 75% ± 5% RH), and independently, after 2 months of storage at cooling conditions (5°C). Stability was found to be extremely high. See Tables 12C-12D, below. See, also, Figures 7E-7F. Due to the small sample size tested at accelerated conditions (less than 2 g), some variability was seen at 1 month time point (Table 12C). It is understood that larger sample sizes in storage act to enhance stability of the product by reducing container headspace. Good stability is expected for at least 6 months storage. Table 12B. Zingerone extract (tincture) quality assessment Content of zingerone (HPLC) 12.8 mg / ml ± 10% Pesticide residues Less than limit of reporting Total aerobic microbial count (cfu / g) < 1 cfu / ml Total yeast and mould count (cfu / g) < 1 cfu / ml Limit of non-volatile residues 440 mg Total ash 3.0% Specific gravity 0.88 Gingerol content ≤ 1% Alcohol content 67%Table 12C. Zingerone extract (tincture) stability assessments Batche Time Zinge Zinge Ginge Ginge Ginge Ginge Shog Shog s / point rone rone rols + rols + rols rols aols aolsCondit (mon (% (mg / Shoga Shoga (% (mg / (% (mg / ions ths) w / w) ml) ols ols w / w) ml) w / w) ml) (% (mg / w / w) ml) 40°C / 0 1.62 13.58 0.18 1.51 0.07 0.58 0.11 0.93 75% 1 1.41 12.39 0.19 1.45 0.07 0.54 0.10 0.91 RH 2 1.59 13.21 0.17 1.45 0.07 0.58 0.10 0.88 Table 12D. Zingerone extract (tincture) stability assessmentsExample 7: Anti-inflammatory activity for zingerone composition

[0513] Overview: These studies were carried out to determine the anti- inflammatory activity of the disclosed botanical extract (ethanolic extract (tincture); see Example 6) standardised to zingerone content. Nitric oxide (NO) and IL-6 levels were assessed. Methodology: The anti-inflammatory activity was determined in lipopolysaccharide (LPS) stimulated murine macrophages, RAW264.7 cells cultured in standard cell culture media (DMEM, foetal bovine serum 5%) and incubated in the presence or absence of different test compounds / extracts and controls. The production of inflammatory mediators, including NO and IL-6 were measured by established methods using commercial ELISA kits (suppliers listed in Table 13). Each sample was tested with at least 6 concentrations (from 20 μg / mL to 0.6 μg / mL). This was done using 3 replicates (maximum concentration was 40 µM zingerone) (n=9), with relevant internal controls. In addition, the cytotoxicity of each sample tested was determined by MTT assay (tetrazolium dye MTT, which is chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The assay parameters for each assay are summarised in Table 13. Table 13. Anti-inflammatory assays and positive controls Assay Plating Stimulation Incubation Kit Positive control time time supplier NO 48 hours LPS 18 hours Griess 1400W 50 ng / ml Reagent (Cayman) IL-6 48 hours LPS 18 hours Peprotech Dexamethasone 50 ng / ml (Sigma-Aldrich)

[0514] The tincture from Example 6 was utilised for these studies. For the NO and IL-6 assays the cultured RAW264.7 cells were counted and plated (0.8 x 105cells / well) in 96 well plates and incubated for 48 hours. The medium was then aspirated and replaced with fresh medium followed by the addition of the test compounds. The compounds were incubated for 1 hour prior to the addition of the stimulant. The plates were then incubated for 18 hours and the supernatant analysed for the mediator of interest, the remaining cell viability was determined by MTT.

[0515] The positive controls were selected based on their widespread use in similar assays, including N-(3-(aminomethyl)benzyl)acetamidine (1400W), a slow, tight binding inhibitor of inducible nitric-oxide synthase (iNOS) (Garvey et al., 1997, J BiolChem 272(8):4959-63) and dexamethasone, a commonly used cytokine inhibitor. The data were presented as means and standard error of means (sem) (n=9).

[0516] The dose response curves were fit using Graph Pad Prism. The 95% confidence intervals (CI) were calculated using the whole data set and gave an estimate error in the IC50 value. The 95% CI for the IC50 where the IC50 was not reached in the testing concentrations, require graphical extrapolation to calculate the IC50. Where the IC50has been estimated by graphicalis used to indicate that it is an estimate and not experimentally determined, unless otherwise stated. The graphical extrapolation results in a wider 95% confidence interval.

[0517] Results: The tincture from Example 6 was assayed to determine anti- inflammatory activity. The anti-inflammatory activity was assessed using key mediators of inflammation: NO, and IL-6. The cytotoxicity of each sample was also determined by MTT assay.

[0518] Cytotoxicity: The effect of the compounds on the viability of the cells was determined by MTT. Potency was monitored to avoid false positives, as dead cells do not produce inflammatory mediators. Cytotoxicity was determined spectrophotometrically as mitochondrial dehydrogenase present in viable cells cleaves the tetrazolium ring of MTT to yield a purple MTT formazan. All doses were tested up to 40 μM zingerone. The results are shown in Figure 9. From the cytotoxicity assay, the EC50for the disclosed tincture was determined as 40 µM zingerone.

[0519] NO assay: NO is a radical metabolite, which has been shown to have numerous physiological functions both as a signalling molecule and as a toxic agent in inflammation (Coleman, 2001). The inhibition of iNOS and reduction in NO levels secreted by immune cells may be a contributing factor to anti-inflammatory activity. The disclosed tincture was therefore assayed to determine if they showed inhibition on the inflammatory signalling molecule NO. The dose dependent effects of the tincture on NO are shown in Figure 10. From the NO assay, the IC50 for the disclosed tincture was determined as 9.2 µM zingerone.

[0520] IL-6 assay: IL-6 is considered a pro-inflammatory cytokine. IL-6 is secreted by T cells and macrophages, which stimulates an immune response. IL-6 is responsible for the increased production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to the differentiation of T cells into regulatory T cells.It can cross the blood-brain barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature set point (Banks, Kastin, & Gutierrez, 1994). The inhibition of IL-6 release by immune cells points to anti-inflammatory activity. The dose dependent effects of the disclosed tincture on IL-6 are shown in Figure 11. From the IL-6 assay, the IC50 for the tincture was determined as 4.6 µM zingerone.

[0521] It can be seen from Figures 10, 11, and 12, that the disclosed tincture inhibited NO and IL-6 in a dose dependent manner. The effective inhibitory levels for NO and IL-6 were significantly lower than the levels required for cytotoxicity, validating the observed anti-inflammatory effects of the tincture. Example 8: Comparative anti-inflammatory testing for zingerone compositions

[0522] Overview: These studies were carried out to determine the anti- inflammatory bioactivity of the disclosed botanical extract (ethanolic extract (tincture); see Example 6) as compared to commercially sourced zingerone in RAW264.7 macrophages.

[0523] Methodology: An extract (ethanolic extract (tincture); Rx7 / 22 / 161) was prepared as described in Example 6. The extract was standardised at 33 mg of zingerone per mL of extract. Commercially available zingerone powder (Vigon #500938) was obtained and dissolved fresh to 33 mg / mL in ethanol on the day of use. Samples in ethanol were further diluted in cell media immediately prior to addition to cell culture. An ethanol solvent control was included in cell culture experiments to exclude any potential solvent effect.

[0524] RAW264.7 cells in growth media (DMEM, 10% FBS, PSN, 2 mM L- glutamine) were plated at 0.8 x 105cells / well in 96-well tissue culture treated plates and incubated at 37°C / 5% CO2 in a humidified environment for 48 hours. The spent media was then aspirated and replaced with media containing 5% FBS. Treatments were added to give final concentrations of 0.625 – 40 µM zingerone (or equivalent dilutions for ethanol solvent control). The cells were incubated with the treatments for 1 hour prior to the addition of 50 ng / mL lipopolysaccharide (LPS; from Escherichia coli O111:B4).

[0525] The cells were co-incubated with treatments and LPS for 18 hours before conditioned media was harvested, centrifuged and cell-free supernatants collected. The viability of the remaining cells was determined by WST-1 assay. Controls included were unstimulated (cells with no LPS), LPS (cells exposed to 50 ng / mL LPS), Dex (cellsexposed to 1 or 10 µg / mL dexamethasone before addition of 50 ng / mL LPS), and media (media containing no cells or samples).

[0526] For the cytotoxicity assay, an equal volume of a 1:5 mixture of WST-1:media was added to each cell well and incubated for 10 minutes. Immediately prior to measurement of absorbance, an equal volume of DPBS was added and absorbance read at 440 nm (and 620 nm for removal of background). Hydrogen peroxide was included as a cytotoxic positive control. Results were normalised to LPS-stimulated control and expressed as percentage cell viability. Sample concentrations where cell viability is below 80% of the unstimulated control were deemed cytotoxic.

[0527] For the IL-6 assay, Interleukin 6 (IL-6) was analysed in cell-free conditioned media that was collected after treatment and LPS-stimulation using a bead- based multiplex assay panel (Legendplex MU Th1 / Th28-plex panel; BioLegend #741054) and measured using a Cytex Aurora Spectral 3 laser flow cytometer. As a follow-up to the IL-6 assay, assays were performed to measure IL-10 and TNF.

[0528] Statistical analysis was conducted using Minitab 18.0. Student’s paired t- tests were conducted to statistical differences between treatments at each timepoint. Data are presented as mean ± standard error of the mean (SEM). Three replicate cell wells were included in each experiment, and three separate cell experiments were performed.

[0529] Results: The viability of the cells after incubation with the compounds / extracts was measured. In the present assay, WST-1 reacts with a mitochondrial enzyme to form a coloured dye which can be measured by absorbance. As such, WST-1 is a measure of cellular metabolism, and a reduction in the WST-1 reaction rate can be indicative of cellular death. Hydrogen peroxide (H2O2) is used as a positive control to induce cell death and ensure the validity of the assay.

[0530] Results for cytotoxicity are shown in Figure 13. This figure depicts cell viability levels following 18 hour treatment or cells with 50 ng / mL lipopolysaccharide (LPS) and LPS with the disclosed botanical extract or zingerone (0.6 to 40 µM) and equivalent ethanol controls. Data are means ± SEM from three independent experiments (n = 3). Assay validation was determined with hydrogen peroxide (H2O2).

[0531] In the left-hand panel, a clear cytotoxic dose-dependent response is seen in the H2O2-exposed cell samples, confirming the assay is working as expected. WST-1 values of <80% of the untreated control (dashed horizontal line) are considered cytotoxic.Cytotoxicity was assessed in RAW264.7 cells after 18 hour of exposure to the disclosed botanical extract or commercially sourced zingerone. Concentrations ranged from 0.3 to 40 µM zingerone. No apparent cell death was detected by the WST-1 assay in RAW264.7 cells exposed to either the ethanolic extract or commercially sourced zingerone at the concentrations tested (Figure 13). That is, none of the data points fell below 80% of WST- 1 response of the untreated LPS-stimulated control. This validated the results from the cytokine assay (results below).

[0532] The cell model used for IL-6 assessments explores the pro-inflammatory response by a mouse macrophage cell line (RAW264.7) when exposed to a bacterial endotoxin (lipopolysaccharide; LPS). Mechanistically, LPS interacts with the membrane- bound TLR4 receptor, triggering a signalling cascade within the cell to activate NF-κB, a transcription factor that regulates the expression of many proinflammatory cytokines, including IL-6.

[0533] Results for IL-6 measurements are shown in Figure 14. This figure depicts IL-6 levels produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS), LPS + 10 µg / mL dexamethasone (Dex) and LPS + disclosed botanical extract or commercially sourced zingerone (0.6 to 40 µM) for 18 hours. Data are means ± SEM from three independent experiments (n = 3). The asterisk denotes significantly lower IL-6 production from zingerone at the corresponding concentration (p < 0.05). Treatments with no error bars at 20,000 pg / mL indicate values ≥20,000 pg / mL.

[0534] It was found that the exposure of RAW264.7 cells to LPS for 18 hours significantly induced IL-6 secretion to concentrations above the assay’s upper limit of accuracy of 20,000 pg / mL (Figure 2). IL-6 secretion by RAW264.7 cells following co- treatment with equivalent ethanol concentrations present in the disclosed botanical extract and commercially sourced zingerone dilutions evaluated were also above the assay’s upper limit. Co-treatment of RAW264.7 cells with LPS + disclosed botanical extract or LPS + commercially sourced zingerone at concentrations ranging from 0.6 to 10 μM were similarly above the assays upper limit of accuracy, making it difficult to assess the anti- inflammatory bioactivity of these samples at these concentrations.

