Methods for preparing zingerone, compositions containing zingerone, and uses thereof

Abstract

Methods for preparing zingerone from ginger, as well as compositions obtained using these methods, and methods for using these compositions, are disclosed. Specifically, beneficial compositions and methods for using them are disclosed.

Description

[Technical Field]

[0001] (Related Applications) This application claims the benefit of U.S. Application No. 63 / 446,013, filed February 15, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to methods for preparing zingerone and compositions containing zingerone. In particular, useful compositions, including pharmaceutical and food compositions, and uses of these compositions are described. [Background technology]

[0003] Ginger (Zingiber officinale) is a flowering plant whose rhizomes are widely used as a spice and in traditional medicine. When consumed in moderate amounts, ginger has few adverse side effects. It is listed as "Generally Recognized as Safe" by the FDA.

[0004] The characteristic aroma and flavor of ginger are due to the volatile oils that comprise 1% to 3% by weight of fresh ginger, consisting mainly of zingerone, shogaols, and gingerols ([6]-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone)) as the main pungent compound. [ka]

[0005] Zingerone (also known as gingerone) has been reported to be produced from gingerol during drying or heat treatment at temperatures of approximately 40°C, as reported by Li et al., 2016, "Chemical Characterization and Antioxidant Activities Comparison in Fresh, Dried, Stir-Frying, and Carbonized Ginger," Journal of Chromatography B Analyt. Technol. Biomed. Life Sci. 1011: 223-232. Zingerone has a mild pungency and a tangy-sweet aroma. Zingerone, also known as vanillylacetone, is a crystalline solid that is reported to be slightly soluble in water and soluble in ether. The water solubility value of zingerone is listed as 0.57 g / L, with a log P of 2.02 and a log S of -2.5, according to the FoodB Compound Database. See https: / / foodb.ca / compounds / FDB010527 [ka]

[0006] Fresh ginger contains very little zingerone, and cooking or drying ginger root is known to produce zingerone through the dehydration of gingerols (through the loss of water molecules) to produce zingerone and hexanal. See, e.g., Gopi et al. 2016, "Study on temperature-dependent conversion of active ingredients of ginger," Int. J. of Pharma Sciences 6(1): 1344-1347.

[0007] Shogaol, which has a more pungent taste and high antioxidant activity, is not found in fresh ginger but is formed from gingerol by heating, storage, or acidification. [ka]

[0008] Zingerone was first isolated from ginger root by Hiroshi Nomura in 1917. Nomura identified and later patented a method for synthesizing zingerone by reacting vanillin with acetone under basic conditions to form dehydrozingerone (US 1,263,796, issued April 23, 1918). This compound was obtained in approximately 95% yield. After this reaction, the intermediate compound was catalytically hydrogenated to produce zingerone, which was obtained in approximately 100% yield.

[0009] Ginger compounds have been shown to be active against enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea. This type of diarrhea is a leading cause of infant mortality in developing countries. It has been reported that zingerone may be the active component responsible for ginger's antidiarrheal effects. This study concluded that bioactive compounds in ginger significantly blocked the binding of enterotoxigenic Escherichia coli heat-labile enterotoxin to the cell surface receptor GM1, resulting in the inhibition of fluid retention in the closed ileal loops of mice. See, for example, Chen et al., 2007, "Ginger and its bioactive component inhibit enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea in mice," Journal of Agricultural and Food Chemistry 55 (21): 8390-7.

[0010] Zingerone has been shown to have anti-inflammatory effects on liver inflammation in a mouse model of peritonitis, as reported by Kumar et al., "Zingerone suppresses liver inflammation induced by antibiotic-mediated endotoxemia through down-regulating hepatic mRNA expression of inflammatory markers in a Pseudomonas aeruginosa peritonitis mouse model," PLOS ONE 9(9): e106536.

[0011] Kumar et al. also reported that zingerone increased the susceptibility of Pseudomonas aeruginosa cells to antibiotics. See Kumar et al., 2014, Life Sciences 117: 24-32. Kumar et al. concluded that zingerone was found to cause changes in the cell surface properties of Pseudomonas aeruginosa, thereby increasing the susceptibility of Pseudomonas aeruginosa cells to antibiotics.

[0012] Limited research has been conducted on optimizing the production of zingerone from natural sources. Given the current emphasis on compositions derived from natural sources, there is a need for new compositions, including plant-based compositions, particularly those with immunomodulatory and anti-inflammatory activity. The present application aims to meet these and other needs. Summary of the Invention

[0013] In one aspect, the present disclosure encompasses a method of producing zingerone by (i) subjecting ginger root to alkaline treatment in an alkaline solution; (ii) subjecting juice obtained from ginger root to alkaline treatment in an alkaline solution; or (iii) subjecting juice and pomace obtained from ginger root to alkaline treatment in an alkaline solution.

[0014] In certain embodiments

[0015] The ginger root is fresh.

[0016] The ginger root is dried.

[0017] The ginger root is dried at about 40 degrees Celsius (about 40°C) to about 70 degrees Celsius, or at about 40 degrees Celsius to about 60 degrees Celsius, or at about 55 degrees Celsius to about 65 degrees Celsius, or at about 60 degrees Celsius.

[0018] The juice is obtained by steeping and / or pressing ginger root.

[0019] The pomace is obtained by juicing, soaking and / or pressing ginger root.

[0020] The ginger root is cut and subjected to an alkaline treatment.

[0021] The ginger root is cut, dried and subjected to an alkaline treatment.

[0022] The alkali treatment is carried out at a temperature of about 40 to about 70 degrees Celsius.

[0023] The alkali treatment is carried out at about 50 to 60 degrees Celsius.

[0024] The alkali treatment is carried out at a temperature of about 55 to about 65 degrees Celsius.

[0025] The alkaline treatment is carried out at about 60 degrees Celsius.

[0026] The alkali treatment is carried out for about 1 to 72 hours.

[0027] The alkali treatment is carried out for about 1 to 48 hours.

[0028] The alkali treatment is carried out for about 1 to 24 hours.

[0029] The alkali treatment is carried out for about 1 to 30 hours, or about 1 to 20 hours, or about 1 to 10 hours, or about 1 to 5 hours.

[0030] The alkali treatment is carried out for about 0.5 hours to about 3 hours, or about 0.5 hours to about 2 hours, or about 1 hour to about 2 hours.

[0031] The alkaline treatment is carried out for about 2 hours.

[0032] The alkaline treatment is carried out for about 1 hour.

[0033] Potassium hydroxide is used.

[0034] Potassium hydroxide (KOH) in liquid form is used.

[0035] Approximately 0.1% to approximately 6% KOH (v / v) is used. Approximately 0.5% to approximately 5.5% KOH (v / v) is used. Approximately 1% to approximately 6% KOH (v / v) is used. Approximately 1.5% to approximately 5.5% KOH (v / v) is used. Approximately 2% to approximately 4% KOH (v / v) is used. Approximately 1.5% to approximately 3.5% KOH (v / v) is used.

[0036] Calcium hydroxide, Ca(OH)2, is used.

[0037] Approximately 0.5% to approximately 4% Ca(OH)2 (v / v) is used. Approximately 1.5% to approximately 3.5% Ca(OH)2 (v / v) is used. Approximately 2% to approximately 3% Ca(OH)2 (v / v) is used.

[0038] After the alkaline treatment, the alkaline solution is neutralized.

[0039] The alkaline solution is neutralized with citric acid.

[0040] The alkaline solution is cooled during neutralization to relieve excess heat.

[0041] The alkaline solution is neutralized to obtain a pH of about 6.5 to about 7.5 or about 7.0 to about 7.3.

[0042] The neutralized solution is lyophilized.

[0043] The neutralized solution is heat dried.

[0044] The neutralized solution is subjected to extraction of zingerone.

[0045] The neutralized solution is dried and optionally subjected to extraction of zingerone.

[0046] The drying is carried out at about 50 degrees Celsius to about 70 degrees Celsius.

[0047] The drying is carried out at about 55 degrees Celsius to about 65 degrees Celsius.

[0048] The drying is carried out at about 60 degrees Celsius.

[0049] The drying is carried out for at least 24 hours.

[0050] The drying is carried out for about 24 hours to about 28 hours.

[0051] The dried material is optionally ground.

[0052] The zingerone is optionally further extracted by one or more alcohol extraction steps.

[0053] The zingerone is optionally further extracted by one or more ethanol extraction steps.

[0054] The ethanol extraction is carried out at a temperature of about 35 degrees Celsius to about 65 degrees Celsius.

[0055] The ethanol extraction is carried out at about 45 degrees Celsius to about 55 degrees Celsius.

[0056] The ethanol extraction is carried out at about 50 degrees Celsius.

[0057] The ethanol extraction is carried out for at least 7 days.

[0058] The ethanol extraction is carried out for 24 hours or less.

[0059] The ethanol extraction is carried out for at least 4 hours.

[0060] The ethanol extraction is carried out for about 4 to 8 hours.

[0061] The ethanol extract is optionally dried.

[0062] The zingerone is extracted using supercritical fluid extraction.

[0063] The zingerone is extracted by a supercritical fluid extraction process followed by an alcohol extraction process.

[0064] The method produces a product that is a composition comprising zingerone.

[0065] The composition is aldehyde-free or substantially aldehyde-free.

[0066] The composition is a plant extract.

[0067] The composition is an ethanol extract.

[0068] The composition is a powder.

[0069] In one aspect, the present disclosure encompasses a method for producing zingerone by subjecting a ginger root extract to alkaline treatment.

[0070] The ginger root extract is obtained by supercritical fluid extraction of ginger root.

[0071] The ginger root extract is obtained by alcohol extraction of ginger root.

[0072] The ginger root extract is obtained by squeezing the juice from ginger root.

[0073] The ginger root extract is obtained by squeezing the juice of ginger root to obtain a liquid juice and a pomace.

[0074] The juicing includes steeping and / or pressing ginger root.

[0075] The alkali treatment is carried out at a temperature of about 30 to about 70 degrees Celsius.

[0076] The alkali treatment is carried out at about 50 to 60 degrees Celsius.

[0077] The alkali treatment is carried out at a temperature of about 55 to about 65 degrees Celsius.

[0078] The alkaline treatment is carried out at about 60 degrees Celsius.

[0079] The alkali treatment is carried out for about 1 to 72 hours.

[0080] The alkali treatment is carried out for about 1 to 48 hours.

[0081] The alkali treatment is carried out for about 1 to 24 hours.

[0082] The alkali treatment is carried out for about 1 to 30 hours, or about 1 to 20 hours, or about 1 to 10 hours, or about 1 to 5 hours.

[0083] The alkali treatment is carried out for about 0.5 hours to about 3 hours, or about 0.5 hours to about 2 hours, or about 1 hour to about 2 hours.

[0084] The alkaline treatment is carried out for about 2 hours.

[0085] The alkaline treatment is carried out for about 1 hour.

[0086] Potassium hydroxide (KOH) is used.

[0087] Potassium hydroxide (KOH) in liquid form is used.

[0088] Approximately 0.1% to approximately 6% KOH (v / v) is used. Approximately 0.5% to approximately 5.5% KOH (v / v) is used. Approximately 1% to approximately 6% KOH (v / v) is used. Approximately 1.5% to approximately 5.5% KOH (v / v) is used. Approximately 2% to approximately 4% KOH (v / v) is used. Approximately 1.5% KOH (v / v) to approximately 3.5% KOH (v / v) is used.

[0089] Calcium hydroxide, Ca(OH)2, is used.

[0090] Approximately 0.5% to approximately 4% Ca(OH)2 (v / v) is used. Approximately 1.5% to approximately 3.5% Ca(OH)2 (v / v) is used. Approximately 2% to approximately 3% Ca(OH)2 (v / v) is used.

[0091] After the alkaline treatment, the alkaline solution is neutralized.

[0092] The alkaline solution is neutralized to obtain a pH of about 6.5 to about 7.5, or a pH of about 7.0 to about 7.3.

[0093] After neutralization of the alkaline solution, the neutralized material is dried.

[0094] The dried material may optionally be ground.

[0095] The dried material is optionally further extracted.

[0096] The zingerone may optionally be further extracted by one or more alcohol extraction steps.

[0097] The zingerone may optionally be further extracted by one or more ethanol extraction steps.

[0098] The ethanol extraction is carried out for at least 7 days.

[0099] The ethanol extraction is carried out for 24 hours or less.

[0100] The ethanol extraction is carried out for at least 4 hours.

[0101] The ethanol extraction is carried out for about 4 to about 8 hours.

[0102] The ethanol extract may optionally be dried.

[0103] The zingerone may optionally be further extracted using supercritical fluid extraction.

[0104] The zingerone may optionally be further extracted by supercritical fluid extraction followed by an alcohol extraction step.

[0105] The method produces a product that is a composition comprising zingerone.

[0106] The composition is aldehyde-free or substantially aldehyde-free.

[0107] The composition is a plant extract.

[0108] The composition is an ethanol extract.

[0109] The composition is a powder.

[0110] The method includes (i) subjecting ginger root to an alkaline treatment in an alkaline solution; or (ii) subjecting juice obtained from the ginger root, and optionally pomace obtained from the ginger root, to an alkaline treatment in an alkaline solution, wherein the alkaline solution contains about 1.5% to about 3.5% KOH (v / v), the alkaline treatment is carried out for about 1 hour to about 2 hours, and after the alkaline treatment, neutralizing the alkaline solution to a pH of about 6.5 to about 7.5.

[0111] Also included is a composition that is a botanical composition comprising zingerone prepared by the method of any one of the preceding aspects.

[0112] The composition is aldehyde-free or substantially aldehyde-free.

[0113] The composition is an ethanol extract.

[0114] The composition is a powder.

[0115] The composition is formulated for administration as a pharmaceutical or food composition.

[0116] The composition is formulated as a dietary supplement.

[0117] Also included is a composition comprising zingerone prepared by the method of any one of the preceding embodiments.

[0118] In one aspect, the disclosure encompasses a method of treating or preventing inflammation, comprising administering to a subject a composition of any one of the preceding aspects, thereby treating or preventing said inflammation.

[0119] In another embodiment, the disclosure encompasses the use of a composition of any one of the preceding embodiments for the manufacture of a medicament for treating or preventing inflammation.

[0120] Also included are compositions comprising zingerone for treating or preventing inflammation.

[0121] In various

[0122] The composition is obtainable by the method according to any one of the preceding aspects.

[0123] The composition is obtained from ginger root.

[0124] The composition is obtained from fresh ginger root.

[0125] The composition is obtained from dried ginger root.

[0126] The composition was obtained from juice prepared from ginger root.

[0127] The juice is prepared by steeping and / or pressing ginger root.

[0128] The composition is obtained using an alkaline conversion process to convert gingerol in ginger root or ginger root juice into zingerone.

[0129] The composition is aldehyde-free or substantially aldehyde-free.

[0130] The composition is formulated as a powder.

[0131] The composition is formulated as a tincture.

[0132] The composition further comprises one or more anti-inflammatory agents.

[0133] The composition further comprises one or more of an analgesic compound, an antipyretic compound, and a psychotropic compound.

[0134] The composition further comprises one or more of a cannabinoid compound, a mushroom compound, a non-steroidal anti-inflammatory drug compound (NSAID), an opioid compound, a salicylate compound, and a steroid compound.

[0135] The composition may comprise any of the following: 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, Further including one or more of pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin and psilocin.

[0136] The inflammation needs to be regulated.

[0137] The inflammation may be acute or chronic inflammation.

[0138] The inflammation is an inflammatory disorder.

[0139] The inflammation is one or more of: an immune disorder; a joint disorder; an infection; a cardiac, circulatory or pulmonary disorder; a neurological disorder; and a neoplastic disorder.

[0140] The inflammation is inflammation affecting one or more of the joints, skin, eyes, ears, nose, mouth, throat, esophagus, kidneys, bladder, liver, spleen, lungs, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

[0141] The inflammation is one or more of Alzheimer's disease, early Alzheimer's disease, ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerative colitis), Crohn's disease, dementia, early 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 Sjogren's syndrome, and systemic lupus erythematosus (SLE; lupus).

[0142] The inflammation is one or more of rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjogren's syndrome arthritis.

[0143] The inflammation is one or more of atherosclerosis, coronary artery disease, pulmonary arterial hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disease, and cytokine storm syndrome.

[0144] The inflammation is inflammation of one or more of breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0145] In various embodiments of the composition:

[0146] The compositions are formulated for topical or oral administration.

[0147] The compositions may be formulated as a solid, semi-solid or liquid.

[0148] The compositions are formulated as solutions, tinctures, gels, jellies, gummies, powders, tablets, or capsules.

[0149] The composition is provided as a sachet.

[0150] The composition comprises zingerone in a dose of about 10 mg to about 3000 mg.

[0151] The composition comprises zingerone in a dose of about 10 mg to about 1500 mg.

[0152] The composition comprises zingerone in a dose of about 10 mg to about 1000 mg.

[0153] The composition comprises zingerone in a dose of about 10 mg to about 500 mg.

[0154] The composition comprises zingerone in a dose of about 10 mg to about 300 mg.

[0155] The composition comprises zingerone in a dose of about 10 mg to about 150 mg.

[0156] The composition comprises zingerone in a dose of about 10 mg to about 100 mg.

[0157] The composition comprises zingerone in a dose of about 10 mg to about 75 mg.

[0158] The composition comprises zingerone in a dose of about 10 mg to about 50 mg.

[0159] The composition is formulated for co-administration with one or more anti-inflammatory agents.

[0160] The composition is formulated for co-administration with one or more of an analgesic compound, an antipyretic compound and a psychotropic compound.

[0161] The compositions are formulated for co-administration with one or more of a cannabinoid compound, a mushroom compound, a non-steroidal anti-inflammatory drug compound (NSAID), an opioid compound, a salicylate compound, and a steroid compound.

[0162] The composition may comprise any of the following: 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, pentazoline, and psilocin, phenazocine, eptazocine, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

[0163] Also included is the use of a composition according to the previous aspect for preparing a medicament for treating or preventing inflammation in a subject.

[0164] Also included is a method of treating or preventing inflammation in a subject, comprising administering to the subject a composition described in the previous aspect.

[0165] The foregoing brief summary broadly outlines the features and technical advantages of certain embodiments of the present disclosure. Additional technical advantages are described in the following detailed description and examples.

[0166] The novel features believed to be characteristic will be better understood from the detailed description when considered in conjunction with the accompanying figures and examples. However, the figures and examples provided herein are intended to help explain or further the understanding of what is disclosed, and are not intended to limit the scope of the present disclosure. [Brief explanation of the drawings]

[0167] [Figure 1] Photo showing fresh ginger root.

[0168] [Figure 2] HPLC UV chromatogram trace (280 nm) of alkali-treated ginger.

[0169] [Figure 3] Schematic diagram showing process comparison

[0170] [Figure 4A] Photograph showing the juicing equipment and raw ginger before juicing.

[0171] [Figure 4B] A photo showing the process of juicing fresh ginger.

[0172] [Figure 5A] Ginger juice was treated with KOH (0.5%) and analyzed by HPLC. The peak areas of zingerone (Z) and gingerol (G) are shown.

[0173] [Figure 5B] Ginger juice was treated with KOH (1%) and analyzed by HPLC. The peak areas of zingerone (Z) and gingerol (G) are shown.

[0174] [Figure 5C]Ginger juice was treated with 2% KOH and analyzed by HPLC. The peak areas of zingerone (Z) and gingerol (G) are shown.

[0175] [Figure 6A] Ginger pomace produced by pressing.

[0176] [Figure 6B] Ginger juice produced by pressing.

[0177] [Figure 7] Schematic showing the ethanol extraction process and evaporation.

[0178] [Figure 8A] GCMS TIC analysis of ethanol extracts

[0179] [Figure 8B] Comparison of the ethanol extract with a hexanal standard. The chromatogram shows the 2-7 min region.

[0180] [Figure 9] Dose-response curves in cytotoxicity assays evaluating botanical extracts of the present disclosure.

[0181] [Figure 10] Dose-response curves in nitric oxide assays evaluating botanical extracts of the present disclosure.

[0182] [Figure 11] Dose-response curves in an IL-6 assay evaluating botanical extracts of the present disclosure.

[0183] [Figure 12] Dose-response curves in cytotoxicity, NO, and IL-6 assays evaluating botanical extracts of the present disclosure.

[0184] [Figure 13]Cell viability of RAW264.7 cells was measured by WST-1 assay.Comparative study of the plant extract of the present disclosure and commercially available zingerone.

[0185] [Figure 14] Interleukins (IL) produced by RAW264.7 cells after lipopolysaccharide (LPS) treatment. Comparative study of the plant extract of the present disclosure with commercially available zingerone.

[0186] [Figure 15] Interleukin (IL)-10 produced by RAW264.7 cells after lipopolysaccharide (LPS) treatment. Comparative study of the plant extract of the present disclosure with commercially available zingerone.

[0187] [Figure 16] Tumor necrosis factor (TNF-α) produced by RAW264.7 cells after lipopolysaccharide (LPS) treatment. Comparative study of the plant extract of the present disclosure with commercially available zingerone.

[0188] [Figures 17A-17B] Both test methods demonstrated the effectiveness of dexamethasone as a positive control. Figure 17A shows the analytical results using Method 1. Figure 17B shows the analytical results using Method 2.

[0189] [Figure 18] Both test methods showed similar results in MTT scoring. Figure 18A shows the analytical results from Method 1. Figure 18B shows the analytical results from Method 2.

[0190] [Figures 19A-19D] The plant extracts of the present disclosure showed cytotoxic effects at 150 μM, 100 μM, 75 μM, and 50 μM. Data are shown as the mean ± SEM of three biological replicates. Nonlinear regression was calculated using GraphPad Prism 9.0. Figure 19A shows the results for synthetic zingerone. Figure 19B shows the results for acetyl zingerone. Figure 19C shows the results for ferulic acid. Figure 19D shows the results for the plant extracts of the present disclosure.

