Process for preparation of zingerone, compositions comprising zingerone and uses thereof

Abstract

Disclosed are methods of preparing zingerone from ginger, as well as compositions obtained using these methods and methods of using these compositions. Beneficial compositions and methods of utilizing these compositions are disclosed.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Patent Application No. 63 / 446,013, filed February 15, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to methods for preparing gingerone and compositions containing gingerone. In particular, it relates to beneficial compositions, including pharmaceutical and dietary compositions, and the uses of these compositions. Background Technology

[0004] ginger( Zingiber officinale Ginger is a flowering plant whose rhizomes are widely used as a spice and traditional medicine. When consumed in moderation, ginger has virtually no negative side effects. It is listed on the FDA's "Generally Recognized as Safe" list.

[0005] The distinctive aroma and flavor of ginger come from volatile oils that make up 1-3% of the weight of fresh ginger. These oils are mainly composed of gingerone, gingerol and gingerol, with [6]-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decone) being the main irritant compound.

[0006]

[0007] For example, 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: As reported in 223-232, gingerone (also known as zingerone) is produced by drying or heat-treating gingerol at a temperature of approximately 40°C. Gingerone has a mild pungent and sweet aroma. Also known as vanillylacetone, gingerone is a crystalline solid, reportedly slightly soluble in water and soluble in ether. The FoodB compound database describes the water solubility of gingerone as 0.57 g / L, with LogP values ​​of 2.02–1.92 and logS of -2.5; see https: / / foodb.ca / compounds / FDB010527.

[0008]

[0009] Fresh ginger contains trace amounts of gingerone, which is known to be produced through steaming or drying ginger roots. This causes gingerol to dehydrate by losing water molecules, producing gingerone and hexanal. See, for example, Gopi et al., 2016, “Study on temperature dependent conversion of active components of ginger”. Int. J. of Pharma Sciences 6(1): 1344-1347.

[0010] Shogaol is more irritating and has higher antioxidant activity. It is not found in raw ginger, but is formed from gingerol during heating, storage, or via acidity.

[0011]

[0012] Shogaol is the dehydrated form of gingerol.

[0013] In 1917, Hiroshi Nomura first isolated gingerone from ginger root. Nomura discovered a method for synthesizing gingerone and later obtained a patent (US 1,263,796, granted April 23, 1918), in which vanillin and acetone react under alkaline conditions to form dehydrogingerone. This compound was obtained in approximately 95% concentration. Following this reaction, the intermediate compound was catalytically hydrogenated to form gingerone, which was obtained in approximately 100% concentration.

[0014] Ginger compounds have been shown to be effective against enterotoxigenic Escherichia coli (E. coli). Escherichia coli Diarrhea caused by heat-sensitive enterotoxins. This type of diarrhea is a leading cause of infant mortality in developing countries. Gingerol has been reported to be the active ingredient responsible for ginger's antidiarrheal effects. This study concluded that the bioactive compounds in ginger significantly blocked the binding of enterotoxin-producing Escherichia coli heat-sensitive enterotoxins to the cell surface receptor GM1, thereby inhibiting fluid accumulation 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-induceddiarrhoea in mice” Journal of Agricultural and Food Chemistry 55 (21): 8390-7.

[0015] As reported by Kumar et al., gingerone has shown anti-inflammatory effects on liver inflammation in a mouse model of peritonitis. See Kumar et al., "Zingerone suppresses liver inflammation induced by antibiotic-mediated endotoxemia through down regulating hepatic mRNA expression of inflammatory markers in..." Pseudomonas aeruginosa peritonitis mouse model PLOS ONE 9(9): e106536.

[0016] Kumar et al. have also reported that gingerone can enhance the activity of Pseudomonas aeruginosa (…). Pseudomonas aeruginosa Cellular sensitivity to antibiotics. See Kumar et al., 2014. Life Sciences 117: 24-32. Kumar et al. concluded that gingerone caused changes in the surface properties of Pseudomonas aeruginosa cells, thereby increasing the sensitivity of Pseudomonas aeruginosa cells to antibiotics.

[0017] Research on optimizing the production of gingerone from natural sources is limited. 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 activities. This application aims to address these and other needs. Summary of the Invention

[0018] On the one hand, this disclosure covers methods for producing gingerone 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 residue obtained from ginger root to alkaline treatment in an alkaline solution.

[0019] In specific aspects: The ginger root is fresh.

[0020] The ginger root is dried.

[0021] Ginger roots are dried at approximately 40°C to approximately 70°C, or at approximately 40°C to approximately 60°C, or at approximately 55°C to approximately 65°C, or at approximately 60°C.

[0022] The juice is obtained by soaking and / or pressing ginger roots.

[0023] The residue is obtained by juicing, soaking, and / or pressing ginger roots.

[0024] Cut the ginger root into small pieces and then treat it with alkali.

[0025] Cut the ginger root into small pieces, dry it, and then treat it with alkali.

[0026] The alkali treatment is carried out at approximately 40 to approximately 70 degrees Celsius.

[0027] The alkali treatment is carried out at approximately 50 to approximately 60 degrees Celsius.

[0028] The alkali treatment is carried out at approximately 55 degrees Celsius to approximately 65 degrees Celsius.

[0029] The alkali treatment is carried out at approximately 60 degrees Celsius.

[0030] The alkali treatment lasts approximately 1-72 hours.

[0031] The alkali treatment takes approximately 1-48 hours.

[0032] The alkali treatment takes approximately 1-24 hours.

[0033] The alkali treatment takes approximately 1-30 hours, or approximately 1-20 hours, or approximately 1-10 hours, or approximately 1-5 hours.

[0034] 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.

[0035] The alkali treatment lasts for about 2 hours.

[0036] The alkali treatment lasts for about 1 hour.

[0037] Use potassium hydroxide (KOH).

[0038] Use potassium hydroxide (KOH) in liquid form.

[0039] Use approximately 0.1% to approximately 6% KOH (v / v). Use approximately 0.5% to approximately 5.5% KOH (v / v). Use approximately 1% to approximately 6% KOH (v / v). Use approximately 1.5% to approximately 5.5% KOH (v / v). Use approximately 2% to approximately 4% KOH (v / v). Use approximately 1.5% to approximately 3.5% KOH (v / v).

[0040] Use calcium hydroxide Ca(OH)2.

[0041] Use approximately 0.5% to approximately 4% Ca(OH)2 (v / v). Use approximately 1.5% to approximately 3.5% Ca(OH)2 (v / v). Use approximately 2% to approximately 3% Ca(OH)2 (v / v).

[0042] After alkali treatment, the alkaline solution is neutralized.

[0043] Neutralize the alkaline solution with citric acid.

[0044] The alkaline solution is cooled during neutralization to mitigate excess heat.

[0045] Neutralize the alkaline solution to obtain a pH of about 6.5 to about 7.5 or about 7.0 to about 7.3.

[0046] Freeze-drying the neutralized solution.

[0047] Heat and dry the neutralized solution.

[0048] The neutralized solution undergoes extraction with gingerone.

[0049] The solution is dried and neutralized, and optionally subjected to gingerone extraction.

[0050] Drying is carried out at approximately 50 to approximately 70 degrees Celsius.

[0051] Drying is carried out at approximately 55 degrees Celsius to approximately 65 degrees Celsius.

[0052] Drying is carried out at approximately 60 degrees Celsius.

[0053] Drying should continue for at least 24 hours.

[0054] The drying process lasts approximately 24 to 28 hours.

[0055] Optional grinding of dried material.

[0056] Gingerone may be further extracted by one or more alcohol extraction steps.

[0057] Gingerone may be further extracted by one or more ethanol extraction steps.

[0058] Ethanol extraction was carried out at approximately 35°C to approximately 65°C.

[0059] Ethanol extraction was carried out at approximately 45°C to approximately 55°C.

[0060] Ethanol extraction was performed at approximately 50 degrees Celsius.

[0061] Ethanol extraction should be carried out for at least 7 days.

[0062] Ethanol extraction is performed for 24 hours or less.

[0063] Ethanol extraction should be performed for at least 4 hours.

[0064] Ethanol extraction takes approximately 4 to 8 hours.

[0065] Optionally dry the ethanol extract.

[0066] Supercritical fluid extraction was used to extract gingerone.

[0067] Gingerone was extracted by supercritical fluid extraction, followed by an alcohol extraction step.

[0068] This method produces a product that is a composition containing gingerone.

[0069] The composition contains no or substantially no aldehydes.

[0070] The composition is a plant extract.

[0071] The composition is an ethanol extract.

[0072] The composition is a powder.

[0073] On the one hand, this disclosure covers a method for producing gingerone by subjecting ginger root extract to alkali treatment.

[0074] Ginger root extract is obtained by supercritical fluid extraction of ginger root.

[0075] Ginger root extract is obtained by alcohol extraction of ginger root.

[0076] Ginger root extract is obtained by juicing ginger roots.

[0077] Ginger root extract is obtained by juicing ginger roots to obtain juice and residue.

[0078] Juicing includes soaking and / or pressing ginger roots.

[0079] The alkali treatment is carried out at approximately 30 to approximately 70 degrees Celsius.

[0080] The alkali treatment is carried out at approximately 50 to approximately 60 degrees Celsius.

[0081] The alkali treatment is carried out at approximately 55 degrees Celsius to approximately 65 degrees Celsius.

[0082] The alkali treatment is carried out at approximately 60 degrees Celsius.

[0083] The alkali treatment lasts approximately 1-72 hours.

[0084] The alkali treatment takes approximately 1-48 hours.

[0085] The alkali treatment takes approximately 1-24 hours.

[0086] The alkali treatment takes approximately 1-30 hours, or approximately 1-20 hours, or approximately 1-10 hours, or approximately 1-5 hours.

[0087] 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.

[0088] The alkali treatment lasts for about 2 hours.

[0089] The alkali treatment lasts for about 1 hour.

[0090] Use potassium hydroxide (KOH).

[0091] Use potassium hydroxide (KOH) in liquid form.

[0092] Use approximately 0.1% to approximately 6% KOH (v / v). Use approximately 0.5% to approximately 5.5% KOH (v / v). Use approximately 1% to approximately 6% KOH (v / v). Use approximately 1.5% to approximately 5.5% KOH (v / v). Use approximately 2% to approximately 4% KOH (v / v). Use approximately 1.5% to approximately 3.5% KOH (v / v).

[0093] Use calcium hydroxide Ca(OH)2.

[0094] Use approximately 0.5% to approximately 4% Ca(OH)2 (v / v). Use approximately 1.5% to approximately 3.5% Ca(OH)2 (v / v). Use approximately 2% to approximately 3% Ca(OH)2 (v / v).