[0535] Notably, co-treatment of RAW 264.7 macrophages with LPS and disclosed botanical extract or commercially sourced zingerone at the highest concentrations evaluated (20 and 40 μM zingerone), resulted in measurable reductions in IL-6 that waswithin the limits of the assay’s accuracy. In particular, co-treatment of RAW264.7 with the disclosed botanical extract at 20 and 40 μM zingerone resulted in significantly (p < 0.05) lower IL-6 production compared with co-treatment with commercially sourced zingerone. These findings indicate that the disclosed botanical extract is more efficacious than commercially sourced zingerone in reducing IL-6 production at these concentrations.

[0536] Specific reductions were also seen for IL-10. Figure 15 depicts IL-10 levels produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS), LPS + 10 µg / mL dexamethasone (Dex) and LPS + disclosed botanical extract or LPS + commercially sourced zingerone (1.25 to 40 µM) for 18 hours. Data are means ± SEM from three independent experiments (n = 3). Carrot denotes significantly lower IL-10 production compared with corresponding ethanol control (p < 0.05). Asterisk denotes significantly lower IL-10 production from zingerone at the corresponding concentration (p < 0.05). Treatments with no error bars at 15,000 pg / mL indicate values 215,000 pg / mL and are above the assays limit of accuracy.

[0537] The results showed that exposure of RAW264.7 macrophages to LPS for 18 hours induced a significant increase in IL-10 that was attenuated by co-treatment with dexamethasone, a known immune suppressant. Co-treatment of cells with 10 and 20 µM zingerone equivalence of the disclosed botanical extract, but not commercially sourced zingerone, significantly attenuated LPS-induced IL-10 secretion by RAW264.7 (p < 0.05). Additionally, IL-10 secretion by RAW264.7 co-treated with the disclosed botanical extract at 20 and 40 µM zingerone was significantly lower compared with commercially sourced zingerone at these concentrations (p < 0.05).

[0538] Results for TNF are shown in Figure 16. This figure depicts TNF levels produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS), LPS + 10 µg / mL dexamethasone (Dex) and LPS + disclosed botanical extract or LPS + commercially sourced zingerone (1.25 to 40 µM) for 18 hours. Data are means ± SEM from three independent experiments (n = 3). Asterisks denotes significantly lower IL-6 production from zingerone at the corresponding concentration (p < 0.05). Treatments with no error bars at 15,000 pg / mL indicate values 215,000 pg / mL and are above the assays limit of accuracy.

[0539] The results showed that exposure of RAW264.7 macrophages to LPS for 18 hours induced a significant increase in TNF that was attenuated by co-treatment withdexamethasone, a known immune suppressant. Co-treatment of cells with the disclosed botanical extract or pure zingerone compound had no significant effect in attenuating LPS- induced TNF secretion (p > 0.05).

[0540] Overall, the anti-inflammatory activities of the disclosed botanical extract (with naturally occurring zingerone) are significant and notably superior to the activities of commercially sourced zingerone, particularly in relation to IL-6 and IL-10 levels. Future assays will be carried out with overall lower induction levels to allow more accurate comparisons.

[0541] On further review of these studies, it was noted that that the concentration of zingerone in the disclosed botanical extract (tincture) was noted incorrectly by the researchers performing the experiments. Although indicated in these studies as 33 mg / mL (0.170 M), the concentration of zingerone in the tincture was actually 33 mg / g (0.134 M). As such, the zingerone concentrations for the tincture dilutions have been incorrectly noted as 0.625, 1.25, 2.5, 5, 10, 20, and 40 µM. Based on the correct starting concentration of 33 mg / g, the zingerone concentrations for the tincture dilutions can be correctly calculated as 0.493, 0.986, 1.97, 3.95, 7.89, 15.8, and 31.6 µM, respectively.

[0542] This means that the results shown for each of IL-6, IL-10, and TNF have all been underestimated in regard to the activity of the disclosed botanical extract, since the commercially sourced zingerone was used at significantly higher concentrations (0.625, 1.25, 2.5, 5, 10, 20, and 40 µM) than the disclosed botanical extract (0.493, 0.986, 1.97, 3.95, 7.89, 15.8, and 31.6 µM). In any case, these initial results are extremely positive, and the studies are currently being repeated using the correct calculations. Table 14. Summary of results for IL-6 inhibition Method 1 Method 2 LPS stimulated 6641.11 ± 503.45 pg / mL 13930.0 ± 340.48 pg / mL LPS + dexamethasone 100 nM 1874.44 ± 394.56 pg / mL 8896.67 ± 241.14 pg / mL LPS + absolute ethanol 5685.56 ± 309.32 pg / mL 13985.56 ± 401.08 pg / mL

[0543] The MTT assay was used identify cytotoxic effects of compound treatment on the RAW264.7 cells. No cytotoxic effect was identified for any compound tested. In the Method 1 conditions, there was a notable drop in the MTT absorbance relative to the unstimulated Method 2 conditions, and both stimulated and unstimulated Method 2 conditions (Figure 18A: Method 1 stimulation; Figure 18B: Method 2 stimulation). Thisdrop is expected as IFN-γ’s mechanism involves stopping cellular proliferation, resulting in less cells present compared to (-) IFN-γ conditions. In particular, fewer cells means fewer mitochondria to metabolise the MTT to formazan, resulting in lower absorbance.

[0544] Conclusions: Dexamethasone at 100 nM was shown to be an effective control due to IL-6 inhibitory activity. The Method 2 set up was chosen for the remaining experiments, as this method showed compatibility to other data obtained. Example 9: Comparing methods for assessing anti-inflammatory activity and viability

[0545] Overview: These studies were carried out: (1) to identify the most effective positive control agent; and (2) to compare two different assessment methods for inflammatory activity. The following outputs were produced for this experiment: viability, measured by MTT; and IL-6 production, measured by ELISA.

[0546] The assessment methods are summarised as follows: Stimulated Day 0 Day 1 Day 2 Day 3 Day 4 Method 1 Plate cells LPS Stim + Harvest MTT and IFN-γ Prime Treatment ELISA finish Method 2 Plate cells LPS Stim + Harvest MTT and Treatment ELISA finish UnstimulatedDay 0 Day 1 Day 2 Day 3 Day 4 Method 1 Plate cells Media + Harvest MTT and Add media Treatment ELISA finish Method 2 Plate cells Media + Harvest MTT and Treatment ELISA finish

[0547] Methodology: Dexamethasone and synthetic zingerone were obtained from Sigma-Aldrich. For these studies, dexamethasone (100 nM) was used as the positive control); absolute ethanol was used as the vehicle control; and synthetic zingerone (20 µM) was used for test treatments. The testing included: (1) IL-6 ELISA – measures production of IL-6 from stimulated / unstimulated RAW264.7 cells; (2) MTT assay – identifies potential cytotoxicity by measuring metabolism of MTT to formazan.

[0548] On Day 0, the flask dedicated to Method 2 conditions was retrieved from 37°C / 5% CO2incubator. The growth media was decanted. Next, 10 mL CTCM was added and the cells were washed. The growth media was again decanted. Then, 10 mL CTCM was added and cells were harvested using a rubber scraper. The resulting cell suspensionwas transferred to a 50 mL falcon tube. This was centrifuged at 400 x g for 5 minutes. Cells were counted after applying a 1 in 10 trypan blue dilution. Samples were then diluted to 1 x 106cells / mL. Cells were then plated or added at 80 µL / well (80,000 cells / well). For stimulated Method 2 samples, 120 uL media was added. This step was repeated for unstimulated Method 2 samples. The cells were returned to the 37°C / 5% CO2 incubator and left for 48-hours.

[0549] On Day 1, the drug dilutions were prepared (see below). The flask dedicated to Method 1 conditions was retrieved from the 37°C / 5% CO2 incubator. The growth media was decanted. Next, 10 mL CTCM was added and the cells were washed. The growth media was again decanted. Then, 10 mL CTCM was added and cells were harvested using a rubber scraper. The resulting cell suspension was transferred to a 50 mL falcon tube. This was centrifuged at 400 x g for 5 minutes. Cells were counted after applying a 1 in 10 trypan blue dilution. Samples were then diluted to 1 x 106cells / mL. Cells were plated at 50,000 / well (50 µL). For stimulated Method 1 samples, 50 µL IFN-γ was added (final concentration 20 U / mL). For unstimulated samples, cells were plated and well volume was made up to 200 µL.

[0550] On Day 2, LPS and treatment conditions were prepared for Method 2 samples. For stimulated Method 2 samples, 50 µL LPS was added (final concentration 20 ng / mL) and 50 µL treatments were added. For unstimulated Method 2 samples, these were centrifuged and 50 µL of CTCM was removed from treatment wells. To these wells, 50 µL of treatment was added. For stimulated Method 1 samples, these were centrifuged and 100 µL media was removed from the wells. To these wells, 50 µL of LPS was added (final concentration 50 ng / mL) and 50 µL treatments were added. For unstimulated Method 1 samples, these were centrifuged and 50 µL of media was removed from the wells. To these wells, 50 µL of treatment was added. The set ups for IL-6 ELISA captures were started.

[0551] On Day 3, ELISA plates were washed and blocking was started. The supernatants for each sample were harvested (170 µL). Next, 50 µL of warmed CTCM was added to wells. Next, 20 µL of 5 mg / mL MTT solution was added. The plates were returned to the 37°C / 5% CO2 incubator for 45 minutes. While the MTT was incubating, ELISA captures were wash off and blocking solution was added. After 45 minutes, 10 μL MTT solubilizer was added. The ELISA process was continued. Cell counting was carried outfor four fields. The cell numbers per field were averaged as follows: (48+58+67+62) / 4 = 58.75 cells in 10 µL. This was then calculated as 58.75 * 10 * 104= 5875000 cells / mL. This was then calculated as 5875000 * 10 = 58750000 cells in 10 mL. Therefore, to make a 1 x 106cells / mL solution, a cell suspension up to 58.75 mL was prepared.

[0552] To prepare dilutions: (1) IFN-γ stock solution was 5,000,000 U / mL. This was diluted to 24,000 U / mL in CTCM; (2) LPS stock solution was 1,000,000 ng / mL. For Method 1, a 80 ng / mL solution was required (final concentration 20 ng / mL in the wells). For Method 2, a 200 ng / mL solution was required (final concentration 50 ng / mL in the wells); (3) Synthetic zingerone stock solution was prepared by adding 2 µL of zingerone into 198 µL CTCM. This gave a 1544.6401 µM stock. From this stock, 25.89 μL was added to 474.11 μL CTCM. This gave an 80 μM stock. The 80 μM stock (50 µL) was added to samples (along with LPS) to achieve a final concentration 20 μM (0.013% ethanol).

[0553] To prepare drug dilutions: (1) Dexamethasone stock solution was prepared by introducing 2 μL of dexamethasone into 998 μL CTCM. This gave a 5096 nM stock solution. From the 5096 nM stock solution, 78.49 μL was added to 921.51 μL CTCM. This gave a 400 nM stock solution. The 400 nM stock solution (50 μL) was added to samples (along with LPS) to achieve a final concentration of 100 nM (0.003% ethanol); (2) Ibuprofen stock solution was prepared by introducing 2 μL ibuprofen into 579.704 μL CTCM. This gave a 1000 μM ibuprofen stock solution. From the 1000 µM ibuprofen stock solution, 400 μL was added into 600 μL CTCM to make a 400 μM stock solution. The 400 µM stock solution (50 μL) was added to the samples (along with LPS; 0.03% ethanol); (3) Risperidone stock solution was prepared by introducing 2 µL of risperidone into 248 μL CTCM. This gave a 584.67 μM stock solution. From the 584.67 μM stock solution, 342.1 μL was added to 157.9 μL CTCM. This gave a 400 μM stock solution. The 400 µM stock solution (50 μL) was added to the samples (along with LPS) to achieve a final concentration of 100 µM (0.14% ethanol).

[0554] For ELISA measurement of IL-6: Reagents from BD Biosciences. Capture: purified NA / LE rat anti-Mouse IL-6; Cat. No. 554398, clone MP5-20F3; Standard: recombinant mouse IL-6; Cat. No. 554582; Detection: biotin rat anti-Mouse IL-6 antibody; Cat. No.554402, clone MP5-32C11; SA-HRP: Streptavidin HRP; Cat. No. 554066; TMB: BD OptEIA™ TMB substrate reagent set; Cat. No. 555214. Two full ELISA plates (each with 96 wells) were utilised when including standards. A total of 11mL volume was prepared for 50 µL / well samples and a total of 22 mL volume was prepared for 100 µL / well samples. For capture, overnight incubation was carried out at 4°C. For the capture antibody, stock was at 1000 µg / mL, with a final concentration desired at 1 µg / mL. The following calculation was performed: 1000 µg / mL * Vi = 1 µg / mL * 11,000 uL. From this calculation, Vi equating to 11 μL rat anti-mouse IL-6 was added into 11 mL pH 9.0 capture buffer. This was introduced to the plates at 50 μL / well. After capture, washing was performed four times.