[0191] [Figure 20] Plant extracts of the present disclosure dose-dependently inhibit IL-6 production from stimulated RAW264.7 cells. Data are presented as the mean ± standard error of the mean (SEM) of three biological replicates.

[0192] [Figure 21] Treatment with the plant extract of the present disclosure at 25 μM produces significantly less IL-6 than treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 μM. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Repeated measures one-way ANOVA with Tukey's correction for multiple comparisons was calculated using GraphPad Prism 9.0. *: P < 0.05, ***: P < 0.001.

[0193] [Figure 22] Treatment with the plant extract of the present disclosure at 25 μM significantly reduced IL-6 levels compared to the solvent control group. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Repeated measures two-way ANOVA with Sidak correction for multiple comparisons was calculated using GraphPad Prism 9.0. *: P<0.05, **: P<0.01.

[0194] [Figures 23A-23D] There was no observable effect on IL-6 production from unstimulated RAW264.7 cells. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Figure 23A shows the results for synthetic zingerone. Figure 23B shows the results for acetyl zingerone. Figure 23C shows the results for ferulic acid. Figure 23D shows the results for a botanical extract of the present disclosure.

[0195] [Figure 24]Plant extracts of the present disclosure reduce TNF (TNF-α) production by stimulated RAW264.7 cells. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates.

[0196] [Figure 25] Treatment with the plant extract of the present disclosure at 25 μM produced significantly less IL-6 than treatment with acetyl zingerone at 150 μM. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Repeated measures one-way ANOVA with Tukey's correction for multiple comparisons was calculated using GraphPad Prism 9.0. *: P < 0.05.

[0197] [Figure 26] Reduction of TNF production by stimulated RAW264.7 cells. Ferulic acid at 150 μM significantly reduces TNF compared to the solvent control. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Repeated measures two-way ANOVA with Sidak correction for multiple comparisons was calculated using GraphPad Prism 9.0. *: P<0.05, **: P<0.01.

[0198] [Figures 27A-27D] There was no observable effect on TNF production from unstimulated RAW264.7 cells. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Figure 27A shows the synthetic zingerone results. Figure 27B shows the acetyl zingerone results. Figure 27C shows the ferulic acid results. Figure 27D shows the results of a botanical extract of the present disclosure.

[0199] [Figure 28] Plant extracts of the present disclosure reduce NO levels in stimulated RAW264.7 cells. Data are presented as the mean ± standard error of the mean (SEM) of three biological replicates.

[0200] [Figure 29] Treatment with the disclosed botanical extract at 25 μM resulted in significantly lower NO levels compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 μM. Data are presented as the mean ± standard error of the mean (SEM) of three biological replicates. Repeated measures one-way ANOVA with Tukey's correction for multiple comparisons was performed in GraphPad Prism 9.0.

[0201] [Figure 30] Treatment with 150 μM synthetic zingerone induced significantly higher NO levels compared to the solvent control. Data are shown as the mean ± standard error of the mean (SEM) of three biological replicates. Repeated measures two-way ANOVA with Sidak correction for multiple comparisons was performed in GraphPad Prism 9.0.

[0202] [Figure 31] Flowchart of large scale ginger juicing process

[0203] [Figure 32] Flowchart of large-scale zingerone extraction process using ginger juice as starting material.

[0204] [Figure 33] Flowchart of large-scale zingerone extraction process using ginger pomace as starting material.

[0205] [Figure 34] Stability of zingerone extract obtained by large-scale production. The test was conducted over a period of two months. Figure 34A shows the zingerone content at 5°C and 40°C. Figure 34B shows the pH level at 5°C and 40°C.

[0206] Detailed Description The following description sets forth numerous example configurations, parameters, etc. However, it should be recognized that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0207] All documents cited in this specification, including patents and patent applications, are hereby incorporated by reference. No admission is made that any document is prior art, and the discussion of any reference is not an admission that such reference forms part of the general knowledge in the art in New Zealand or anywhere else.

[0208] definition When a range is specified in the specification, e.g., a temperature range, a time range, a composition range, etc., all intermediate ranges, subranges, and individual values ​​within the specified range are intended to be included in the disclosure. Thus, each specified range (e.g., 1 to 10) also includes all possible combinations of numerical values ​​between the recited minimum and maximum values ​​(e.g., 1, 1.1, 2, 3, 3.3, 4, 5.5, 6, 7, 8.9, 9, and 10), as well as any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, 3.1 to 4.9). Accordingly, all subranges of all ranges explicitly disclosed herein are hereby expressly disclosed. Numerical values ​​listed in parentheses are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the recited minimum and maximum values ​​are considered to be expressly disclosed herein in the same manner.

[0209] In each instance herein, in the descriptions, embodiments, and examples of the present disclosure, the terms "comprising," "including," and the like are to be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise," "comprising," and the like are to be interpreted in an inclusive sense, i.e., "including, but not limited to," as opposed to an exclusive sense.

[0210] As used herein, "and / or" means additionally or alternatively.

[0211] As used herein, 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" may mean one or more elements.

[0212] Throughout this specification, the term "about" is used to indicate that a value includes the standard deviation of error for the method employed to determine, for example, compound levels or dosage levels, as described in detail herein. In particular, the term "about" encompasses deviations (positive and negative) of up to 10% in the stated value or range.

[0213] As used herein, the term "comprising" may refer to the presence of zingerone or zingerone extract in a composition. Illustratively, zingerone or zingerone extract may comprise at least 1% by weight, 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, zingerone or zingerone extract may comprise at least 1% by volume, 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 (% w / v or % v / v).

[0214] The term "substantially free" with respect to aldehydes refers to a product having negligible levels of aldehyde content. The product may be, for example, a composition described herein or a product produced by a method described herein. Illustratively, the aldehyde level 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.

[0215] As used herein, the term "alkaline treatment" refers to exposing a sample (e.g., ginger, ginger juice, ginger pomace, or any combination thereof) to an aqueous solution containing alkali having a pH greater than 7. Solutions include, but are not limited to, solutions containing sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and any combination thereof. It is understood that alkaline treatment, as described herein, can occur at various temperatures, and that the alkaline solution can be heated before or during exposure to a sample containing ginger or a ginger extract. The alkaline solution contains a chemically effective amount of alkali to convert at least a portion of the gingerol present in the sample to zingerone. Specific methodologies are described in detail herein.

[0216] Generally, the term "extract" in this disclosure refers to a plant extract (also referred to as a botanical drug). More specifically, "extract" refers to a composition in which one or more liquid, solid, or chemical components of a plant or plant part are isolated or concentrated. For example, a liquid, solid, or semi-solid extract can be obtained. The extract can be obtained by one or more of juicing, pressing, soaking, mashing, grinding, or other standard processes. Solvent extraction is also included. Solid extracts include, for example, powders obtained by drying or evaporation. As a specific example, the extract can be prepared in a dry form or in the form of a solution. "Zingerone extract" refers to an extract containing zingerone prepared / produced from ginger root (i.e., the rhizome of ginger, which can also be referred to as "ginger"). Specific extracts and methods for their preparation are described in detail herein.

[0217] As used herein, the term "composition" encompasses a product containing one or more active ingredients (e.g., a combination described herein) and one or more suitable excipients containing other ingredients. These excipients may be physiologically acceptable. The term "composition" also encompasses any product produced, directly or indirectly, by the binding, complexation, or aggregation of two or more active ingredients. In certain embodiments, the composition may include a suitable solvate or salt of each compound. Specifically, the extracts disclosed herein may be prepared as compositions suitable for administration to a subject or as compositions suitable for formulation for administration to a subject. Various exemplary compositions are described in detail herein.

[0218] By "pharmaceutical composition" is meant a composition that is administered to a subject, e.g., to treat or prevent inflammation. By "food composition" is meant a composition that is ingested by a subject, e.g., to reduce or prevent inflammation.

[0219] As used herein, "administration" or "administering" means providing an extract of the present disclosure or a composition prepared from the extract of the present disclosure to a subject. The extract of the present disclosure and the composition of the present disclosure can be administered via any suitable route and any suitable formulation. In some cases, it may be useful to use different administration routes and / or different formulations in the same subject. For example, one or more oral formulations may be used, one or more topical formulations may be used, or one or more oral formulations may be used in combination with one or more topical formulations. Non-limiting examples of administration routes and administration formulations are provided herein.

[0220] "Anti-inflammatory agents" refer to ingredients that relieve one or more symptoms of inflammation. These include drugs, phytochemicals, botanicals, plant extracts and essential oils, as well as tisanes and other infusions, and various other ingredients that help reduce inflammation. These may be used in combination with the compositions and extracts herein to help modulate the immune response.

[0221] "Co-administration" or "co-administering" refers to the combined use of active ingredients, for example, for therapeutic or cosmetic enhancement, and includes the administration of co-formulations (i.e., combinations) as well as the simultaneous, sequential, or separate administration of separate formulations. Similarly, "in conjunction" refers to the combined use of one or more active ingredients and a device / procedure. This can include the use of an active ingredient prior to, concurrently with, and / or after the use of a device / procedure.

[0222] The term "inflammation" includes any degree of inflamed tissue in a subject, lasting for any period of time. Specific examples include acute inflammation or chronic inflammation. Inflammation can be painful. Inflammation can be in the joints, skin, lungs, heart, circulatory system, gastrointestinal tract, genitourinary tract, etc. These and other types of inflammation are encompassed herein.

[0223] Symptoms of inflammation include one or more of pain, heat, redness, swelling, and loss of function. Markers of inflammation include the production of proinflammatory cytokines (e.g., IL-6, TNF-α) and / or proinflammatory small molecules (e.g., NO), and the activation and / or accumulation of immune cells.

[0224] As used herein, a "subject" can be a human or a non-human animal, particularly a mammal, including cattle, sheep, goats, pigs, horses, and other livestock, as well as dogs, cats, and other domesticated pets. In certain embodiments, the subject is a human.

[0225] As used herein, "preventing" means stopping or slowing the onset or progression of inflammation or a disorder involving inflammation. Preventive measures can result in stopping or slowing the onset of inflammation or a disorder or its symptom(s), preventing the progression of inflammation or a disorder or its symptoms, or alleviating inflammation or a disorder or its symptoms once it has occurred. Preventive measures can also affect the support, maintenance, and / or protection of a body system. It should be understood that the term "treatment or prevention" does not exclude the possibility of obtaining both treatment and prevention of a disorder. A "therapeutic" effect or "therapeutic" method can include treatment or prevention, or both.

[0226] As used herein, "treating" means improving or eliminating inflammation or a disorder involving inflammation. Treatment results in a reduction, e.g., improvement or elimination, of the inflammation or disorder or one or more symptoms of the inflammation or disorder. Elimination in the context of treatment includes partial or complete resolution of the inflammation or disorder or its symptoms. Partial or complete healing is encompassed, for example, by an improvement in one or more relevant health parameters. Treatment can include a reduction in the onset of inflammation or disorder or its symptoms. Treatment can also suppress an existing inflammation / inflammatory disorder or its symptoms, or ameliorate an existing inflammation / inflammatory disorder or its symptoms. In the context of "treatment," healing of wounds and rashes is particularly mentioned.

[0227] "Alleviation" means improving inflammation or a disorder involving inflammation. Alleviation results in a decrease, e.g., an improvement, of the inflammation or disorder, or one or more symptoms of the inflammation or disorder. Healing is specifically encompassed, e.g., an improvement in one or more related health parameters. Alleviation includes a decrease in the occurrence of inflammation or disease, or symptoms thereof. Alleviation may also be the suppression of an existing inflammation / inflammatory disorder or its symptoms. Particular mention is made of alleviation of inflammation in wounds and rashes.

[0228] The term "effective amount" means a sufficient amount of the active ingredient in a suitable composition and in a suitable dosage form to treat or prevent a particular disorder or at least one symptom thereof. The "effective amount" varies depending on the ingredients used, the type of treatment, and the species, age, weight, health, etc., of the subject being treated.

[0229] "Combination" refers to the use of two or more components (e.g., two or more active ingredients) together. Methods of use include when the components are formulated together (i.e., a combined formulation) or when the components are used simultaneously, sequentially, or separately (e.g., via different formulations, the same formulation, or co-formulation). These and other specific combinations are included in the present disclosure.

[0230] Methods for preparing compositions The present inventors have found that zingerone compositions prepared from ginger root according to the methods of the present disclosure have significant anti-inflammatory activity that exceeds the activity of commercially available zingerone compositions. Accordingly, the present disclosure generally relates to zingerone compositions prepared from ginger root, and methods for preparing such compositions.

[0231] In one aspect, the present disclosure provides a method for producing zingerone from ginger root by subjecting the ginger root to alkaline treatment. The alkaline treatment may include incubation in an alkaline solution as described herein. As a starting material, the ginger root may be fresh. For example, to aid in preparation, it may be useful to optimize the period that the ginger root is kept in soil before harvesting. In this way, the ginger root utilized is fresh and retains the advantageous properties of fresh ginger root.

[0232] The preparation method of the present disclosure produces a highly effective plant extract. By way of example, to optimize freshness, ginger root can be harvested less than 48 hours, less than 24 hours, less than 12 hours, less than 6 hours, or less than 3 hours before processing. For example, fresh ginger can have a moisture content of about 80% to about 95%, about 81% to about 95%, about 82% to about 95%, about 83% to about 95%, or about 85% to about 95% on a wet basis.

[0233] Alternatively, the ginger root may be dried before treatment. For example, the ginger root may be dried at about 40°C to about 70°C, or about 55°C to about 65°C, or about 60°C. Drying may be carried out for about 1 to 72 hours, or about 1 to 48 hours, or about 1 to 24 hours, or about 1 to 20 hours, or about 1 to 18 hours, or about 1 to 10 hours, or about 1 to 5 hours.

[0234] In certain embodiments, ginger root selected for use in the disclosed methods may have a minimum level of gingerol, e.g., 6-gingerol. For example, ginger root (e.g., fresh ginger root) may have about 0.3 mg / g to about 10 mg / g, or about 0.3 mg / g to about 9 mg / g, or about 0.3 mg / g to about 8 mg / g, or about 0.3 mg / g to about 7 mg / g, or about 0.3 mg / g to about 6 mg / g, or about 0.4 mg / g to about 5 mg / g of 6-gingerol. By way of further example, 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. Therefore, in certain circumstances, it may be advantageous to test the level of gingerol, e.g., 6-gingerol, in the starting material before initiating the methods disclosed herein.

[0235] In one embodiment, the method includes subjecting juice and / or pomace from ginger root to alkaline treatment. Ginger juice and / or ginger pomace can be obtained by steeping and / or pressing. Steeping can involve homogenizing using a blender, food processor, or similar device. Pressing can involve the use of a mechanical or hand press, with screw presses being particularly popular. The solid material remaining after juicing (ginger pomace) can be re-juiced to obtain ginger juice. This can be repeated as necessary. The various juice and pomace samples can be combined prior to alkaline treatment, e.g., juice sample A + juice sample B, or pomace sample A + pomace sample B, or juice samples A, B + pomace samples A, B.

[0236] Optionally, diluted juice can be obtained by subjecting ginger pomace to hot water treatment. For example, water may be added to the pomace in a weight ratio of about 6 to about 1 (about 6:1), or about 5 to about 1 (about 5:1), or about 4 to about 1 (about 4:1), or about 3 to about 1 (about 3:1). The water may be, for example, about 40°C to about 80°C, or about 50°C to about 70°C, or about 55°C to about 65°C, or about 60°C. The incubation time in 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 sample may then be subjected to alkaline treatment. The diluted juice sample may be combined with other juice samples before alkaline treatment.

[0237] In one embodiment, potassium hydroxide (KOH) can be used for the alkaline treatment. For example, solid KOH, such as KOH pellets, can be used. For example, the solid KOH can have an initial concentration of about 100%, or about 90% to about 100%. Alternatively, liquid KOH can be used. For example, the liquid KOH can have an initial concentration of about 50%, or about 40% to about 60%, or about 45% to about 55%. The concentration (e.g., final concentration) of KOH used in the treatment mixture can be, for example, about 0.1% to about 6% (v / v), or about 0.5% to about 5.5% (v / v), or about 1% to about 6% (v / v), or about 1.5% to about 5.5% (v / v), or about 2% to about 4% (v / v), or about 1.5% to about 3.5% (v / v), or about 2% (v / v). Alternatively, calcium hydroxide (Ca(OH)2) may be used for the alkaline treatment. For example, liquid Ca(OH)2 may 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% (v / v), about 1.5% to about 3.5% (v / v), about 1% to about 2% (v / v), or about 3.0% (v / v). Examples of liquid forms include stock solutions of about 25% to about 65%, about 30% to about 60%, about 35% to about 55%, about 45% to about 55%, or about 50%.

[0238] In certain embodiments, the alkali treatment can achieve a pH level of 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 at least pH 13. The alkali treatment can be carried out for a sufficient time and at a sufficiently elevated temperature to obtain the desired level of zingerone. For example, the alkali treatment can be carried out for about 1 to 72 hours, or about 1 to 48 hours, or about 1 to 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 a specific example, the alkali treatment can be carried out at about 40°C to about 70°C, or about 50°C to about 60°C, or about 55°C to about 65°C, or about 60°C. It is understood that lower temperatures may allow for longer treatment periods. For example, an alkali treatment carried out at room temperature can be carried out for about 3 days to about 9 days, or about 5 days to about 9 days, or about 5 days to about 7 days.

[0239] After alkaline treatment, the treated mixture may be further processed, for example, by one or more of neutralization, extraction, and drying. Neutralization may utilize citric acid or other acid compositions. Illustratively, neutralization 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 of citric acid may be used, 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 of citric acid may be used.

[0240] Regarding extraction, zingerone extraction can be achieved by one or more alcoholic extractions, for example, one or more ethanolic extractions. Illustratively, the alcoholic extraction, e.g., ethanolic extraction, can be performed for about 1 to 72 hours, or about 1 to 48 hours, or about 1 to 24 hours, or about 6 to 24 hours, or about 8 to 24 hours, or about 12 to 24 hours, or about 18 to 24 hours. In a specific embodiment, the alcoholic extraction can be performed for 24 hours or less, for example, at least 4 hours, or about 4 to about 12 hours, or about 4 to about 8 hours. The alcoholic extraction can be performed, for example, at about 35°C to about 65°C, or about 45°C to about 55°C, or at least 50°C, or about 50°C.

[0241] 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 utilized. In particular, the amount of ethanol in the extract composition may be reduced by air drying, rotary evaporation, lyophilization, or other techniques. Certain drying methods (e.g., rotary drying) may involve increasing the temperature and pressure to remove residual liquid. For example, drying may be performed under vacuum, e.g., at 50 mBar or less, 45 mBar or less, or 40 mBar or less. As a further example, drying may be performed at about 35°C to about 50°C, or about 35°C to about 45°C, or at least 30°C, or at least 35°C, or at least 40°C, or about 40°C. If the material is dried after neutralization and before alcohol extraction, drying may be performed for, for example, 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. Drying can be carried out, for example, at about 45° C. to about 75° C., or about 55° C. to about 65° C., or at a temperature below 60° C., or about 60° C. Other alternative methods of drying are also described herein.

[0242] As part of the initial process, ginger root may be washed or sterilized. Plant components (e.g., fruit or seeds) may be passed through an assembly with one or more roller brushes to remove any adhering foreign matter. Next, conventional cleaning techniques may be utilized. For example, components may be washed using a series of spray nozzles. Cleaning additives to aid in cleaning or reduce the microbial count of plant components may be utilized in accordance with local regulations and requirements. For example, plant components may be cleaned using a chlorine wash and / or an ozone-impregnated water wash, followed by a rinse with fresh water.

[0243] As mentioned above, it may be desirable to prepare a liquid or semi-solid zingerone composition from ginger root. As described herein, zingerone components may be extracted by chemical means (e.g., solvent extraction). Solvent extraction may utilize one or more of water extraction, methanol extraction, ethanol extraction, or 2-propanol extraction. Supercritical fluid extraction, such as CO2 extraction, may also be used to extract zingerone. Emulsions, pastes, suspensions, and syrups are also suitable. For example, in certain embodiments, it may be desirable to use a paste from ginger root or from ginger root components (e.g., zingerone or zingerone extract). As an example, ginger root may be heated for several hours and filtered to produce a thick, concentrated form. The thickened paste may be spread onto a flat sheet or transferred to packaging, such as a bag, tube, jar, bottle, or other container. The paste may be transferred aseptically. It may also be desirable to prepare a paste from mature plant components. The paste may be a smooth preparation.

[0244] In certain embodiments, the present disclosure encompasses mechanical means (e.g., juicing means, such as soaking and / or squeezing) for extracting zingerone from ginger root. In one embodiment, a squeezing assembly can be adapted to perform a pulping or comminution process. Such a process is relatively mild and gentle compared to traditional fruit pulping techniques (soft pulping). Soft pulping does not utilize significant cell disruption or lysis. The press belt may be multiple loops rotating around a series of pulleys. The distance separating the press belts may decrease in the direction of travel of the plant components. In this manner, greater force may be exerted on the plant components as they travel along the length of the squeezing assembly. In certain embodiments, a squeezing assembly or mechanical press may be used to obtain juice from ginger root as described herein. Alternatively, or in addition, mechanical soaking may be used to obtain juice. For example, commercially available juicing equipment may be utilized.

[0245] Ginger root ingredients (e.g., zingerone or zingerone extract) can be processed by a freezing process. This process can be followed by or combined with a drying or evaporation process. In alternative embodiments, the ingredients are dried or evaporated and then processed into a powder without a freezing process. Methods including air drying or heat-assisted drying (e.g., oven drying) can be used. Drying can be achieved, for example, by one or more of sun or solar drying, hot air drying, batch drying, rotary drying, tunnel drying, belt drying, fluidized 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 to 70°C, about 55°C to 65°C, or at least 50°C, at least 55°C, at least 60°C, or at least 65°C. Evaporation can be achieved, 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 can be used. For example, filtration followed by freeze-drying can be used.