[0095] After alkali treatment, the alkaline solution is neutralized.

[0096] Neutralize the alkaline solution to obtain a pH of about 6.5 to about 7.5 or about 7.0 to about 7.3.

[0097] After neutralizing the alkaline solution, dry the neutralized material.

[0098] Optional grinding of dried material.

[0099] Optionally, the dried material can be further extracted.

[0100] Gingerone may be further extracted by one or more alcohol extraction steps.

[0101] Gingerone may be further extracted by one or more ethanol extraction steps.

[0102] Ethanol extraction should be carried out for at least 7 days.

[0103] Ethanol extraction is performed for 24 hours or less.

[0104] Ethanol extraction should be performed for at least 4 hours.

[0105] Ethanol extraction takes approximately 4 to 8 hours.

[0106] Optionally dry the ethanol extract.

[0107] Gingerone may be further extracted using supercritical fluid extraction.

[0108] Gingerone was optionally extracted by supercritical fluid extraction, followed by a further extraction step by alcohol extraction.

[0109] This method produces a product that is a composition containing gingerone.

[0110] The composition contains no or substantially no aldehydes.

[0111] The composition is a plant extract.

[0112] The composition is an ethanol extract.

[0113] The composition is a powder.

[0114] The method includes: (i) subjecting ginger root to alkaline treatment in an alkaline solution; or (ii) subjecting juice obtained from ginger root and optionally residue obtained from ginger root to alkaline treatment in an alkaline solution, wherein the alkaline solution contains about 1.5% to about 3.5% KOH (v / v), wherein the alkaline treatment is carried out for about 1 hour to about 2 hours, and wherein after the alkaline treatment, the pH of the alkaline solution is neutralized to about 6.5 to about 7.5.

[0115] It also covers compositions that are plant extracts containing gingerone, which are prepared by any of the methods in the preceding aspects.

[0116] The composition contains no or substantially no aldehydes.

[0117] The composition is an ethanol extract.

[0118] The composition is a powder.

[0119] The composition is formulated for use as a pharmaceutical composition or a dietary composition.

[0120] The composition was formulated as a dietary supplement.

[0121] Compositions containing gingerone are also included, which are prepared by any of the methods in the preceding aspects.

[0122] On the one hand, this disclosure covers methods for treating or preventing inflammation, which include: applying to an individual a composition of any of the preceding aspects, thereby treating or preventing inflammation.

[0123] On the other hand, this disclosure covers the use of compositions of any of the preceding aspects for the preparation of medicaments for the treatment or prevention of inflammation.

[0124] It also covers compositions containing gingerone for the treatment or prevention of inflammation.

[0125] In various aspects: The composition was obtained by any of the methods described in the preceding aspects.

[0126] The composition was obtained from ginger root.

[0127] The composition was obtained from fresh ginger roots.

[0128] The composition was obtained from dried ginger root.

[0129] The composition was obtained from the juice prepared from ginger root.

[0130] The juice is prepared by soaking and / or pressing ginger roots.

[0131] The composition is obtained by using an alkaline conversion step to convert gingerol in ginger root or gingerol from ginger root juice into gingerone.

[0132] The composition contains no or substantially no aldehydes.

[0133] The composition was formulated as a powder.

[0134] The composition was formulated as a tincture.

[0135] The composition also contains one or more anti-inflammatory agents.

[0136] The composition also includes one or more of the following: analgesic compounds, antipyretic compounds, and psychotropic compounds.

[0137] The composition also includes one or more of the following: cannabinoid compounds, fungal compounds, nonsteroidal anti-inflammatory drug compounds (NSAIDs), opioid compounds, salicylates, and steroid compounds.

[0138] The composition also contains one or more of the following: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, and mefenamic acid. acid), meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocine, betamethasone, cortisone Sone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, cannabigerol, tetrahydrocannabinol, psilocybin, and psilocin.

[0139] Inflammation needs to be regulated.

[0140] Inflammation can be acute or chronic.

[0141] Inflammation is an inflammatory condition.

[0142] Inflammation is one or more of the following: immune disorders; arthritis; infection; heart, circulatory, or lung diseases; neurological disorders; and neoplasmic disorders.

[0143] Inflammation is inflammation affecting one or more of the following: 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.

[0144] Inflammation is one or more of the following: 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, Sjögren's syndrome, and systemic lupus erythematosus. erythematosus (SLE; lupus).

[0145] Inflammation is one or more of the following: rheumatoid arthritis, ankylosing spondylitis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjögren's syndrome arthritis.

[0146] Inflammation is one or more of the following: atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disorder, and cytokine storm syndrome.

[0147] Inflammation is one or more of the following: breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0148] In various aspects of the composition: The composition is formulated for topical or oral administration.

[0149] The composition is formulated as a solid, semi-solid, or liquid.

[0150] The composition is formulated as a solution, tincture, gel, jelly, gummy, powder, tablet or capsule.

[0151] The composition is supplied in sachets.

[0152] The composition contains gingerone in doses of about 10 mg to about 3000 mg.

[0153] The composition contains a dose of gingerone of about 10 mg to about 1500 mg.

[0154] The composition contains a dose of gingerone of about 10 mg to about 1000 mg.

[0155] The composition contains a dose of gingerone of about 10 mg to about 500 mg.

[0156] The composition contains a dose of gingerone of about 10 mg to about 300 mg.

[0157] The composition contains a dose of gingerone of about 10 mg to about 150 mg.

[0158] The composition contains a dose of gingerone of about 10 mg to about 100 mg.

[0159] The composition contains a dose of gingerone of about 10 mg to about 75 mg.

[0160] The composition contains a dose of gingerone of about 10 mg to about 50 mg.

[0161] The composition is formulated for co-administration with one or more anti-inflammatory agents.

[0162] The composition is formulated for co-administration with one or more of the following: analgesic compounds, antipyretic compounds, and psychotropic compounds.

[0163] The composition is formulated for co-administration with one or more of the following: cannabinoid compounds, fungal compounds, nonsteroidal anti-inflammatory drug compounds (NSAIDs), opioid compounds, salicylates, and steroid compounds.

[0164] The composition is formulated for co-administration with one or more of the following: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etoricoxib, etoricoxib, biphenylacetic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxapazine, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levonorphanol, levonorphanone, pentazocine, phenazocine, etanertin, betamethasone, cortisone, dexamethasone, dexamethasone, ethammethasone, hydrocortisone, methylprednisolone, prednisolone, prednisolone, triamcinolone, cannabidiol, cannabinol, tetrahydrocannabinol, psilocybin, and dephosphated psilocybin.

[0165] It also covers the use of the compositions of the preceding aspects in the preparation of a medicament for treating or preventing inflammation in an individual.

[0166] It also covers methods for treating or preventing inflammation in an individual, which involve applying a previously described composition to the individual.

[0167] The foregoing brief overview broadly describes the features and technical advantages of certain embodiments of this disclosure. Further technical advantages will be described in the detailed description and examples below.

[0168] The novel features considered peculiar will be better understood from the detailed description when considered in conjunction with any of the accompanying drawings and examples. However, the drawings and examples provided herein are intended to help illustrate or enhance the understanding of the disclosure and are not intended to limit the scope of this disclosure. Attached Figure Description

[0169] Figure 1 A photograph depicting fresh ginger root.

[0170] Figure 2HPLC UV chromatogram of alkali-treated ginger (280 nm).

[0171] Figure 3 : A schematic diagram showing the comparison process.

[0172] Figure 4A : A photo depicting a juicer and the raw ginger before juicing.

[0173] Figure 4B Photo depicting the juicing of raw ginger.

[0174] Figure 5A Ginger juice was treated with KOH (0.5%) and analyzed by HPLC. The peak areas of gingerone (Z) and gingerol (G) are shown.

[0175] Figure 5B Ginger juice was treated with KOH (1%) and analyzed by HPLC. The peak areas of gingerone (Z) and gingerol (G) are shown.

[0176] Figure 5C Ginger juice was treated with KOH (2%) and analyzed by HPLC. The peak areas of gingerone (Z) and gingerol (G) are shown.

[0177] Figure 6A Ginger residue produced through pressing.

[0178] Figure 6B Ginger juice produced by pressing.

[0179] Figure 7 : A schematic diagram showing the ethanol extraction process and evaporation.

[0180] Figure 8A GCMS TIC analysis of ethanol extract.

[0181] Figure 8B Comparison of ethanol extract and hexanal standard. The chromatogram shows the 2-7 minute region.

[0182] Figure 9 : Dose-response curves for cytotoxicity assays. The disclosed plant extracts were evaluated.

[0183] Figure 10 Dose-response curves for nitric oxide determination. The disclosed plant extracts were evaluated.

[0184] Figure 11 Dose-response curves for IL-6 assays. The disclosed plant extracts were evaluated.

[0185] Figure 12Dose-response curves for cytotoxicity, NO, and IL-6 assays. The disclosed plant extracts were evaluated.

[0186] Figure 13 Cell viability of RAW264.7 cells was measured using WST-1. A comparative study of the disclosed plant extracts and commercially available gingerone was conducted.

[0187] Figure 14 Interleukin (IL)-6 produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). A comparative study of the disclosed plant extract and commercially available gingerone.

[0188] Figure 15 Interleukin (IL)-10 produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). A comparative study of the disclosed plant extract and commercially available gingerone.

[0189] Figure 16 Tumor necrosis factor (TNF)-α produced by RAW264.7 cells treated with lipopolysaccharide (LPS). A comparative study of the disclosed plant extract and commercially available gingerone.

[0190] Figure 17A and Figure 17B Both test methods showed the efficacy of dexamethasone as a positive control. Figure 17A The results of the analysis using Method 1 are shown. Figure 17B The results of the analysis using Method 2 are shown.

[0191] Figure 18A and 18B Both testing methods showed similar MTT scores. Figure 18A The results of the analysis using Method 1 are shown. Figure 18B The results of the analysis using Method 2 are shown.

[0192] Figures 19A to 19D The disclosed plant extracts exhibited cytotoxic effects at 150 µM, 100 µM, 75 µM, and 50 µM. Data are presented as mean ± SEM of three biological replicates. Nonlinear regression was calculated using GraphPad Prism 9.0. Figure 19A The results of the synthesis of gingerone are shown. Figure 19B The results for acetyl gingerone were shown. Figure 19C The results for ferulic acid were shown. Figure 19D The results of the disclosed plant extracts are shown.

[0193] Figure 20The disclosed plant extracts produced a dose-dependent inhibition of IL-6 production in stimulated RAW264.7 cells. Data are presented as mean ± SEM of three biological replicates.