[0555] To perform blocking, a 2 hour incubation was carried out at room temperature. For this, 100 µL of 10% FCS was added to each well. This was calculated as: 10% FCS = 5 mL FCS in 50 mL 1X PBS. After blocking, washing was performed three times. The plates were incubated for 2 hours at room temperature. The stimulated samples were diluted as 10 μL in 100 µL 5% FCS.5% FCS, equated to 2.5 mL FCS into 50 mL 1X PBS. This was introduced to the plates at 50 µL / well. Standards were prepared as follows: 16 µL was added into 784 µL 5% FCS. The highest concentration was 4 ng / mL. This was serially diluted by diluting 100 µL into 100 µL down to 7.8125 pg / mL. A sample with 0 pg / mL also included. The serial dilutions were produced in duplicate After standard preparation, washing was performed four times.

[0556] For the initial detection step, a 1 hour incubation was carried out at room temperature. Stock was at 500 µg / mL, with a final concentration desired at 0.5 µg / mL. The following calculation was performed: 500 µg / mL * Vi = 0.5 µg / mL * 11,000 µL. From this calculation, the Vi equating to 11 µL biotin rat anti-mouse IL-6 was added into 11 mL 5% FCS / PBS. This was introduced to the plates at 10 µL / well. After this initial detection step, washing was performed six times. Incubation with streptavidin-horseradish peroxidase (SA-HRP) was carried out for 1 hour at room temperature. For this, a 1 in 2000 dilution for SA-HRP was prepared. The calculation was performed as follows: 11,000 / 2000 = 5.5 µL. From this calculation, 5.5 µL SA-HRP was added into 11 mL 5% FCS / PBS. This was introduced to the plates at 50 µL / well. After the SA-HRP step, washing was performed eight times.

[0557] For the final detection step, incubation with TMB (3, 3', 5, 5'- tetramethylbenzidine) was carried out. The TMB was prepared by introducing 11 mL TMB-A and 11 mL TMB-B into separate 15 mL falcon tubes covered in tinfoil. After the washing following SA-HRP incubation, the TMB-A and TMB-B samples were combinedand this was introduced to the plates at 100 µL / well. Colour was allowed to develop. Colour development was stopped by adding 100 µL H2SO4 to wells. The plates were read on a plate reader.

[0558] Results: Interleukin 6 (IL-6) was produced in response to LPS in both experimental set ups, with greater levels produced using the Method 2 set up (Figure 17A: Method 1 stimulation; Figure 17B: Method 2 stimulation). The unstimulated conditions for both set ups did not noticeably change with any treatment. Dexamethasone (100 nM) was the only control compound capable of inhibiting IL-6 in either set up. In the Method 1 set up, dexamethasone had a greater inhibition of IL-6 (29.32% of the stimulated / untreated condition and 33.42% of the vehicle) compared to the Method 2 set up (64.03% of the stimulated / untreated condition and 63.62% of the vehicle). A summary of the IL-6 levels is presented in Table 14, below. Example 10: Anti-inflammatory activity and cell viability analysis in comparative testing

[0559] Overview: The methodology as noted in the previous example was used to assess various markers, including: (1) viability: Identifies concentrations which have a cytotoxic effect, measured by MTT assay; (2) IL-6: Proinflammatory cytokine, measured by ELISA; (3) TNF: Proinflammatory cytokine, measured by ELISA; (6) Nitric oxide: Proinflammatory small molecule, measured by Griess assay.

[0560] Certain data has been presented at a log of the concentration tested to make data visualisation easier and to perform non-linear regression where appropriate. The testing concentrations and their corresponding log10value are listed below. Table 15. Testing concentrations and calculated Log10 concentrations Concentration (µM) Log10Concentration 3.125 0.49485 6.25 0.79588 12.5 1.09691 25 1.39794 50 1.69897 75 1.875061 100 2 150 2.176091

[0561] Methodology: Anti-inflammatory activity and viability were assessed as follows.

[0562] At Day 0, a dedicated T75 flask was retrieved for replicate from incubator. In the laminar flow cabinet, the flask was gently tapped to detach any non-adherent cells. The growth media was decanted and replaced with 10 mL CTCM. The flask was gently swayed to wash the cells. The wash media was decanted and replaced with 10 mL CTCM. Using a rubber scraper, cells were detached. The suspension was transferred to a 50 mL Falcon tube. The cells were centrifuged at 400 x g for 5 minutes. The cells were resuspended in 10 mL CTCM. Cells were counted after applying a 1 in 10 trypan blue dilution. Cells were diluted to 1 x 106cells / mL. Cells were plated at 80 µL / well. To this, we added 120 µL to bring the volume to 200 µL. The plate was returned to the incubator. Cells were continued to be plated as indicated. In this way, plates were dedicated to stimulated and unstimulated samples. Plates were left for 48 hours before any treatment occurred.

[0563] Round 1 counting (stimulated / unstimulated): Average number of cells in four fields = 54 -> x 10 x 104to give 5,400,000 cells in 1 mL, which equated to 54,000,000 cells in 10 mL. For the dilution, 44 mL CTCM was added to bring to 1 million cells / mL.

[0564] Round 2 counting (stimulated): Average number of cells in four fields = 15.7 -> x 10 x 104to give 1,570,000 cells in 1 mL, which equated to 15,700,000 cells in 10 mL. For the dilution, 5.70 mL CTCM was added to bring to 1 million cells / mL.

[0565] Round 2 counting (unstimulated): Average number of cells in four fields = 25.75 -> x 10 x 104to give 2,575,000 cells in 1 mL, which equated to 25,750,000 cells in 10 mL. For the dilution, 15.75 mL CTCM was added to bring to 1 million cells / mL.

[0566] Round 3 counting (stimulated): Average number of cells in four fields = 25 -> x 10 x 104to give 2,500,000 cells in 1 mL, which equated to 25,000,000 cells in 10 mL. For the dilution, 15 mL CTCM was added to bring to 1 million cells / mL.

[0567] Round 3 counting (unstimulated): Average number of cells in four fields = 32 -> x 10 x 104to give 3,200,000 cells in 1 mL, which equated to 32,000,000 cells in 10 mL. For the dilution, 22 mL CTCM was added to bring to 1 million cells / mL.

[0568] At Day 1, the treatments were prepared. Synthetic zingerone, acetyl zingerone, and ferulic acid were utilised in stock solutions at 360,416.0231 µM. Acetylzingerone was obtained from Sytheon Ltd. Synthetic zingerone and ferulic acid were obtained from Sigma-Aldrich. To generate the required dosages, 1199.4 µL CTCM was added to a 2 µL aliquot of each stock solution. This produced a 600 µM test solution of each compound. When added to the cells the concentration was 150 µM – 50 µL in 200 µL. The disclosed botanical extract was utilised in a stock solution at 134,000 µM. To generate the required dosages, 6 µL of the extract was added with 1334 µL CTCM. This produced a 600 µM test solution. See summary tables, below. Zingerone, Acetyl Zingerone, Ferulic AcidCf (Well) Cf (stock) Ci Vi Vf V CTCM EtOH % EtOH % in well150 600 360416 2 1201.4 1199.4 0.166472 0.041618112100 400 600 433.33333 650 216.6666667 0.110982 0.02774540875 300 600 325 650 325 0.055491 0.01387270450 200 400 325 650 325 0.027745 0.00693635225 100 300 216.66667 650 433.3333333 0.009248 0.00231211712.5 50 200 162.5 650 487.5 0.002312 0.0005780296.25 25 100 162.5 650 487.5 0.000578 0.0001445073.125 12.5 50 162.5 650 487.5 0.000145 3.61268E-05Conc (Well) Conc to make Ci Vi Vf V CTCM EtOH % EtOH % in well150 600 134000 6 1340 1334 0.447761 0.111940299100 400 600 433.3333 650 216.6667 0.298507 0.07462686675 300 600 325 650 325 0.149254 0.03731343350 200 400 325 650 325 0.074627 0.01865671625 100 300 216.6667 650 433.3333 0.024876 0.00621890512.5 50 200 162.5 650 487.5 0.006219 0.0015547266.25 25 100 162.5 650 487.5 0.001555 0.0003886823.125 12.5 50 162.5 650 487.5 0.000389 9.71704E-05

[0569] At Day 2, stimulation and treatments were started. The plates were retrieved from the incubator. The plates were centrifuged at 400 x g for 5 minutes. The media (150 µl) was removed and replaced with 50 µL CTCM. The test solution for each treatment was added (50 µl) to the appropriate wells. The plates were returned to the incubator for 1 hour. An LPS solution was prepared. For this, a 2 µL aliquot (1,000,000 ng / mL) was added to 9,998 µL CTCM to give a 200 ng / mL solution (final concentration 50 ng / mL). The LPS solution was added at 50 µL / well. For unstimulated conditions, the treatments were added at 50 µL / well followed by 50 µL of CTCM. The plates were returned to the incubator for 18 hours. All ELISA plates were coated. The MTT reagent was prepared. This was done by mixing 50 mg MTT into 10 mL 1X PBS. This produced a 5 mg / mL reagent solution for use with 6 plates.

[0570] At Day 3, supernatant harvesting, MTT assays, and ELISA were carried out. For supernatant harvesting, plates were centrifuges for 5 minutes at 400 x g. A multichannel pipette was set to 170 µL. This was used to remove supernatant. The supernatant was transferred to a labelled UB-96WP. For the MTT assays, CTCM was prewarmed in a 37°C water bath. After harvesting supernatant, 50 µL of warmed CTCM was added to cells. Next, 20 µL of MTT reagent solution was added to cells. The plates were returned to the incubator. After MTT development, 100 µL MTT solubilising solution was added. This was incubated overnight.

[0571] ELISA reagents: Reagents from BD Biosciences. Capture: purified NA / LE rat anti-Mouse IL-6; Cat. No. 554398, clone MP5-20F3; Standard: recombinant mouse IL-6; Cat. No. 554582; Detection: biotin rat anti-Mouse IL-6 antibody; Cat. No. 554402, clone MP5-32C11; SA-HRP: Streptavidin HRP; Cat. No. 554066; TMB: BD OptEIA™ TMB substrate reagent set; Cat. No. 555214. Solutions included: (1) ELISA capture buffer pH 6: 14.196 Na2HPO4in 1 L mqH2O. The pH was adjusted with HCl. (2) ELISA capture buffer pH 9: 14.196 Na2HPO4 in 1 L mqH2O. The pH was adjusted with NaOH. (3) ELISA wash solution: 1 mL Tween®20, 200 mL 10X PBS, and 1800 mL mqH2O. (4) 5% FCS in PBS: 2.5 mL FCS in 47.5 mL 1X PBS. (5) 10% FCS in PBS: 5 mL FCS in 45 mL 1X PBS. (6) 0.18 M H2SO4: 9.78 mL concentrated M H2SO4 in 1 L mL H2O.

[0572] ELISA assays: ELISA plates were coated with a capture antibody suspended in an ELISA capture buffer at 50 µL / well. The pH was adjusted based on the cytokine being tested (see summary tables, below). The coated ELISA plates were incubated at 4°C. overnight. The following day, the plates were retrieved from the refrigerator. The plates were washed four times in ELISA wash buffer. Next, 100 µL / well blocking solution was added to the plates. The plates were incubated at room temperature for 2 hours. The plates were washed three times. The supernatant was diluted depending on the cytokine being tested. The test sample was added along with a standard curve of the cytokine to quantify at 50 µL / well (see summary tables, below). The plates were incubated for 2 hours at room temperature or overnight at 4°C. For sequential ELISA, the supernatant was transferred to another plate before washing. Washing was carried out four times.