[0246] If freezing is used, it may be desirable to freeze the ginger root ingredients (e.g., zingerone or zingerone extract) as soon as possible after production to maintain freshness. However, freezing can occur within 24 or 48 hours, if necessary. Standard freezing methods can be utilized. Blast freezing is particularly desirable for use with the present disclosure. The ingredients can be frozen in standard-sized pails used to collect the frozen product after processing. The ingredients can be stored frozen (e.g., at -18°C) until needed, for example. Optionally, the ingredients can then be freeze-dried, i.e., lyophilized. Freeze-drying techniques are widely used. The freeze-drying cycle can be up to 48 hours. In certain embodiments, the process can be carried out in a manner that avoids water formation and minimizes moisture content during the process. It is understood that freeze-drying / lyophilizing does not preclude the use of higher temperatures (i.e., temperatures higher than the freezing temperature). For example, higher temperatures may be used to remove residual moisture in the secondary drying step of a lyophilization (lyophilization) / freeze drying procedure.

[0247] The dried or evaporated ingredients (e.g., zingerone or zingerone extract) obtained from ginger root can then be ground into a powder and used as appropriate. Standard grinding methods can be used. Standard mesh sizes can be used to produce the powder, such as US 20, US 23, US 30, US 35, US 40, US 45, or US 50 mesh sizes. The sieve size of the powder can range from 1.0 to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or 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.

[0248] The composition may be prepared as a pharmaceutical composition. The composition may also be prepared as a food composition, such as a functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement). In various embodiments, the composition may be prepared in a liquid or solid form, or a semi-solid form. Various formulations are encompassed by the present disclosure. In certain embodiments, it may be desirable to formulate the composition into a powder. The powder may be provided in a free-flowing form or as a solid cake. The composition may be provided as a suspension-forming powder, a solution-forming powder, a bulk granule, or a bulk powder. The powder may be prepared as a tablet or capsule, or other formulation, as described in detail herein.

[0249] For any liquid or semi-solid product obtained from ginger root, it will be understood that 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 or food composition, as described herein. Similarly, for any solid product obtained from ginger root, it will be understood that the solid may be used as such (e.g., through a grinding process, sieving process, or other process) or may be resuspended to obtain a liquid or semi-solid form for use as a pharmaceutical or food composition, as described herein.

[0250] composition The inventors have discovered that zingerone compositions prepared from ginger root according to the methods disclosed herein have significant anti-inflammatory and immunomodulatory properties useful for reducing cellular inflammatory markers, reducing tissue damage, antioxidant effects, and treating or preventing inflammation and inflammatory diseases in a subject.

[0251] The compositions of the present disclosure may be formulated as one or more formulations, including pharmaceutical and food compositions. By way of non-limiting example, the percentage of zingerone or zingerone extract in the composition may be from about 0.01% to about 30%, or from about 1% to about 30%, or from about 1% to about 15%, or from about 1% to about 10%, or from about 1% to about 9%, or from about 1% to about 8%, or from about 0.1% to about 7%, or from about 0.1% to about 6%, or from about 0.1% to about 5%, or from about 0.1% to about 4%, or from about 0.1% to about 4%, or from about 0.1% to about 3%, or at least about 1%, at least about 4%, or at least about 5%. The percentage may be 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 about 6.25%, about 12.5%, or about 25%, where these percentages represent v / v values ​​for liquid compositions, w / w values ​​for solid compositions, or w / v values ​​for liquid or semi-solid compositions. In any of the various forms (e.g., liquid, solid, semi-solid, etc.) disclosed herein, the composition may be free or substantially free of aldehydes.

[0252] Illustratively, the solid composition may contain from about 0.5 to about 300 mg / g of zingerone, from about 1 to about 150 mg / g of zingerone, from about 1 to about 100 mg / g of zingerone, or from about 1 to about 80 mg / g, or from about 1 to about 60 mg / g, or from about 1 to about 50 mg / g, or from about 1 to about 40 mg / g, or from about 1 to about 20 mg / g, or from about 1 to about 15 mg / g, or from about 1 to about 10 mg / g, or from about 10 to about 60 mg / g of gingerone. or about 10 to about 50 mg / g zingerone, or about 10 to about 40 mg / g zingerone, or about 10 to about 30 mg / g zingerone, or about 10 to about 20 mg / g zingerone, or about 10 to about 15 mg / g zingerone, or at least about 50 mg / g zingerone, or at least about 40 mg / g zingerone, or at least about 10 mg / g zingerone (w / w).

[0253] Similarly, by way of further example, the liquid or semi-solid composition may contain from about 0.5 to about 300 mg / ml of zingerone, from about 1 to about 150 mg / ml of zingerone, from about 1 to about 100 mg / ml of zingerone, or from about 1 to about 80 mg / ml, or from about 1 to about 60 mg / ml, or from about 1 to about 50 mg / ml, or from about 1 to about 40 mg / ml, or from about 1 to about 20 mg / ml, or from about 1 to about 15 mg / ml, or from about 1 to about 10 mg / ml, or from about 10 to about 60 mg / ml. g / ml zingerone, or about 10 to about 50 mg / ml zingerone, or about 10 to about 40 mg / ml zingerone, or about 10 to about 30 mg / ml zingerone, or about 10 to about 20 mg / ml zingerone, or about 10 to about 15 mg / ml zingerone, or at least about 50 mg / ml zingerone, or at least about 40 mg / ml zingerone, or at least about 10 mg / ml zingerone (w / v).

[0254] In various embodiments, topical compositions can be prepared for use on, for example, hands (e.g., hand cream), pre-operative tissues (e.g., surgical preparations of the skin), mucous membranes (e.g., treating inflammation of the bladder, urethra, or vagina, or irrigating these cavities before a medical procedure), wounds or burns (e.g., ointments, bandages, or dressings), mouth or throat (e.g., mouthwash or lozenges), or eyes (e.g., eye drops or ointments).

[0255] By way of non-limiting example, topical compositions may contain one or more diluents (e.g., ethanol or other alcohols), emollients (e.g., PEG-45, palm kernel glyceride, or isopropyl myristate), humectants (e.g., glycerin 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., amphoacetates, isethionates, sulfosuccinates, etc.)). Carriers may include, in particular, sodium lauroamphoacetate, sodium cocoyl isethionate, disodium oleamidosulfosuccinate, 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 combinations thereof. Other possible carriers include lecithin (e.g., in liquid form) and propylene glycol. Combinations of any of the carriers described herein are also permitted.

[0256] In yet another embodiment, the compositions can be prepared for various routes of administration, including oral formulations. 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, ocular, vaginal, intralesional, transdermal, and transmucosal formulations. Standard methods for formulating pharmaceutical compositions are available. See, for example, Remington: Essentials of Pharmaceutics, 2013, Pharmaceutical Press, London.

[0257] In certain embodiments, the compositions of the present disclosure can be prepared as powder or any other suitable dosage form.Topical formulations can be prepared as, for example, aerosol, balm, cream, dressing, drops, emulsion, thin film, foam, gel, jelly, liquid, lotion, mask, oil, ointment, paste, powder, ointment, soap, spray, suspension, solution, tincture and vapor.Additional topical formulations include bandages, dressings, patches, pads, sponges, strips, tapes and others as mentioned herein.

[0258] As described herein, the compositions may be formulated, for example, as semi-solid or liquid compositions for oral administration (e.g., taken directly by mouth or enclosed in a capsule or other form), or for enteral or parenteral administration (e.g., taken by injection, feeding tube, or other form). Alternatively, the compositions may be formulated as powders for encapsulation, tableting, or addition to or incorporation into other products.

[0259] Oral formulations can be prepared, for example, as draughts, drops, elixirs, emulsions, liquids, linctuses, solutions, sprays, suspensions, syrups, tonics, or as films, gels, jellies, gummies, troches, nuggets, pastes, purees, pressed cakes, powders, pills, or strips. In other embodiments, oral formulations can be prepared as tablets or capsules, e.g., with liquid, semisolid, or solid contents. Oral formulations can be provided in sachet form, e.g., powder sachets, or gel or jelly sachets. Also included are oral formulations comprising thin strips or solids in capsules to be mixed with food or beverages. Oral formulations can be provided as shooters or shots (to be taken orally), e.g., liquid shots, gel or jelly shots, paste shots, or powder shots.

[0260] Particularly included are delayed-release formulations, sustained-release formulations, and rapid disintegration formulations.Particularly included are capsules, such as gel capsules, and also include sachets and chewable tablets.Furthermore, also included are combination formulations in which the powder of the present disclosure is mixed with other beneficial agents, such as one or more antimicrobial agents.As described herein, other formulations are also possible.

[0261] The dissolution time of oral formulations can be modified for rapid effect or sustained release. Oral formulations can also contain a mixture of slow-release and fast-release particles to provide rapid and sustained absorption at the same dose. Oral formulations such as tablets and capsules can be coated with special coatings to make them resistant to stomach acid. Oral formulations can also be coated with sugars, varnishes, or waxes to improve taste.

[0262] Thus, tablets can be prepared as fast-dissolving tablets, and capsules can be prepared as sustained-release capsules. Tablets can be divided, chewable, effervescent, orally disintegrating, or tablets for forming suspensions. Capsules can be gel capsules, e.g., containing solid, semi-solid, or liquid contents. This includes gel capsules made by one-piece and two-piece gel encapsulation. Particular mention is made of hard and soft capsules. Gelatin-free capsules and caplets are also mentioned.

[0263] It will be understood that certain formulations are suitable for either topical or other application. Particular formulations of interest are ophthalmic formulations (e.g., drops, ointments), otic formulations (e.g., drops, ointments), nasal or respiratory tract formulations (e.g., drops, sprays, insufflation compositions, inhalation compositions, nebulization compositions), dermal formulations (e.g., soaps, sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, patches, tapes, bandages, dressings, sponges, vapors), throat or mouth formulations (e.g., drops, lozenges, mouthwashes, toothpastes, sprays, pastes, gels, jellies, gummies), mucosal formulations (e.g., sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, dressings, sponges).

[0264] Solid, semi-solid, and liquid compositions can be prepared by combining zingerone or zingerone extract with one or more compounds to ensure a stable and active composition. For example, oral formulations such as tablets or capsules can contain about 5 to about 50% w / w zingerone or zingerone extract; up to about 80% w / w of one or more fillers, lubricants, glidants, or binders; and up to about 10% w / w of compounds to ensure easy disintegration, disintegration, and dissolution of the tablet in the stomach or intestines. In one particular example, the extract (e.g., an ethanolic tincture) of the present disclosure can be mixed with one or more carrier substances, such as glycerol, glyceryl esters, hydrogenated oils, polyethylene glycol, or poloxamer, and contained within a capsule (e.g., a gel capsule).

[0265] Thus, the compositions may include one or more of a variety of excipients, such as solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, thickeners, viscosity enhancers / reducers, emollients, surfactants, chelating agents, adjuvants, anti-adherents, anti-caking agents, binders, coating agents, disintegrants, lubricants, glidants, flow agents, adsorbents, flavors, taste masking agents, colors, sweeteners, or preservatives.

[0266] Illustratively, the composition may contain less than 1% preservative, e.g., about 0.005% to about 0.5%, or about 0.05% to about 0.15%, or 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%, where these percentages are representative of w / v 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, including its sodium salts, e.g., sodium benzoate.

[0267] Other useful excipients include, but are not limited to, stearin, magnesium stearate, and stearic acid; saccharides and derivatives thereof, such as disaccharides: sucrose, lactose; polysaccharides and derivatives thereof, such as starch, cellulose or modified cellulose, 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 thickening agents, such as acacia gum, gellan gum, guar gum, locust bean gum, xanthan gum. Gum, agar, arrowroot carrageenan, gelatin, glycerin, kudzu, lecithin, starch; synthetic polymers such as polyvinylpyrrolidone, polyethylene glycol; fatty acids, plant-based surfactants such as sunflower lecithin, wax, shellac, plastics, plant fibers such as corn protein zein; hydroxypropyl methylcellulose; cross-linked polymers such as cross-linked polyvinylpyrrolidone (crospovidone) and cross-linked sodium carboxymethylcellulose (croscarmellose sodium); sodium starch glycolate; silicon dioxide, fumed silica, talc, and magnesium carbonate. Combinations of any excipients can be used.

[0268] A variety of delivery systems are available, including 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, inorganic nanoparticles (e.g., metal nanoparticles, dendrimers, etc.), etc. For example, phytosomes containing lecithin can be used to enhance the topical and oral absorption of zingerone or zingerone extract.

[0269] Liquid composition can be stored as tincture in vial, bag, ampoule, cartridge or pre-filled syringe, for example.Composition can be transferred from vial to larger container and mixed with other materials.Dry or evaporated composition can be stored in vial, cartridge, dual chamber syringe or pre-filled mixing system, for example.Also, before administration, dry composition can be reconstituted as liquid and then administered.

[0270] Exemplary unit dosages of the composition include about 0.1 mg to about 1000 mg of zingerone or zingerone extract, about 1 mg to about 500 mg of zingerone or zingerone extract, about 1 mg to about 250 mg of zingerone or zingerone extract, about 1 mg to about 200 mg of zingerone or zingerone extract, about 1 mg to about 100 mg of zingerone or zingerone extract, about 1 mg to about 50 mg of zingerone or zingerone extract, or about 1 mg to about 25 mg of zingerone or zingerone extract. Dosages can be formulated for administration once a week, twice a week, three times a week, every other day, once a day, twice a day, three times a day, or more frequently as needed. Dosages can be adjusted as needed for pediatric, elderly, overweight, underweight, and other patients. Dosages can be adjusted according to standard methods. Therefore, a wide range of unit dosage forms can be envisioned and prepared.

[0271] Method of using the composition As mentioned above, the composition of the present disclosure can be used to treat or prevent inflammation and various health conditions related to inflammation.For example, the composition of the present disclosure can be used to reduce the level of pro-inflammatory cytokines or pro-inflammatory small molecules in a subject.In certain embodiments, the pro-inflammatory cytokine can be an interleukin cytokine such as IL-6 and / or IL-10.The pro-inflammatory cytokine can be tumor necrosis factor such as TNF.The pro-inflammatory small molecule can be nitric oxide.

[0272] In certain embodiments, the composition can comprise zingerone or zingerone extract produced by the method described herein.The composition of the present disclosure can also be prepared in one or more pharmaceutical forms.In addition, or alternatively, the composition can also be prepared in one or more food forms, such as functional foods or beverages, natural ingredients (e.g., natural additives), or natural supplements (e.g., dietary supplements).

[0273] In various embodiments, the compositions of the present disclosure can be used to target one or more inflammatory disorders affecting one or more of the joints, skin, eyes, ears, nose, mouth, throat, esophagus, kidneys, bladder, liver, spleen, lungs, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

[0274] Diseases of interest include immune system disorders (e.g., autoimmune disorders), such as Alzheimer's disease (e.g., early Alzheimer's disease), ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerative colitis), Crohn's disease, dementia (e.g., early dementia), depression, diabetes, fibromyalgia, gout, immune-mediated inflammatory diseases, infections (e.g., microbial infections), inflammatory bowel disease (IBD), interstitial cystitis, multiple sclerosis (MS), and polymyalgia. These include, but are not limited to, psoriasis, scleroderma, and Sjogren's syndrome and systemic lupus erythematosus (SLE; lupus); joint disorders such as rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, Sjogren's syndrome arthritis; cardiac, circulatory, and pulmonary disorders such as atherosclerosis, coronary artery disease, pulmonary arterial 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 tumors, such as breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0275] Other diseases of interest include musculoskeletal disorders (e.g., disorders of bones, cartilage, fingers, joints, limbs, muscles, tendons, etc.) and back disorders (e.g., conditions of the soft tissues of the spine or back). Also mentioned are inflammatory diseases associated with infections, such as gingivitis. In particular, anti-inflammatory therapy for the skin is mentioned. By way of non-limiting example, the compositions of the present disclosure may be used for one or more of blisters, dermatitis, eczema, hives, lesions, papules, plaques, psoriasis, rashes, rosacea, ulcers, and wounds. The wounds may be acute or chronic (e.g., non-healing or recurrent wounds). In particular, surgical wounds and scars are mentioned.

[0276] In certain embodiments, the compositions of the present disclosure can be used in conjunction with one or more anti-inflammatory agents. For example, the compositions can be prepared as a combined formulation with one or more anti-inflammatory agents. Alternatively, the compositions can be used in separate formulations with one or more anti-inflammatory agents. When two or more active ingredients (e.g., a zingerone composition and an anti-inflammatory agent) are used, their use can be coordinated by simultaneous, sequential, or separate administration. Furthermore, the compositions described herein can be used in conjunction with various medical or non-medical treatments. The compositions can be used before, during, or after the treatment, or any combination thereof.

[0277] By way of example, the anti-inflammatory agent may include one or more components obtained from a plant, such as one or more plant compounds, preparations, extracts, and / or oils. These include manuka (e.g., L. scoparium), hohere (e.g., Hoheria angustifolia, Hoheria glabrata, Hoheria lyallii, Hoheria populnea, Hoheria sexstylosa), horopito (e.g., Pseudowintera colorata), kawakawa (e.g., Piper excelsum), koromico (e.g., Hebe stricta, Hebe salicifolia, or Hebe elliptica), elliptica), poroporo (e.g., Solanum aviculare), and pukatea (e.g., Laurelia novae-zelandiae). Particular mention is made of manuka extracts (e.g., manuka oil) and kawakawa extracts (e.g., leaf extracts). Also mentioned are ingredients derived from Psilocybe spp., such as P. azurescens, P. semilanceata, and P. cyanescens, and Cannabis spp., such as C. sativa.

[0278] Also included are essential oils 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, etc. Also included are honey (e.g., manuka honey), arnica (e.g., arnica oil, creams, gels), activated charcoal, capsaicin, sesame, yarrow (e.g., for various skin care products), and comfrey (e.g., for ointments or creams).

[0279] As a further example, the anti-inflammatory agent may include one or more drug compounds. These include analgesics, antipyretics, and psychotropic drugs. Examples 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 particularly mentioned. Opioid compounds (e.g., KOR inhibitors) and steroid compounds (e.g., corticosteroids) are also mentioned. Examples include butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, and eptazocinem. Further examples include betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone.Cannabinoid compounds and mushroom compounds are also mentioned.Examples include cannabidiol, cannabigerol and tetrahydrocannabinol.Further examples include psilocybin and psilocin.

[0280] A variety of anti-inflammatory agents can be adapted and utilized in accordance with the present disclosure. Any combination of anti-inflammatory agents can be used. Any medical device or procedure can also be used in conjunction with the compositions and extracts of the present disclosure.

[0281] The compositions of the present disclosure described herein are useful as anti-inflammatory formulations. In certain embodiments, the compositions can be used in methods to reduce or delay inflammation in specific tissues, including joints, skin, ears, eyes, nose, mouth, gums, throat, digestive tract, heart, circulatory system, lungs, vagina, urinary tract tissue, and other tissues described herein. The compositions can be applied, for example, to burns to reduce the likelihood of inflammation, or to the skin before surgery to reduce inflammation around a surgical site. The compositions can also be used as hand cleansers (e.g., soaps or hand sanitizers) applied with or without water. The compositions can be used for minor skin irritations, cuts, or scrapes. The compositions can be used as mouthwashes or gargles to reduce inflammation caused by, for example, stomatitis or gingivitis. The compositions can also be utilized as lozenges or throat sprays, for example, to soothe a sore throat. Eye drops or ointments can be used to reduce inflammation in the eyes, including the eyelids.

[0282] The compositions of the present disclosure are also used as formulations that can be used in the methods of treating or preventing inflammation and inflammatory diseases described herein. The inflammation can 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.

[0283] As described herein, the compositions may be administered via a variety of routes, including parenteral (e.g., topical) and enteral (e.g., oral) administration. Enteral administration may be via a duodenal or gastric tube, including a nasogastric tube, or other standard methods. Oral administration may be via tablets, capsules, sachets, drops, elixirs, linkages, solutions, emulsions, suspensions, drafts, purees, pastes, pomaces, syrups, gels, jellies, gummies, tonics, or various other methods. Topical administration may be via drops, sprays, ointments, soaps, pads, sponges, dressings, bandages, or various other methods. Standard administration methods may be utilized by those skilled in the art. The compositions disclosed herein are not limited to a particular dosage form.

[0284] As exemplary dosages, the compositions may be administered in doses of about 1 to about 3000 mg of zingerone or zingerone extract, or about 100 to about 3000 mg of zingerone or zingerone extract, or about 100 to about 2500 mg of zingerone or zingerone extract, or about 100 to about 2000 mg of zingerone or zingerone extract, or about 1 to about 1800 mg of zingerone or zingerone extract, or about 100 to about 1600 mg of zingerone or zingerone extract, or about 100 to about 1400 mg of zingerone or zingerone extract, or about 100 to about 1200 mg of zingerone or zingerone extract, or about 100 to about 1000 mg of zingerone or zingerone extract. Additional exemplary dosages include about 10 to about 300 mg of gingerone or zingerone extract, or about 10 to about 200 mg of zingerone or zingerone extract, or about 10 to about 150 mg of zingerone or zingerone extract, or about 10 to about 100 mg of zingerone or zingerone extract, or about 10 to about 80 mg of zingerone or zingerone extract, or about 10 to about 60 mg of zingerone or zingerone extract, or about 10 to about 55 mg of zingerone or zingerone extract, or about 10 to about 50 mg of zingerone or zingerone extract, or about 10 to about 40 mg of gingerone or zingerone extract. These dosage ranges are particularly useful for ginger components (such as gingerone or zingerone extract) that have been dried and ground into a powder.