[0194] Figure 21 Treatment with the disclosed plant extract at 25 µM produced significantly less IL-6 compared to treatment with 150 µM synthetic gingerone, acetylshogaol, or ferulic acid. Data are presented as mean ± SEM of three biological replicates. Repeated measures one-way ANOVA with Turkey correction was calculated in GraphPad Prism 9.0 for multiple comparisons. P < 0.05 P < 0.001.

[0195] Figure 22 Treatment with the disclosed plant extract at 25 µM significantly reduced IL-6 levels compared to the control. Data are presented as mean ± SEM of three biological replicates. Repeated measures two-way ANOVA with Sidak correction was calculated in GraphPad Prism 9.0 for multiple comparisons. P < 0.05 P < 0.01.

[0196] Figures 23A to 23D No effect was observed on IL-6 production in unstimulated RAW264.7 cells. Data are presented as mean ± SEM of three biological replicates. Figure 23A The results of the synthesis of gingerone are shown. Figure 23B The results for acetyl gingerone were shown. Figure 23C The results for ferulic acid were shown. Figure 23D The results of the disclosed plant extracts are shown.

[0197] Figure 24 The disclosed plant extracts reduced TNF (TNF-α) production in stimulated RAW264.7 cells. Data are presented as mean ± SEM from three biological replicates.

[0198] Figure 25Treatment with the disclosed plant extract at 25 µM produced significantly less IL-6 compared to treatment with 150 µM acetylshogaol. Data are presented as mean ± SEM of three biological replicates. Repeated measures one-way ANOVA with Turkey correction was calculated in GraphPad Prism 9.0 for multiple comparisons. P < 0.05.

[0199] Figure 26 Reduced TNF production in stimulated RAW264.7 cells. 150 µM ferulic acid produced a significantly reduced TNF production compared to the mediator. Data are presented as mean ± SEM of three biological replicates. Repeated measures two-way ANOVA with Sidak correction was calculated in GraphPad Prism 9.0 for multiple comparisons. P < 0.05 P < 0.01.

[0200] Figures 27A to 27D No effect was observed on TNF production in unstimulated RAW264.7 cells. Data are presented as mean ± SEM from three biological replicates. Figure 27A The results of the synthesis of gingerone are shown. Figure 27B The results for acetyl gingerone were shown. Figure 27C The results for ferulic acid were shown. Figure 27D The results of the disclosed plant extracts are shown.

[0201] Figure 28 The disclosed plant extract reduced NO levels in stimulated RAW264.7 cells. Data are presented as mean ± SEM from three biological replicates.

[0202] Figure 29 Treatment with the disclosed plant extract at 25 µM produced significantly lower NO levels compared to treatment with 150 µM synthetic gingerone, acetylshogaol, or ferulic acid. Data are presented as mean ± SEM of three biological replicates. Repeated measures one-way ANOVA with Turkey correction was calculated in GraphPad Prism 9.0 for multiple comparisons.

[0203] Figure 30 Treatment with 150 µM synthetic gingerone produced significantly higher NO levels compared to treatment with the medium. Data are presented as mean ± SEM of three biological replicates. Repeated measures two-way ANOVA with Sidak correction was calculated in GraphPad Prism 9.0 for multiple comparisons.

[0204] Figure 31 Flowchart of a large-scale ginger juicing method.

[0205] Figure 32 : Flowchart of a large-scale gingerone extraction method using ginger juice as the starting material.

[0206] Figure 33 : Flowchart of a large-scale gingerone extraction method using ginger residue as a starting material.

[0207] Figure 34A and Figure 34B Stability of gingerone extract obtained through large-scale production. Tests were conducted over a two-month period. Figure 34A The gingerone content was shown at 5°C and 40°C. Figure 34B The pH levels are shown at 5°C and 40°C. Detailed Implementation

[0208] The following description illustrates many exemplary configurations, parameters, etc. However, it should be understood that such description is not intended to limit the scope of this disclosure, but is provided as a description of exemplary embodiments.

[0209] All references cited in this specification (including patents and patent applications) are incorporated herein by reference. No reference is acknowledged to constitute prior art. Discussion of any reference does not constitute an acknowledgment that such reference is part of general knowledge in the art in New Zealand or any other country.

[0210] definition

[0211] When ranges (e.g., temperature ranges, time ranges, or composition ranges) are given in the specification, all intermediate ranges and subranges, as well as all individual values ​​included within a given range, are intended to be included in this disclosure. Thus, specific ranges (e.g., 1 to 10) include all possible numerical combinations between the listed minimum and maximum values ​​(e.g., 1, 1.1, 2, 3, 3.3, 4, 5.5, 6, 7, 8.9, 9, and 10), and ranges of any rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.9), and therefore all subranges of all ranges explicitly disclosed herein are explicitly disclosed herein. The numerical values ​​provided in parentheses are merely examples of specific indications, and all possible numerical combinations between the listed minimum and maximum values ​​should be considered as being expressly stated in a similar manner in this disclosure.

[0212] In each example herein, in the description, embodiments, and instances of this disclosure, the terms “comprising,” “including,” etc., should be interpreted broadly and without limitation. Therefore, unless the context explicitly requires otherwise, throughout the specification and claims, the words “comprising,” “including,” etc., should be interpreted in an inclusive rather than exclusive sense, that is, in the sense of “including but not limited to.”

[0213] As used in this article, "and / or" means additionally or alternatively.

[0214] In this specification, the use of the articles “a” and “an” means that the grammatical individual of the article is one or more (i.e., at least one). For example, “an element” can be used to refer to one element or more elements.

[0215] Throughout this specification, the term "about" is used to indicate that a numerical value includes the standard deviation of the error of the method used to determine that value, such as the level or dosage level of a compound as described in detail herein. Specifically, the term "about" includes a deviation (positive and negative) of up to 10% from the stated numerical value or range.

[0216] As used herein, the term "comprising" can mean the presence of gingerone or gingerone extract in the composition. As an example, gingerone or gingerone extract may constitute at least 1%, at least 2%, at least 4%, at least 5%, at least 10%, at least 12%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% (% w / w) of the composition by weight. Alternatively, gingerone or gingerone extract may constitute at least 1%, at least 2%, at least 3%, 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% (% v / v) of the composition by volume.

[0217] The term "substantially free" regarding aldehydes means a product having a negligible aldehyde content. The product can be, for example, the composition described herein, or a product generated, for example, by the methods described herein. As an example, aldehyde levels can 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.

[0218] As used herein, the term "alkali treatment" means exposing a sample (e.g., ginger, ginger juice, ginger pomace, or any combination thereof) to an alkaline aqueous solution with a pH greater than 7. This includes, but is not limited to, solutions containing sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or any combination thereof. It should be understood that alkali treatment can be performed within the temperature range described herein, and the alkaline solution can be heated before or during exposure to a sample containing ginger or an extract from ginger. The alkaline solution will have a chemically effective amount of alkali to convert at least some of the gingerols present in the sample into gingerones. Specific methodologies are described in detail herein.

[0219] Generally, the term "extract" in this disclosure refers to plant extracts (also known as "phytomedicines"). More specifically, an "extract" is a composition in which one or more liquid, solid, or chemical components of a plant or plant part have been separated or concentrated. For example, liquid, solid, or semi-solid extracts can be obtained. Extracts can be obtained by one or more of the following methods: juicing, pressing, maceration, crushing, grinding, or other standard methods. Solvent-based extraction is also included. Solid extracts are particularly indicated, for example, powders obtained by drying or evaporation. As a specific example, extracts can be prepared in a dried form or in a solution form. "Gingerone extract" refers to an extract containing gingerone prepared / produced from ginger root (i.e., ginger rhizome, also known as "ginger"). Specific extracts and methods of their production are described in detail herein.

[0220] As used herein, the term "composition" encompasses a product comprising one or more active ingredients (e.g., combinations as described herein) and one or more suitable excipients comprising other adjuvants. These may be physiologically acceptable excipients. It encompasses any product directly or indirectly resulting from the combination, complexation, or aggregation of two or more active ingredients. In certain aspects, the composition may comprise any suitable solvate or salt of each compound. As a specific example, the extracts of this disclosure may be prepared into compositions suitable for individual administration, or formulations suitable for individual administration. Various exemplary compositions are described in detail herein.

[0221] "Pharmaceutical composition" refers to a composition administered to an individual for, for example, to treat or prevent inflammation. "Dietary composition" refers to a composition ingested by an individual for, for example, to alleviate or prevent inflammation.

[0222] As used herein, “administration of” or “administering” refers to providing an individual with the disclosed extract or a composition formulated from the disclosed extract. The disclosed extract and the disclosed composition may be administered via any suitable route and via any suitable formulation. In some cases, it may be useful to use different routes of administration and / or different formulations in the same individual. For example, one or more oral formulations may be used, or 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. This document provides non-limiting exemplary descriptions of routes of administration and administration formulations.

[0223] "Anti-inflammatory agent" refers to a component that reduces one or more symptoms of inflammation. This includes pharmaceuticals, phytochemicals, plant components, plant extracts, and essential oils, as well as herbs and other infusions, along with various other ingredients, to help reduce inflammation. Such components can be used in combination with the compositions and extracts disclosed herein and can be used to assist in the regulation of the immune response.

[0224] "Co-administration" or "co-administering" refers to the combined use of active ingredients (e.g., for treatment or to improve appearance) and includes the administration of co-preparations (i.e., combination preparations) as well as the simultaneous, sequential, or separate administration of different preparations. Similarly, "in conjunction" refers to the combined use of one or more active ingredients with a device / procedure. This can include the use of the active ingredient before, during, and / or after the use of the device / procedure.

[0225] The term "inflammation" encompasses any degree of inflammation in an individual's tissues that persists for any period of time. Specific examples include acute or chronic inflammation. Inflammation can be associated with pain. Inflammation can occur in the joints, skin, lungs, heart, circulatory system, digestive tract, genitourinary tract, and more. This article covers these and other types of inflammation.

[0226] Symptoms of inflammation include one or more of the following: pain, fever, redness, swelling, and loss of function. Other markers of inflammation include the production of pro-inflammatory cytokines (e.g., IL-6, TNF-α) and / or pro-inflammatory small molecules (e.g., NO), as well as the activation and / or accumulation of immune cells.

[0227] As used in this article, "subject" can be human or non-human animal, particularly mammals, including cattle, sheep, goats, pigs, horses and other livestock, as well as dogs, cats and other domestic pets. In certain respects, the subject is human.