[0573] For detection, a biotinylated detection antibody was suspended in a solution of FCS and PBS and added to the plates at 50 µL / well. The plates were incubatedfor 1 hour at room temperature. The plates were washed six times. Streptavidin-horseradish peroxidase was suspended in a solution of FCS and PBS. This was introduced to the plates at 50 µL / well. The plate was incubated at room temperature, in darkness, for 1 hour. The required volumes of TMB-A and TMB-B were placed into separate falcon tubes. These were stored at room temperature, in darkness, during the SA-HRP incubation. The plates were washed eight times. The TMB solution was introduced to the plates at 100 µL / well. Colour was allowed to develop. To stop the reaction, 0.18 M H2SO4solution was added at 100 µL / well. See tables, below. Plates were read using a PerkinElmer EnSpire® plate reader at 450 nm. Stimulated conditions IL-6 Capture TNF Capture Day 0 Day 1: 8:00am 10:00 am 4X Wash 4X Wash Add block (10% FCS) Add block (10% FCS)12:00 pm 3X Wash 3X Wash Add IL-6 dilution and Add TNF dilution and standards standards 1:00 pm 2:00 pm 4X Wash 4X Wash Add detection Add detection 3:00 pm 6X Wash 6X Wash Add SA-HRP Add SA-HRP 4:00 pm 8X Wash 8X Wash Add TMB reagent. Add TMB reagent. Add H2SO4. Add H2SO4. Unstimulated conditions TNF Capture IL-6 Capture Day 0 Day 1: 4X Wash 8:00am Add block (10% FCS) 10:00 am 3X Wash 4X Wash Samples and Standards Add block (10% FCS) 12:00 pm 4X Wash 3X Wash Add detection Add IL-6 sample and standards 1:00 pm 6X Wash Add SA-HRP 2:00 pm 8X Wash 4X Wash Add TMB reagent. Add H2SO4. Add detection 3:00 pm 6X Wash Add SA-HRP 4:00 pm 8X Wash Add TMB reagent. Add H2SO4.

[0574] MTT assays: On the day preceding the assay, MTT powder was dissolved in 1X dPBS to a concentration of 5 mg / mL. Each plate required 20 μL / well. For 96 wells, this required 1920 μL MTT solution to be prepared. The concentration was 5 mg of MTT per mL of solution. This equated to 10 mg MTT per 96 well plate. On the day of the assay, CTCM was warmed in a 37°C water bath. While media was warming, cell cultures (RAW264.7) were centrifuged at 400 x g for 5 minutes. Using a multichannel pipette set to 180 μL, supernatant was removed and transferred to a labelled U-bottom 96 well plate. The supernatant was stored at -20 °C until analysis. To the remaining cells, 60 μL warmed CTCM was added to bring the volume to 80 µL / well. To this, 20 µL MTT solution wasadded. The plate was returned to the 37°C / 5% CO2incubator for 45 minutes. After 45 minutes, 50 μL MTT solubilizing solution was added. This included 10% SDS (w / v), 0.01 M HCl – pH adjusted to 4.0 with sodium hydroxide; or 10% SDS (w / v), 45% DMF – pH adjusted to 4.0 with acetic acid. The plate was covered with tinfoil and left overnight. After overnight incubation, the plate was read using a PerkinElmer EnSpire® plate reader at 580 nm.

[0575] NO assays: A Griess reaction protocol was utilised. NO production was measured via NaNO2 product in a 96 well format. Reagents included: (1) Greiss solution A (50 mL): 1% (w / v) sulphanilamide (500 mg), 2.5% phosphoric acid, stored at 40°C and protected from light; (2) Greiss solution B (50 mL): 0.1% (w / v) N-(1- naphthyl)ethylenediamine (50 mg), 2.5% phosphoric acid, stored at 40°C and protected from light; (3) 2.5% phosphoric acid (100 mL): 2.94 mL 85% phosphoric acid, 97.6 mL ddH2O. For the plate standards, rows 1 and 2 (across) of each plate included 1:1 dilutions of NaNO2from 500 μM to 0 μM. In rows A and B (down), 50 μl of culture media was added to each well. The first well in each row received 95 μl. To the first wells, 5 μl of 10 mM NaNO2was added. From these wells, 50 μL was taken and dilutions made down the row excluding the last well of each row (base line). For the test samples, 170 μl supernatant was removed from the wells without disturbing the cellular monolayer on the bottom of each well. From this 170 μl sample, 50 μl samples (in triplicate) were transferred to a flat- bottomed 96 well plate. For the Greiss reaction, equal volumes of Greiss solution A and Greiss solution B were mixed to the desired volume (about 5 mL total per 96 well plate). The mixed Greis solution A+B was added to each well (50 μl per well). Any bubbles were removed using a hair dryer. Absorbance was read at 570 nm.

[0576] Results – viability: The MTT assay was used to identify any drops in metabolism which may be indicative of a cytotoxic effect of any of the compounds. The only compound found to have a cytotoxic effect was the disclosed botanical extract at and above 50 µM. The data is summarised in Figures 19A-19D and Tables 16 and 17, below. Table 16. IC50Calculation for the MTT analysis Extract Stimulated Unstimulated IC50 (µM) 50.52 48.53 R20.978 0.936Table 17. Closest concentration to IC50 Botanical Extract Stimulated Unstimulated Viability at 50 µM 64.69 ± 11.31% 51.61 ± 21.94 %

[0577] Results – IL-6: IL-6 is a common proinflammatory cytokine produced by macrophages upon stimulation with LPS. IL-6 is involved in propagating the early innate immune response. The only substantial inhibition of IL-6 production from stimulated RAW264.7 macrophages was from the disclosed botanical extract (Figure 20). The most potent IL-6 inhibition was seen with the botanical extract at 25 µM, reducing the IL-6 production to 41% of the absolute ethanol control (Figure 20). Considerable inhibition of IL-6 production was also seen with the botanical extract at 6.25 µM and 12.5 µM (Figure 20). Surprisingly, the botanical extract at 25 µM showed significantly greater inhibition of IL-6 than the synthetic zingerone at 150 µM (Figures 20 and 21). A summary of the dose dependent inhibition by the disclosed botanical extract is set out in Table 18, below. Potential inhibition was observed from treatment with synthetic zingerone or acetyl zingerone at 150 µM. This treatment with synthetic zingerone or acetyl zingerone reduced IL-6 to 64% and 69% of the absolute ethanol control respectively. These results are summarised in Table 18, below. Ferulic acid did not have an effect on IL-6 production at any concentration tested. Concentrations of the disclosed botanical extract associated with cytotoxicity were omitted to avoid confounding data. Table 18. Summary of results for IL-6 inhibition Compound Concentration IL-6 (% Vehicle) ± SEM Botanical extract 25 µM 41.66 ± 7.94 % 12.5 µM 61.58 ± 2.51 % 6.25 µM 76.64 ± 1.23 % Synthetic zingerone 150 µM 64.15 ± 4.01 % Acetyl zingerone 150 µM 69.90 ± 2.64 % Dexamethasone (control) 100 nM 54.32 ± 11.03 %

[0578] To determine if RAW264.7 cells treated with the disclosed botanical extract had significantly lower levels of IL-6 compared to cells treated with synthetic zingerone, acetyl zingerone, or ferulic acid, a One-Way Analysis of Variance (One-Way ANOVA) was used. LPS-Stimulated RAW264.7 cells treated with the disclosed botanical extract at 25 µM had significantly lower levels of IL-6 compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 µM (Figure 21). A Two-WayANOVA was also performed to determine if the IL-6 levels observed in the compound treated conditions were significantly different from the vehicle treated conditions. Treatment with the disclosed botanical extract at 25 µM or synthetic zingerone / acetyl zingerone at 150 µM produced significantly lower IL-6 compared to treatment with the respective vehicles (Figure 22).

[0579] Under unstimulated conditions (Figures 23A-23D), the levels of IL-6 were very low relative to the stimulated conditions which was expected. Due to the low levels of cytokine produced in unstimulated conditions no trend was able to be observed. This provides confidence that the testing set is not inducing the production of IL-6. Because the levels of IL-6 in unstimulated conditions were low and often at 0 pg / mL, this data is not normalised to the absolute ethanol control.

[0580] Results – TNF: TNF is also a proinflammatory cytokine produced by macrophages when stimulated with LPS. The greatest inhibition of TNF from stimulated RAW264.7 cells was observed after treatment with ferulic acid at 150 µM. This treatment reduced TNF to 61% of the absolute ethanol control (Figure 24). At concentrations of 50 µM, the inhibition from ferulic acid treatment tapered off to the level of the absolute ethanol control. Synthetic zingerone had an apparently modest inhibitory effect on TNF production at 150 µM, reducing the level of TNF to around 80% of the absolute ethanol control. The disclosed botanical extract also had a modest inhibitory effect on TNF, reducing the level of TNF to around 80% of the absolute ethanol control at 25, 12.5 and 6.25 µM. acetyl zingerone had no effect on TNF production. Concentrations of the disclosed botanical extract associated with cytotoxicity were omitted to avoid confounding data. Results are summarised in Table 19, below. Table 19. Summary of results for TNF inhibition Compound Concentration TNF (% Vehicle) ± SEM Ferulic acid 150 µM 61.09 ± 8.99 % 100 µM 73.33 ± 11.49 % Synthetic zingerone 150 µM 78.95 ± 8.04 % Botanical extract 25 µM 80.44 ± 3.94 % 12.5 µM 75.24 ± 4.11 % 6.25 µM 79.31 ± 2.67 % Dexamethasone (control) 100 nM 12.49 ± 2.05 %

[0581] No significant differences were observed from treatment with the disclosed botanical extract at 25 µM when compared to treatment with synthetic zingerone or ferulic acid at 150 µM (Figure 25). A statistically significant difference was observed between treatment with the disclosed botanical extract at 25 µM and treatment with acetyl zingerone at 150 µM. A Two-Way ANOVA was also performed to determine if the TNF levels in the compound treated conditions were statistically significant from the vehicle treated conditions. From this analysis, treatment with ferulic acid at 150 µM showed significantly lower TNF levels compared to treatment with vehicle (Figure 26).

[0582] In unstimulated conditions (Figures 27A-27D), the levels of TNF were much lower and often not above the background reading of the ELISA. No trend in TNF production was observed in the unstimulated conditions. This provides confidence that the testing set is not inducing the production of TNF. Because the levels of TNF in unstimulated conditions were low and often at 0 pg / mL, this data is not normalised to the absolute ethanol control.

[0583] Results – NO: Nitric oxide (NO) is a small molecule released by macrophages during the inflammatory response which is capable of killing invading pathogens. Only the disclosed botanical extract had an inhibitory effect on NO, reducing it to 77% of the absolute ethanol control (Figure 28). Synthetic zingerone caused elevations at the higher range of concentrations tested (50, 75, 100 and 150 µM) when compared to the absolute ethanol control. Acetyl zingerone at 150 µM also caused a slight elevation in NO levels. Ferulic acid did not appear to alter NO production. Concentrations of the disclosed botanical extract associated with cytotoxicity were omitted to avoid confounding data. Results are summarised in Table 20, below. Table 20. Summary of results for NO production Compound Concentration (µM) % NO Production ± SEM Synthetic zingerone 150 137 ± 14.87 % 100 140.44 ± 14.41 % 75 135.95 ± 9.78 % 50 128.64 ± 3.55 % Acetyl zingerone 150 125.22 ± 7.69 % Botanical extract 25 77.40 ± 6.64 % Dexamethasone (control) 100 90 ±3.98 %

[0584] Noting that nitric oxide measurement was only able to be performed in stimulated conditions due to the volume of supernatant required to measure it.

[0585] To determine if RAW 264.7 cells treated with the disclosed botanical extract at 25 µM had significantly lower NO levels compared to the other treatment conditions, a One-Way ANOVA was used. Treatment with the disclosed botanical extract at 25 µM produced significantly lower levels of NO compared to treatment with synthetic zingerone, acetyl zingerone and ferulic acid at 150 µM (Figure 29). To determine if the changes in NO observed were significantly different from its vehicle, a Two-Way ANOVA was used. Treatment with the disclosed botanical extract at 25 µM did not produce significantly lower NO compared to treatment with vehicle. However, treatment with synthetic zingerone at 150 µM produced significantly greater NO levels than treatment with vehicle (Figure 30).

[0586] Conclusions: Regarding the disclosed botanical extract, cytotoxicity was observed at concentrations about 50 µM. The disclosed extract showed the greatest inhibition of IL-6 from stimulated RAW264.7 cells. Treatment with the disclosed botanical extract at 25 µM reduced IL-6 levels to below 50% of the absolute ethanol control. This inhibition of IL-6 by the disclosed extract was statistically significant when compared to the vehicle control and when compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid. Treatment with the disclosed botanical extract also produced a modest inhibition of TNF from stimulated RAW264.7 cells. In addition, the disclosed botanical extract at 25 µM a inhibited NO production from stimulated RAW264.7 cells. This inhibition of NO by the disclosed botanical extract was statistically significant when compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid.

[0587] The other test compounds produced mixed results and, in most cases, substantially lower efficacy. Treatment with synthetic zingerone or acetyl zingerone at 150 µM produced a modest inhibition of IL-6. These reductions were statistically significant when compared to treatment with vehicle. Treatment with synthetic zingerone at 150 µM produced a modest reduction in TNF levels. Ferulic acid at 150 µM also inhibited TNF production, reducing levels to around 60% of the absolute ethanol control. This reduction was deemed to be statistically significant when compared to treatment with vehicle. Ferulic acid showed no effect on NO production. Synthetic zingerone and acetylzingerone both increased NO production. The increase in NO by synthetic zingerone was deemed to be statistically significant when compared to treatment with vehicle.