[0285] The European Food Safety Authority (EFSA) has classified zingerone as safe for animal (e.g., rat) consumption, with a no-observed-adverse-effect level (NOAEL) based on a dose of up to 128 mg / kg / day (EFSA, 2016, 14(8):4557). Exemplary dosages can be determined, for example, based on an average 70 kg human subject. Exemplary dosages include, for example, for zingerone or zingerone extract, 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. Additional exemplary dosages include, for example, for zingerone or zingerone extract, from about 0.1 mg / kg to about 20 mg / kg, or from about 0.1 mg / kg to about 15 mg / kg, or from about 0.1 mg / kg to about 10 mg / kg, or from about 0.1 mg / kg to about 8 mg / kg, or from about 0.5 mg / kg to about 6 mg / kg, or from about 0.1 mg / kg to about 4 mg / kg, or from about 0.1 mg / kg to about 2 mg / kg, or from about 0.1 mg / kg to about 1 mg / kg.

[0286] The dosages described herein can be administered once a week, every other day, once a day, twice a day, three times a day, or less frequently or more frequently, as needed. Administration can be performed during or before meals. Those skilled in the art can easily determine the appropriate dosage and dosage form. [Example]

[0287] The examples described herein are provided for the purpose of illustrating particular embodiments and are not intended to limit the disclosure in any way.

[0288] Example 1: Preparation of Zingerone An initial sample of fresh ginger (400 gm), as shown in Figure 1, was sourced from New Zealand and was cleaned of any dirt or soil. The washed ginger was then diced or finely chopped and subjected to an alkaline treatment (800 g of 0.5% potassium hydroxide in distilled water). The resulting mixture was stirred and placed in an oven at 60°C for 22 hours. It is understood that the resulting mixture could also be placed in a water bath and maintained at approximately 60°C for a desired period of time.

[0289] The pH of the mixture was then adjusted to pH 7 by adding concentrated citric acid, and the treated plant material was spread on a metal tray and dried in an oven at 60°C for 20 hours. The resulting dried material weighed 35 grams, representing a dry yield of 8.75%. This dried material was scraped into a flask, covered with 95% ethanol (210 ml), and extracted. The flask was shaken and placed in an oven at 40°C for 16 hours. The extract (Extract 1) was filtered using a glass funnel with a glass wool plug. The remaining plant material was extracted again with 95% ethanol (Extract 2), and then two additional times with 50% ethanol (Extracts 3 and 4).

[0290] The extract was analyzed directly for zingerone content. A sample of 294 mg of freshly chopped ginger was also extracted with 2 ml of ethanol, and this extract was also analyzed. The results are shown in Table 1. [Table 1]

[0291] result A 400g supply of fresh ginger root was shown to contain 260mg of 6-gingerol (i.e., 0.65mg / g). This means that the theoretical maximum yield of zingerone from the processed material would be approximately 171mg (weight loss due to the lower molecular weight of zingerone compared to 6-gingerol). Approximately 50% of the 6-gingerol was not converted. Further research is expected to increase the alkaline conversion rate of 6-gingerol to zingerone.

[0292] Ethanol extractions were performed using the minimum volume necessary to cover the treated plant material. The most concentrated extract, extract 1, contained 0.47 mg / mL of zingerone. This concentration could, in principle, be increased by a multi-step process or by evaporating part of the ethanol. Reducing the volume of ethanol by 80% yielded a 2.35 mg / mL zingerone solution.

[0293] It is expected that a concentration of approximately 16 mg / g of solid extract will be obtained if the extract is completely dried. Higher concentrations are expected if starting with a higher initial 6-gingerol content and / or more complete conversion. For example, the theoretical 25 mg dose could potentially be achieved by formulating the dried extract directly into an oil or glycerol carrier, providing the required dose of zingerone in one or two 500 mg capsules.

[0294] The majority of zingerone was extracted in the first extraction. Double extractions may be effective. The third and fourth extractions resulted in a slight increase in total amount. Overall, ethanol extraction was found to be very effective and to be a very efficient and inexpensive preparation method.

[0295] Example 2: Preparation of Zingerone Summary: These studies demonstrate that freeze-drying fresh ginger after aqueous alkali treatment significantly improved conversion, reaching a zingerone content of approximately 1% in dried ginger. The treated ginger was then extracted with supercritical CO2 and CO2 + ethanol co-solvents, resulting in a combined extraction yield of 3% and an average zingerone concentration of approximately 12% in the extracted oleoresin. Furthermore, drying fresh ginger at a moderate temperature (60°C) and then extracting it with supercritical CO2 resulted in an extraction yield of 4.6%. The extracted oleoresin was then alkali-treated, and the final product contained approximately 15% zingerone.

[0296] Drying: Fresh ginger material samples imported from Fiji were sliced ​​into 2-5 mm pieces and placed in a single layer on perforated oven trays. Drying was performed by forced convection. Drying was performed at moderate temperature (60°C) with the goal of removing moisture without converting gingerols. The process was terminated when the moisture content of the ginger reached 7%. The resulting dried ginger was stored refrigerated until use in extraction studies.

[0297] Catalytic Conversion: A small-scale preliminary study was performed by treating approximately 1.6 g of chopped fresh Fiji ginger with an aqueous solution 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 added reagent to ginger was approximately 3:1. The sample was then shaken and placed at room temperature or in an oven at 37°C or 60°C overnight before analysis. It should be understood that a suitable water bath can be used to hold the sample at the desired temperature for the desired period of time.

[0298] After the alkaline treatment was selected, 5.2 kg of fresh ginger was chopped using a vertical cutter mixer (RobotCoupe® 45). The ginger was then mixed with 0.5% KOH (approximately 0.1 N) in a 3:1 liquid:solid ratio (volume:weight) to achieve a pH of approximately 12.5. The mixture was manually stirred and placed in a 60°C oven for 24 hours. After this time, the mixture was neutralized to a pH of approximately 7.2 by adding concentrated citric acid (625 g / L). The neutralized mixture was then freeze-dried, and the ginger obtained from this process was stored refrigerated until use in extraction studies.

[0299] Catalytic Conversion Results: As previously mentioned, 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 zingerone and 6-gingerol contents were quantified for these experiments (Table 2). It was concluded that treatment with KOH at 60°C was the most efficient and resulted in the highest zingerone concentration. Table 2 shows the amount of 6-gingerol (mg / g) and zingerone (mg / g) obtained under each condition. The results are expressed on a wet basis. The HPLC traces are shown in Figure 2. From the HPLC traces, the 1% Ca(OH)2 treatment resulted in the highest zingerone concentration. )2 It can be seen that 0.5% KOH exhibits similar performance. In the case of 0.5% KOH at 60°C, the ratio of zingerone to 6-gingerol is 6.5. In the case of 1.0% Ca(OH) at 60°C, )2 In this case, the zingerone:6-gingerol ratio is 4.0. [Table 2]

[0300] This process was applied to a larger sample of fresh ginger (5.2 kg), and the resulting treated ginger was then neutralized and freeze-dried. The yield of freeze-dried ginger was 17%, or 17 g of treated freeze-dried ginger per 100 g of chopped fresh ginger. The zingerone and 6-gingerol contents of the freeze-dried ginger were measured to be 10.2 mg / g and 3.3 mg / g, respectively (dry weight basis). See Table 2-1. The zingerone content was at least 10 times that of oven-dried ginger. [Table 3]

[0301] Extraction: A supercritical extraction experiment was performed using alkali-treated ginger. Alkali-treated freeze-dried ginger was lightly crushed by hand and placed in a 2-L extraction vessel fitted with sintered filter discs on both ends, completely filling the vessel. The extraction was performed as described above until a sudden drop in the extraction rate was observed, corresponding to 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 at a rate of approximately 10 wt% (i.e., 10 g ethanol per 100 g CO2). The ethanol pump was stopped after a 2:1 ethanol:feed ratio (2 g ethanol per 1 g feed) was introduced. The CO2 was then circulated to flush out any remaining ethanol from the bed. Upon completion of the extraction, the apparatus was depressurized, and the residue was allowed to degas overnight before being removed. The ethanol present in the extract was removed by rotary evaporation under vacuum. Extraction parameters are shown in Table 3. [Table 4]

[0302] Analysis: Samples were prepared for analysis by adding methanol, after neutralization if necessary. The extract was dissolved directly in methanol. Analysis was performed by HPLC using an acetonitrile / 0.1% formic acid gradient. Detection was at 280 nm, and the column used was a Phenomenex Kinetex C18 (150 x 2.1 mm). Zingerone eluted at approximately 2 minutes, and 6-gingerol at 5.2 minutes. Quantitation of zingerone and 6-gingerol was obtained from standard curves constructed using analytical standards of these compounds.

[0303] Extraction Results: As previously described, ginger, freeze-dried after alkali treatment, was extracted with CO2, followed by CO2 + ethanol co-solvent. No free water was observed in the CO2 extract, and the ethanol in the CO2 + ethanol extract was removed by rotary evaporation under vacuum. The extract had a sweet, caramel-like aroma. The CO2 yield was 1.5%. An additional 1.5% could be extracted by adding 10% ethanol co-solvent. The composition of the different fractions is shown in Tables 4 and 5. See also Figure 3. [Table 5] [Table 6]

[0304] The results confirm that the CO2 extract of the alkali-treated sample contained 153 mg / g zingerone (15.3%) and 103 mg / g 6-gingerol (10.3%), yielding a zingerone / gingerol ratio of approximately 1.5. The CO2 + ethanol extract contained 91 mg / g zingerone and 52 mg / g gingerol (a zingerone / gingerol ratio of approximately 1.75). The post-extraction ginger pomace or residue was also analyzed and found to contain 5.4 mg / g zingerone. Considering the masses of the feed, extract, and pomace (Table 4), the mass balance for zingerone can be calculated to be 90.6%. This indicates that 90.6% of the zingerone originally present in the feed is present in the extract and pomace. This difference may be due to degradation during extraction or the ethanol removal step. The extraction yield of zingerone (i.e., grams of zingerone extracted per 100 g of zingerone in the feed) was only 37% when calculated based on the extract. However, because 54% of the zingerone initially present in the feed remained unextracted in the pomace, the zingerone extraction yield calculated based on the pomace results was 46%. This accounts for the "missing" zingerone. Because the proportion of unextracted zingerone is significant, further improvements to the extraction process can be made to reduce it. The extraction yield of gingerol is higher than that of zingerone (82% for the untreated sample and 71% for the treated sample) because gingerol is more soluble in CO2.

[0305] In a separate experiment, the conversion of gingerol to zingerone was tested by subjecting CO2 extracts obtained from unprocessed ginger root to alkaline treatment. Overnight treatment with both 0.1N and 1N KOH at 60°C worked well, resulting in a zingerone concentration of approximately 15% in the processed ginger. The resulting material appeared much cleaner than the alkali-treated crude ginger, making this an interesting alternative that could offer a more cost-effective extraction process overall, since the conversion process requires less material. In fact, starting with the same amount of fresh ginger (100 kg), and based on the results obtained in this work, extracting the unprocessed ginger and then alkali-treating the extract yields nearly twice as much zingerone in the final product as alternative processes (see comparison in the table below). However, even in this case, the total zingerone yield of the process is approximately 0.1% (0.1 kg zingerone per 100 kg fresh ginger). Further optimization is anticipated.

[0306] In a separate experiment, a sample of raw ginger CO2 extract was mixed with KOH and converted to a zingerone-rich extract. A post-processing neutralization step involved the isolation of zingerone from the aqueous reaction mixture as a zingerone-rich resin. Three grams of oleoresin was taken twice, and 9 ml of 1N KOH was added to the sample in a plastic vial. The sample was then shaken and placed in a 60°C oven overnight. The treated sample was then neutralized by adding 10 ml of 1N HCl. A slight excess of acid was added to ensure all the KOH was neutralized. The addition was performed in two stages, with mixing after each stage. The sample was centrifuged at 2000 rpm to separate the water from the oleoresin. After centrifugation, most of the water was removed with a pipette. The resulting resin was then removed. A tincture was made by adding absolute alcohol to one of the resin samples (approximately 3 g of resin to 5 ml of ethanol) (Sample 1). The other sample was stored in resin form (Sample 2). Samples 1 and 2 were analyzed for zingerone content. The concentration of zingerone in the tincture (sample 1) was calculated to be 23 mg / g of tincture, and the concentration of zingerone in the treated resin (sample 2) was 52 mg / g.

[0307] For both samples 1 and 2, these values ​​were lower than those obtained in previous treatments, where the zingerone content was estimated at 150 mg / g. In this work, resin sample 2 was separated from the water and analyzed. The separated water was then analyzed and estimated to contain an additional 50–55 mg (approximately 25%) of zingerone for each 3 g batch. This result was quite unexpected, as zingerone has been reported to have fairly limited water solubility. This method contrasts with the direct addition of alcohol to the crude neutralized product, as a significant portion of the zingerone remains in the water and is not separated from the resin.

[0308] Since water can extract some of the zingerone from the resin, drying the entire neutralized alkali-treated product may also produce a high yield of zingerone after treatment. Considering that alkali-treated zingerone was found to be more soluble in water than previously reported, further process optimization will be performed to improve the yield.

[0309] Example 3: Further reaction methods and comparisons Abstract: Zingerone is not naturally occurring in ginger, but is a product of conversion from gingerol during processing. In these studies, alkali-treated and dried ginger samples were obtained from Samoa and extracted with ethanol using two different extraction conditions: 20 hours at 40°C (Extract A) and 7 days at room temperature (Extract B). The zingerone concentrations in the resulting extracts were 41.4 mg / g for Extract A and 43.8 mg / g for Extract B.

[0310] Extraction A: Processed ginger, produced by the Scientific Research Organization of Samoa (SROS), was supplied in two separate plastic bags. The contents of both bags were combined and frozen at -80°C. The contents were then ground using a knife mill (Wiley) fitted with a 2 mm mesh. The ground material (782.3 g) was then placed in a round-bottom flask with food-grade ethanol in a weight ratio of approximately 5:1. The flask was then placed in a water bath at 40°C and 5 rpm and stirred overnight (total extraction time 20 h). The mixture was then filtered under vacuum, and the ethanol was removed by rotary evaporation under vacuum, yielding 43.8 g of a highly viscous, dark brown resin with a characteristic ginger aroma. The extraction yield was 5.6%. 10 g of this resin (Extract A) was removed and stored refrigerated under a nitrogen flush for future bioassays.

[0311] Extraction B: Ginger was frozen and crushed as described above. The crushed material (785.9 g) was placed in a bucket with food-grade ethanol in a weight ratio of approximately 5:1. The ginger was soaked in ethanol at room temperature (22–29 °C) for 7 days. Samples were collected 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, yielding 49.3 g of a highly viscous, dark brown resin with a characteristic ginger aroma, very similar to that obtained in Extraction A (Figure 1). The extraction yield was 6.3%.

[0312] Zingerone and aldehyde analysis: Zingerone quantification was performed by HPLC on the starting material (i.e., processed ginger) and the final two resins, as well as on samples from Extraction B on days 1, 3, and 7 (note that the liquid sample from day 7 corresponds to the final resin sample). For the HPLC quantification method, methanol was added and the samples were ground prior to analysis. The zingerone content of all fractions is shown in Table 6. The mass balance and yield of zingerone are shown in Table 7. [Table 7] [Table 8]

[0313] As noted above, Extract A was obtained by treatment at 40° C. for 20 hours, and Extract B was obtained by treatment at room temperature for 7 days. The results in Tables 6 and 7 show that although increasing the temperature to 40° C. also resulted in high yields, longer treatment at room temperature resulted in higher levels of zingerone.

[0314] To determine the zingerone content in the starting material, it was first extracted with a suitable solvent. In one process, extraction with ethanol resulted in a low zingerone content (1.44 mg / g). In a second process, extraction with methanol was performed, and the ginger, along with the solvent, was ground in a mortar and pestle, resulting in a high zingerone content (3.1 mg / g). For reference, the zingerone content of this material reported by a Samoan laboratory was 1.86 mg / g.

[0315] On this basis, the zingerone yield (i.e., the amount of zingerone in the extract relative to the amount in the feed) was estimated to be 75% for Extract A and 89% for Extract B. The 6-gingerol peak observed in HPLC analysis to determine the zingerone content was consistently observed at approximately 1 / 8 the area of ​​the zingerone peak, suggesting that the extraction had little effect on the zingerone to gingerol ratio.

[0316] Samples of both final resins were taken, placed in ethanol, and tested by GCMS for aldehyde analysis. Very low levels of hexanal were observed (too low for quantitation). Hexanal is a by-product of the reaction that forms zingerone, but is somewhat volatile. The identity of the hexanal peak was confirmed by library matching of the MS data and the preparation of a hexanal standard.

[0317] Consider Each of the tests described herein was effective in producing zingerone. A comparison of the results of Examples 2 and 3 is shown in Table 8. [Table 9]

[0318] Similar values ​​were obtained for fresh and dried ginger 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, goes much further than the standard retroaldol reaction. Example 3, as directly described above, provides further advances, utilizing temperature and pH adjustments and extraction. Further advances are provided in Examples 5, 6, and 12, as described herein.

[0319] It has been noted that the Samoan ginger was not harvested at the required time (9 months in the soil), which affected the level of gingerols present in the ginger and, therefore, the zingerone content in the final product. Therefore, further benefits are expected. The Fijian ginger had a significantly higher zingerone content than the Samoan ginger (10.2:1.44 = 7.08-fold). This means that, when applying the experimental conditions of Example 3, the total yield of Fijian ginger can be estimated to be 310 mg / g. That is, 43.8 mg / g (amount obtained from Samoan ginger in Example 3) × 7.08 (higher starting content in Fijian ginger) = 310 mg / g.

[0320] Table 5 in Example 2 shows the results of pH-treated ginger and CO2 extraction. 322 grams of fresh ginger was found to provide 153 mg / g zingerone. In comparison, Example 3 utilized 785.9 grams (2.4 times the amount of product used in Example 2), providing 43.8 mg / g zingerone. However, this lower yield can be explained by the lower level of Samoan starting material (1.44 mg / g zingerone).

[0321] Example 4: Processing method using juicing and alkaline treatment Summary: Ginger root was mechanically sauerkraut, and the sauerkraut and residual solids were assayed for 6-gingerol levels. The majority of 6-gingerol was present in the sauerkraut. Treatment of the sauerkraut with alkali demonstrated that virtually all of the 6-gingerol was converted to zingerone at 60°C for 5-6 hours.

[0322] Juice extraction summary: Fresh ginger (500-1000 g) was pre-processed by blending / steeping and pressing. The liquid fraction was retained, and the pomace was washed in warm water (1 part ginger to 4 parts water) at a temperature of 55-60°C for 10-15 minutes. This was done in a covered container. It was then pressed again. Each fraction was analyzed for 6-gingerol content (a total of five analyses): 1) a fresh ginger sample immediately before processing; 2) the liquid fraction after the first blending / steeping; 3) the ginger pomace after the first blending / steeping; 4) the second liquid fraction collected after further washing of the first pomace; and 5) the final ginger pomace after washing and pressing as above. Moisture content readings were taken on the ginger pomace from each lot after the final pressing (samples 3 and 5 above).

[0323] Alkali treatment summary: The liquid fraction was subjected to an alkali treatment to establish the conversion to zingerone. Samples were taken at various time points and analyzed for zingerone content. Samples were subjected to small-scale treatment with KOH (conventional 5%). Treatments were performed at room temperature, 30°C, and 60°C for 1 hour, 2 hours, 3 hours, and 5 hours using 1 ml samples. In addition, one sample was treated at 60°C for 24 hours. Up to 15 samples were analyzed.

[0324] Juice Extraction Method: Two samples of fresh ginger root were obtained: one locally (organic from Ebisu, Pitone) and the other received from Phil Rasmussen in Auckland. Both samples appeared plumper and juicier than regular supermarket ginger root. Moisture content was measured by slicing approximately 10 g of each root, freezing it in liquid air, and then freeze-drying it. The roots were extracted with methanol and the 6-gingerol content was measured. For this, approximately 5 g of each sample was cut into 4-5 pieces and crushed using a small kitchen garlic press. The crushed roots and juice were extracted with methanol (2 x 15 ml) at 60 °C for 20 minutes. HPLC analysis with detection at 280 nm was then performed. The results are shown in Table 9 below. [Table 10]

[0325] A sample from Oakland was selected for juicing. To do this, 635 g of ginger root was processed using a domestic juice extractor. This included a rotating screw drive with a mesh juice filter and an adjustable solids nozzle (see Figures 4A-4B). The extractor removed 516.3 g (81%) of liquid (Juice 1, J1) and collected 101.5 g of solids (Pomace 1, M1). A portion of the dried solids was removed for analysis, and 90.5 g was extracted with 360 ml of hot tap water. After allowing this to stand for 15 minutes, the material was passed through the juice extractor. This process yielded 350 g of juice (Juice 2, J2) along with 70.2 g of solids (Pomace 1, M2).

[0326] The two liquids were refrigerated overnight. Both were cloudy due to the precipitation of solids. These samples were shaken before analysis or processing. The juice samples were analyzed for 6-gingerol content by mixing the samples with ethanol (1:1), centrifuging, and directly injecting the supernatant. The solid contents of the two pressed solids, M1 and M2, were 37% and 26.3%, respectively.

[0327] The results are shown in Table 10 below. The gingerol values ​​were multiplied by weight to obtain the total amount of gingerol in each material. The juice was found to contain 81.6% of the measured gingerols. The calculated total gingerols for the feed were lower than the recovered amount, suggesting partial extraction of gingerols from the roots. The gingerol percentages were based on the total measured gingerols for the pomace and juice (rather than measurements for the feed). [Table 11]

[0328] Alkaline treatment: This procedure was performed on the first juice (J1) recovered from the juice extraction process (described above). Juice samples (after shaking to suspend all solids) were reacted with KOH at RT, 30°C, or 60°C for 1, 2, 3, or 5 hours, and at 60°C for 24 hours. Three concentrations of KOH (0.5%, 1.0%, and 2.0%) were also tested. A 2N KOH solution was prepared (5.6 g KOH in 50 ml water). To produce 0.5%, 1%, and 2% KOH concentrations in each sample, 0.25 ml, 0.5 ml, or 1 ml of 2N KOH was added to 5.5 ml of juice and shaken. The samples were then placed at RT (laboratory), 30°C (water bath), or 60°C (drying oven). Sampling for HPLC analysis was performed by removing 200 μl from each sample and adding 200 μl of 1N HCl followed by 500 μl of ethanol. After centrifugation, the samples were directly injected into the HPLC. The peak areas of zingerone and gingerol were compared (see below).