[0228] As used herein, “preventing” refers to stopping or delaying the onset or progression of inflammation or a condition involving inflammation. Preventive measures may result in the cessation or delay of the development of inflammation or a condition or its symptoms, the prevention of the progression of inflammation or a condition or its symptoms, or the reduction of inflammation or a condition or its symptoms that have already occurred (if such a thing happens). Preventive measures may also play a role in supporting, maintaining, and / or protecting bodily systems. It should be understood that the term “treating or preventing” does not preclude the possibility of both treatment and prevention of a condition. A “therapeutic” effect or “therapeutic” approach may include treatment or prevention or both.

[0229] As used herein, “treating” means to improve or resolve inflammation or a condition involving inflammation. Treatment will result in a reduction (e.g., improvement or resolution) of inflammation or a condition, or one or more symptoms of inflammation or a condition. Resolution in treatment includes partial or complete reversal of inflammation or a condition or its symptoms. This covers partial or complete healing, such as improvement in one or more related health parameters. Treatment may include manifestations of reducing inflammation or a condition or its symptoms. Treatment may also suppress existing inflammation / inflammatory condition or its symptoms, or provide relief from existing inflammation / inflammatory condition or its symptoms. In the context of “treatment,” it specifically refers to the healing of wounds and rashes.

[0230] "Alleviation" refers to the improvement of inflammation or a condition involving inflammation. Alleviation will result in a reduction (e.g., improvement) of inflammation or a condition, or one or more symptoms of inflammation or a condition. It specifically encompasses healing, such as improvement in one or more related health parameters. Alleviation includes the relief of inflammation or a condition or its symptoms. Alleviation can also play a role in suppressing existing inflammation / inflammatory conditions or their symptoms. It specifically refers to the relief of inflammation in wounds and rashes.

[0231] The term "effective amount" refers to a sufficient amount of active ingredient in a suitable composition and in a suitable dosage form to treat or prevent the indicated condition or at least one symptom thereof. The "effective amount" will vary depending on the ingredient used, the type of treatment, and the species, age, weight, health status, etc., of the individual being treated.

[0232] "Combination" refers to the combined use of two or more components (e.g., two or more active components). Use can be by co-formulating the components (i.e., a combination formulation), or by using the components simultaneously, sequentially, or separately (e.g., via different formulations, identical formulations, or co-formulations). These and other specific combinations are covered in this disclosure.

[0233] Method for preparing the composition

[0234] The inventors of this invention have discovered that gingerone compositions prepared from ginger root according to the disclosed method possess significant anti-inflammatory activity, exceeding that of commercially available gingerone compositions. Therefore, this disclosure generally relates to gingerone compositions prepared from ginger root and methods thereof.

[0235] On one hand, this disclosure provides a method for producing gingerone from ginger roots by subjecting them to alkaline treatment. As described herein, alkaline treatment may include incubation in an alkaline solution. The ginger roots used as raw material can be fresh ginger roots. For example, optimizing the retention time of the ginger roots in the soil before harvest may be helpful in preparation. In this way, the ginger roots used will be fresh, and the advantageous characteristics of fresh ginger roots will be retained.

[0236] The disclosed preparation method produces highly effective plant extracts. As an example, to optimize freshness, ginger roots 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 treatment. For example, fresh ginger can have a moisture content of about 80% to about 95%, about 81% to about 95%, or about 82% to about 95%, or about 83% to about 95%, or about 85% to about 95% on a wet basis.

[0237] Alternatively, the ginger root can be dried before processing. For example, the ginger root can be dried at about 40°C to about 70°C, or at about 55°C to about 65°C, or at about 60°C. The drying can be carried out for about 1-72 hours, or about 1-48 hours, or about 1-24 hours, or about 1-20 hours, or about 1-18 hours, or about 1-10 hours, or about 1-5 hours.

[0238] In some respects, the ginger root selected for 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. As another example, ginger root may contain 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 some cases, it may be advantageous to test the level of gingerol (e.g., 6-gingerol) in the raw material before starting the methods disclosed herein.

[0239] On one hand, the method involves subjecting the juice and / or residue from ginger roots to alkali treatment. The ginger juice and / or residue can be obtained through soaking and / or pressing. Soaking may include homogenization using a mixer, food processor, or similar machine. For pressing, machine or manual pressing can be used, specifically screw pressing. The solid matter remaining after pressing (ginger residue) can be pressed again to obtain ginger juice. This can be repeated as needed. Various juice and residue samples can be mixed before alkali treatment, for example, juice sample A + juice sample B, or residue sample A + residue sample B, or juice samples A and B + residue samples A and B.

[0240] Optionally, the ginger residue can be subjected to hot water treatment to obtain a diluted juice. For example, water can be added to the residue in a 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) by weight. The water can be added at, for example, from about 40°C to about 80°C, or from about 50°C to about 70°C, or from about 55°C to about 65°C, or at about 60°C. The incubation time in the water can be from about 5 minutes to about 60 minutes, or from about 10 minutes to about 30 minutes, or from about 15 minutes to about 20 minutes, or about 15 minutes. The diluted juice sample can then be subjected to alkali treatment. Prior to alkali treatment, the diluted juice sample can be mixed with other juice samples.

[0241] On one hand, potassium hydroxide (KOH) can be used for alkaline treatment. For example, solid KOH, such as KOH granules, can be used. As an example, solid KOH can be at an initial concentration of about 100%, or about 90% to about 100%. Alternatively, liquid KOH can be used. As an example, liquid KOH can be at an initial concentration of about 50%, or about 40% to about 60%, or about 45% to about 55%. The concentration of KOH used when treating mixtures (e.g., the final concentration) can be, for example, about 0.1% to about 6%, or about 0.5% to about 5.5%, or about 1% to about 6%, or about 1.5% to about 5.5%, or about 2% to about 4%, or about 1.5% to about 3.5%, or about 2% (v / v). As an alternative, calcium hydroxide (Ca(OH)₂) can be used in alkaline treatment. For example, liquid Ca(OH)₂ can be used in alkaline solutions. The concentration of Ca(OH)2 used when processing the mixture can be, for example, from about 0.5% to about 4%, from about 1.5% to about 3.5%, from about 1% to about 2%, or about 3.0% (v / v). The liquid form can include, for example, a stock solution of about 25% to about 65%, or about 30% to about 60%, or about 35% to about 55%, or about 45% to about 55%, or about 50%.

[0242] In some respects, alkaline treatment can bring the pH level of the treated solution to approximately pH 9 to approximately pH 14, or approximately pH 9.5 to approximately pH 13.5, or approximately pH 10 to approximately pH 13.5, or approximately pH 10.5 to approximately pH 13.5, or approximately pH 11.5 to approximately pH 13.5, or approximately pH 12.5 to approximately pH 13.5, or at least pH 13. Alkaline treatment can be carried out at sufficiently high temperatures for a sufficient duration to obtain the desired level of gingerone. For example, alkaline treatment can be carried out for approximately 1–72 hours, or approximately 1–48 hours, or approximately 1–24 hours. Other examples include treatments of approximately 1 hour to approximately 30 hours, or approximately 1 hour to approximately 20 hours, or approximately 1 hour to approximately 10 hours, or approximately 1 hour to approximately 15 hours, or approximately 1 hour to approximately 7 hours, or approximately 1 hour to approximately 6 hours, or approximately 1 hour to approximately 5 hours, or approximately 1 hour to approximately 4 hours, or approximately 0.5 hours to approximately 3 hours, or approximately 0.5 hours to approximately 2 hours, or approximately 1 hour to approximately 2 hours, or at least 1 hour, or approximately 2 hours, or approximately 1 hour. As a specific example, alkali treatment can be carried out at approximately 40°C to approximately 70°C, or approximately 50°C to approximately 60°C, or approximately 55°C to approximately 65°C, or approximately 60°C. It should be understood that lower temperatures may allow for longer treatment times. For example, alkali treatment carried out at room temperature may last approximately 3 days to approximately 9 days, or approximately 5 days to approximately 9 days, or approximately 5 days to approximately 7 days.

[0243] Following alkali treatment, the mixture may be further processed, for example, by one or more of the following: neutralization, extraction, and drying. For neutralization, citric acid or other acid compositions may be used. As an example, 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, citric acid of about 10 g / L to about 700 g / L can be used, or citric acid of about 10 g / L to about 600 g / L, or about 10 g / L to about 500 g / L, or about 10 g / L to about 400 g / L, or about 10 g / L to about 300 g / L, or about 10 g / L to about 200 g / L, or about 10 g / L to about 100 g / L, or about 10 g / L to about 50 g / L, or about 10 g / L to about 40 g / L, or about 10 g / L to about 30 g / L, or about 10 g / L to about 20 g / L, or about 15 g / L to about 16 g / L can be used.

[0244] For extraction, gingerone extraction can be achieved by one or more alcohol extractions (e.g., one or more ethanol extractions). As an example, alcohol extraction (e.g., ethanol extraction) can be carried out for about 1-72 hours, or about 1-48 hours, or about 1-24 hours, or about 6-24 hours, or about 8-24 hours, or about 12-24 hours, or about 18-24 hours. In one particular aspect, alcohol extraction can be carried out 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. Alcohol extraction can be carried out, for example, at about 35°C to about 65°C, or about 45°C to about 55°C, or at least 50°C, or at about 50°C.

[0245] One or more drying steps may be used before and / or after extraction (e.g., alcohol extraction). For example, freeze-drying or thermal drying may be employed. Specifically, the amount of ethanol in the extract composition can be reduced by air drying, rotary evaporation, freeze-drying, or other techniques. For some drying methods (e.g., rotary drying), heat and pressure may be increased to remove residual liquid. For example, drying may be carried out under vacuum, such as at 50 mBar or less, 45 mBar or less, or 40 mBar or less. As another example, drying may be carried out at about 35°C to about 50°C, or at least 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. When the material is dried after neutralization but before alcohol extraction, drying may be carried out 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 at about 55°C to about 65°C, or not greater than 60°C, or at about 60°C. Other alternative drying methods are described herein.

[0246] As part of the initial treatment, ginger roots can be washed or disinfected. Plant components (e.g., fruits or seeds) can be cleaned using an assembly with one or more roller brushes to remove any attached foreign matter. Conventional washing techniques can then be used. For example, a series of nozzles can be used to wash the components. Depending on local regulations and requirements, washing additives that help clean or reduce the microbial count on the plant components may be used. For example, the plant components can be washed with water by chlorine washing and / or ozone impregnation, followed by rinsing with clean water.

[0247] As noted, it may be ideal to prepare liquid or semi-solid gingerone compositions from ginger root. As described herein, gingerone components can be extracted by chemical methods (e.g., solvent-based extraction). Solvent-based extraction can utilize one or more of the following: water, methanol, ethanol, or 2-propanol. Supercritical fluid extraction, such as CO2 extraction, can also be used to extract gingerone. Emulsions, pastes, suspensions, and syrups are also suitable. For example, in some aspects, it may be necessary to use pastes (e.g., gingerone or gingerone extracts) from ginger root or ginger root components. As an example, ginger root can be heated for several hours, filtered, and concentrated into a viscous concentrate. Upon thickening, the paste can be spread on a plate or transferred to packaging (e.g., bags, tubes, cans, bottles, or other containers). The paste can be transferred aseptically. It may be necessary to prepare the paste from mature plant components. The paste can be a smooth formulation.