[0588] In summary, it was observed that the disclosed botanical extract significantly out-performed the commercially available compounds tested. The botanical extract showed potent inhibition of proinflammatory cytokine expression and proinflammatory small molecule production when compared to commercially sourced zingerone and commercially sourced acetyl zingerone. These results demonstrate the superior treatment efficacy of the botanical extract for blockade of / interference with inflammatory pathways.

[0589] It should be recognised that the superior inhibitory activity of the botanical extract is particularly surprising, especially given that the comparative testing utilised the botanical extract at significantly lower concentrations (e.g., 25 µM) than the commercially available compounds (e.g., 100 µM or 150 µM). Example 11: Comparative anti-inflammatory testing for individual treatments and cotreatments

[0590] Overview: These studies were carried out to assess the effects of cotreatment of ferulic acid with either synthetic zingerone or acetyl zingerone. The upper and lower concentrations of synthetic zingerone, acetyl zingerone, and ferulic acid were selected as 75 µM and 150 µM. The following factors were measured: (1) viability -- identifying concentrations with a cytotoxic effect, measured by MTT assay; (2) IL-6 -- proinflammatory cytokine, measured by ELISA; (3) TNF -- proinflammatory cytokine, measured by ELISA; nitric oxide (NO) -- proinflammatory small molecule, measured by Griess Assay.

[0591] Results – MTT: In stimulated cells, individual treatments with zingerone or ferulic acid did not alter the MTT score in a way which may indicate cytotoxicity at either 75 or 150 µM (Figure 31). This was expected as neither of these compounds were reported to lower the MTT score in Example 10. In stimulated cells, zingerone at 75 µM combined with ferulic acid at 75 µM or 150 µM resulted in a drop in MTT score to around 80% of the absolute ethanol control. However, when the concentration of zingerone was raised to 150 µM and combined with ferulic acid at 75 µM or 150 µM, the MTT scores sat above 80% of the absolute ethanol control. Although cytotoxicity would be expected to occur in a dose dependent manner, this was not observed. Therefore, the lower MTT scoreat the lower zingerone concentration could point to other mechanisms involved. For example, it is possible that there could be a change in cellular metabolism.

[0592] As expected, individual treatments with acetyl zingerone did not reduce the MTT score in any substantial way (Figure 32). Cotreatment with acetyl zingerone at 75 µM and ferulic acid at 75 µM or 150 µM resulted in a drop in MTT score to around 80% of the absolute ethanol control. However, when the concentration of acetyl zingerone was raised to 150 µM and combined with ferulic acid at 75 µM or 150 µM, the MTT score was seen at above 80% of the absolute ethanol control. Further investigation will be able to confirm if the lower MTT score at the lower acetyl zingerone concentration is due to a metabolic change or other possible effects. Summaries of the mean MTT scores for stimulated conditions are detailed in Tables 21 and 22. In these tables, the MTT scores are shown as a percentage of the absolute ethanol control. Table 21. Summary of MTT scores for stimulated cells Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0.00 % 128.47 ± 12.31 % 125.20 ± 3.56 % Concentration 75 106.06 ± 7.74 % 78.41 ± 4.32 % 87.12 ± 2.44 % (µM) 150 104.92 ± 5.94 % 72.54 ± 4.62 % 83.29 ± 4.41 % Table 22. Summary of MTT scores for stimulated cells Acetyl Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0.00 % 123.51 ± 6.27 % 125.76 ± 2.30 % Concentration 75 108.01 ± 6.16 % 83.03 ± 5.64 % 94.00 ± 2.03 % (µM) 150 99.02 ± 4.84 % 81.90 ± 4.61 % 94.96 ± 1.38 %

[0593] Similar trends in MTT scores were observed for unstimulated cells. Individual treatments with zingerone, acetyl zingerone, or ferulic acid produced no substantial reductions in MTT scores. In unstimulated cells, zingerone at 75 µM (Figure 33) or acetyl zingerone at 75 µM (Figure 34) combined with ferulic acid at 75 µM or 150 µM reduced the MTT score of the cells to around 80% of the absolute ethanol control. This reduction in MTT score was not seen with combinations containing zingerone or acetyl zingerone at 150 µM. Summaries of the mean MTT scores for the unstimulated conditions are set out in Tables 23 and 24. In these tables, the MTT scores are shown as a percentage of the absolute ethanol control.Table 23. Summary of MTT scores for unstimulated cells Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0.00 % 117.21 ± 2.15 % 126.63 ± 4.35 % Concentration 75 108.37 ± 6.08 % 84.28 ± 3.90 % 95.38 ± 2.30 % (µM) 150 113.20 ± 4.42 % 82.68 ± 3.50 % 97.45 ± 2.93 % Table 24. Summary of MTT scores for unstimulated cells Acetyl Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0.00 % 114.28 ± 4.75 % 125.09 ± 10.79 % Concentration 75 107.01 ± 0.01 % 84.78 ± 3.94 % 93.48 ± 1.25 % (µM) 150 117.10 ± 8.45 % 83.85 ± 0.36 % 96.11 ± 2.08 %

[0594] Results – IL-6: Individual treatment with zingerone at 75 µM or 150 µM reduced IL-6 production from stimulated RAW264.7 cells (Figure 35). Less of an effect or no effect was observed from individual treatment with ferulic acid. This was consistent with the results set out in Example 10. Cotreatment with zingerone and ferulic acid appeared to reduce IL-6 production more than the individual treatments (Figure 35). A summary of the effect of zingerone and ferulic acid cotreatment on IL-6 production is set out in Table 25. In the table, IL-6 production is shown as a percentage of the absolute ethanol control. Table 25. Summary of IL-6 production from stimulated cells Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0 % 68.16 ± 7.47 % 60.29 ± 13.32 Concentration 75 96.67 ± 8.12 % 74.76 ± 10.19 % 53.42 ± 18.07 % (µM) 150 89.11 ± 13.25 62.19 ± 15.81 % 44.1518.95 %

[0595] Synergy is present where the effect of cotreatment is greater than the sum of the individual treatments. To determine potential synergy, the individual percentages of inhibition were summed and displayed as the calculated sum (e.g., represented graphically as a dotted line), and the actual effect of cotreatment was plotted next to the calculated sum. Where the actual effect was greater than the calculated sum, this pointed to a synergistic result.

[0596] To assess the effect of cotreatment, the values displayed in Figure 35 were converted to percent inhibition values by subtracting them from 100, and this data wasdisplayed in Figures 36A-36D. Cotreatment with zingerone at 150 µM and ferulic acid at 150 µM reduced IL-6 levels to 44.15% of the absolute ethanol control, which equates to a 55.85% inhibition of IL-6. The analysis showed a synergistic effect, where the effect of cotreatment is greater than the sum of the individual treatments (Figures 36A-36D). One- Way ANOVA was performed to determine the significance of the differences. Cotreatment with zingerone at 150 µM and ferulic acid at 75 or 150 µM was significantly greater than ferulic acid treatment alone (Figures 36C-36D).

[0597] Individual treatment with acetyl zingerone at 75 µM or 150 µM reduced IL-6 production from stimulated RAW264.7 cells (Figure 37). Some reduction in IL-6 levels was observed from individual treatment with ferulic acid. Cotreatment with acetyl zingerone and ferulic acid also appeared to reduce IL-6 levels (Figure 37). However, this reduction occurred primarily due to inclusion of acetyl zingerone. A summary of the effect of acetyl zingerone and ferulic acid cotreatment on IL-6 production is set out in Table 26. In the table, IL-6 production is shown as a percentage of the absolute ethanol control. Table 26. Summary of IL-6 production from stimulated cells Acetyl Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0 % 65.23 ± 2.94 % 64.78 ± 9.62 % Concentration 75 89.85 ± 9.72 % 76.05 ± 14.69 % 64.01 ± 6.29 % (µM) 150 76.95 ± 12.28 % 58.86 ± 12.67 % 56.278.17 %

[0598] To assess the effect of cotreatment, the values displayed in Figure 37 were converted to percent inhibition values by subtracting them from 100, and this data was displayed in Figures 38A-38D. The calculated sum of the individual treatments was displayed as a dotted line of each of the plots in Figures 38A-38D. This analysis showed that, although cotreatment reduced IL-6 levels, the effect of the cotreatments was not greater than the individual treatments. Additionally, none of the cotreatments were deemed significantly different from the individual treatments by One-Way ANOVA. In unstimulated cells, no treatment induced the production of IL-6 (Figures 39A-39C). This confirmed that the individual treatments and cotreatment were not activating the cells.

[0599] Results – TNF: Cotreatment with zingerone and ferulic acid outperformed single compound treatment in TNF inhibition (Figure 40). Individual treatment with zingerone or ferulic acid produced a dose dependent decrease in TNF production, and cotreatment with zingerone and ferulic acid enhanced this effect (Figure 40). For example,cotreatment with zingerone at 75 µM and ferulic acid at 75 µM produced a substantially greater inhibition of TNF as compared to the individual treatments. Increasing the concentration of either treatment increased the TNF inhibition. A summary of the effect of zingerone and ferulic acid cotreatment on TNF production is set out in Table 27. In the table, TNF production is shown as a percentage of the absolute ethanol control. Table 27. Summary of TNF production from stimulated cells Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0 % 88.54 ± 19.05 % 56.58 ± 28.07 % Concentration 75 87.90 ± 10.84 % 48.51 ± 7.19 % 29.35 ± 11.67 % (µM) 150 72.36 ± 22 % 38.53 ± 9.67 % 25.0813.67 %

[0600] To assess the effect of cotreatment, the values displayed in Figure 40 were converted to percent inhibition values by subtracting them from 100, and this data was displayed in Figures 41A-41D. The calculated sum of the individual treatments was displayed as a dotted line of each of the plots in Figures 41A-41D. The analysis showed that cotreatment with zingerone and ferulic acid produced a synergistic effect (Figures 41A-41D). The cotreatments of zingerone and ferulic acid (at all concentrations tested) surpassed the sum of the individual treatments.

[0601] The most striking effect was observed for cotreatment with zingerone at 75 µM and ferulic acid at 75 µM (Figure 41A). This cotreatment produced 53% inhibition as compared to ~11% inhibition for zingerone alone and 12% inhibition for ferulic acid alone. Cotreatment at the other concentrations also outperformed the calculated sum for individual treatments. In addition to this, One-Way ANOVA showed that zingerone / ferulic acid cotreatments had a significantly greater inhibition of TNF compared to individual treatments. In particular, cotreatment with zingerone at 75 µM and ferulic acid at 75 µM had a significantly greater TNF inhibition compared to both the individual treatments (Figure 41A). This reinforces the synergistic effect seen with cotreatment.

[0602] Cotreatment with acetyl zingerone and ferulic acid also outperformed single individual treatments for TNF inhibition (Figure 42). Individual treatment with acetyl zingerone had little effect on TNF levels. Ferulic acid had a higher inhibitory effect. However, cotreatment with acetyl zingerone and ferulic acid showed even greater inhibition. The greatest effect was noted for cotreatment with acetyl zingerone at 75 µM and ferulic acid at 75 µM, which reduced TNF levels down to around 50% of the vehiclecontrol. Increasing the concentration of either treatment increased the TNF inhibition. A summary of the effect of acetyl zingerone and ferulic acid cotreatment on TNF production is set out in Table 28. In the table, TNF production is shown as a percentage of the absolute ethanol control. Table 28. Summary of TNF production from stimulated cells Acetyl Zingerone Concentration (µM) 0 75 150 Ferulic Acid 0 100 ± 0 % 101.89 ± 17.09 % 82.36 ± 19.14 % Concentration 75 78.82 ± 13.4 % 55.15 ± 5.80 % 48.17 ± 7.48 % (µM) 150 57.51 ± 12.24 % 39.24 ± 6.95 % 35.44 ± 9.20 %

[0603] To assess the effect of cotreatment, the values displayed in Figure 42 were converted to percent inhibition values by subtracting them from 100, and this data was displayed in Figures 43A-43D. The calculated sum of the individual treatments was displayed as a dotted line of each of the plots in Figures 43A-43D. The analysis showed that cotreatment with acetyl zingerone and ferulic acid produced a synergistic effect (Figures 43A-43D). Cotreatment with acetyl zingerone and ferulic acid (at all concentrations tested) surpassed the sum of the individual treatments.