[0329] These results demonstrated that treatment with 2% KOH could achieve complete conversion to zingerone within 5 hours (see Figure 5C). Incubation at 60°C was particularly successful (see Figure 5C). Results were presented as peak areas for zingerone (Z) and gingerol (G). It should be noted that the KOH-treated sample had solids that precipitated in the tube. For analysis, the tube was shaken, and samples were taken at the wide-bore tip to avoid clogging.

[0330] Example 5: Additional Processing Methods Using Juice and Alkaline Treatment Abstract: The objective is to produce an extract from ginger with 6-gingerol converted to zingerone via an alkali-catalyzed retro-aldol reaction. The current process aims to reduce processing time and water usage. The process was tested at approximately 40 kg scale before further production at 200 kg scale was undertaken.

[0331] Briefly, fresh ginger was received, treated with alkali, and then processed by freeze-drying to produce 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 described in detail in Example 6.

[0332] Methodology: Imported fresh ginger was subjected to alkaline pretreatment and drying. To this end, fresh ginger (36.67 kg) was pressed in a Vincent Corporation CP-4 screw press, producing two streams: ginger juice and pomace. The screw press settings were a VSD speed of 50% and a cone air pressure of 2 bar. The pomace from the first press was pressed a second time to remove any remaining juice.

[0333] 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 alkalized juice was kept at 60°C for 5 hours to convert gingerol to zingerone. The pH was neutralized to 7.2 by adding anhydrous citric acid. The juice containing zingerone was freeze-dried and pulverized.

[0334] Samples were taken: fresh ginger (ZINGO); two pomaces (GMARC and GMARC2); juice before KOH addition (GKOH0); juice after 2, 3 and 5 hours of treatment (GKOH2, GKOH3, GKOH5); and the final dry extract (GPE).

[0335] Additionally, a sample of GMARC2 was extracted with hot water as follows: Water was added to GMARC2 in a 5:1 w:w ratio. The mixture was heated to 60°C and held at this temperature for 15 minutes. The extract was separated from the solids using a screw press with the same settings as above. Samples were taken: extract (GMARC2 HWEX); pomace (HW MARC). Each sample was analyzed by HPLC for total solids (LOD, 16 hours at 100°C) and gingerol or zingerone content.

[0336] 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 in the presence of alkali were diluted 1:1:1 with 1N HCl and ethanol. (2) Solid samples, such as fresh ginger or ginger pomace, were double-extracted with ethanol (sonication, heating at 60°C for 20 minutes, vortexing, centrifugation), and the supernatants were combined. Solid extracts (approximately 5 g) were typically prepared to 50 mL for analysis. Raw fresh ginger was coarsely chopped and then blended with ethanol using an ULTRA-TURRAX® mixer. Quantitation was performed by comparison with a standard curve generated using zingerone. Molecular weight correction was performed for gingerol.

[0337] Pressing: 36.67 kg of Fijian fresh ginger was received and pressed. Pressing was effective, producing a large amount of pale green juice and a fibrous pomace. 28.92 kg of juice was recovered in the first press. 6.6 kg of pomace was then pressed a second time, recovering an additional 2.18 kg of juice. The total juice yield was 31.1 kg, representing 86% of the incoming fresh ginger mass. The final recovered pomace mass was 4.05 kg. There was typically a 1-2 kg holdup in the screw press at the end of the run. This resulted in a small difference between the feed mass and the combined mass of pomace and juice.

[0338] Hot water extraction: 3.62 kg of GMARC2 and 18.1 kg of water were heated to 60°C, then extracted at 60°C for 15 minutes, followed by separation in a screw press. 21.18 kg of the mixture was then pressed. It should be noted that approximately 500 g of water was lost as evaporation during the extraction. From this, 17.62 kg of extract was collected and subsampled for analysis. Additionally, 2.69 kg of pomace was collected.

[0339] Alkaline treatment: For this, 1.236 kg of 50% KOH solution was added to 31.1 kg of ginger juice to reach a target KOH concentration of 2%. The pH after the KOH addition was 12.18. Once the KOH was added, the color of the juice changed from light green to reddish brown. The juice was kept at 60°C for 5 hours and then neutralized by adding 500 g of anhydrous citric acid. The pH after the addition of citric acid was 7.23.

[0340] Freeze Drying: The processed juice was transferred to freeze dryer trays and frozen overnight before being transferred to a Cuddon FD 80 freeze dryer. A total of 28.51 kg of juice was loaded onto the trays and dried. Approximately 3 kg of juice was lost prior to freeze drying due to manual handling. After drying, 2.86 kg of dried extract (a total of 10% of the dried juice mass) was collected. This was milled and subsampled. After milling, subsampling and loss handling, a total of 2.19 kg was packaged in foil bags and stored for further processing. Approximately 1.6 kg of this was sent for ethanol extraction (see Example 6).

[0341] A summary of the mass balance is shown below. [Table 12] [Table 13] [Table 14]

[0342] Gingerol and zingerone levels were measured as described above, and the results are shown in Table 11 below. [Table 15]

[0343] This table shows the gingerol content of various fractions in the pretreatment and processing processes. From these measurements, there was a total of 19.1 g of 6-gingerol in the 36.67 kg raw ginger feed. The zingerone content of the dried ginger extract (GPE) was 5.7 mg / g. Therefore, the total zingerone in the 2.86 kg dry powder (before grinding and milling losses) was 16.30 g. The gingerol concentration in the juice before conversion (GKOH-0) was 6.9 mg / g on a dry basis. Converting to a wet basis using a solids concentration of 6.67% (before the total solids content was increased to approximately 10% by the addition of KOH and anhydrous citric acid), the total gingerols in the juice were 14.32 g. The GKOH-2, -3, and -5 values ​​for 6-gingerol were confirmed.

[0344] The mass balance between gingerols in the juice and zingerone in the final powder extract does not exactly match. This is likely due to measurement variability. The pomace count appears to be somewhat inflated when the total mass balance calculation is taken into account. When the pomace was extracted with hot water, the 6-gingerol content in the extract was 0.22 mg / g, corresponding to a total of 5.7 g of 6-gingerol in this extract, approximately 25% of the 6-gingerol in the feed. A total of 18 kg of water was required for the water extraction. This resulted in increased mass of KOH and citric acid, further increasing the dry load by 58%. Therefore, in certain situations, it may be desirable to omit the water extraction.

[0345] Notably, these experiments demonstrated that nearly complete recovery of 6-gingerol (in the form of zingerone) in the final product was achieved. Samples taken during the conversion reaction (GKOH2, GKOH3, and GKOH5) indicated that the conversion of 6-gingerol to zingerone occurred within the first 2 hours of treatment, with no significant increase in zingerone levels in samples taken after 2 hours of treatment. If the conversion were complete after 2 hours, this incubation time (or even shorter) would be sufficient.

[0346] It was concluded that the process involving KOH treatment of the juice phase after expression was a viable manufacturing method. This represents a significant advance over the standard alkali-catalyzed retro-aldol reaction. Further experiments utilized over 200 kg of ginger as starting material. The 6-gingerol level in this batch was 1.1 mg / g, which was expected to reflect the corresponding zingerone level.

[0347] Example 6: Ethanol Extraction Process and Analysis Summary: Fresh ginger was received, treated with alkali, and then freeze-dried to produce 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.

[0348] Methodology: The processed ginger prepared as described above (Example 5) was kept refrigerated until use. Approximately half of the received ginger was extracted with XNS food-grade ethanol at room temperature. The processed ginger was placed in a 10 L glass vessel equipped with an overhead stainless steel stirrer along with ethanol (using a 1:5 weight ratio of ginger to ethanol). The mixture was stirred for 72 hours at a speed sufficient to prevent solids from settling on the bottom.

[0349] After 24 hours, the agitator was stopped and the solids were allowed to settle for 10 minutes, after which a 50 mL sample was taken from the top. After another 24 hours, a second sample was taken using the same procedure. After a total of 72 hours, the agitator was stopped and the mixture was filtered under vacuum using filter paper. Samples were taken from the cake, and the final tincture sample was taken from the 72-hour filtrate. The remaining filtrate was labeled ZINGOEE. Approximately 300 mL of ZINGOEE sample was taken and placed in a glass bottle and stored refrigerated. In a further step, all of the ZINGOEE was evaporated, producing a total resin weight of 43.5 g at 88.9 mg / g zingerone.

[0350] 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 times was achieved. The resulting concentrated extract (ZINGOCE) was then analyzed, and once the zingerone content was confirmed, a small sample of this concentrated extract was used to produce a standard-grade tincture containing approximately 12 mg / g zingerone by dilution with food-grade ethanol. Two separate samples of this standardized tincture were sent to SCU (Australia) for analysis, and a third sample was kept on-site for zingerone analysis.

[0351] Results: The process and results are shown in Figure 7. In these experiments, 801.5 g of received processed ginger was used for ethanol extraction with 4007.3 g of food-grade ethanol. After 72 h at room temperature (16–20°C) under stirring, the mixture was filtered, yielding 3556.7 g of a clear, brown, aromatic ethanolic tincture [ZINGOEE] and 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 the resulting ethanolic tincture, including samples taken at 24 and 48 h, was 3631.7 g.

[0352] Zingerone quantification was performed by HPLC on the starting material (i.e., treated ginger, GPE) and samples after ethanol extraction for 24, 48, and 72 hours at room temperature. The spent ginger solid (i.e., the cake from the filtration process) was also analyzed. The zingerone content of all fractions is shown in Table 12. The HPLC results showed little difference among the three extraction times, indicating that 24 hours was sufficient for extraction. [Table 16]

[0353] The zingerone concentration in the filtrate [ZINGOEE] was 1.1 mg / g, i.e., 3.88 g of zingerone in the liquid, or 85% of the starting zingerone, indicating a reasonable recovery. The zingerone concentration in the cake was 0.61 mg / g, i.e., 0.63 g of zingerone in the cake. However, it should be noted that a certain amount of ethanol solution was still present in the cake. In future processing, the cake could be washed with clean ethanol to remove as much of the extractables as possible.

[0354] The zingerone mass balance for the ethanol extraction process is 98.5%. This is calculated by dividing 3.88g in the extract and 0.63g in the cake by 4.58g in the feed. Room temperature extraction was found to be advantageous for zingerone recovery. While increasing the extraction temperature may result in higher zingerone recovery, room temperature extraction is clearly effective.

[0355] After collecting approximately 300 mL of Zingoee sample, the remaining extract (3249 g) was evaporated under vacuum to yield 104.9 g of concentrated extract [Zingoee] with a zingerone content of 33 mg / g (3.46 g zingerone). The total solids content of this concentrated extract was 37.1% (measured as loss on drying at 110°C), indicating a final oleoresin weight of 38.9 g if all ethanol was removed. Extrapolating this figure to the total Zingoee produced, the extraction yield for this process is approximately 5.4%. A standardized tincture containing 12 mg / g zingerone was prepared by mixing 25.5 g of Zingoee with 44.5 g of food-grade ethanol. A sample of this tincture was sent for further testing.

[0356] Additionally, further analysis indicated the absence of aldehydes in the final product. See Figures 8A-8B. For these evaluations, samples of the processed ginger ethanol extract were analyzed using GCMS for the presence of aldehydes. If present, this is expected to be (primarily) hexanal derived from 6-gingerol. Powder preparations (e.g., pre-extraction powders, as in Examples 4 and 5 above) are also expected to be aldehyde-free. As a next step, the ethanolic tincture is evaporated to produce a thick ethanolic paste.

[0357] Conclusion: The proposed method, which involves screw pressing, KOH treatment at the juice stage, neutralization with citric acid, and then freeze-drying, proved highly effective, recovering almost all of the 6-gingerol at the juice stage and converting it entirely to zingerone after treatment with 2% KOH at 60°C for 5 hours. The dry powder before the process now contained 5.32 mg / g zingerone, which is at least twice as efficient as previous production techniques.

[0358] Ethanol extraction at room temperature for 24 hours is an optional step that achieves at least 85% recovery of zingerone. Recovery can be further enhanced by washing the solid with fresh ethanol after extraction. In these methods, the ethanol extract [ZINGOEE] was evaporated to significantly reduce the volume, and the concentrated extract [ZINGOCE] was then reconstituted with fresh ethanol to produce a standardized tincture containing a target dose of 12 mg / g zingerone.

[0359] The extraction process resulted in an overall mass balance (out / in) of 98.5%. The zingerone content (ZINGOEE) in the filtrate was 1.1 mg / g, and the zingerone content (ZINGOCE) in the concentrated extract was 33 mg / g. This method produced 104.9 g of ZINGOCE at 33 mg / g, leaving 3.46 g of zingerone. This figure is lower than the 3.88 g of ZINGOCE because 400 g of ZINGOCE was removed before evaporation (plus a retentate sample of approximately 300 mL for testing). Taking this into account, the calculation is a good fit. The overall zingerone mass balance before evaporation was calculated to be 98.5% (= (0.63 + 3.88) / 4.58).

[0360] The results of this study show that 100 kg of fresh ginger with a 6-gingerol content of 0.5 mg / g can produce 3.6 kg of standard tincture with a zingerone content of 12 mg / g, suggesting that higher levels of 6-gingerol will result in higher zingerone yields.

[0361] Example 7: Anti-inflammatory Activity of Zingerone Compositions Summary: These studies were conducted to determine the anti-inflammatory activity of plant extracts of the present disclosure (ethanol extracts; see Example 6) normalized for zingerone content. Nitric oxide (NO) and IL-6 levels were assessed. Methodology: Anti-inflammatory activity was assessed using lipopolysaccharide (LPS)-stimulated murine macrophages, RAW264.7 cells, cultured in standard cell culture medium (DMEM, 5% fetal bovine serum) and incubated in the presence or absence of different test compounds / extracts and controls. Production of inflammatory mediators, including NO and IL-6, was measured by established methods using commercially available ELISA kits (suppliers listed in Table 13). Each sample was tested at at least six concentrations (20 μg / mL to 0.6 μg / mL). This was performed in triplicate (maximum concentration 40 μM zingerone) (n = 9), along with relevant internal controls. Additionally, cytotoxicity of each tested sample was measured by MTT assay (tetrazolium dye MTT, chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Assay parameters for each assay are summarized in Table 13. [Table 17]

[0362] The tincture of Example 6 was used in these studies. For NO and IL-6 assays, cultured RAW264.7 cells were counted and seeded (0.8 × 10) in 96-well plates. 5 The plates were incubated for 48 hours (cells / well). The medium was then aspirated and replaced with fresh medium, followed by the addition of test compounds. These compounds were incubated for 1 hour before the addition of stimulants. The plates were then incubated for 18 hours, the supernatants were analyzed for the mediators of interest, and remaining cell viability was measured by MTT.

[0363] 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 Biol Chem 272(8):4959-63), and dexamethasone, a commonly used cytokine inhibitor. Data are presented as mean and standard error of the mean (s.e.m.) (n=9).

[0364] Dose-response curves were fitted using Graph Pad Prism. 95% confidence intervals (CI) were calculated using the entire data set, and IC 50 The estimated error of the IC value was given. 50 If IC is not reached 50 The 95% CI of IC 50 requires extrapolation of the graph to calculate IC 50 Where is estimated by graphical extrapolation, "about" is used to indicate that it is an estimate and not experimentally determined, unless otherwise stated. Graphical extrapolation resulted in a wider 95% confidence interval.

[0365] Results: The tinctures of Example 6 were assayed to measure anti-inflammatory activity. Anti-inflammatory activity was assessed using NO and IL-6, which are key mediators of inflammation. Cytotoxicity of each sample was also measured using the MTT assay.

[0366] Cytotoxicity: The effect of compounds on cell viability was measured by MTT. Potency was monitored to avoid false positives, as dead cells do not produce inflammatory mediators. Cytotoxicity was measured spectrophotometrically, as mitochondrial dehydrogenases present in viable cells cleave the tetrazolium ring of MTT to produce purple MTT formazan. All doses were tested up to 40 μM zingerone. The results are shown in Figure 9. The cytotoxicity assay showed that the EC 50 was determined to be 40 μM zingerone.

[0367] NO Assay: NO is a radical metabolite that has been shown to have many physiological functions as both a signaling molecule and a toxic substance in inflammation (Coleman, 2001). Inhibition of iNOS secreted by immune cells and reduction of NO levels may be contributing factors to anti-inflammatory activity. Therefore, the tincture of the present disclosure was assayed to determine whether it exhibited inhibition of the inflammatory signaling molecule NO. The dose-dependent effect of the tincture on NO is shown in Figure 10. From the NO assay, the IC of the tincture of the present disclosure was 50 was determined to be 9.2 μM zingerone.

[0368] IL-6 Assay: IL-6 is considered a pro-inflammatory cytokine. IL-6 is secreted by T cells and macrophages and stimulates immune responses. IL-6 is involved in increasing neutrophil production in the bone marrow. It supports B cell proliferation and antagonizes T cell differentiation into regulatory T cells. It crosses the blood-brain barrier and initiates the synthesis of PGE2 in the hypothalamus, thereby altering the body temperature set point (Banks, Kastin, & Gutierrez, 1994). Inhibition of IL-6 release by immune cells indicates anti-inflammatory activity. The dose-dependent effect of the tincture of the present disclosure on IL-6 is shown in Figure 11. From the IL-6 assay, the IC50 of the tincture was determined to be 4.6 μM zingerone.

[0369] 10, 11, and 12 confirm that the tincture of the present disclosure inhibited NO and IL-6 in a dose-dependent manner. The effective levels of inhibition of NO and IL-6 were significantly lower than those required for cytotoxicity, supporting the observed anti-inflammatory effects of the tincture.

[0370] Example 8: Comparative study of the anti-inflammatory properties of zingerone compositions Summary: These studies were conducted to determine the anti-inflammatory bioactivity of a plant extract of the present disclosure (ethanol extract; see Example 6) compared to commercially available zingerone in RAW264.7 macrophages.

[0371] Methodology: An extract (ethanol extract (tincture); Rx7 / 22 / 161) was prepared as described in Example 6. The extract was standardized at 33 mg of zingerone per mL of extract. Commercially available zingerone powder (Vigon #500938) was obtained and dissolved fresh in ethanol to 33 mg / mL on the day of use. Samples in ethanol were further diluted in cell culture medium immediately before addition to cell culture. An ethanol solvent control was included in cell culture experiments to rule out possible solvent effects.

[0372] RAW264.7 cells in growth medium (DMEM, 10% FBS, PSN, 2 mM L-glutamine) were plated at 0.8 × 10 in 96-well tissue culture-treated plates. 5 Cells were plated per well and incubated for 48 hours at 37°C / 5% CO2 in a humidified environment. Spent medium was then aspirated and replaced with medium containing 5% FBS. Treatments were added to a final concentration of 0.625-40 μM zingerone (or equivalent dilutions for ethanol vehicle control). Cells were incubated with treatments for 1 hour before adding 50 ng / mL lipopolysaccharide (LPS; from E. coli O111:B4).

[0373] After 18 hours of incubation of cells with treatments and LPS, the conditioned medium was collected, centrifuged, and the cell-free supernatant was collected. The viability of the remaining cells was measured using the WST-1 assay. Controls included unstimulated (cells without LPS), LPS (cells exposed to 50 ng / mL LPS), Dex (cells exposed to 1 or 10 μg / mL dexamethasone before the addition of 50 ng / mL LPS), and medium (medium without cells or sample).

[0374] For the cytotoxicity assay, an equal volume of a 1:5 mixture of WST-1 and medium was added to each cell well and incubated for 10 minutes. Immediately before measuring absorbance, an equal volume of DPBS was added, and absorbance was read at 440 nm (and 620 nm for background subtraction). Hydrogen peroxide was included as a positive cytotoxicity control. Results were normalized to LPS-stimulated controls and expressed as percent cell viability. Sample concentrations that resulted in cell viability less than 80% of the unstimulated control were considered cytotoxic.

[0375] For the IL-6 assay, interleukin 6 (IL-6) was analyzed in cell-free conditioned medium collected after treatment and LPS stimulation using a bead-based multiplex assay panel (Legendplex MU Th1 / Th2 8-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 to measure IL-10 and TNF were performed.

[0376] Statistical analysis was performed using Minitab 18.0. Student's paired t-test was performed for statistical differences between treatments at each time point. 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.

[0377] Results: Cell viability after incubation with compounds / extracts was measured. In this assay, WST-1 reacts with mitochondrial enzymes to form a colored pigment that can be measured by absorbance. Thus, WST-1 is a measure of cellular metabolism, and a decrease in WST-1 reaction rate can indicate cell death. Hydrogen peroxide (H2O2) was used as a positive control to induce cell death and ensure the validity of the assay.

[0378] Cytotoxicity results are shown in Figure 13. This figure shows cell viability levels after 18 hours of treatment with 50 ng / mL lipopolysaccharide (LPS), LPS plus the disclosed plant extract, zingerone (0.6-40 μM), and the corresponding ethanol control. Data are the mean ± standard error of three independent experiments (n=3). Assay validation was performed using hydrogen peroxide (HO).

[0379] The left panel shows a clear cytotoxic dose-dependent response in cell samples exposed to H2O2, confirming that the assay was functioning as expected. WST-1 values ​​less than 80% of the untreated control (horizontal dashed line) were considered cytotoxic. Cytotoxicity was assessed in RAW264.7 cells after 18 hours of exposure to the plant extracts disclosed herein or commercially available 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 the ethanol extract or commercially available zingerone at the concentrations tested (Figure 13). That is, no data points were below 80% of the WST-1 response of the untreated LPS-stimulated control. This validated the cytokine assay results (see results below).