[0248] In a specific aspect, this disclosure includes mechanical methods (e.g., juicing methods such as maceration and / or pressing) for extracting gingerone from ginger roots. In one embodiment, the pressing assembly may be adapted to perform a pulping or pulverizing process. Such a method can be relatively gentle and mild (soft pulping) compared to conventional fruit pulping techniques. For soft pulping, no significant decomposition or lysis of cells is utilized. The pressing belt may be multiple loops rotating around a series of pulleys. The distance separating the pressing belts may decrease along the direction of travel of the plant component. In this way, increased forces can be applied to the plant component as it travels along the length of the pressing assembly. In a particular aspect, as described herein, juice can be obtained from ginger roots using a pressing assembly or a mechanical press. Alternatively, or in addition, mechanical maceration may also be used to obtain juice. For example, commercial juicing equipment may be used.

[0249] Ginger root components (e.g., gingerone or gingerone extract) can be processed via a freezing step. This can be followed by a drying or evaporation step, or a combination of both. In an alternative embodiment, the component is dried or evaporated and then processed into a powder without an intermediate freezing step. Methods involving air drying or heat-assisted drying (e.g., oven drying) can be used. Drying can be achieved, for example, by one or more of the following: sunlight or solar drying, hot air drying, batch drying, rotary drying, tunnel drying, belt drying, fluidized bed drying, impact drying, puffing drying, drum drying, spray drying, vacuum drying, freeze drying, or permeation drying. Exemplary temperatures for drying include about 50°C–70°C, about 55°C–65°C, or at least 50°C, at least 55°C, at least 60°C, or at least 65°C. Evaporation can be achieved, for example, by one or more of the following: disc evaporation, batch evaporation, tubular evaporation, rising film evaporation, falling film evaporation, rising-falling film evaporation, or stirred film evaporation. Combinations of various drying and evaporation methods can be used. For example, filtration can be used, followed by freeze drying.

[0250] If freezing is used, it may be necessary to freeze the ginger root components (e.g., gingerone or gingerone extract) as soon as possible after their formation to maintain freshness. However, freezing can be carried out within 24 or 48 hours, depending on the requirements. Standard freezing methodologies can be utilized. Blast freezing is particularly suitable for this disclosure. The components can be frozen in standard-sized pallets used to collect the frozen product after processing. For example, the components can be cryopreserved (e.g., at -18°C) until needed. Optionally, the components can then be freeze-dried, i.e., lyophilized. Freeze-drying technology is widely used. Freeze-drying cycles can be up to 48 hours. In certain aspects, such processes can be carried out to avoid water formation and to minimize moisture content during processing. It should be understood that freeze-drying / lyophilization does not preclude the use of higher temperatures (i.e., above freezing temperatures). For example, higher temperatures can be used during the secondary drying stage of a freeze-drying / lyophilization process to remove residual moisture.

[0251] The resulting dried or evaporated components from ginger root (e.g., gingerone or gingerone extract) can then be ground into powder and used as appropriate. Standard grinding methods can be used. Standard sieve sizes can be used to produce the powder, for example, US 20, US 23, US 30, US 35, US 40, US 45, or US 50 sieve sizes can be used. The particle size of the powder can be 1.0 mm to 0.3 mm; or 0.84 mm to 0.4 mm; or 0.71 mm to 0.5 mm; or can be about 1.0 mm, about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47 mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm.

[0252] The composition can be formulated as a pharmaceutical composition. The composition can also be formulated as a dietary 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 respects, the composition can be prepared in liquid or solid form, or in a semi-solid form. This disclosure includes various formulations. In some respects, it may be desirable to formulate the composition as a powder. The powder can be provided in a free-flowing form or as a solid mass. The composition can be provided as a powder for forming a suspension, a powder for forming a solution, a bulk granule, or a bulk powder. As described in detail herein, the powder can be formulated as a tablet or capsule or other formulation.

[0253] It should be understood that for any liquid or semi-solid product obtained from ginger root, the liquid / semi-solid may be used in this form, or may be dried or evaporated to obtain a powder form for use as a pharmaceutical composition or dietary composition, as described herein. Similarly, it should be understood that for any solid product obtained from ginger root, the solid may be used as is (e.g., by grinding, sieving, or other treatment), or may be resuspended to obtain a liquid or semi-solid form for use as a pharmaceutical composition or dietary composition, as described herein.

[0254] Composition

[0255] The inventors of this invention have discovered that gingerone compositions prepared from ginger root according to the methods disclosed herein have significant anti-inflammatory and immunomodulatory properties, which can be used to reduce cellular inflammatory markers, reduce tissue damage, have antioxidant effects, and treat or prevent inflammation and inflammatory conditions in individuals.

[0256] The compositions disclosed herein can be prepared as one or more formulations, including pharmaceutical compositions and dietary compositions. As a non-limiting example, the percentage of gingerone or gingerone extract in the composition may be from about 0.01% to about 30%, or about 1% to about 30%, or about 1% to about 15%, or about 1% to about 10%, or about 1% to about 9%, or about 1% to about 8%, or about 0.1% to about 7%, or about 0.1% to about 6%, or about 0.1% to about 5%, or about 0.1% to about 4%, or about 0.1% to about 4%, or about 0.1% to about 3%, or a percentage of at least 1%, at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, or at least about 50%, or a percentage of about 6.25%, about 12.5%, or about 25%, these percentages representing the v / v value of the liquid composition, or the w / w value of the solid composition, or the w / v value of the liquid or semi-solid composition. In any of the various forms disclosed herein (e.g., liquid, solid, semi-solid, etc.), the composition may be free of or substantially free of aldehydes.

[0257] As an example, the solid composition may include about 0.5 mg / g to about 300 mg / g gingerone, about 1 mg / g to about 150 mg / g gingerone, about 1 mg / g to about 100 mg / g gingerone, or about 1 mg / g to about 80 mg / g, or about 1 mg / g to about 60 mg / g, or about 1 mg / g to about 50 mg / g, or about 1 mg / g to about 40 mg / g, or about 1 mg / g to about 20 mg / g, about 1 mg / g to about 15 mg / g, or about 1 mg / g to about 10 mg / g, or about 10 mg / g to about 60 mg / g gingerone, or about 10 mg / g to about 50 mg / g gingerone, or about 10 mg / g to about 40 mg / g gingerone, or about 10 mg / g to about 30 mg / g gingerone, or about 10 mg / g to about 20 mg / g gingerone, or about 10 mg / g to about 15 mg / g gingerone. mg / g gingerone, or at least about 50 mg / g gingerone, or at least about 40 mg / g gingerone, or at least about 10 mg / g gingerone (w / w).

[0258] Similarly, as another example, the liquid or semi-solid composition may include about 0.5 mg / ml to about 300 mg / ml gingerone, about 1 mg / ml to about 150 mg / ml gingerone, about 1 mg / ml to about 100 mg / ml gingerone, or about 1 mg / ml to about 80 mg / ml, or about 1 mg / ml to about 60 mg / ml, about 1 mg / ml to about 50 mg / ml, or about 1 mg / ml to about 40 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 15 mg / ml, or about 1 mg / ml to about 10 mg / ml, or about 10 mg / ml to about 60 mg / ml gingerone, or about 10 mg / ml to about 50 mg / ml gingerone, or about 10 mg / ml to about 40 mg / ml gingerone, or about 10 mg / ml to about 30 mg / ml gingerone, or about 10 mg / ml to about 2 ...30 mg / ml gingerone, or about 10 mg / ml to about 30 mg / ml gingerone, or about 10 mg / ml to about 30 mg / ml gingerone, or about 10 mg / ml to about 30 mg / ml gingerone, or about 10 mg / ml to about 30 mg / ml gingerone, or about 10 mg / ml to about 15 mg / ml gingerone, or at least about 50 mg / ml gingerone, or at least about 40 mg / ml gingerone, or at least about 10 mg / ml gingerone (w / v).

[0259] Topical compositions can be prepared for various purposes, such as for the hands (e.g., hand cream), preoperative tissues (e.g., surgical preparation of the skin), mucous membranes (e.g., treatment of inflammation of the bladder, urethra, or vagina, or cleaning of these cavities before medical procedures), wounds or burns (e.g., ointments, bandages, or dressings), the mouth or throat (e.g., mouthwash or lozenges), or the eyes (e.g., eye drops or ointments).

[0260] As a non-limiting example, a topical composition may comprise one or more of the following: a diluent (e.g., ethanol or other alcohols), an emollient (e.g., PEG-45, palm kernel glyceride, or isopropyl myristate), a humectant (e.g., glycerin or methyl propylene glycol), a carrier (e.g., one or more oils), an occlusive agent (e.g., mineral oil or dimethyl silicone oil), other conditioning agents (e.g., behenyltrimethylammonium methyl sulfate or polyquaternium-7), and a surfactant (e.g., a mild surfactant (e.g., amphoteric acid salt, hydroxyethyl...). Sulfonates, sulfosuccinates, particularly sodium lauroamphoacetate, sodium cocoyl hydroxyethyl sulfonate, disodium oleoylamino sulfosuccinate, sodium dodecyl sulfate, and C14-16 alkenyl sulfonate. Exemplary oils include olive oil, coconut oil (e.g., coconut-derived MCT oil), palm oil (e.g., palm kernel-derived MCT oil), any other MCT oil (medium-chain triglyceride oil), and any combination thereof. Other possible carriers include lecithin (e.g., in liquid form) and propylene glycol. Also refers to any combination of carriers described herein.

[0261] In other respects, the composition can be formulated for various routes of administration, including oral formulations. Compositions prepared for other enteral or parenteral administration routes are also included. Enteral formulations include, but are not limited to, oral, rectal, sublingual, sublipal, and oral formulations. Parenteral formulations include, but are not limited to, nasal, intraocular, vaginal, intralesional, transdermal, and transmucosal formulations. Standard methods can be used to formulate pharmaceutical compositions. See, for example, Remington: Essentials of Pharmaceutics, 2013, Pharmaceutical Press, London .

[0262] In certain aspects, the compositions disclosed herein can be prepared as powders or any other suitable dosage form. Topical formulations can be prepared as, for example, aerosols, balms, creams, dressings, drops, emulsions, films, foams, gels, jelly gels, liquids, lotions, masks, oils, ointments, pastes, powders, ointments, soaps, sprays, suspensions, solutions, tinctures, and vapors. Other topical formulations include bandages, dressings, patches, pads, sponges, strips, bandages, and other topical formulations indicated herein.