[0604] The most pronounced inhibition occurred in cotreatment with acetyl zingerone at 75 µM and ferulic acid at 75 µM (Figure 43A). This cotreatment produced a 44% inhibition of TNF as compared to no inhibition by acetyl zingerone and 21% inhibition by ferulic acid. One-Way ANOVA showed that acetyl zingerone / ferulic acid cotreatments had a significantly greater inhibition of TNF compared to acetyl zingerone alone (Figures 43A-43D). A full summary of the effect of zingerone / acetyl zingerone and ferulic acid cotreatment on TNF production is set out in Table 29. Table 29. Summary of TNF inhibition (± SEM) Treatment Treatment 1 % Treatment 2 % Calculated Actual Effect Inhibition Inhibition Sum 5 µM Zingerone & 11.45 ± 19.04 % 12.10 ± 10.84 % 23.55 % 53.49 ± 7.19 % 5 µM Ferulic Acid 5 µM Zingerone & 11.45 ± 19.04 % 27.64 ± 22.71 % 39.10 % 61.47 ± 9.67 % 50 µM Ferulic Acid 50 µM Zingerone & 43.42 ± 28.07 % 12.10 ± 10.84 % 55.51 % 70.65 ± 11.67 % 5 µM Ferulic Acid 50 µM Zingerone & 43.42 ± 28.07 % 27.64 ± 22.71 % 71.06 % 74.92 ± 13.67 % 50 µM Ferulic Acid75 µM Acetyl Zingerone -1.88 ± 17.09 % 21.18 ± 13.40 % 19.29 % 44.85 ± 5.80 % & 75 µM Ferulic Acid 75 µM Acetyl Zingerone -1.88 ± 17.09 % 42.49 ± 12.24 % 40.60 % 60.76 ± 6.94 % & 150 µM Ferulic Acid 150 µM Acetyl 17.64 ± 19.14 % 21.18 ± 13.40 % 38.83 % 51.84 ± 7.47 % Zingerone & 75 µM Ferulic Acid 150 µM Acetyl 17.64 ± 19.14 % 42.49 ± 12.24 % 60.13 % 64.56 ± 9.20 % Zingerone & 150 µM Ferulic Acid Dexamethasone 76.79 ± 2.32 % 100 nM (Control)

[0605] In unstimulated cells, TNF levels were very low, calculated at over a thousand-fold lower than TNF levels in stimulated cells. In any case, no treatment condition was observed to induce activation of the RAW264.7 cells (Figure 44).

[0606] Results – NO: Analysis of nitric oxide (NO) was only able to be performed in LPS stimulated conditions due to the volume of supernatant required for the analysis. In Example 10, we did not observe any inhibition of NO in response to individual compounds but rather we saw an increase in NO in response to zingerone treatment. This was confirmed in the present studies, which found that increasing concentrations of zingerone produced an elevation in nitric oxide levels. When ferulic acid was introduced with zingerone, NO levels returned to that of the absolute ethanol control (Figure 45). Similarly, individual treatment with acetyl zingerone produced increased NO levels, whereas cotreatment with acetyl zingerone and ferulic acid lead to a reduction in NO levels (Figure 46).

[0607] Conclusions: Regarding IL-6 production, cotreatment with zingerone and ferulic acid reduced IL-6 levels to 44.15% of the absolute ethanol control, i.e., a 55.85% inhibition of IL-6. Synergy in IL-6 reduction was observed, where cotreatment with zingerone and ferulic acid produced a greater effect than the sum of the individual treatments. In addition, cotreatment with zingerone and ferulic acid was significantly greater than ferulic acid treatment alone.

[0608] Regarding TNF production, cotreatment with zingerone (or acetyl zingerone) and ferulic acid outperformed single compound treatment in TNF inhibition. Increasing the concentration of the individual treatment increased the TNF inhibition. Synergy in TNF reduction was observed, where cotreatment with zingerone (or acetyl zingerone) and ferulic acid produced a greater effect than the sum of the individualtreatments. In addition, cotreatment with zingerone (or acetyl zingerone) and ferulic acid had a significantly greater inhibition of TNF compared to both the individual treatments.

[0609] Regarding NO measurements, individual treatment with zingerone or acetyl zingerone produced increases in NO levels. In contrast, cotreatment with zingerone (or acetyl zingerone) and ferulic acid reduced NO levels to that of the absolute ethanol control. This also pointed to synergistic effects.

[0610] No cotreatment induced the production of IL-6 or TNF from unstimulated cells, providing confirmation that the compounds were not inducing activation of the cells and that there were no contaminants in compounds.

[0611] Concluding remarks for Examples 9-11: The botanical extract as disclosed herein produced a dose dependent inhibition of IL-6, and also inhibition of TNF and nitric oxide. The botanical extract’s inhibition of IL-6 was statistically significant where compared to treatment with vehicle, zingerone, acetyl zingerone, or ferulic acid. Although less potent than the botanic extract, zingerone and acetyl zingerone (used individually) also inhibited IL-6. On the other hand, treatment with zingerone or acetyl zingerone enhanced NO production. Combining zingerone or acetyl zingerone with ferulic acid reversed this enhancement.

[0612] Ferulic acid acted as an effective inhibitor of TNF. However, significantly greater inhibition of TNF was observed when zingerone or acetyl zingerone was combined with ferulic acid. This synergy was observed at all concentrations tested, and increasing concentrations produced higher levels of inhibition of TNF. A synergistic effect was also observed in relation to IL-6, where the effect of the combined treatments was greater than the sum of the individual treatments. It was noted, however, that the inhibitory effects of the cotreatments on TNF appeared to be greater than the inhibitory effects of cotreatments on IL-6. Without wishing to be bound by theory, this could point to blockade of / interruption of different biological pathways. Example 12: Preliminary testing of combination treatments in animals

[0613] Overview: Zingerone and ferulic acid, individually and in combination, were investigated for anti-inflammatory effects in an experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis.

[0614] Materials and methods: Zingerone and ferulic acid were obtained from Sigma-Aldrich. Six groups of female C57BL / 6J mice (9-11 weeks, 5 per group) were administered a vehicle control, zingerone (10 mg / kg), zingerone (10 mg / kg) and ferulic acid (10 mg / kg), zingerone (5 mg / kg) and ferulic acid (5 mg / kg), and ferulic acid (10 mg / kg) alongside a healthy control group via oral gavage. Doses were administered daily from days 5-30, with disease induction conducted on day 1.

[0615] Results: In these studies, induction of the disease model was not successful. However, body weights of the mice were monitored as an indication of safety and tolerability. On average, body weight increased by 0.3 g over the treatment period, with an average weight of 19.7 g at the beginning of the treatment period and an average weight of 20.0 g at the end of the treatment period. No significant change in body weight was observed for any of the zingerone or ferulic acid treatments, which indicated the dosages used were safe and tolerable. Example 13: Comparative testing in an animal immunisation model for rheumatoid arthritis

[0616] Overview: After our preliminary analyses, we have observed that both the botanical extracts and the zingerone / ferulic acid combination have distinct immunomodulatory effects in the immunisation model. These effects appear to be distinct from dexamethasone. The key points and results for each of the treatment approaches are set out further below.

[0617] Animals: Adult female and male C57BL6 / J mice were bred and housed at the Victoria University of Wellington (VUW) Small Animal Facility (Wellington, New Zealand). Mice were housed in a temperature-controlled (20-22ºC) environment, exposed to a 14 h dark–10 h light cycle, and provided ad libitum with food and water. All animal experiments in this study were approved by the VUW Animal Ethics Committee (31251) and adhered to the ARRIVE reporting guidelines.

[0618] Compounds for in vivo use: The botanical extract resin (BE) was supplied by the Ferrier Research Institute and delivered in sweetened condensed milk (SCM; Nestlé® Highlander®). Animals were administered 1.9 mg (50 ^L), 3.8 mg (100 ^L), or 5.6 mg (150 ^L) directly to the mouth daily. The vehicle control group received an equal volume (150 ^L) of SCM as the high BE group. Zingerone and ferulic acid were suppliedby Vigon. Dexamethasone was sourced from Merck. Zingerone, ferulic acid, the combination of zingerone+ferulic acid, and dexamethasone were formulated and delivered in 0.4% methylcellulose (Merck) / 8% ethanol. The vehicle control group received an equal volume (100 ^L) of 0.4% methylcellulose / 8% ethanol.

[0619] Immunisations: Female and male C57BL / 6 mice, aged 10 to 14 weeks, were immunized with chicken type 2 collagen (100 μg / mouse; Merck) emulsified on ice with Freund’s complete adjuvant (Merck) containing heat-killed Mycobacterium tuberculosis H37Ra (500 μg / mouse; Fort Richard) subcutaneously in the hind legs (50 μL / hind leg). Mice were weighted daily.

[0620] Treatments were started 3 days post immunisation. The BE were delivered orally directly to the mouth and zingerone, ferulic acid, the combination of zingerone+ferulic acid, and dexamethasone were delivered by oral gavage in 100 μL volume. Treatment groups were dispersed through multiple cages to account for cage and litter differences. Each experiment included male and female mice, and 2 independent experiments were performed. The use of experimental replicates was used to address both individual and experimental variability. The results from the 2 independent experiments were pooled creating a final group size of 8 / group.

[0621] Plasma collection: Blood was collected on day 7 by tail bleed and day 14 by cardiac puncture into tubes containing EDTA. Plasma was collected and stored at -20^C until use.

[0622] Cytokine analyses: IL-6, IL-1^, IL-17A, and MCP-1 in d7 and d14 plasma samples were analysed with the Legendplex mouse inflammation cytometric bead array kit. Kit was purchased from BioLegend and used as per the manufacturer’s instructions. TNF in plasma samples was analysed by sandwich ELISA. All ELISA reagents were purchased from BD Biosciences and used as per the manufacturer’s instructions.

[0623] Antibody analyses: IgM levels in the plasma were measured by ELISA. All ELISA components were purchased from BD Biosciences and used as per the manufacturer’s instructions. Collagen-specific mouse IgG was quantified in plasma using the mouse anti-chick type II collagen IgG antibody assay kit (2031T; Chondrex) as per the manufacturer’s instructions.

[0624] Disease parameter calculations for heatmap: Individual parameters were normalised as follows to generate the heatmap:

[0625] % effect = (mean experimental group value - mean lowest group value) / (mean top group value - mean lowest group value)x100

[0626] These calculations standardised the range of values (0-100) to equalise the weighting of the individual outcomes.

[0627] Statistical analyses: Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA). Statistical tests are indicated in the figure legends. Differences of P<0.05 were considered statistically significant. P values between 0.05 and 0.10 are indicated in the figures as indicators of significance.

[0628] Results for botanical extract study: The botanical extracts (BE) were administered orally (by droplet to mouth) in sweetened condensed milk (SCM). Mice receiving the low dose received one 50 ^l droplet (1.9 mg of resin), mid dose two droplets (100 ^l; 3.88 mg), and the highest dose 3 droplets (150 ^l; 5.69 mg). The dose of extract administered was calculated for the experiments. Vehicle treated controls were administered three droplets (150 ^l) of SCM alone.

[0629] Weight recovery – Immunisation caused significant weight loss in mice compared to healthy controls (Figure 47a). Mice receiving the highest BE dose regained the weight lost by immunisation more quickly than the vehicle controls administered the same amount of SCM (Figure 47b). The BE was administered from day 3; thus the consistent improvement in the BE group on weight change is a positive preliminary finding.

[0630] Plasma IgM – As previously observed, immunisation induced an increase in plasma IgM in the vehicle (SCM) treated group at day 7 (shown as “0” dose; Figure 48). All doses of BE appeared to impair this increase with the medium dose having the most consistent effect (Figure 48). By day 14, none of the groups had elevated IgM; this was as expected (Figure 48).

[0631] Plasma collagen-specific IgG – The presence of collagen-specific antibody was induced by the collagen immunisation (Figure 49). In the mouse model of rheumatoid arthritis, collagen-specific IgG antibody is responsible for driving disease. By day 14, highlevels of collagen-specific antibody can be detected in the plasma (vehicle group shown at “0” dose; dashed line) with all doses of BE causing a similar reduction with the low BE dose having the best effect (Figure 49).

[0632] Plasma cytokines – Mice receiving BE had a dose-dependent reduction in plasma TNF at day 14 (Figure 50). This reduction approaches statistical significance.

[0633] Summary: We detected consistent effects on plasma TNF, IgM and collagen-specific IgG. These effects were similar between all BE groups. Certain groups appeared close to statistical significance. The observed differences may be attributable to variability in the individual animal responses.

[0634] Results for combined zingerone and ferulic acid: Zingerone and ferulic acid were administered by oral gavage at 25 mg / kg / day. A combination of zingerone and ferulic acid (Z&FA) was administered by oral gavage and contained 12.5 mg / kg / day. The vehicle used for the oral gavage was 0.4% methylcellulose / 8% ethanol.