[0380] The cellular model used to assess IL-6 expression was a proinflammatory response by a murine macrophage cell line (RAW264.7) upon exposure to bacterial endotoxin (lipopolysaccharide; LPS). Mechanistically, LPS interacts with the membrane-bound TLR4 receptor, triggering an intracellular signaling cascade that activates NF-κB, a transcription factor that regulates the expression of many proinflammatory cytokines, including IL-6.

[0381] The results of IL-6 measurements are shown in Figure 14. This figure shows the IL-6 levels produced by RAW264.7 cells after 18 hours of treatment with lipopolysaccharide (LPS), LPS plus 10 μg / mL dexamethasone (Dex), and LPS plus the disclosed plant extract or commercially available zingerone (0.6-40 μM). Data are means ± SEM from three independent experiments (n=3). Asterisks indicate that treatment with the corresponding concentration of zingerone significantly reduced IL-6 production (p<0.05). Treatments without error bars at the 20,000 pg / mL level indicate values ​​above 20,000 pg / mL.

[0382] Exposure of RAW264.7 cells to LPS for 18 hours was found to significantly induce IL-6 secretion up to concentrations exceeding the upper limit of assay precision of 20,000 pg / mL (Figure 2). IL-6 secretion by RAW264.7 cells after co-treatment with the plant extract of the present disclosure and equivalent ethanol concentrations present in the evaluated commercial zingerone dilutions also exceeded the upper limit of assay precision. Co-treatment of RAW264.7 cells with LPS plus the plant extract of the present disclosure or LPS plus commercial zingerone at concentrations ranging from 0.6 to 10 μM similarly exceeded the upper limit of assay precision, making it difficult to assess the anti-inflammatory bioactivity of these samples at these concentrations.

[0383] In particular, co-treatment of RAW 264.7 macrophages with LPS and the plant extract of the present disclosure or commercially available zingerone at the highest concentrations evaluated (20 and 40 μM zingerone) resulted in a measurable reduction in IL-6 that was within the limits of assay precision. In particular, co-treatment of RAW 264.7 with the plant extract of the present disclosure and 20 and 40 μM zingerone resulted in significantly (p<0.05) lower IL-6 production compared to co-treatment with commercially available zingerone. These findings indicate that the plant extract of the present disclosure is more effective than commercially available zingerone in reducing IL-6 production at these concentrations.

[0384] A specific reduction in IL-10 was also observed. Figure 15 shows the IL-10 levels produced by RAW264.7 cells after 18 hours of treatment with lipopolysaccharide (LPS), LPS + 10 μg / mL dexamethasone (Dex), and LPS + disclosed plant extract or LPS + commercial zingerone (1.25–40 μM). Data are means ± SEM from three independent experiments (n=3). Carrots show significantly lower IL-10 production compared to the corresponding ethanol control (p<0.05). Asterisks indicate a significant reduction in IL-10 production by zingerone at the corresponding concentration (p<0.05). The treatment without an error bar at 15,000 pg / mL shows an IL-10 value of 21,5000 pg / mL, which is beyond the quantitation limit of the assay.

[0385] The results show that 18 hours of exposure of RAW264.7 macrophages to LPS induced a significant increase in IL-10, which was attenuated by co-treatment with dexamethasone, a known immunosuppressant. Co-treatment of cells with 10 and 20 μM zingerone equivalents of the plant extract of the present disclosure, but not commercially available zingerone, significantly attenuated LPS-induced IL-10 secretion by RAW264.7 (p<0.05). Furthermore, IL-10 secretion by RAW264.7 co-treated with the plant extract of the present disclosure at 20 and 40 μM zingerone was significantly lower than that of commercially available zingerone at these concentrations (p<0.05).

[0386] The TNF results are shown in Figure 16. This figure shows the TNF levels produced by RAW264.7 cells after 18 hours of treatment with lipopolysaccharide (LPS), LPS + 10 μg / mL dexamethasone (Dex), and LPS + disclosed plant extract or LPS + commercial zingerone (1.25–40 μM). Data are means ± SEM from three independent experiments (n=3). Asterisks indicate a significant reduction in IL-6 production by zingerone at the corresponding concentration (p<0.05). The treatment without an error bar at 15,000 pg / mL indicates a value of 21,5000 pg / mL, which is beyond the precision limit of the assay.

[0387] The results show that exposure of RAW264.7 macrophages to LPS for 18 hours induced a significant increase in TNF, which was attenuated by co-treatment with dexamethasone, a known immunosuppressant. Co-treatment of cells with the plant extracts or pure zingerone compounds of the present disclosure did not show a significant effect in attenuating LPS-induced TNF secretion (p>0.05).

[0388] Overall, the anti-inflammatory activity of the plant extracts of the present disclosure (including naturally occurring zingerone) is significantly and significantly superior to that of commercially available zingerone, particularly with respect to IL-6 and IL-10 levels. Future assays will be performed at lower levels of inflammation induction overall to allow for more accurate comparisons.

[0389] A review of these studies revealed that the researchers who conducted the experiments incorrectly reported the concentration of zingerone in the plant extracts (tinctures) of the present disclosure. These studies listed a concentration of 33 mg / mL (0.170 M), but the actual concentration of zingerone in the tincture was 33 mg / g (0.134 M). Therefore, the concentrations of zingerone in the tincture dilutions were incorrectly recorded as 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM. Based on the correct initial concentration of 33 mg / g, the correct concentrations of zingerone in each dilution are 0.493 μM, 0.986 μM, 1.97 μM, 3.95 μM, 7.89 μM, 15.8 μM, and 31.6 μM, respectively.

[0390] This means that the results shown for IL-6, IL-10, and TNF, respectively, are all underestimates of the activity of the plant extract of the present disclosure, because commercially available zingerone was used at significantly higher concentrations (0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM) than the plant extract of the present disclosure (0.493 μM, 0.986 μM, 1.97 μM, 3.95 μM, 7.89 μM, 15.8 μM, and 31.6 μM). In any event, these initial results are very positive, and the study is currently being repeated using correct calculations. [Table 18]

[0391] An MTT assay was used to identify the cytotoxic effects of compound treatment on RAW264.7 cells. No cytotoxic effects were observed for any of the compounds tested. There was a significant decrease in MTT uptake in Method 1 conditions compared to both the unstimulated Method 2 conditions and the stimulated and unstimulated Method 2 conditions (Figure 18A: stimulated Method 1; Figure 18B: stimulated Method 2). This decrease is expected because the mechanism of IFN-γ involves arresting cell proliferation, resulting in fewer cells present compared to the (-)IFN-γ condition. Notably, fewer cells mean fewer mitochondria metabolizing MTT to formazan, resulting in lower uptake.

[0392] Conclusion: 100 nM dexamethasone was shown to be an effective control due to its IL-6 inhibitory activity. For the remaining experiments, we chose to use Method 2 as this method was compatible with other data obtained.

[0393] Example 9: Comparison of methods to assess anti-inflammatory activity and viability Summary: These studies were conducted to: (1) identify the most effective positive control agent; and (2) compare two different assays for assessing inflammatory activity. The following results were obtained: viability measured by MTT; and IL-6 production measured by ELISA.

[0394] The evaluation method is summarized as follows: [Table 19] [Table 20]

[0395] Methodology: Dexamethasone and synthetic zingerone were obtained from Sigma-Aldrich. In these studies, dexamethasone (100 nM) was used as a positive control; absolute ethanol was used as a solvent control; and synthetic zingerone (20 μM) was used as a test treatment. Tests included: (1) IL-6 ELISA—measures IL-6 production from stimulated and unstimulated RAW264.7 cells; (2) MTT assay—measures the metabolism of MTT to formazan to identify potential cytotoxicity.

[0396] On day 0, the flasks for the Method 2 condition were removed from the 37°C / 5% CO2 incubator. The growth medium was decanted. 10 mL of CTCM was then added to wash the cells. The growth medium was decanted again. 10 mL of CTCM was then added and the cells were collected using a rubber scraper. The resulting cell suspension was transferred to a 50 mL Falcon tube, which was centrifuged at 400 x g for 5 minutes. The cells were counted after applying a 1:10 trypan blue dilution. The sample was then diluted to 1 x 10 6 The medium was diluted to 1000 cells / mL. Cells were then plated or added at 80 μL / well (80,000 cells / well). For stimulated Method 2 samples, 120 μL of medium was added. This step was repeated for unstimulated Method 2 samples. Cells were returned to a 37°C / 5% CO2 incubator and left for 48 hours.

[0397] Drug dilutions were prepared on day 1 (see below). Flasks dedicated to the Method 1 condition were harvested from the 37°C / 5% CO2 incubator. The growth medium was decanted. 10 mL of CTCM was then added and the cells were washed. The growth medium was decanted again. 10 mL of CTCM was then added and the cells were harvested using a rubber scraper. The resulting cell suspension was transferred to a 50 mL Falcon tube, which was centrifuged at 400 x g for 5 minutes. Cells were counted after applying a 1:10 trypan blue dilution. Samples were then diluted to 1 x 10 6The cells were diluted to 1000 cells / mL. Cells were seeded at 50,000 / well (50 μL). For stimulated Method 1 samples, 50 μL of IFN-γ was added (final concentration 20 U / mL). For unstimulated samples, cells were seeded and the well volume was brought up to 200 μL.

[0398] On day 2, LPS and treatment conditions were prepared for Method 2 samples. For stimulated Method 2 samples, 50 μL of LPS was added (final concentration 20 ng / mL) and 50 μL of treatment was added. For unstimulated Method 2 samples, these were centrifuged and 50 μL of CTCM was removed from the treatment wells. To these wells, 50 μL of treatment was added. For stimulated Method 1 samples, these were centrifuged and 100 μL of media was removed from the wells. To these wells, 50 μL of LPS (final concentration 50 ng / mL) was added and 50 μL of treatment was 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 LPS was added (final concentration 50 ng / mL) and 50 μL of treatment was 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. IL-6 ELISA capture setup was initiated.

[0399] On the third day, the ELISA plate was washed and blocking began. The supernatant of each sample was collected (170 μL). 50 μL of pre-warmed CTCM was then added to the wells. 20 μL of 5 mg / mL MTT solution was then added. The plate was returned to a 37°C / 5% CO2 incubator for 45 minutes. While the MTT was incubating, the ELISA capture was washed away and blocking solution was added. After 45 minutes, 10 μL of MTT solubilizer was added. The ELISA process continued. Cell counts were performed in 4 fields. The cell count per field was averaged as follows: (48 + 58 + 67 + 62) / 4 = 58.75 cells in 10 μL. This was then calculated as 58.75 * 10 * 10 4 = 5,875,000 cells / mL. This was calculated as 5,875,000 x 10 = 58,750,000 cells in 10 mL. Therefore, 1 x 10 6 To make a cells / mL solution, up to 58.75 mL of cell suspension was prepared.

[0400] Preparation of dilutions: (1) The IFN-γ stock solution was 5,000,000 / mL. This was diluted to 24,000 U / mL in CTCM; 2) The LPS stock solution was 1,000,000 ng / mL. For Method 1, an 80 ng / mL solution was required (final concentration in the well: 20 ng / mL). For Method 2, a 200 ng / mL solution was required (final concentration in the well: 50 ng / mL). (3) The synthetic zingerone stock solution was prepared by adding 2 μL of zingerone to 198 μL of CTCM. This resulted in a 1544.6401 μM stock. From this stock, 25.89 μL was added to 474.11 μL of CTCM. This resulted in an 80 μM stock. The 80 μM stock (50 μL) was added to the sample (along with LPS) to achieve a final concentration of 20 μM (0.013% ethanol).

[0401] Preparation of drug dilutions: (1) A dexamethasone stock solution was prepared by adding 2 μL of dexamethasone to 998 μL of CTCM. This resulted in a 5096 nM stock solution. From the 5096 nM stock solution, 78.49 μL was added to 921.51 μL of CTCM. This resulted in a 400 nM stock solution. 50 μL of the 400 nM stock solution was added to the sample (along with LPS) to obtain a final concentration of 100 nM (0.003% ethanol). (2) An ibuprofen stock solution was prepared by adding 2 μL of ibuprofen to 579.704 μL of CTCM. This resulted in a 1000 μM ibuprofen stock solution. From the 1000 μM ibuprofen stock solution, 400 μL was added to 600 μL of CTCM to make a 400 μM stock solution. A 400 μM stock solution (50 μL) was added to the sample (with LPS; 0.03% ethanol). (3) A risperidone stock solution was prepared by adding 2 μL of risperidone to 248 μL of CTCM. This resulted in a 584.67 μM stock solution. From the 584.67 μM stock solution, 342.1 μL was added to 157.9 μL of CTCM. This resulted in a 400 μM stock solution. A 400 μM stock solution (50 μL) was added to the sample (with LPS) to achieve a final concentration of 100 μM (0.14% ethanol).

[0402] IL-6 ELISA: 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. TMTMB Substrate Reagent Set; Cat. No. 555214. When standards were included, two complete ELISA plates (96 wells each) were used. A total of 11 mL was prepared for the 50 μL / well samples, and a total of 22 mL was prepared for the 100 μL / well samples. Capture was incubated overnight at 4°C. For the capture antibody, the stock was 1000 μg / mL, with a desired final concentration of 1 μg / mL. The following calculation was made: 1000 μg / mL * Vi = 1 μg / mL * 11,000 μL. From this calculation, a Vi equivalent to 11 μL of rat anti-mouse IL-6 was added to 11 mL of pH 9.0 capture buffer. This was delivered to the plate at 50 μL / well. Four washes were performed after capture.

[0403] Blocking was performed by incubation at room temperature for 2 hours. To do this, 100 μL of 10% FCS was added to each well. This was calculated as 10% FCS = 5 mL of FCS in 50 mL of 1X PBS. After blocking, the plate was washed three times. The plate was incubated at room temperature for 2 hours. 10 μL of stimulated sample was diluted in 100 μL of 5% FCS. 5% FCS corresponds to 2.5 mL of FCS, which was diluted in 50 mL of 1X PBS. 50 μL of this was introduced into the plate per well. The standard substance was prepared as follows: 16 μL was added to 784 μL of 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 0 pg / mL sample was also included. Serial dilutions were made in duplicate and washed four times after standard preparation.

[0404] The first detection step was incubated at room temperature for 1 hour. The stock was 500 μg / mL, and the desired final concentration was 0.5 μg / mL. The following calculation was made: 500 μg / mL * Vi = 0.5 μg / mL * 11,000 μL. From this calculation, Vi corresponding to 11 μL of biotin rat anti-mouse IL-6 was added to 11 mL of 5% FCS / PBS. This was introduced to the plate at 10 μL / well. This first detection step was followed by six washes. Incubation with streptavidin-horseradish peroxidase (SA-HRP) was carried out for 1 hour at room temperature. For this, a 1:2000 dilution of SA-HRP was prepared. The calculation was made as follows: 11,000 / 2000 = 5.5 μL. From this calculation, 5.5 μL of SA-HRP was added to 11 mL of 5% FCS / PBS. This was introduced to the plate at 50 μL / well. After the SA-HRP step, eight washes were performed.

[0405] The final detection step was incubation with TMB (3,3',5,5'-tetramethylbenzidine). TMB was prepared by adding 11 mL of TMB-A and 11 mL of TMB-B to separate 15 mL Falcon tubes covered with tin foil. After washing after the SA-HRP incubation, the TMB-A and TMB-B samples were combined and added to the plate at 100 μL / well. Color development was then stopped by adding 100 μL of hydrogen peroxide to the wells. The plate was read on a plate reader.

[0406] Results: Interleukin 6 (IL-6) was produced in response to LPS in both experimental settings, with higher levels produced using the Method 2 setting (Figure 17A: Method 1 stimulation; Figure 17B: Method 2 stimulation). The unstimulated condition in both settings was not significantly changed by either treatment. Dexamethasone (100 nM) was the only control compound capable of inhibiting IL-6 in either setting. In the Method 1 setting, dexamethasone had a greater inhibition of IL-6 (29.32% of stimulated / untreated condition and 33.42% of vehicle) compared to the Method 2 setting (64.03% of stimulated / untreated condition and 63.62% of vehicle). A summary of IL-6 levels is shown in Table 14 below.

[0407] Example 10: Anti-inflammatory activity and cell viability analysis in comparative studies Summary: The methodology described in the previous example was used to evaluate various markers, including: (1) Viability: an indicator for identifying cytotoxic concentrations, measured by MTT assay; (2) IL-6: a pro-inflammatory cytokine, measured by ELISA; (3) TNF: a pro-inflammatory cytokine, measured by ELISA; and (6) Nitric oxide: a pro-inflammatory small molecule, measured by Griess assay.

[0408] To facilitate data visualization and, where necessary, nonlinear regression, certain data are presented as the logarithm of the tested concentration. 10 The values ​​are shown below. [Table 21]

[0409] Methodology: Anti-inflammatory activity and viability were assessed as follows.

[0410] On day 0, dedicated T75 flasks were harvested from the incubator and replicated. In a laminar flow cabinet, the flasks were gently tapped to detach any non-adherent cells. The growth medium was decanted and replaced with 10 mL of CTCM. The flasks were gently rocked to wash the cells. The wash medium was decanted and replaced with 10 mL of CTCM. A rubber scraper was used to detach the cells. 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 of CTCM. The cells were counted after applying a 1:10 trypan blue dilution. The cells were collected at 1 x 10 6 The plate was diluted to 100 cells / mL. Cells were seeded at 80 μL / well. To this, 120 μL was added to bring the volume to 200 μL. The plate was returned to the incubator. Cells continued to be seeded as indicated. In this way, plates were assigned to stimulated and unstimulated samples. The plate was left for 48 hours before treatment.

[0411] First round count (stimulated / unstimulated): average cell count in 4 fields = 54 -> x 10x10 4 This yielded 5,400,000 cells in 1 mL, which corresponds to 54,000,000 cells in 10 mL. For dilution, 44 mL of CTCM was added to achieve 1 million cells / mL.

[0412] Second round count (stimulation): average cell count in 4 fields = 15.7 -> x 10 x 10 4 This yielded 1,570,000 cells in 1 mL, which corresponds to 15,700,000 cells in 10 mL. For dilution, 5.70 mL of CTCM was added to achieve 1 million cells / mL.

[0413] Second round counting (unstimulated): average cell count in 4 fields = 25.75 -> x 10 x 10 4 This yielded 2,575,000 cells in 1 mL, which corresponds to 25,750,000 cells in 10 mL. For dilution, 15.75 mL of CTCM was added to achieve 1 million cells / mL.

[0414] Third round count (stimulation): average cell count of 4 fields = 25 -> x 10 x 10 4 This yielded 2,500,000 cells in 1 mL, which corresponds to 25,000,000 cells in 10 mL. For dilution, 15 mL of CTCM was added to achieve 1 million cells / mL.

[0415] Third round count (unstimulated): average cell count in 4 fields = 32 -> x 10 x 10 4 This yielded 3,200,000 cells in 1 mL, which corresponds to 32,000,000 cells in 10 mL. For dilution, 22 mL of CTCM was added to achieve 1 million cells / mL.

[0416] On day 1, treatments were prepared. Synthetic zingerone, acetyl zingerone, and ferulic acid were utilized in a 360,416.0231 μM stock solution. Acetyl zingerone was obtained from Sytheon Ltd. Synthetic zingerone and ferulic acid were obtained from Sigma-Aldrich. To generate the required doses, 1199.4 μL of CTCM was added to a 2 μL aliquot of each stock solution. This resulted in a 600 μM test solution for each compound. When added to cells, concentrations ranged from 150 μM to 50 μL in 200 μL. Plant extracts of the present disclosure were utilized in a 134,000 μM stock solution. To generate the required doses, 6 μL of extract was added with 1334 μL of CTCM. This resulted in a 600 μM test solution. See the summary table below. [Table 22] [Table 23]

[0417] Stimulation and treatment began on day 2. The plates were removed from the incubator and centrifuged at 400xg for 5 minutes. The medium (150μl) was removed, and 50μl of CTCM was added. 50μl of each treatment test solution was added to the appropriate wells. The plates were returned to the incubator and incubated for 1 hour. An LPS solution was prepared. To do this, a 2μL aliquot (1,000,000ng / mL) was added to 9,998μL of CTCM to create a 200ng / mL solution (final concentration 50ng / mL). 50μL of LPS solution was added per well. For unstimulated conditions, 50μL of treatment was added per well, followed by 50μL of CTCM. The plates were returned to the incubator and incubated for 18 hours. All ELISA plates were coated. MTT reagent was prepared by mixing 50mg of MTT in 10mL of 1X PBS. This produced a 5 mg / mL reagent solution for use on six plates.

[0418] On day 3, supernatant collection, MTT assay, and ELISA were performed. For supernatant collection, the plate was centrifuged at 400 x g for 5 minutes. A multichannel pipette set to 170 μL was used to remove the supernatant. The supernatant was transferred to a labeled UB-96WP. For the MTT assay, CTCM was preheated in a 37°C water bath. After supernatant collection, 50 μL of warmed CTCM was added to the cells. Next, 20 μL of MTT reagent solution was added to the cells. The plate was returned to the incubator. After MTT development, 100 μL of MTT solubilization solution was added and incubated overnight.

[0419] ELISA reagents: BD Biosciences. Capture: Purified NA / LE rat anti-mouse IL-6 antibody, 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. TMTMB Substrate Reagent Set; Cat. No. 555214. Contains solutions: (1) ELISA Capture Buffer pH 6: 14.196 Na2HPO4 in 1 L mq HO. pH adjusted with HCl. (2) ELISA Capture Buffer pH 9: 14.196 Na2HPO4 in 1 L mq HO. pH adjusted with NaOH. (3) ELISA Wash Solution: 1 mL Tween® 20, 200 mL 10X PBS, 1800 mL mq HO. (4) 5% FCS in PBS: Dissolve 2.5 mL FCS in 47.5 mL 1X PBS. (5) 10% FCS in PBS: Dissolve 5 mL FCS in 45 mL 1X PBS. (6) 0.18 M H2SO4: Dissolve 9.78 mL of concentrated M H2SO4 in 1 L mL HO.