[0263] As described herein, the composition may be formulated as a semi-solid or liquid composition, for example for oral administration (e.g., directly through the mouth or via encapsulation or other forms), or for enteral or parenteral administration (e.g., via injection, esophagus, or other forms). Alternatively, the composition may be formulated as a powder for encapsulation, tableting, or addition or incorporation into other products.

[0264] Oral preparations can be formulated as, for example, drinks, drops, elixirs, emulsions, liquids, linctus, solutions, sprays, suspensions, syrups, tonics, or films, gels, jelly, gummies, lozenges, blocks, pastes, purees, pomace, powders, pills, or strips. In other respects, oral preparations can be formulated as tablets or capsules, for example, tablets or capsules containing liquid, semi-solid, or solid contents. Oral preparations can be provided in sachets, for example, powder sachets or gel or jelly sachets. Also included are oral preparations containing strips or containing solids from capsules for mixing with food or beverages. Oral preparations can be provided as a shooter or shot (by oral administration), for example, liquid injections, gel or jelly injections, paste injections, or powder injections.

[0265] Specifically, this includes delayed-release formulations, extended-release formulations, and rapidly disintegrating formulations. Specifically, it includes capsules (e.g., gel capsules), as well as sachets and chewable tablets. Furthermore, it includes combination formulations, which comprise the powder of this disclosure mixed with other beneficial drugs (e.g., one or more anti-inflammatory agents). Other formulations are also possible as described herein.

[0266] To achieve rapid onset of action or sustained release, the dissolution time of oral formulations can be altered. Oral formulations may also contain a mixture of sustained-release and immediate-release particles to produce rapid and sustained absorption at the same dosage. Special coatings can be used for oral formulations such as tablets and capsules to provide resistance to stomach acid. Oral formulations may also be coated with sugar, varnish, or wax to improve taste.

[0267] Therefore, tablets can be formulated as fast-dissolving tablets, and capsules can be formulated as sustained-release capsules. Tablets can be scored tablets, chewable tablets, effervescent tablets, orally disintegrating tablets, or tablets used to form a suspension. Capsules can be, for example, gel capsules, and can contain solid, semi-solid, or liquid contents. This includes gel capsules made by encapsulating a single gel capsule and two gel capsules. Hard-shell capsules and soft-shell capsules are particularly relevant. Non-gelatin capsules and caplets are also included.

[0268] It should be understood that certain formulations will be suitable for topical or other applications. Target-specific formulations are: ocular formulations (e.g., drops, ointments), ear formulations (e.g., drops, ointments), nasal or respiratory formulations (e.g., drops, sprays, inhaled compositions, aspirated compositions, nebulized compositions), skin formulations (e.g., soaps, sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, patches, strips, bandages, dressings, sponges, vapors), throat or oral formulations (e.g., drops, lozenges, mouthwash, toothpaste, sprays, pastes, gels, jelly, gummies), and mucosal formulations (e.g., sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, dressings, sponges).

[0269] Solid, semi-solid, and liquid compositions may combine gingerone or gingerone extract with one or more compounds to ensure a stable and active composition. For example, oral formulations (e.g., tablets or capsules) may contain: about 5% to about 50% w / w gingerone or gingerone extract; up to about 80% w / w one or more fillers, lubricants, flow aids, or binders; and up to about 10% w / w compounds to ensure that the tablet readily disintegrates, depolymerizes, and dissolves in the stomach or intestine. In a particular example, the extract of this disclosure (e.g., an ethanol tincture) may be mixed with one or more carrier substances (e.g., glycerol, glyceryl esters, hydrogenated oils, polyethylene glycol, poloxamer, etc.) and included in a capsule (e.g., a gel capsule).

[0270] Therefore, the composition may contain a variety of excipients, such as one or more of the following: solubilizers, stabilizers, buffers, tension modifiers, fillers, thickeners, viscous / thickness reducers, emollients, surfactants, chelating agents, adjuvants, anti-adhesives, anti-caking agents, binders, coatings, disintegrants, lubricants, flow aids, flow agents, adsorbents, flavoring agents, flavor masking agents, coloring agents, sweeteners, or preservatives.

[0271] As an example, the composition may contain less than 1% of a preservative, for example, about 0.005% to about 0.5%, or about 0.05% to about 0.15%, or may contain about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, or about 0.2% of a preservative, these percentages representing w / v values ​​or w / w values. Useful preservatives include, but are not limited to, sorbic acid, sodium sorbate, potassium sorbate, citric acid, ascorbic acid, malic acid, tartaric acid, propionic acid, and benzoic acid, for example, benzoic acid in the form of its sodium salt, such as sodium benzoate.

[0272] Other useful excipients include, but are not limited to: glyceryl stearate, magnesium stearate, and stearic acid; sugars and their derivatives, such as disaccharides: sucrose, lactose; polysaccharides and their derivatives, such as starch, cellulose or modified cellulose (e.g., microcrystalline cellulose) and cellulose ethers (e.g., hydroxypropyl cellulose); sugar alcohols, such as isomalt, xylitol, sorbitol, and maltitol; proteins, such as gelatin; polysaccharides, such as pectin; gums and other thickeners, such as gum arabic, gellan gum, guar gum, locust bean gum, saffron gum, agar, and arrowroot gum. Excipients include: carrageenan, gelatin, glycerin, kudzu root, lecithin, starch; synthetic polymers such as polyvinylpyrrolidone, polyethylene glycol; fatty acids, plant-based surfactants such as sunflower lecithin, waxes, shellac, plastics, and plant fibers (e.g., corn gluten); hydroxypropyl methylcellulose; crosslinked polymers such as crospovidone (crosslinked polyvinylpyrrolidone) and crospovidone sodium carboxymethyl cellulose (crospovidone sodium carboxymethyl cellulose); sodium carboxyacetic acid starch; silica, fumed silica, talc, and magnesium carbonate. Any combination of excipients may be used.

[0273] Various delivery systems can be utilized, such as nanoparticle delivery systems, including polymer nanoparticles (e.g., PEG, PLGA, PLA, chitosan, etc.), lipid-based nanoparticles (e.g., liposomes, micelle nanoparticles, phytosomes, etc.), nanocrystals / nanoshells, and inorganic nanoparticles (e.g., metal nanoparticles, dendritic polymers, etc.). For example, phytosomes that may include lecithin can be used to increase the absorption of gingerone or gingerone extracts, both topically and orally.

[0274] Liquid compositions can be stored as tinctures, for example in vials, pouches, ampoules, cartridges, or drug-loaded syringes. Compositions can also be transferred from vials to larger containers and mixed with other materials. Dry or evaporated compositions can be stored, for example in vials, cartridges, dual-chamber syringes, or pre-filled mixing systems. Before application, the dry form of the composition can be reconstituted into a liquid.

[0275] Exemplary unit doses of the composition comprise: about 0.1 mg to about 1000 mg of gingerone or gingerone extract, about 1 mg to about 500 mg of gingerone or gingerone extract, about 1 mg to about 250 mg of gingerone or gingerone extract, about 1 mg to about 200 mg of gingerone or gingerone extract, about 1 mg to about 100 mg of gingerone or gingerone extract, about 1 mg to about 50 mg of gingerone or gingerone extract, or about 1 mg to about 25 mg of gingerone or gingerone extract. The doses can be formulated for once-weekly, twice-weekly, three times-weekly, every other day, once-daily, twice-daily, or three times-daily, or more times as needed. If necessary, the dose can be adjusted for children, the elderly, overweight individuals, underweight individuals, or other patients. Dosage changes can be made according to standard methods. Therefore, it should be understood that a wide range of unit dosage forms can be conceived and prepared.

[0276] Using the composition method

[0277] As noted above, the disclosed compositions can be used to treat or prevent inflammation and various inflammation-related health conditions. For example, the disclosed compositions can be used to reduce the levels of pro-inflammatory cytokines or pro-inflammatory small molecules in an individual. In specific aspects, pro-inflammatory cytokines can be interleukin cytokines, such as IL-6 and / or IL-10. Pro-inflammatory cytokines can be tumor necrosis factor, such as TNF. Pro-inflammatory small molecules can be nitric oxide.

[0278] In some aspects, the composition may comprise gingerone or gingerone extract produced by the methods described herein. The compositions disclosed herein may also be prepared in one or more pharmaceutical forms. Furthermore, or alternatively, the composition may be prepared in one or more dietary forms, such as functional foods or beverages, natural adjuvants (e.g., natural additives), or natural supplements (e.g., dietary supplements).

[0279] In various aspects, the disclosed compositions can be used to target one or more inflammatory conditions. Inflammation is inflammation affecting one or more of the following: 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.

[0280] Target conditions include, but are not limited to: immune system disorders (e.g., autoimmune disorders), such as Alzheimer's disease (e.g., early Alzheimer's), 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), polymyalgia psoriasis, scleroderma, Sjögren's syndrome, and systemic lupus erythematosus (SLE); arthritis conditions, such as rheumatoid arthritis, ankylosing spondylitis, and arthritis. Arthritis, fibromyalgia arthritis, gouty arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, and Sjögren's syndrome arthritis; heart, circulatory, and lung conditions such as atherosclerosis, coronary artery disease, pulmonary hypertension, hypoxic pulmonary hypertension, pneumonia, acute respiratory distress syndrome, and coronavirus (e.g., Covid-19) respiratory conditions, cytokine storm syndrome; and proliferative conditions, including various cancers and tumors such as breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

[0281] Other target conditions include musculoskeletal conditions (e.g., conditions of bones, cartilage, fingers, joints, limbs, muscles, tendons, etc.) and back conditions (e.g., conditions of the spine or soft tissues of the back). It also refers to inflammatory conditions related to infection, such as gingivitis. Specifically, it refers to anti-inflammatory treatment of the skin. As a non-limiting example, the disclosed compositions can be used for one or more of the following: blisters, dermatitis, eczema, urticaria, lesions, papules, plaques, psoriasis, rashes, rosacea, ulcers, and wounds. Wounds can be acute or chronic (e.g., wounds that do not heal or recur). Specifically, it refers to surgical wounds and scars.

[0282] In certain aspects, the disclosed compositions can be used with one or more anti-inflammatory agents. For example, the compositions can be prepared as a combination formulation with one or more anti-inflammatory agents. Alternatively, the compositions can be used as separate formulations with one or more anti-inflammatory agents. In the case of using two or more active agents (e.g., gingerone compositions and anti-inflammatory agents), they can be used in a coordinated manner by simultaneous, sequential, or separate administration. Furthermore, the compositions described herein can be used in conjunction with a variety of medical or non-medical procedures. The use of the compositions can be performed before, during, or after the procedure, or any combination thereof.