[0635] Weight recovery – Mice receiving Z&FA regained the weight lost by immunisation more quickly than the vehicle controls or dexamethasone (Figure 51). Z&FA was administered from day 3; thus the effect of Z&FA on weight change is a very positive preliminary finding. This finding shows distinct effects of Z&FA compared to dexamethasone.

[0636] Plasma IgM – As previously observed, treatment with the vehicle did not induce a significant increase in plasma IgM at day 14 (Figure 48). However, while none of the compounds altered plasma IgM levels at day 7, by day 14, the Z&FA group showed reduced IgM levels in the plasma (Figure 52). Moreover, the reduced levels of IgM in the Z&FA group were significantly different from dexamethasone (Figure 52). This finding shows distinct effects of Z&FA compared to dexamethasone.

[0637] Plasma collagen-specific IgG – The presence of collagen-specific antibody has been induced by the collagen immunisation (Figure 53). In the mouse model of rheumatoid arthritis, collagen-specific IgG antibody is responsible for driving disease. By day 14, high levels of collagen-specific antibody can be detected in the plasma of vehicle treated mice (Figure 53). Both zingerone and Z&FA treatments reduced the level of anti- collagen antibody in the plasma with the Z&FA showing statistically significant effects compared to vehicle (Figure 53). Ferulic acid and dexamethasone did not cause any notablereduction (Figure 53). This finding shows distinct effects of Z&FA and zingerone compared to ferulic acid alone or dexamethasone.

[0638] Plasma IL-6 – Immunisation induced significant IL-6 levels in the plasma in the vehicle treated group at both day 7 (Figure 54a) and day 14 (Figure 54b) compared to healthy mice. At day 7, dexamethasone significantly reduced IL-6 while ferulic acid alone also reduced IL-6 levels compared to vehicle (Figure 54a). By day 14, the Z&FA combination had the greatest effect on reducing IL-6 levels (Figure 54b). In contrast, dexamethasone did not alter IL-6 levels compared to vehicle alone (Figure 54b). This finding shows distinct effects of Z&FA compared to dexamethasone.

[0639] Plasma IL-17A – Immunisation induced significant IL-17A levels in the plasma in the vehicle treated group at both day 7 (Figure 55a) and day 14 (Figure 55b) compared to healthy mice. At day 7, only dexamethasone significantly reduced IL-17A compared to vehicle (Figure 55a). By day 14, the zingerone and Z&FA treated groups had significantly reduced IL-17A levels similar to dexamethasone while ferulic acid did not alter IL-17A (Figure 55b). This finding shows distinct effects of Z&FA and zingerone compared to ferulic acid alone.

[0640] Plasma IL-1^ – Immunisation induced significant IL-1^ levels in the plasma in the vehicle treated group at day 14 (Figure 56b) but not day 7 (Figure 56a) compared to healthy mice. At day 7, only Z&FA showed reduced IL-1^ compared to vehicle (Figure 56a), and at day 14, the Z&FA treated group maintained the reduced IL- 1^ levels. Zingerone, ferulic acid and dexamethasone did not alter IL-1^ at day 14 (Figure 56b). This finding shows distinct effects of Z&FA compared to zingerone, ferulic acid and dexamethasone.

[0641] Plasma MCP-1 – Immunisation alone did not induce significant MCP-1 levels in the plasma in the vehicle treated group at either day 7 Figure 57a) or day 14 (Figure 57b) compared to healthy mice. However, at day 7, significantly elevated MCP-1 was detected in the zingerone alone treated mice compared to Z&FA (Figure 57a), and a modest reduction in MCP-1 was maintained by day 14 in the Z&FA treated group (Figure 57b). Ferulic acid, Z&FA and dexamethasone did not alter MCP-1 at either day 7 (Figure 57a) or day 14 (Figure 56b) compared to vehicle alone. This finding shows distinct effects of Z&FA compared to zingerone alone.

[0642] Plasma cytokine profile – Using a heatmap to illustrate the unique pattern of effects induced by Z&FA in the immunisation model, Z&FA is shown to be the most consistent at reducing IL-1^, MCP-1 and collagen-specific IgG (Figure 58). Dexamethasone has the greatest effects on IL-17A with Z&FA showing similar effects at day 14 and significantly reduces early IL-6 while Z&FA reduce IL-6 at the later timepoint (Figure 58). Together, these findings illustrate the unique combination of effects of Z&FA compared to zingerone, ferulic acid, and dexamethasone in this immunisation model.

[0643] Summary: We detected consistent effects of the Z&FA combination treatment on plasma IgM and collagen-specific IgG at day 14 post immunisation. The reduction in anti-collagen IgG was also observed in the zingerone treated group. It is important to note that the combination contains only 50% of the dose of zingerone as the zingerone alone, and yet it is similarly effective. The Z&FA combination also shows effects on IgM that are not observed in either the single zingerone or ferulic acid treatments suggesting beneficial combinatorial effects that can be synergistic. We also detected distinct changes to the plasma cytokines IL-6, IL-17A, IL-1^, and MCP-1, which are all cytokines associated with rheumatoid arthritis pathogenesis. Z&FA showed a distinct pattern of changes to these cytokines compared to zingerone, ferulic acid, and dexamethasone.

[0644] Discussion: In our immunisation model, we use collagen to induce autoantibody-like responses. Additionally, this model induced the key inflammatory cytokines that drive early stages of inflammation in rheumatoid arthritis (see Burmester et al., 2014, Nat Rev Rheumatol 10(2):77-88. doi: 10.1038 / nrrheum.2013.168). These cytokines and autoantibodies are important targets for current disease modifying therapies in rheumatoid arthritis (see, e.g., Huang et al., 2021, Front Immunol, 12:686155, doi: 10.3389 / fimmu.2021.686155). To summarise briefly: IL-1 and IL-6 actions lead to the differentiation of Th17 cells, which are pro-inflammatory and pathogenic in rheumatoid arthritis. These cytokines are important therapeutic targets in rheumatoid arthritis (e.g., TNF, IL-1, IL-6). IL-1 and IL-6 promote the production of IL-17 which is produced by Th17 cells and involved in the pathogenesis of rheumatoid arthritis. MCP-1 (also called CCL2) attracts monocytes to migrate into tissue. This allows activated monocytes to get into the synovium during rheumatoid arthritis. Regarding plasma IgM, IgM is the first antibody made in a response. Thus, a small rise in IgM around day 7 is a good indicator ofB cell activation. After that time point, IgG is predominantly made. Collagen-specific IgG (e.g., autoantibodies) are pathogenic in rheumatoid arthritis. Targeting the production of these antibodies by B cells is a validated therapeutic strategy. This is the approach utilised in our animal model.

[0645] Persons of ordinary skill can utilise the disclosures and teachings herein to produce other embodiments and variations without undue experimentation. All such embodiments and variations are considered to be part of this disclosure.

[0646] Accordingly, one of ordinary skill in the art will readily appreciate from the present disclosure that later modifications, substitutions, and / or variations performing substantially the same function or achieving substantially the same result as embodiments described herein may be utilised according to such related embodiments. Thus, the present disclosure is intended to encompass, within its scope, the modifications, substitutions, and variations to processes, manufactures, compositions of matter, compounds, means, methods, and / or steps disclosed herein.

[0647] The description herein may contain subject matter that falls outside of the scope of the claimed invention. This subject matter is included to aid understanding of the invention.

[0648] In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of this disclosure. Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

Claims

CLAIMS 1. A combination comprising: compound I and compound II, these compounds being of formula I and formula II, respectively:, wherein: R1is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3;R2 is -H, or when taken together with R4 and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3is -H or when taken together with R2forms =O; and R4 is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, and ; and wherein: R10is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R11 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R12is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R13 is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds, wherein compound I and compound II are not the same.

2. The combination of claim 1, wherein compound I is selected from the group consisting of:acetyl zingerone zingeronezingerol ethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone 4-hydroxy-3-methoxycinnamic acid ethyl ester6-gingerolhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acidconiferyl aldehyde eugenol4-hydroxy-3-methoxybenzoic acid and any salts, solvates, hydrates, and protected forms thereof.

3. The combination of claim 1, wherein compound I is selected from the group consisting of: zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

4. The combination of claim 1, wherein compound II is selected from the group consisting of:ferulic acid hydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatevanilpyruvic acid methyl-3-methoxy-4-hydroxystyryl ketonesinapic acid 3,4,5-trihydroxycinnamic acidcinnamic acid p-coumaric acid and any salts, solvates, hydrates, and protected forms thereof.

5. The combination of claim 1, wherein compound II is selected from the group consisting of: ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

6. The combination of any one of claims 1 to 5, wherein the combination consists essentially of compound I and compound II.

7. The combination of any one of claims 1 to 6, wherein compound I or compound II is: (i) a synthetic compound; (ii) a naturally occurring compound; (iii) included as a botanical extract.

8. The combination of any one of claims 1 to 7, used in treating or preventing inflammation in a subject.

9. The combination of claim 8, wherein compound I and compound II are administered to the subject (i) simultaneously; (ii) sequentially; (iii) separately; or (iv) as a co-formulation.

10. A kit comprising compound I and compound II, these compounds being of formula I and formula II, respectively:, wherein: R1is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3;R2 is -H, or when taken together with R4 and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3is -H or when taken together with R2forms =O; and R4 is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, andwherein: R10is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R11 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R12is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R13 is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds, wherein compound I and compound II are not the same.

11. The kit of claim 10, wherein compound I is selected from the group consisting of:acetyl zingerone zingeronezingerol ethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone 4-hydroxy-3-methoxycinnamic acid ethyl ester6-gingerolhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acidconiferyl aldehyde eugenol4-hydroxy-3-methoxybenzoic acid and any salts, solvates, hydrates, and protected forms thereof.

12. The kit of claim 10, wherein compound I is selected from the group consisting of: zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

13. The kit of claim 10, wherein compound II is selected from the group consisting of:ferulic acid hydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatevanilpyruvic acid methyl-3-methoxy-4-hydroxystyryl ketone3,4,5-trihydroxycinnamic acid sinapic acidcinnamic acid p-coumaric acid and any salts, solvates, hydrates, and protected forms thereof.

14. The kit of claim 10, wherein compound II is selected from the group consisting of: ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

15. The kit of any one of claims 10 to 14, wherein compound I or compound II is: (i) a synthetic compound; (ii) a naturally occurring compound; (iii) included as a botanical extract.

16. The kit of any one of claims 10 to 15, wherein the kit comprises: (i) compound I and compound II in separate containers; (ii) compound I and compound II in the same container; (iii) compound I and compound II formulated as one or more pharmaceutical compositions or dietary compositions; (iv) compound I and compound II in different formulations for administration; (v) compound I and compound II in the same formulations for administration; or (vi) compound I and compound II co-formulated for administration.

17. The kit of any one of claims 10 to 16, wherein the kit comprises compound I and / or compound II: (i) formulated as a solid, semi-solid, or liquid; (ii) formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule; or (iii) formulated to be provided in a sachet.

18. The kit of any one of claims 10 to 17, used in treating or preventing inflammation.

19. The kit of claim 18, wherein compound I and compound II are administered to the subject (i) simultaneously; (ii) sequentially; (iii) separately; or (iv) as a co-formulation.

20. A composition comprising compound I and compound II, these compounds being of formula I and formula II, respectively:, wherein: R1is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3;R2 is -H, or when taken together with R4 and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3is -H or when taken together with R2forms =O; and R4 is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, andwherein: R10 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R11 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R12 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R13is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds, wherein compound I and compound II are not the same.

21. The composition of claim 20, wherein compound I is selected from the group consisting of:acetyl zingerone zingeronezingerol ethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone 4-hydroxy-3-methoxycinnamic acid ethyl esterhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acidconiferyl aldehyde eugenol4-hydroxy-3-methoxybenzoic acid and any salts, solvates, hydrates, and protected forms thereof.

22. The composition of claim 20, wherein compound I is selected from the group consisting of: zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

23. The composition of claim 20, wherein compound II is selected from the group consisting of:ferulic acid hydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatemethyl-3-methoxy-4-hydroxystyryl ketone vanilpyruvic acid3,4,5-trihydroxycinnamic acid sinapic acidcinnamic acid p-coumaric acid and any salts, solvates, hydrates, and protected forms thereof.

24. The composition of claim 20, wherein compound II is selected from the group consisting of: ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

25. The composition of any one of claims 20 to 24, which consists essentially of compound I and compound II.

26. The composition of any one of claims 20 to 25, wherein compound I or compound II is: (i) a synthetic compound; (ii) a naturally occurring compound; (iii) included as a botanical extract.

27. The composition of any one of claims 20 to 26, wherein: (i) the composition is a pharmaceutical composition; (ii) the composition is a dietary composition; (iii) the composition is a food; (iv) the composition is a beverage; (v) the composition is an additive; or (vi) the composition is a dietary supplement.