[0420] ELISA assay: ELISA plates were coated with 50 μL / well of capture antibody suspended in ELISA capture buffer. The pH was adjusted based on the cytokine being tested (see summary table below). The coated ELISA plates were incubated overnight at 4°C. The next day, the plates were removed from the refrigerator. The plates were washed four times with ELISA wash buffer. Next, 100 μL / well of blocking solution was added to the plates. The plates were incubated at room temperature for 2 hours. The plates were washed three times. Supernatants were diluted according to the cytokine being tested. Test samples were added at 50 μL / well along with a standard curve of the cytokine being quantified (see summary table below). The plates were incubated at room temperature for 2 hours or overnight at 4°C. For sequential ELISAs, the supernatant was transferred to a separate plate before washing. Washing was performed four times.

[0421] For detection, biotinylated detection antibodies were suspended in a solution of FCS and PBS and added to the plate at 50 μL / well. The plate was incubated at room temperature for 1 hour. The plate was washed six times. Streptavidin-horseradish peroxidase was suspended in a solution of FCS and PBS and added to the plate at 50 μL / well. The plate was incubated at room temperature in the dark for 1 hour. The required amounts of TMB-A and TMB-B were placed in separate Falcon tubes. These were stored at room temperature in the dark during the SA-HRP incubation. The plate was washed eight times. TMB solution was added to the plate at 100 μL / well. Color development was allowed. To stop the reaction, 100 μL / well of 0.18 M H2SO4 solution was added. See the table below. The plate was read at 450 nm using a PerkinElmer EnSpire® plate reader. [Table 24] [Table 25] [Table 26] [Table 27]

[0422] MTT Assay: The day before the assay, MTT powder was dissolved in 1X dPBS to a concentration of 5 mg / mL. 20 μL per well was required for each plate. For a 96-well plate, 1920 μL of MTT solution was required. The concentration was 5 mg MTT per mL of solution. This corresponds to 10 mg MTT per 96-well plate. On the day of the assay, CTCM was warmed in a 37°C water bath. While the medium was warming, cell cultures (RAW264.7) were centrifuged at 400 x g for 5 minutes. The supernatant was removed using a multichannel pipette set to 180 μL and transferred to a labeled U-bottom 96-well plate. The supernatant was stored at -20°C until analysis. 60 μL of warmed CTCM was added to the remaining cells to bring the volume to 80 μL per well. 20 μL of MTT solution was then added. The plate was returned to a 37°C / 5% CO2 incubator and incubated for 45 minutes. After 45 minutes, 50 μL of MTT solubilization solution was added. This solution contained either 10% SDS (w / v), 0.01 M HCl (adjusted to pH 4.0 with sodium hydroxide), or 10% SDS (w / v), 45% DMF (adjusted to pH 4.0 with acetic acid). The plate was covered with aluminum foil and left overnight. After overnight incubation, readings were taken at 580 nm using a PerkinElmer EnSpire® plate reader.

[0423] NO assay: A Griess reaction protocol was used. NO production was measured via NaNO2 production in a 96-well format. Reagents were as follows: (1) Greiss solution A (50 mL): 1% (w / v) sulfanilamide (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 of 85% phosphoric acid, 97.6 mL of ddH2O. For the plate standard, rows 1 and 2 (across) of each plate contained a 1:1 dilution of NaNO2 from 500 μM to 0 μM. In rows A and B (bottom), 50 μl of culture medium was added to each well. 95 μl was added to the first well of each row. To the first well, 5 μl of 10 mM NaNO2 was added. 50 μL was removed from these wells and diluted down the row, except for the last well in each row (baseline). For the test samples, 170 μl of supernatant was removed from the wells, avoiding disturbing the cell monolayer at the bottom of each well. From this 170 μl sample, 50 μl samples (in triplicate) were transferred to a flat-bottom 96-well plate. For the Greiss reaction, equal volumes of Greiss solution A and Greiss solution B were mixed to the required volume (approximately 5 mL total per 96-well plate). The combined Greiss solution A+B was added to each well (50 μl per well). Absorbance was read at 570 nm.

[0424] Results—Viability: An MTT assay was used to identify metabolic decline that may indicate a cytotoxic effect of the compound. Only plant extracts of the present disclosure at concentrations of 50 μM or higher exhibited cytotoxic effects. The data are summarized in Figures 19A-19D and Tables 16 and 17 below. [Table 28] [Table 29]

[0425] Results—IL-6: IL-6 is a common pro-inflammatory cytokine produced by LPS-stimulated macrophages. IL-6 is involved in the propagation of early innate immune responses. The only substantial inhibition of IL-6 production from stimulated RAW264.7 macrophages was observed with the plant extract of the present disclosure (Figure 20). The most potent IL-6 inhibition was observed with 25 μM of the plant extract, which reduced IL-6 production to 41% of the absolute ethanol control (Figure 20). Significant inhibition of IL-6 production was also observed with 6.25 μM and 12.5 μM of the plant extract (Figure 20). Surprisingly, 25 μM of the plant extract exhibited significantly greater IL-6 inhibition than 150 μM of synthetic zingerone (Figures 20 and 21). A summary of the dose-dependent inhibition by the plant extract of the present disclosure is shown in Table 18 below. Potential inhibition was observed with 150 μM of synthetic zingerone or acetyl zingerone treatment. Treatment with synthetic zingerone or acetyl zingerone reduced IL-6 to 64% and 69% of the absolute ethanol control, respectively. These results are summarized in Table 18 below. Ferulic acid had no effect on IL-6 production at any of the concentrations tested. Concentrations of plant extracts of the present disclosure associated with cytotoxicity were omitted to avoid confounding data. [Table 30]

[0426] One-way ANOVA was used to determine whether IL-6 levels were significantly lower in RAW264.7 cells compared to cells treated with the plant extracts of the present disclosure. Treatment of LPS-stimulated RAW264.7 cells with 25 μM of the plant extracts of the present disclosure resulted in significantly lower IL-6 levels compared to treatment with 150 μM of synthetic zingerone, acetyl zingerone, or ferulic acid (Figure 21). Two-way ANOVA was also performed to determine whether the IL-6 levels observed in the compound-treated conditions were significantly different from the vehicle-treated conditions. Treatment with 25 μM of the plant extracts of the present disclosure or 150 μM of synthetic zingerone / acetyl zingerone significantly reduced IL-6 compared to treatment with the respective vehicles (Figure 22).

[0427] Under unstimulated conditions (Figures 23A-23D), IL-6 levels were very low compared to stimulated conditions, as expected. Because cytokine production levels were low under unstimulated conditions, no trend was observed. This confirms that the test set did not induce IL-6 production. Because IL-6 levels were low, often 0 pg / mL, under unstimulated conditions, the data were not normalized to the absolute ethanol control.

[0428] Results—TNF: TNF is also a pro-inflammatory cytokine produced by macrophages stimulated with LPS. Maximum inhibition of TNF production from stimulated RAW264.7 cells was observed after treatment with 150 μM ferulic acid. This treatment reduced TNF to 61% of the absolute ethanol control (Figure 24). At a concentration of 50 μM, the inhibition by ferulic acid treatment gradually decreased to the level of the absolute ethanol control. Synthetic zingerone clearly had a moderate inhibitory effect on TNF production at 150 μM, reducing TNF levels to approximately 80% of the absolute ethanol control. Plant extracts of the present disclosure also had a moderate inhibitory effect on TNF, reducing TNF levels to approximately 80% of the absolute ethanol control at 25 μM, 12.5 μM, and 6.25 μM. Acetyl zingerone had no effect on TNF production. Concentrations of plant extracts of the present disclosure related to cytotoxicity were omitted to avoid confounding data. The results are summarized in Table 19 below. [Table 31]

[0429] No significant difference was observed with 25 μM of the plant extract of the present disclosure compared to 150 μM of synthetic zingerone or ferulic acid (FIG. 25). A statistically significant difference was observed between 25 μM of the plant extract of the present disclosure and 150 μM of acetyl zingerone. A two-way ANOVA was also performed to determine whether TNF levels in the compound-treated conditions were statistically significant compared to the vehicle-treated condition. This analysis showed that 150 μM of ferulic acid treatment resulted in significantly lower TNF levels compared to the vehicle treatment (FIG. 26).

[0430] In unstimulated conditions (Figures 27A-27D), TNF levels were much lower and rarely exceeded the ELISA background value. No trend toward TNF production was observed in unstimulated conditions, suggesting that the test set did not induce TNF production. Because TNF levels in unstimulated conditions were low, often 0 pg / mL, the data were not normalized to the absolute ethanol control.

[0431] Results—NO: Nitric oxide (NO) is a small molecule released by macrophages during inflammatory responses and has the ability to kill invading pathogens. Only the plant extracts of the present disclosure demonstrated an inhibitory effect on NO, reducing NO to 77% of the absolute ethanol control (Figure 28). Synthetic zingerone caused an increase compared to the absolute ethanol control at the high concentration range tested (50 μM, 75 μM, 100 μM, and 150 μM). Acetyl zingerone at 150 μM also caused a slight increase in NO levels. Ferulic acid did not appear to alter NO production. Concentrations of the plant extracts of the present disclosure related to cytotoxicity were omitted to avoid confounding data. Results are summarized in Table 20 below. [Table 32]

[0432] It should be noted that nitric oxide measurements can only be performed in stimulated conditions due to the amount of supernatant required for the measurements.

[0433] One-way analysis of variance (ANOVA) was used to determine whether treatment of RAW 264.7 cells with 25 μM of the plant extract of the present disclosure significantly reduced NO levels compared to other treatment conditions. Treatment with 25 μM of the plant extract of the present disclosure significantly reduced NO levels compared to treatment with 150 μM of synthetic zingerone, acetyl zingerone, and ferulic acid (Figure 29). Two-way analysis of variance (ANOVA) was used to determine whether the observed changes in NO were significantly different from vehicle treatment. Treatment with 25 μM of the plant extract of the present disclosure significantly reduced NO levels compared to vehicle treatment. However, treatment with 150 μM of synthetic zingerone significantly increased NO levels compared to vehicle treatment (Figure 30).

[0434] Conclusion: Cytotoxicity was observed for the plant extract of the present disclosure at a concentration of approximately 50 μM. The extract of the present disclosure most strongly inhibited IL-6 production from stimulated RAW264.7 cells. Treatment with 25 μM of the plant extract of the present disclosure reduced IL-6 levels to less than 50% of the absolute ethanol control. This inhibition of IL-6 by the extract of the present disclosure 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 plant extract of the present disclosure also slightly inhibited TNF production from stimulated RAW264.7 cells. Furthermore, 25 μM of the plant extract of the present disclosure inhibited NO production from stimulated RAW264.7 cells. This inhibition of NO by the plant extract of the present disclosure was statistically significant when compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid.

[0435] Other test compounds produced mixed results, with significant reductions in efficacy in most cases. Treatment with synthetic zingerone or acetyl zingerone at 150 μM slightly inhibited IL-6. These reductions were statistically significant compared to vehicle treatment. Treatment with synthetic zingerone at 150 μM slightly reduced TNF levels. Ferulic acid at 150 μM also inhibited TNF production, reducing levels to approximately 60% of the absolute ethanol control. This reduction was determined to be statistically significant compared to vehicle treatment. Ferulic acid had no effect on NO production. Both synthetic zingerone and acetyl zingerone increased NO production. The increase in NO by synthetic zingerone was determined to be statistically significant compared to vehicle treatment.

[0436] In summary, it was observed that the plant extract of the present disclosure exhibits significantly superior effects to the commercially available compounds tested.Compared to commercially available zingerone and commercially available acetyl zingerone, the plant extract strongly inhibited the expression of pro-inflammatory cytokines and the production of pro-inflammatory small molecules.These results demonstrate the excellent therapeutic effect of the plant extract in blocking / inhibiting inflammatory pathways.

[0437] The superior inhibitory activity of the plant extracts is particularly surprising, given that the comparative studies used significantly lower concentrations of the plant extracts (e.g., 25 μM) than the commercially available compounds (e.g., 100 μM or 150 μM).

[0438] Example 11: Preliminary studies in animals Abstract: We investigated the administration of zingerone in an animal model of acute encephalomyelitis (EAE).

[0439] Materials and Methods: Six groups of female C57BL / 6J mice (9-11 weeks old, 5 mice per group) were orally administered control groups (vehicle control, zingerone (10 mg / kg), or other test compounds) along with a healthy control group. Induction of EAE began on day 1. Test compounds were administered daily from day 5 to day 30.

[0440] Results: EAE was not successfully induced in this study (data not shown). However, mouse body weight was monitored as an indicator of safety and tolerability. During the treatment period, there was an average gain of 0.3 g, with the average body weight at the beginning of the treatment period being 19.7 g and at the end of the treatment period being 20.0 g. No significant changes in body weight were observed with zingerone administration, suggesting that the dose was safe and well tolerated.

[0441] Example 12: Large-scale production process Summary: A large-scale production process was implemented. This makes it possible to produce up to 700±50 kg of fresh ginger rhizomes in one production run. The summary is as follows:

[0442] Step 1: Initial juice extraction of ginger rhizome.

[0443] Introduction: A total of 1360 kg of fresh ginger was delivered to Phytex's Synergy Food group manufacturing plant in Brookvale, Sydney. The material required refrigerated conditions (4°C ± 3°C) for overnight storage. During processing, the material was returned to refrigerated conditions where possible to reduce the risk of deterioration. The ginger juice was collected in a 200 L blue poly drum and returned to the Phytex plant. The solid pomace was collected in another 200 L blue poly drum (see Figure 31 for an example). The equipment and juicing area were cleaned and equipment maintenance / calibration was carried out. Clean tags or cleaning logs were used where necessary.

[0444] Details of Run A: Ginger rhizomes were harvested in two equal batches of approximately 700 kg each. The second 700 kg batch was refrigerated until use the following week. The first 700 kg batch was run through a belt press juicer (Voran EBP500). All juice from the juicer was stored in a 200 L blue HDPE drum (provided by Phytex) on a wooden pallet. All solids / pomace from the juicer was placed in another 200 L blue HDPE drum (provided by Phytex). The solid pomace was run through the belt press juicer (Voran EBP500) again to remove more liquid from the solid pomace. The liquid from the second press was combined with the original 200 L blue HDPE drum that contained the liquid from the first press.

[0445] Run B Details: The following week, juicing of the second 700 kg batch began. The second 700 kg batch was run through a belt press juicer (Voran EBP500). All juice from the juicer was stored in a 200 L blue HDPE drum (Phytex) mounted on a wooden pallet. All solids / pomace from the juicer were collected in another 200 L blue HDPE drum (Phytex). The solid pomace was run through the belt press juicer (Voran EBP500) again to remove more liquid from the solid pomace. The second pomace was mixed with the original 200 L blue HDPE drum that contained the pomace from the first run.

[0446] The entire amount of ginger juice and solid pomace was transferred for in-process sampling and preparation for ultrafiltration (UF). Mass balances and yields were calculated and recorded. At the end of Process 1, each batch of 700 kg ± 50 kg of ginger rhizome yielded approximately 600 L ± 50 L of ginger juice and approximately 200 kg ± 50 kg of ginger pomace / solids.

[0447] Step 2(i) : Alkaline treatment of ginger juice and ginger pomace. See flow chart in Figure 32.

[0448] Introduction: Runs A and B were performed with two equal volumes of KOH. Because the maximum capacity of the extraction vessel was 400 L, each run (A or B) was divided into two parts (Runs A1 / A2 and Runs B1 / B2). Before starting the process, the equipment maintenance / calibration and cleaning status were checked. Clean tags or cleaning logs were used, if necessary.

[0449] Ginger juice processing (300 L per batch): The volume of ginger juice obtained in multiple 200 L HDPE drums was measured. The net weight of all HDPE drums was recorded. The remaining ginger juice / settled solids in each 200 L HDPE drum were mixed using a 1 m plastic mixing paddle to ensure the solids were resuspended. Clamps were attached to the decantation lids (lids with 25 mm poly valves) of each 200 L HDPE drum. Using a drum lifter, all solids were poured into the extraction vessel and mixed with the UF concentrate. The purple propeller of a wall-mounted agitator, equipped with two 100 mm stainless steel propellers, was inserted into the extraction vessel. The propeller height was set to an appropriate height to mix the juice volume. The wall-mounted agitator was connected to the utility board and agitation was initiated in a clockwise direction at 50 Hz. The sludge that had settled at the bottom of each drum was skimmed and transferred to the extraction vessel where it was dissolved by heating and alkaline treatment. One liter of water was hosed into each drum to remove any remaining ginger juice, which was then added to the extraction vessel.

[0450] Addition of potassium hydroxide to ginger juice: While stirring was initiated, KOH was added at 2.0% w / w. This was based on the total amount of juice obtained from the ginger rhizome (raw material). Before adding the KOH, the volume of ginger juice and the initial pH level were recorded. The blue HDPE drum was tared on a 150 kg scale. The total amount of ginger juice to be included in the alkaline treatment was weighed. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffers and the calibration was confirmed with pH 2.0 buffer. In the extraction vessel, 50% KOH was added to the ginger juice extract at the following rate: wt. KOH (kg) = wt. ginger juice (kg) x 0.03 kg (50%) KOH. At this rate, the pH rose to 13.0 ± 1.0. All pH adjustments were performed with an automatic temperature-compensated Mettler-Toledo pH meter. After the pH reached the specified range, stirring was continued for a minimum of 15 minutes, and the final pH and amount of KOH added were recorded.

[0451] Alkaline treatment procedure for ginger juice: A stainless steel steam coil was inserted into the extraction vessel and connected to the steam inlet in the utility wall using a dedicated steam hose. The steam valve was turned on at the wall to heat the coil with steam. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffer solutions and the calibration was confirmed with a pH 2.0 buffer solution. The ginger juice (pH >13) was heated to 60°C ± 2°C with stirring for 60 minutes. The steam flow rate was adjusted to maintain the alkaline liquid within the temperature range (60°C ± 2°C). A sample (20 mL) was taken from the stirred alkaline ginger juice to monitor the conversion of 6-gingerol to zingerone. The volume of alkaline ginger juice was recorded.

[0452] Neutralization Procedure for Ginger Juice: After stirring the alkaline ginger juice for an additional 5 minutes, the solution was neutralized with >98.0% citric acid. The stainless steel coil in the extraction vessel was connected to chilled water, which was circulated. The addition of citric acid (>98%) to the alkaline juice resulted in a strong acid-base reaction, which was exothermic and generated heat. Chilled water was used to reduce excess heat in the solution. Citric acid was prepared prior to use. This was based on a starting raw material weight of 1,340 kg. Citric acid granules (18.0 kg) were weighed into a 20 L purple polyethylene bucket. A citric acid aqueous solution (50% w / v) was further prepared by dissolving 1.0 kg of citric acid granules in 1.0 L of purified water in a 20 L purple polyethylene bucket. This was dissolved using a stainless steel hand stirrer. While stirring the alkaline ginger juice in the extraction vessel, the citric acid granules were added. This was accomplished by adding 10 kg and waiting for the granules to dissolve. The pH of the solution was monitored throughout this process. Citric acid was added slowly, using 500 ml spoonfuls at a time, waiting 30 seconds before adding the next spoonful. Once all of the citric acid (18.0 kg) had been added and dissolved, the pH was read. Next, 50% aqueous citric acid was added until the pH was in the 7.0-7.3 range. Once this desired range was reached, stirring was continued for an additional 15 minutes. All pH adjustments were performed using an automatic temperature-compensated Mettler Toledo pH meter. The resulting solution was referred to as "zingerone solution." The final pH, the amount of citric acid added (>98%), and the volume of zingerone solution were recorded. A 20 mL sample was taken from the zingerone solution to monitor the conversion of 6-gingerol to zingerone.

[0453] Step 2(ii) : Alkali treatment of ginger pomace. See flow chart in Figure 33.

[0454] Processing of ginger pomace / solids (50 kg per batch): A wall-mounted agitator was inserted into the extraction vessel and fitted with three large 150 mm diameter propellers. Agitation was initiated at 50 Hz. A total of 50 kg of solid pomace was added to the extraction vessel from a 200 L HDPE drum. The solids were continuously drawn into the liquid, and the solution was kept moving freely by the agitator. If the solution became too viscous, 20 L of water was added.

[0455] Potassium hydroxide addition to ginger pomace / solids: KOH was added at 2% w / w. This was based on the weight of the total juice extract obtained from the ginger rhizome material. A blue HDPE drum was tared on a 150 kg scale. The total amount of pomace to be included in the alkaline treatment was weighed. A pH electrode was calibrated with pH 7.0 and pH 4.0 buffers and the calibration was confirmed with a pH 2.0 buffer. In the extraction vessel, 50% KOH was added to the ginger juice extract at the following rate: wt. (kg) KOH = wt. (kg) ginger pomace x 0.03 kg (50%) KOH. The calculation took into account the weight of the ginger pomace (50 kg) and the added water (20 kg). At this rate, the pH rose to 13.0 ± 1.0. After reaching the pH range, stirring was continued for an additional 15 minutes (minimum). The final pH and amount of KOH added were recorded.

[0456] Alkaline treatment procedure for ginger pomace / solids: A SS steam coil was inserted into the extraction vessel and connected to the steam inlet in the utility wall using a dedicated steam hose. The steam valve was turned on at the wall to heat the coil with steam. The pH electrode was calibrated with pH 7.0 and pH 4.0 buffer solutions, and the calibration was confirmed with pH 2.0 buffer solution. The ginger pomace (pH >13) was heated to 60°C ± 2°C with stirring for 60 minutes. The steam flow rate was adjusted to maintain the alkaline liquid within the temperature range (60°C ± 2°C). A sample (20 mL) was taken from the stirred alkaline ginger pomace to monitor the conversion of 6-gingerol to zingerone. The volume of the alkaline ginger pomace was recorded.