[0283] As an example, anti-inflammatory agents may include one or more components obtained from plants, such as one or more plant compounds, mixtures, extracts, and / or oils. These include components derived from: manuka (e.g., manuka tree (manuka)). L. scoparium Malvaceae evergreen trees (e.g., narrow-leaved ribbon tree) Hoheria angustifolia ), Hoheria glabrata , Hoheria lyallii , Hoheria populnea , Hoheria sexstylosa ), Horopito (for example, Horopito) Pseudowintera colorata Kawakawa (e.g., kawakawa pepper) Piper Excelsum ), similar to arrowhead (koromiko) (e.g., upright long-stemmed flower ( Hebe stricta ), willow-leaf long-step flower ( Hebe salicifolia ) or elliptical leaves and long-flowered florets ( Hebe elliptica )), Poroporo (for example, Poroporo ( Solanum aviculare ), New Zealand laurel (pukatea) (for example, New Zealand laurel ( Laurelia novae-zelandiae Extracts from *Manuka* (e.g., *Manuka* oil) and *Peppermint* (e.g., leaf extract) are specifically mentioned. Extracts from the *Psilocybe* genus are also noted. Psilocybe (e.g., Dark Blue Naked Mushroom) P. azurescens ), Psilocybe nippon ( P. semilanceata ) and Blue-stemmed Psilocybe ( P. cyanescens The components of the cannabis plant ( ) Cannabis (e.g., Capsella spp.) (e.g., Capsella spp.) C. sativa )) of the ingredients.

[0284] This also includes essential oils, such as those from basil, bergamot, chamomile (e.g., Roman chamomile), clary sage, clove, copaiba, eucalyptus, fennel, frankincense, helichrysum, hops, lavender, marjoram (e.g., sweet marjoram), patchouli, peppermint, rose, rosemary, tea tree, thyme, and turmeric. It also includes honey (e.g., manuka honey), arnica (e.g., arnica oil, creams, or gels), activated charcoal, capsaicin, sesame, yarrow (e.g., used in various skin preparations), and comfrey (e.g., used in ointments or creams).

[0285] As another example, anti-inflammatory agents can include one or more pharmaceutical compounds. These include analgesics, antipyretics, and psychotropic agents. Examples include acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodoxacin, etoricoxib, biphenylacetic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxapazine, piroxicam, sulindac, tenoxicam, and others. Specifically, this refers to NSAID compounds and salicylates. It also refers to opioid compounds (e.g., KOR inhibitors) and steroid compounds (e.g., corticosteroids). Examples include butorphanol, nalbuphine, levonorphanol, levorphine, pentazocine, finazocine, and etazocine. Other examples include betamethasone, cortisone, defoscolate, dexamethasone, ethylamine, hydrocortisone, methylprednisolone, prednisolone, prednisolone, and triamcinolone. It also refers to cannabinoid compounds and fungal compounds. Examples include cannabidiol, cannabinol, and tetrahydrocannabinol. Other examples include psilocybin and dephosphorylated psilocybin.

[0286] Various anti-inflammatory agents can be modified and utilized according to this disclosure. Any combination of anti-inflammatory agents can be used. Any medical device and procedure can also be used in conjunction with the disclosed compositions and extracts.

[0287] As described herein, the compositions disclosed herein can be used as anti-inflammatory agents. In certain aspects, the compositions can be used in methods of reducing or delaying inflammation in certain tissues. These tissues include: joints, skin, ears, eyes, nose, mouth, gums, pharynx, digestive tract, heart, circulatory system, lungs, vagina, and urinary tract tissues, as well as other tissues indicated herein. The compositions can be applied, for example, to burns to reduce the chance of inflammation, or applied to the skin prior to surgery to prevent inflammation of the skin around the surgical site. The compositions can be used as hand cleansers (e.g., soaps or hand disinfectants), applied with or without water. The compositions can be used for minor skin irritations, cuts, or abrasions. The compositions can be used as mouthwashes or gargles, for example, to prevent inflammation caused by oral ulcers or gingivitis. The compositions can also be used as lozenges and throat sprays, for example, to relieve sore throats. Eye drops or ointments can be used to combat inflammation of the eyes (including the eyelids).

[0288] As described herein, the compositions disclosed herein can also be used as formulations for the treatment or prevention of inflammation and inflammatory conditions. Inflammation may affect one or more physiological components, including one or more of the following: the joint system, circulatory system, respiratory system, digestive system, renal system, excretory system, genitourinary system, skin system, nervous system, lymphatic system, endocrine system, muscular system, skeletal system, and sensory system.

[0289] As described herein, various routes of administration can be used for the compositions, including parenteral (e.g., topical) and enteral (e.g., oral) administration. Enteral administration can be performed via a duodenal tube or gastric tube (including a nasogastric tube) or other standard methods. Oral administration can be performed in tablets, capsules, sachets, drops, elixirs, medicated sugars, solutions, emulsions, suspensions, beverages, sauces, pastes, residues, syrups, gels, jelly, gummies, tonics, or various other methods. Topical application can be performed via drops, sprays, ointments, soaps, pads, sponges, dressings, bandages, or various other methods. Standard modes of administration can be used by a skilled technician. The compositions disclosed herein are not limited to specific forms of administration.

[0290] As an exemplary dosage, the composition may be administered in doses of about 1 mg to about 3000 mg of gingerone or gingerone extract, or about 100 mg to about 3000 mg of gingerone or gingerone extract, or about 100 mg to about 2500 mg of gingerone or gingerone extract, or about 100 mg to about 2000 mg of gingerone or gingerone extract, or about 1 mg to about 1800 mg of gingerone or gingerone extract, or about 100 mg to about 1600 mg of gingerone or gingerone extract, or about 100 mg to about 1400 mg of gingerone or gingerone extract, or about 100 mg to about 1200 mg of gingerone or gingerone extract, or about 100 mg to about 1000 mg of gingerone or gingerone extract. Additional examples include gingerone or gingerone extract in doses of about 10 mg to about 300 mg, or about 10 mg to about 200 mg, or about 10 mg to about 150 mg, or about 10 mg to about 100 mg, or about 10 mg to about 80 mg, or about 10 mg to about 60 mg, or about 10 mg to about 55 mg, or about 10 mg to about 50 mg, or about 10 mg to about 40 mg. These dose ranges are particularly applicable to ginger components (e.g., gingerone or gingerone extract) that have been dried and ground into powder.

[0291] The European Food Safety Authority (EFSA) has classified gingerone as safe for consumption in animals (e.g., rats) where no adverse effects (NOAEL) have been observed based on doses up to 128 mg / kg / day (EFSA, 2016, 14(8):4557). Exemplary doses can be determined, for example, for human individuals averaging 70 kg. Such exemplary doses may include, for example, gingerone or gingerone extracts at doses of 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 doses may include, for example, gingerone or gingerone extract at doses of about 0.1 mg / kg to about 20 mg / kg, or about 0.1 mg / kg to about 15 mg / kg, or about 0.1 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 8 mg / kg, or about 0.5 mg / kg to about 6 mg / kg, or about 0.1 mg / kg to about 4 mg / kg, or about 0.1 mg / kg to about 2 mg / kg, or about 0.1 mg / kg to about 1 mg / kg.

[0292] The dosage indicated herein may be administered once a week, every other day, once a day, twice a day, three times a day, or fewer or more times as needed. Administration may be with food or before meals. A technician will readily determine the appropriate dosage and formulation.

[0293] Example

[0294] The embodiments described herein are provided for the purpose of illustrating specific implementations and are not intended to limit this disclosure in any way.

[0295] Example 1: Preparation of gingerone

[0296] like Figure 1The initial sample of fresh ginger shown (400 gm) was from New Zealand and cleaned of impurities and dirt. The washed ginger was then cut into small pieces or chopped and subjected to an alkali treatment (800 g of a 0.5% potassium hydroxide distilled aqueous solution). The resulting mixture was stirred and placed in an oven at 60°C for 22 hours. It should be understood that the resulting mixture can also be placed in a water bath and maintained at approximately 60°C for the desired time.

[0297] The pH of the mixture was then adjusted to pH 7 by adding concentrated citric acid. 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 g, yielding a dry yield of 8.75%. The dried material was then scraped into a flask and extracted with 95% ethanol (210 ml). The flask was shaken and placed in an oven at 40°C for 16 hours. The extract was filtered through a glass funnel with a cotton stopper (extract 1). The remaining plant material was extracted again with 95% ethanol (extract 2), and then extracted more than twice with 50% ethanol (extracts 3 and 4).

[0298] The content of gingerone in the extract was directly analyzed. 294 mg of chopped fresh ginger sample was also extracted with 2 ml of ethanol, and the extract was analyzed. The results are shown in Table 1.

[0299] Table 1. Composition of the extract The dry weights of extract 1 and extract 2 were estimated by drying 5 ml portions of each extract.

[0300] result

[0301] The 400 g of fresh ginger root provided showed a content of 260 mg of 6-gingerol (i.e., 0.65 mg / g). This implies a theoretical maximum yield of gingerone from the treated material of approximately 171 mg (weight loss is due to the lower molecular weight of gingerone relative to 6-gingerol). Approximately 50% of the 6-gingerol remained unconverted. Further research is anticipated to increase the alkaline conversion of 6-gingerol to gingerone.

[0302] Ethanol extraction was performed using the minimum volume required to cover the treated plant material. This means that extract 1 (the most concentrated extract) contained 0.47 mg / mL of gingerone. It is foreseeable that, in principle, this concentration could be increased using multiple soaking methods or by evaporating some of the ethanol. Reducing the ethanol volume by 80% would yield a solution containing 2.35 mg / mL of gingerone.

[0303] A completely dried extract is expected to yield a solid extract concentration of approximately 16 mg / g. Higher concentrations can be expected if starting with a higher initial 6-gingerol content and / or a more complete conversion. For example, a theoretical dose of 25 mg could be achieved by directly formulating the dried extract into an oil or glycerol carrier to provide the desired dose of gingerone in one or two 500 mg capsules.

[0304] Most gingerone is extracted in the first extraction. In some cases, a second extraction is beneficial. The third and fourth extractions slightly increase the total amount. Overall, ethanol extraction is considered a very effective, efficient, and inexpensive preparation method.