28. The composition of any one of claims 20 to 27, which is: (i) formulated as a solid, semi-solid, or liquid; (ii) formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule; (iii) formulated to be provided in a sachet; and / or (iv) formulated to include one or more anti-inflammatory agents.

29. The composition of any one of claims 20 to 28, used in treating or preventing inflammation.

30. A method of treating or preventing inflammation comprising administering to a subject compound I and compound II, these compounds being of formula I and formula II, respectively:, wherein: R1is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3;R2 is -H, or when taken together with R4 and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3 is -H or when taken together with R2 forms =O; and R4is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, and ; and wherein:R10is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R11 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R12is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R13 is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds, wherein compound I and compound II are not the same.

31. The method of claim 30, wherein compound I is selected from the group consisting of:acetyl zingerone zingeroneethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone4-hydroxy-3-methoxycinnamic acid ethyl esterhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acid-hydroxy-3-methoxybenzoic acidand any salts, solvates, hydrates, and protected forms thereof.

32. The method of claim 30, wherein compound I is selected from the group consisting of: zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

33. The method of claim 30, wherein compound II is selected from the group consistingferulic acid hydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatevanilpyruvic acid methyl-3-methoxy-4-hydroxystyryl ketone3,4,5-trihydroxycinnamic acid sinapic acidcinnamic acid p-coumaric acid and any salts, solvates, hydrates, and protected forms thereof.

34. The method of claim 30, wherein compound II is selected from the group consisting of: ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

35. The method of any one of claims 30 to 34, wherein compound I or compound II is: (i) a synthetic compound; (ii) a naturally occurring compound; (iii) included as a botanical extract.

36. The method of any one of claims 30 to 35, wherein: (i) compound I and compound II are administered as different formulations; (ii) compound I and compound II are administered as the same formulations; or (iii) compound I and compound II are administered as a co-formulation.

37. The method of any one of claims 30 to 36, wherein: (i) compound I and compound II are administered simultaneously; or (ii) compound I and compound II are administered sequentially.

38. The method of any one of claims 30 to 37, wherein the subject is administered: (i) a pharmaceutical composition which comprises compound I and compound II; (ii) a pharmaceutical composition which comprises compound I and another pharmaceutical composition which comprises compound II; (iii) a pharmaceutical composition which consists essentially of compound I and compound II; (iv) a pharmaceutical composition which consists essentially of compound I and another pharmaceutical composition which consists essentially of compound II; (v) a dietary composition which comprises compound I and compound II; (vi) a dietary composition which comprises compound I and another dietary composition which comprises compound II; (vii) a dietary composition which consists essentially of compound I and compound II; or (vii) a dietary composition which consists essentially of compound I and another dietary composition which consists essentially of compound II.

39. The method of any one of claims 30 to 38, wherein the subject is administered compound I and / or compound II: (i) as a solid, semi-solid, or liquid (ii) as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule; or (iii) as contents of a sachet.

40. The method of any one of claims 30 to 39, where in the subject is further administered one or more anti-inflammatory agents.

41. The method of any one of claims 30 to 40, where in the subject is further administered: (i) one or more of: an analgesic compound, antipyretic compound, and psychotropic compound; (ii) one or more of: a cannabinoid compound. mushroomcompound, non-steroid anti-inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound; or (iii) one or more of: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

42. The method of any one of claims 30 to 41, wherein: (i) the composition is formulated as a dosage form comprising about 1 mg to about 5000 mg of compound I or compound II; (ii) the composition is formulated as a dosage form comprising about 1 mg to about 1500 mg of compound I or compound II; (iii) the composition is formulated as a dosage form comprising about 5 mg to about 500 mg of compound I or compound II; (iv) the composition is formulated as a dosage form comprising about 1 mg to about 150 mg of compound I or compound II; (v) the composition is formulated as a dosage form comprising about 5 mg to about 50 mg of compound I or compound II; (vi) the composition is formulated as a dosage form comprising about 1 mg to about 15 mg of compound I or compound II; or (vii) the composition is formulated as a dosage form comprising about 1 mg to about 10 mg of compound I or compound II.

43. The method of any one of claims 30 to 42, wherein: (a) the inflammation is an inflammatory disorder; (b) the inflammation requires modulation; (c) the inflammation is associated with an immune disorder; (d) the inflammation is associated with an arthritic disorder; (e) the inflammation is associated with an infection; (f) the inflammation is associated with a cardiac, circulatory, or pulmonary disorder;(g) the inflammation is associated with a neurological disorder; and / or (h) the inflammation is associated with a neoplastic disorder.

44. The method of any one of claims 30 to 43, wherein: (i) the inflammation is of one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen, lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system; (ii) the inflammation is associated with one or more of: Alzheimer’s disease, early stage Alzheimer’s disease, ankylosing spondylitis, arthritis, asthma, colitis, Crohn's disease, dementia, early stage dementia, depression, diabetes, fibromyalgia, gout, infection, immune mediated inflammatory disease, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma, and Sjögren’s syndrome, and systemic lupus erythematosus. (iii) the inflammation is associated with one or more of: rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjögren’s syndrome arthritis; (iv) the inflammation is associated with one or more of: atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disorder, and cytokine storm syndrome; or from one or more of: breast cancer, leukaemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer. (v) the inflammation is associated with one or more of: a blister, dermatitis, eczema, hive, lesion, papule, plaque, psoriasis, rash, rosacea, ulcer, and wound.

45. Use of compound I and compound II, for preparing a combination therapy for treating or preventing inflammation in a subject, these compounds being of formula I and formula II, respectively:, wherein: R1is C1-C3alkyl, preferably -CH3, -CH2CH3¸or -CH2CH2CH3;R2 is -H, or when taken together with R4 and the carbon to which it is attached is -C=C-B, B being selected from -C(O)-H, -C(O)-CH3, -C(O)-O-CH3, and -C(O)-O-CH2CH3; R3 is -H or when taken together with R2 forms =O; and R4is selected from one of the following: -H, -OH, -CH3, -CH2-C(O)-CH3, -C(O)-OH, -C(O)-C(O)OH, C(O)-OCH2CH3, CH2-C(OH)-CH3, -CH=CH2, -CH2-C(O)-CH2-C(O)-(CH2)4-CH3, andwherein: R10 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R11is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3;R12 is -H, -OH or -O-C1-C3alkyl, preferably -CH3, -CH2CH3¸-CH2CH2CH3; R13is selected from one of the following: -CH=CH-C(O)OH, -CH=CHCH2OH, -CH2-CH2C(O)OH, -CH=CH-C(O)OCH3, -CH=CH-C(O)OCH(CH3)2, -CH=CH-C(O)CH3, and -CH2-C(O)-C(O)OH; or salts, solvates, hydrates, or protected forms of these compounds, wherein compound I and compound II are not the same.

46. The use of claim 45, wherein compound I is selected from the group consisting of:acetyl zingerone zingeronezingerol ethyl zingeronemethyl-3-methoxy-4-hydroxystyryl ketone 4-hydroxy-3-methoxycinnamic acid ethyl ester6-gingerolhomovanillic acid acetovanilloneethyl vanillate vanilpyruvic acidconiferyl aldehyde eugenol4-hydroxy-3-methoxybenzoic acid and any salts, solvates, hydrates, and protected forms thereof.

47. The use of claim 45, wherein compound I is selected from the group consisting of: zingerone, acetyl zingerone, ethyl zingerone, and zingerol, and any salts, solvates, hydrates, and protected forms thereof.

49. The use of claim 46, wherein compound II is selected from the group consisting of:ferulic acid hydroferulic acidcaffeic acid 4-hydroxy-3-methoxycinnamylic alcoholmethyl 4-hydroxy-3-methoxy-cinnamate 4-methoxy cinnamic acid3,4-dimethoxycinnamic acid isopropyl 4-hydroxy-3-methoxycinnamatevanilpyruvic acid methyl-3-methoxy-4-hydroxystyryl ketone3,4,5-trihydroxycinnamic acid sinapic acidcinnamic acid p-coumaric acid and any salts, solvates, hydrates, and protected forms thereof.

49. The use of claim 45, wherein compound II is selected from the group consisting of: ferulic acid, hydroferulic acid, and caffeic acid, and any salts, solvates, hydrates, and protected forms thereof.

50. The use of any one of claims 45 to 49, wherein compound I or compound II is: (i) a synthetic compound; (ii) a naturally occurring compound; (iii) included as a botanical extract.

51. The use of any one of claims 45 to 50, wherein: (i) compound I and compound II are formulated as different forms for administration; (ii) compound I and compound II are formulated as the same forms for administration; or (iii) compound I and compound II are co-formulated for administration.

52. The use of any one of claims 45 to 51, wherein: (i) compound I and compound II are formulated for simultaneous administration; or (ii) compound I and compound II are formulated for sequential administration.

53. The use of any one of claims 46 to 52, which utilises: (i) a pharmaceutical composition which comprises compound I and compound II; (ii) a pharmaceutical composition which comprises compound I and another pharmaceutical composition which comprises compound II; (iii) a pharmaceutical composition which consists essentially of compound I and compound II;(iv) a pharmaceutical composition which consists essentially of compound I and another pharmaceutical composition which consists essentially of compound II; (v) a dietary composition which comprises compound I and compound II; (vi) a dietary composition which comprises compound I and another dietary composition which comprises compound II; (vii) a dietary composition which consists essentially of compound I and compound II; and (viii) a dietary composition which consists essentially of compound I and another dietary composition which consists essentially of compound II.

54. The use of any one of claims 45 to 53, wherein compound I and / or compound II is formulated for administration: (i) as a solid, semi-solid, or liquid (ii) as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule; or (iii) as contents of a sachet.

55. The use of any one of claims 45 to 54, wherein compound I and / or compound II is formulated for administration with one or more anti-inflammatory agents.

56. The use of any one of claims 45 to 55, wherein compound I and / or compound II is formulated for administration with: (i) one or more of: an analgesic compound, antipyretic compound, and psychotropic compound; (ii) one or more of: a cannabinoid compound. mushroom compound, non-steroid anti-inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound; or (iii) one or more of: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

57. The use of any one of claims 45 to 56, which utilises: (i) a dosage form comprising about 1 mg to about 5000 mg of compound I or compound II; (ii) a dosage form comprising about 1 mg to about 1500 mg of compound I or compound II; (iii) a dosage form comprising about 5 mg to about 500 mg of compound I or compound II; (iv) a dosage form comprising about 1 mg to about 150 mg of compound I or compound II; (v) a dosage form comprising about 5 mg to about 50 mg of compound I or compound II; (vi) a dosage form comprising about 1 mg to about 15 mg of compound I or compound II; or (vii) a dosage form comprising about 1 mg to about 10 mg of compound I or compound II.

58. The use of any one of claims 45 to 57, wherein: (i) the inflammation is an inflammatory disorder; (j) the inflammation requires modulation; (k) the inflammation is associated with an immune disorder; (l) the inflammation is associated with an arthritic disorder; (m) the inflammation is associated with an infection; (n) the inflammation is associated with a cardiac, circulatory, or pulmonary disorder; (o) the inflammation is associated with a neurological disorder; and / or (p) the inflammation is associated with a neoplastic disorder.

59. The use of any one of claims 45 to 58, wherein: (vi) the inflammation is of one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen, lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system; (vii) the inflammation is associated with one or more of: Alzheimer’s disease, early stage Alzheimer’s disease, ankylosing spondylitis, arthritis, asthma, colitis, Crohn's disease, dementia, early stage dementia, depression, diabetes, fibromyalgia, gout, infection, immune mediated inflammatory disease, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgiapsoriasis, scleroderma, and Sjögren’s syndrome, and systemic lupus erythematosus. (viii) the inflammation is associated with one or more of: rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjögren’s syndrome arthritis; (ix) the inflammation is associated with one or more of: atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disorder, and cytokine storm syndrome; or from one or more of: breast cancer, leukaemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer. (x) the inflammation is associated with one or more of: a blister, dermatitis, eczema, hive, lesion, papule, plaque, psoriasis, rash, rosacea, ulcer, and wound.

60. The combination of claim 7, the kit of claim 15, the composition of claim 26, the method of claim 35, the use of claim 50, wherein the botanical extract is produced by a method comprising: (i) subjecting ginger root to an alkaline treatment in alkaline solution; or (ii) subjecting juice, and optionally marc, obtained from ginger root to an alkaline treatment in an alkaline solution, wherein the alkaline solution comprises about 1% to about 6% KOH (v / v), wherein the alkaline treatment is carried out for about 1 to about 2 hours, and wherein following alkaline treatment, the alkaline solution is neutralised to a pH of about 6.5 to about 7.5.

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