[0457] Neutralization of the ginger pomace / solids: After stirring the alkaline ginger pomace for an additional 5 minutes, the solution was neutralized with 98.0% citric acid. The stainless steel coil in the extraction vessel was connected to a chilled water supply for circulation. This was done to reduce excess heat from the exothermic reaction (acid + base). Prior to use, citric acid was prepared as described above. While the alkaline ginger pomace was being stirred 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 throughout this process. Citric acid was added slowly in 500 ml increments, waiting 30 seconds before adding the next scoop. Once all the citric acid had been added and dissolved (18 kg total), a pH reading was taken. Next, 50% aqueous citric acid was added until the pH was in the 7.0-7.3 range. Once this target range was reached, stirring was continued for an additional 15 minutes. All pH reading adjustments were integrated into an automatic temperature-compensated Mettler Toledo pH meter. The resulting solution was recorded as "zingerone pomace." The final pH, amount of citric acid added (>98%), and amount of zingerone pomace were recorded. A sample (20 mL) was taken from the zingerone pomace to monitor the conversion of 6-gingerol to zingerone. Mass balance and yield were calculated.

[0458] Process 3 : Preparation of zingerone powder by drying and grinding.

[0459] Oven drying: Equipment maintenance / calibration and cleaning were verified. Drying was set up using a calibrated COMARK digital logger with a stainless steel probe. The time was recorded for each drum of zingerone concentrate removed from the refrigerator in preparation for drying. The Brix% and theoretical zingerone content were recorded. All trays with Teflon mats were loaded into the oven for drying. The drums were mixed using a polypropylene paddle, and then a 4-L container was used to remove a certain amount of liquid. This was then transferred to a 2-L container and placed in each tray. 3 L was then poured onto the Teflon mats of each tray. Trays were loaded onto each shelf, ensuring sufficient gaps (at least 30 mm) between trays to allow airflow and break down the dried material. The oven temperature was set to 60°C. After 24 hours, each tray was inspected, and any loose material was scraped off the Teflon mats, if necessary. The product was placed reverse-side down on the inverted tray. To facilitate drying, the product was gently broken up using a plastic spatula. The trays were returned to the oven and allowed to dry for a further 4 hours (28 hours total). Once sufficiently dry (after 24 or 28 hours), the product was manually crushed using a stainless steel spatula and collected in 100 micron plastic bags. The weight of each bag was measured and recorded and placed into a 100 L rectangular transfer bin. The weight of the rectangular transfer bin was measured and recorded. The product was now ready for milling.

[0460] Milling: Prior to milling, the stainless steel hammer mill was ensured to be clean and equipped with a clean 1 mm mesh screen ("fine zingerone screen"). A 3 mm rubber mat seal was attached to the lid, and the thumbscrew was tightened securely to secure the lid. The hammer mill was run for 10 seconds to ensure proper rotation and clearance. A 100 micron plastic bag was attached to the mill chamber outlet and secured with an adjustable clamp. The overhead dust collector was activated. A plastic scoop was used to slowly transfer the coarse, dry zingerone material into the feed chute. Two scoops at a time were emptied into the chute, and the inlet flap was opened every few seconds. All material in the chute was passed through the stainless steel screen and collected in a plastic bag collector. All bags were weighed, and the yield was calculated using a mass balance. A representative sample (20 g) was removed from each bag and measured for moisture content. All 20 kg bags were placed in a 100 micron plastic bag. After removing any remaining air, the bags were double-sealed using an impulse heat sealer. The bags were stored in a refrigerator. A quality analysis of the final product (zingerone powder) was carried out. See Table 21 below. All quality indicators were good. [Table 33]

[0461] Process 4 : Ethanol extract of zingerone powder.

[0462] Introduction: As outlined below, up to 75 kg of dry powder can be extracted in a single extraction run. The equipment, including the extraction vessel, was maintained, calibrated, and cleaned. A 40 mm diameter stainless steel strainer was attached to the bottom outlet of the extraction vessel. This was hand-tightened onto a 25 mm thread at the bottom to filter solids after extraction stirring. A dedicated stainless steel heating coil was installed within the extraction vessel and connected to steam later in the process. A glass condenser coil was attached to the lid of the extraction tank, with the inlet / outlet connected to a cooling water inlet / outlet on the wall. Cooling water was circulated through the glass condenser coil.

[0463] Single Extraction: For extraction, 95% ethanol was added to a clean, lidded extraction vessel. The addition rate was: volume of 95% ethanol = amount of zingerone powder (kg) x 5 kg. The purple WMS shaft was inserted into the bearing gland in the extraction vessel lid. Stirring was initiated at 200-300 rpm to ensure complete mixing of the ethanol solution. 75 kg of dried zingerone powder from step 3 was slowly added to the extraction vessel through the 100 mm access port in the vessel lid. Stirring continued throughout the addition. After all the powder was added, the access port was closed and the lid was sealed. All initial temperatures were recorded before heating. A steam supply was then connected to the stainless steel coil in the extraction vessel. The temperature of the ethanol solution was monitored during stirring and maintained above 50°C for at least 4 hours. The stirrer was stopped and the solution was allowed to settle for 20 minutes. The final temperature was recorded before filtering the ethanol extract. A 20 mL sample was taken from the ethanol extract and tested for zingerone.

[0464] Filtration of residual solids from the ethanol extract: The contents of the ethanol extract were discharged into two clean 200 L HDPE drums. Stirring was continued during discharge. The ethanol extract was allowed to settle for 30 minutes to allow the solids to settle. A hopper plate filter was fitted with a Z1 pad (5 micron), which was attached and compressed with a stainless steel plunger tool. The top layer of the ethanol extract was decanted from the hopper plate filter using a drum lifter. The decanted solution was poured onto a 55 micron prefilter and collected in a bucket. The filtered ethanol extract was collected from the hopper plate filter into two clean blue HDPE drums. Once the hopper plate filter was filled with solids, a vacuum was applied to the entire system for 10 minutes to collect all liquids. After each use of the vacuum filter, the hopper plate filter was emptied and transferred to a clean plastic bucket. This procedure was repeated until all of the ethanol extract had passed through the Z1 pad. The volume of the 95% ethanol wash was recorded. A sample (20 mL) was taken from the entire filtered ethanol extract and tested for zingerone. The two drums containing the filtered ethanol extract were stored in a refrigerator.

[0465] Rotary evaporation of the ethanol extract: Three 15-L buckets of filtered ethanol extract were removed from the refrigerator and allowed to warm to room temperature. A Heidolph round-bottom flask was prepared, dried, and weighed. 1 L of fresh 95% ethanol was added to the RBF of a 20-L Heidolph rotary evaporator. The chilled water inlet / outlet lines were connected to the appropriate valves on the wall, and the flow rate was adjusted as needed by adjusting the valves. The rotary evaporator was connected to a Heidolph vacuum pump. White, 4-mm HDPE tubing soaked in 95% ethanol was connected to the inlet tap of the rotary evaporator. A chilled water circulation was initiated through the glass condenser. Residual water was removed during the evaporation phase by operating at a minimum of 50 mBar and 40°C. A vacuum was then applied, and the round-bottom flask was slowly rotated (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 drained into the rotary evaporator and collected in the condenser flask. Pressure, color, etc. were observed.

[0466] Standardization of the ethanol extract: The 45 L of filtered ethanol extract was concentrated in a rotary evaporator (45 L divided into three buckets) to the 4 L volume line marked on the outside of the RBF. This line indicates the concentration of the 45 L ethanol extract to 4 L (12.5 mg / ml). At that point, a sample of the concentrated tincture (10 mL) was taken and the zingerone concentration was measured. If the zingerone concentration was greater than 12.5 mg / ml, 1 L of the filtered ethanol extract from step 4 was added and the concentration was continued until the appropriate concentration was reached. If the zingerone concentration was less than 12.5 mg / ml, the concentration was continued until the appropriate concentration was reached. Twelve amber 4 L glass bottles were washed with fresh 95% ethanol and allowed to drain for 30 minutes. After standardization to 12.5 mg / ml, the RBF was removed from the rotary evaporator and transferred to a 15 L bucket. The standardized gingerone tincture was transferred into cleaned and drained bottles, leaving 30 mm of clearance from the bottle bottom threads. The filled bottles were capped with Teflon-sealed plastic lids and hand-tightened. Parafilm was wrapped around the bottle necks and lids. The sealed bottles were placed in 100-micron plastic bags and double-heat sealed. These were then placed in boxes, which were then stored in a refrigerator. A quality analysis of the final product (ethanol extract) was performed. See Table 22 below. Quality indicators were found to be excellent. The stability of the final product was evaluated and confirmed after two months of storage at accelerated conditions (40°C ± 2°C / 75% ± 5% RH) and separately after two months of storage at refrigerated conditions (5°C). It was found to be very stable. See Tables 23 and 24 below. Due to the small sample size (less than 2 g) tested at accelerated conditions, some variability was observed at the one-month time point (Table 23). It is believed that a larger sample size during storage reduces the headspace in the container and improves product stability. Good stability is expected for at least six months of storage. [Table 34] [Table 35] [Table 36]

[0467] Those skilled in the art, using the disclosure and teachings herein, will be able to devise other embodiments and modifications without undue experimentation, and all such embodiments and modifications are considered to be part of this disclosure.

[0468] Thus, those skilled in the art will readily appreciate from this disclosure that subsequent modifications, substitutions, and / or variations that perform substantially the same function or achieve substantially the same results as the embodiments described herein are available in accordance with such related embodiments. Accordingly, the present disclosure is intended to include within its scope such modifications, substitutions, and variations of the processes, manufacture, compositions, compounds, means, methods, and / or steps disclosed herein.

[0469] This description may include subject matter that is outside the scope of the claimed invention. This subject matter is included to aid in understanding the present invention.

[0470] Any references herein to external sources, such as patents or other literature, are generally made for the purpose of providing context for explaining features of the present disclosure, and unless expressly stated, the references to such sources should not be construed or interpreted in any jurisdiction as indicating prior art or public knowledge in the art.

Claims

1. 1. A method for producing zingerone, comprising: (i) subjecting ginger root to an alkaline treatment in an alkaline solution; or (ii) subjecting the juice and / or pomace obtained from the ginger root to alkaline treatment in an alkaline solution; thereby producing zingerone.

2. 10. The method of claim 1, wherein the ginger root is fresh.

3. (a) (i) Chop the ginger root; (b) chopping and drying the ginger root of (i); or (c) obtaining the juice and / or pomace of (ii) by steeping and / or squeezing the ginger root; 3. The method according to claim 1 or 2.

4. (a) potassium hydroxide (KOH) is used in the alkaline solution; or (b) the alkaline solution uses potassium hydroxide (KOH) in liquid form; The method according to any one of claims 1 to 3.

5. The alkaline solution (a) about 1% to about 6% KOH (v / v); (b) about 1.5% to about 5.5% KOH (v / v); (c) about 2% to about 4% KOH (v / v); (d) about 1.5% to about 3.5% KOH (v / v); or (e) Approximately 2% KOH (v / v) 5. The method of claim 4, comprising:

6. The alkaline solution contains calcium hydroxide (Ca(OH) 2 4. The method according to claim 1, wherein a

7. The alkaline solution (a) Approximately 0.5% to approximately 4% Ca(OH) 2 (v / v); (b) Approximately 1.5% to approximately 3.5% Ca(OH) 2 (v / v); or (b) Approximately 2.0% to approximately 3.0% Ca(OH) 2 (v / v) 7. The method of claim 6, comprising:

8. (a) the alkaline treatment is carried out at a temperature of about 40 degrees Celsius to about 70 degrees Celsius; (b) the alkaline treatment is carried out at about 50 degrees Celsius to about 60 degrees Celsius; (c) the alkaline treatment is carried out at about 55 degrees Celsius to about 65 degrees Celsius; or (d) the alkaline treatment is carried out at about 60 degrees Celsius; The method according to any one of claims 1 to 7.

9. (a) the alkaline treatment is carried out for about 1 to 30 hours, about 1 to 20 hours, about 1 to 10 hours, or about 1 to 5 hours; (b) the alkaline treatment is carried out for about 0.5 hours to about 3 hours, or about 0.75 hours to about 2.5 hours, or about 1 hour to about 2 hours; or (c) the alkaline treatment is carried out for about 1 hour or about 2 hours; The method according to any one of claims 1 to 8.

10. (a) Neutralizing alkaline solutions; (b) Neutralizing the alkaline solution and then drying; or (c) neutralizing the alkaline solution and optionally drying, followed by one or more additional extraction steps; The method of any one of claims 1 to 9, further comprising:

11. said one or more additional extraction steps (a) one or more ethanol extraction steps; (b) supercritical fluid extraction; or (c) supercritical fluid extraction followed by one or more ethanol extraction steps; 11. The method of claim 10, comprising:

12. 12. The method of any one of claims 1 to 11, wherein the method produces a composition comprising zingerone.

13. 13. The method of any one of claims 1 to 12, wherein the method produces an aldehyde-free or substantially aldehyde-free composition.

14. 14. A composition comprising zingerone, wherein the zingerone is obtainable by the method according to any one of claims 1 to 13.

15. 15. The composition of claim 14, wherein the composition is formulated as a pharmaceutical composition or a dietary supplement.

16. 16. The composition of claim 14 or 15, wherein the composition is formulated as a liquid, solid, or semi-solid.

17. 17. The composition of any one of claims 14 to 16, formulated for topical or oral administration.

18. 18. The composition of any one of claims 14 to 17, formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule.

19. (a) zingerone in a dose of about 10 mg to about 1500 mg; (b) zingerone in a dose of about 10 mg to about 1000 mg; (c) zingerone in a dose of about 10 mg to about 150 mg; (d) zingerone in a dose of about 10 mg to about 100 mg; or (e) zingerone in a dose of about 10 mg to about 50 mg; 19. The composition of any one of claims 14 to 18, formulated to comprise:

20. (a) formulated for co-administration with an additional anti-inflammatory agent; (b) formulated for co-administration with one or more of an analgesic compound, an antipyretic compound, and a psychotropic compound; (c) formulated for co-administration with one or more of a cannabinoid compound, a mushroom compound, a nonsteroidal anti-inflammatory drug compound (NSAID), an opioid compound, a salicylate compound, and a steroid compound; (d) 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, including one or more of pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin, 20. The composition according to any one of claims 14 to 19.

21. A composition according to any one of claims 14 to 20 for use in the treatment or prevention of inflammation.

22. 22. The composition of claim 21, wherein the inflammation is acute inflammation or chronic inflammation.

23. (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 joint disorders; (e) the inflammation is associated with an infection; (f) the inflammation is associated with cardiac, circulatory, or pulmonary disorders; (g) the inflammation is associated with a neurological disorder; and / or (h) the inflammation is associated with a neoplastic disorder; 23. The composition of claim 22.

24. 24. The composition of any one of claims 21 to 23, wherein the inflammation is inflammation of one or more of the joints, skin, eyes, ears, nose, mouth, throat, esophagus, kidneys, bladder, liver, spleen, lungs, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

25. 25. The composition of any one of claims 21 to 24, wherein the inflammation is associated with one or more of Alzheimer's disease, early Alzheimer's disease, ankylosing spondylitis, arthritis, asthma, colitis, Crohn's disease, dementia, early dementia, depression, diabetes, fibromyalgia, gout, infection, immune-mediated inflammatory disease, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma, and Sjogren's syndrome, and systemic lupus erythematosus.

26. 25. The composition of any one of claims 21 to 24, wherein the inflammation is associated with one or more of rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjogren's syndrome arthritis.

27. 25. The composition of any one of claims 21 to 24, wherein the inflammation is associated with one or more of atherosclerosis, coronary artery disease, pulmonary arterial hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disease, and cytokine storm syndrome; or one or more of breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

28. 25. The composition of any one of claims 21 to 24, wherein the inflammation is associated with one or more of blisters, dermatitis, eczema, hives, lesions, papules, plaques, psoriasis, rash, rosacea, ulcers, and wounds.

29. 15. Use of the composition of claim 14 for preparing a medicament for treating or preventing inflammation in a subject.

30. 30. The use of claim 29, wherein the medicament reduces or slows the progression of the inflammation.

31. (a) the medicament is formulated as a solid, semi-solid, or liquid; and / or (b) the medicament is formulated for topical or oral administration; 31. Use according to claim 29 or 30.

32. 32. The use according to any one of claims 29 to 31, wherein the medicament is formulated as a solution, gel, jelly, gummy, powder, tablet or capsule.

33. The pharmaceutical (a) zingerone in a dose of about 10 mg to about 1500 mg; (b) zingerone in a dose of about 10 mg to about 1000 mg; (c) zingerone in a dose of about 10 mg to about 150 mg; (d) zingerone in a dose of about 10 mg to about 100 mg; or (e) zingerone in a dose of about 10 mg to about 50 mg 33. The use of claim 32, formulated to comprise: 。

34. (a) the composition is formulated for co-administration with an additional anti-inflammatory agent; (b) the composition is formulated for co-administration with one or more of an analgesic compound, an antipyretic compound, and a psychotropic compound; (c) formulated for co-administration with one or more of a cannabinoid compound, a mushroom compound, a nonsteroidal anti-inflammatory drug compound (NSAID), an opioid compound, a salicylate compound, and a steroid compound; (d) The medicine is 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, or levallorphan , including one or more of pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin, Use according to any one of claims 29 to 33.

35. The use according to any one of claims 29 to 34, wherein the inflammation is acute inflammation or chronic inflammation.

36. (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 joint disorders; (e) the inflammation is associated with an infection; (f) the inflammation is associated with cardiac, circulatory, or pulmonary disorders; (g) the inflammation is associated with a neurological disorder; and / or (h) the inflammation is associated with a neoplastic disorder; 36. The use according to claim 35.

37. 37. The use of claim 35 or 36, wherein the inflammation is inflammation of one or more of the joints, skin, eyes, ears, nose, mouth, throat, esophagus, kidneys, bladder, liver, spleen, lungs, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system and skeletal system.

38. 38. The use of any one of claims 35 to 37, wherein the inflammation is associated with one or more of Alzheimer's disease, early Alzheimer's disease, ankylosing spondylitis, arthritis, asthma, colitis, Crohn's disease, dementia, early dementia, depression, diabetes, fibromyalgia, gout, infection, immune-mediated inflammatory disease, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma and Sjogren's syndrome and systemic lupus erythematosus.

39. 38. The use of any one of claims 35 to 37, wherein the inflammation is associated with one or more of rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis and Sjogren's syndrome arthritis.

40. The inflammation is associated with one or more of atherosclerosis, coronary artery disease, pulmonary arterial hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disease, and cytokine storm syndrome; or one or more of breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer. Use according to any one of claims 35 to 37.

41. 38. The use according to any one of claims 35 to 37, wherein the inflammation is associated with one or more of blisters, dermatitis, eczema, hives, lesions, papules, plaques, psoriasis, rash, rosacea, ulcers and wounds.

42. 16. A method for treating or preventing inflammation, comprising administering to a subject the composition of claim 15, thereby treating or preventing said inflammation.

43. 43. The method of claim 42, wherein said administering reduces or slows the progression of said inflammation.

44. (a) the composition is administered as a solid, semi-solid, or liquid; and / or (b) the composition is administered topically or orally; 44. The method of claim 42 or 43.

45. 45. The method of any one of claims 42 to 44, wherein the composition is administered as a solution, gel, jelly, gummy, powder, tablet, or capsule.

46. The composition comprises: (a) zingerone in a dose of about 1 mg to about 5000 mg; (b) zingerone in a dose of about 1 mg to about 1500 mg; (c) zingerone in a dose of about 5 mg to about 500 mg; (d) zingerone in a dose of about 1 mg to about 15 mg; or (e) zingerone in a dose of about 1 mg to about 10 mg.

46. ​​The method of claim 45, wherein the

47. (a) the composition is co-administered with an additional anti-inflammatory agent; (b) the composition is administered simultaneously with one or more of an analgesic compound, an antipyretic compound, and a psychotropic compound; (c) the composition is administered simultaneously with one or more of a cannabinoid compound, a mushroom compound, a nonsteroidal anti-inflammatory drug compound (NSAID), an opioid compound, a salicylate compound, and a steroid compound; (d) The composition is selected from the group consisting 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, and penicillin. Concomitantly administered with one or more of the following: methadone, phenazocine, eptazocine, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin 47. The method of any one of claims 42 to 46.

48. 48. The method of any one of claims 42 to 47, wherein the inflammation is acute inflammation or chronic inflammation.

49. (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 joint disorders; (e) the inflammation is associated with an infection; (f) the inflammation is associated with cardiac, circulatory, or pulmonary disorders; (g) the inflammation is associated with a neurological disorder; and / or (h) the inflammation is associated with a neoplastic disorder; 49. The method of claim 48.

50. 50. The method of claim 48 or 49, wherein the inflammation is inflammation of one or more of the joints, skin, eyes, ears, nose, mouth, throat, esophagus, kidneys, bladder, liver, spleen, lungs, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.

51. 51. The method of any one of claims 48 to 50, wherein the inflammation is associated with one or more of Alzheimer's disease, early Alzheimer's disease, ankylosing spondylitis, arthritis, asthma, colitis, Crohn's disease, dementia, early dementia, depression, diabetes, fibromyalgia, gout, infection, immune-mediated inflammatory disease, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma, and Sjogren's syndrome, and systemic lupus erythematosus.

52. 51. The method of any one of claims 48-50, wherein the inflammation is associated with one or more of rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjogren's syndrome arthritis.

53. 51. The method of any one of claims 48-50, wherein the inflammation is associated with one or more of atherosclerosis, coronary artery disease, pulmonary arterial hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disease, and cytokine storm syndrome; or one or more of breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

54. 51. The method of any one of claims 48 to 50, wherein the inflammation is associated with one or more of blisters, dermatitis, eczema, hives, lesions, papules, plaques, psoriasis, rash, rosacea, ulcers, and wounds.

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