[0305] Example 2: Preparation of gingerone

[0306] Overview: These studies show that aqueous alkali treatment followed by freeze-drying of fresh ginger significantly improves conversion rates, resulting in gingerone content of approximately 1% in the dried ginger. The treated ginger was then extracted with a supercritical CO2 and CO2 + ethanol co-solvent, achieving a combined extraction rate of 3% and an average gingerone concentration of approximately 12% in the extracted oleoresin. Furthermore, drying fresh ginger at a moderate temperature (60°C) followed by supercritical CO2 extraction yielded an extraction rate of 4.6%. Alkali treatment of the extracted oleoresin then produced a final product containing approximately 15% gingerone.

[0307] Drying: Fresh ginger samples imported from Fiji were sliced ​​into 2-5 mm pieces and placed in a single layer on a porous oven tray. Drying was carried out under forced convection. Drying was conducted at a moderate temperature (60°C) to remove moisture without converting gingerol. The process was considered complete when the moisture content of the ginger reached 7%. The obtained dried ginger was refrigerated until used in extraction experiments.

[0308] Catalytic conversion: A small-scale preliminary experiment was conducted by treating approximately 1.6 g of freshly chopped Fijian 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-to-weight ratio of the reagents to the ginger was approximately 3:1. The sample was then shaken and placed in a fume hood at room temperature or in an oven at 37°C or 60°C overnight prior to analysis. It should be understood that a suitable water bath can be used to maintain the sample at the desired temperature for the desired time.

[0309] Once the alkali treatment was selected, 5.2 kg of fresh ginger was chopped using a vertical chopper mixer (RobotCoupe R45). The ginger was then mixed with 0.5% KOH (approximately 0.1 N) at a liquid-to-solid ratio of 3:1 (volume:weight) to obtain a pH of approximately 12.5. The mixture was manually stirred and placed in an oven at 60°C for 24 hours. Subsequently, 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 in this process was refrigerated until used in extraction experiments.

[0310] Catalytic conversion results: As noted, different bases 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 contents of gingerone and 6-gingerol in these experiments were quantified (Table 2), and it was concluded that treatment with KOH at 60°C was the most effective and produced the highest concentration of gingerone. Table 2 shows the amounts of 6-gingerol (mg / g) and gingerone (mg / g) obtained under the corresponding conditions, and the results are expressed on wet basis. HPLC traces are shown below. Figure 2 As shown in the HPLC traces, 1% Ca(OH)₂ and 0.5% KOH exhibit similar performance. At 60°C, the ratio of gingerone to 6-gingerol is 6.5 for 0.5% KOH. At 60°C, the ratio of gingerone to 6-gingerol is 4.0 for 1.0% Ca(OH)₂.

[0311] Table 2. Peak areas of gingerone and 6-gingerol at 280 nm in the treated samples

[0312] This treatment 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%, meaning that there were 17 g of treated freeze-dried ginger per 100 g of chopped raw ginger. The contents of gingerone and 6-gingerol in the freeze-dried ginger were determined to be 10.2 mg / g and 3.3 mg / g (dry basis), respectively. See Table 2-1. The gingerone content is at least 10 times higher than that in dried ginger.

[0313] Table 2-1: Composition of processed and freeze-dried ginger

[0314] Extraction: Supercritical extraction experiments were conducted using alkali-treated ginger. Alkali-treated freeze-dried ginger was gently crushed by hand and placed into a 2 L extraction vessel with sintered filter plates at both ends, filling the vessel completely. Extraction was performed as described above until a sharp 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 ratio of approximately 10 wt% (i.e., 10 g ethanol / 100 g CO2). After introducing an ethanol:feed ratio of 2:1 (2 g ethanol / g feed), the ethanol pump was stopped. CO2 was then circulated to flush away any remaining ethanol in the bed. After extraction, the equipment was depressurized, and the residue was allowed to degas overnight before unloading. Ethanol present in the extract was removed by vacuum rotary evaporation. The extraction parameters are listed in Table 3.

[0315] Table 3. Summary of Extraction Parameters

[0316] Analysis: Samples for analysis were prepared by adding methanol after neutralization, as needed. The extract was dissolved directly in methanol. Analysis was performed by HPLC using an acetonitrile / 0.1% formic acid gradient. Detection was performed at 280 nm using a Phenomenex Kinetex C18 column (150 x 2.1 mm). Gingerone eluted at approximately 2 min, and 6-gingerol eluted at 5.2 min. Quantifications of gingerone and 6-gingerol were obtained from standard curves prepared using analytical standards employing these compounds.

[0317] Extraction Results: As noted, ginger treated with alkali and subsequently freeze-dried was extracted with CO2, followed by extraction with a CO2 + ethanol co-solvent. No free water was observed in the CO2 extract, and the ethanol obtained in the CO2 + ethanol extract was removed by vacuum rotary evaporation. The extract had a sweet caramel aroma. The yield obtained with CO2 was 1.5%. Adding 10% ethanol co-solvent resulted in an additional 1.5% extraction. The composition of the different fractions is shown in Tables 4 and 5. See also Figure 3 .

[0318] Table 4. Composition of extracts obtained from alkali-treated ginger

[0319] Table 5. Mass balance of gingerone (Z) extracted from alkali-treated ginger

[0320] The results show that for the alkali-treated samples, the CO2 extract contained 153 mg / g gingerone (15.3%) and 103 mg / g (10.3%) 6-gingerol, with a gingerone / gingerol ratio of approximately 1.5. The CO2+ethanol extract contained 91 mg / g gingerone and 52 mg / g gingerol (a gingerone / gingerol ratio of approximately 1.75). The residue or ginger after extraction was also analyzed and found to contain 5.4 mg / g gingerone. When considering the masses of the feed, extract, and residue (Table 4), the gingerone mass balance could be calculated to be 90.6%. This indicates that 90.6% of the gingerone initially present in the feed is present in both the extract and residue. This difference may be due to degradation during extraction or in the ethanol removal step. When calculated based on the extract, the gingerone extraction yield (i.e., the number of grams of gingerone extracted per 100 g of gingerone in the feed) was only 37%. However, 54% of the initial gingerone present in the feed residue remained unextracted in the residue, therefore the gingerone extraction rate could also be calculated as 46% based on the residue results. This explains the "missing" gingerone. Since the proportion of unextracted gingerone is significant, the extraction method can be further improved to reduce this situation. The extraction rate of gingerol was higher than that of gingerone (82% in the untreated sample and 71% in the treated sample) because it is more soluble in CO2.

[0321] In another experiment, CO2 extracts obtained from untreated ginger roots were subjected to alkali treatment to investigate the conversion of gingerol to gingerone. Overnight treatment at 60°C with 0.1 N and 1 N KOH yielded good results, resulting in gingerone concentrations of approximately 15% in the treated ginger. The resulting material appeared cleaner than alkali-treated crude ginger, making this a potentially more cost-effective extraction method due to the conversion occurring on a smaller volume of material. Indeed, for the same starting amount of fresh ginger (100 kg), based on the results obtained in this study, extracting untreated ginger followed by alkali treatment of the extract resulted in nearly twice the gingerone yield of the final product compared to other alternative processes (see comparison in the table below). However, even under these conditions, the total gingerone yield of this process was only approximately 0.1% (0.1 kg gingerone / 100 kg fresh ginger). Further optimization is expected to be feasible.

[0322] In another experiment, a CO2 extract sample of untreated ginger was mixed with KOH and converted into a gingerone-rich extract. The post-treatment neutralization step involved separating the gingerone from the aqueous reaction mixture in the form of a gingerone-rich resin. Three grams of oleoresin were taken in duplicate, and 9 ml of 1 N KOH was added to the sample in a plastic vial. The sample was then shaken and placed in an oven at 60°C overnight. The treated sample was then neutralized by adding 10 ml of 1 N HCl. This was a small excess of acid to ensure all KOH was neutralized. The addition was performed in two steps, with mixing after each step. The sample was centrifuged at 2000 rpm to separate water from the oleoresin. After centrifugation, most of the water was removed using a pipette. The resulting resin was then removed. Anhydrous ethanol (approximately 5 ml ethanol per 3 grams of resin) was added to one of the resin samples to produce a tincture (Sample 1). The other sample was preserved in resin form (Sample 2). The gingerone content of Sample 1 and Sample 2 was analyzed. The concentration of gingerone in the tincture (sample 1) was calculated to be 23 mg / g tincture, while the concentration of gingerone in the treated resin (sample 2) was 52 mg / g.

[0323] These values ​​for Samples 1 and 2 are lower than those obtained using the earlier treatment, for which the gingerone content was estimated at 150 mg / g. In this work, resin sample 2 was separated from the water and analyzed. Subsequently, the separated water was also analyzed, and an estimate of 50-55 mg (approximately 25%) of gingerone was obtained per 3 g batch. This result is quite unexpected, as gingerone has been reported to have very limited water solubility. This method contrasts sharply with the direct addition of alcohol to the crude neutralized product, as a significant portion of the gingerone appears to remain in the water and is not separated from the resin.

[0324] Because water can extract some gingerone from the resin, high yields of gingerone may also be obtained after processing the product by drying the completely neutralized alkali. Given that gingerone appears to be more soluble in water after alkali treatment than previously reported, further process optimization will be conducted to improve the yield.

[0325] Example 3: Further reaction methods and comparisons

[0326] Overview: Gingerone is not naturally occurring in ginger, but rather a product derived from gingerol through processing. In these studies, alkali-treated and dried ginger samples were obtained from Samoa and extracted with ethanol using two different extraction conditions: extraction at 40°C for 20 hours (extract A) and extraction at room temperature for 7 days (extract B). The results showed that the gingerone concentration obtained from extract A was 41.4 mg / g, and the gingerone concentration obtained from extract B was 43.8 mg / g.

[0327] Extraction A: Processed ginger from SROS (Scientific Research Organisation of Samoa) was placed in two separate plastic bags. The contents of the two bags were combined and frozen at -80°C, then ground using a Wiley grinder with a 2 mm sieve. The ground material (782.3 g) was then placed in a round-bottom flask with food-grade ethanol at a ratio of approximately 5:1 by weight. The flask was then placed in a 40°C water bath and stirred at 5 rpm overnight (total extraction time 20 hours). After this, the mixture was filtered under vacuum, and the ethanol was removed by rotary evaporation under vacuum to produce 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 collected and stored under refrigeration after nitrogen purging for possible future bioassays.

[0328] Extraction B: As described above, ginger was frozen and ground. The ground material (785.9 g) was placed in a container with food-grade ethanol at a ratio of approximately 5:1 by weight. The ginger was infused in ethanol at room temperature (22-29°C) for 7 days. Samples were taken on days 1, 3, and 7. After 7 days, the mixture was filtered under vacuum, and the ethanol was removed by rotary evaporation under vacuum, yielding 49.3 g of a highly viscous, dark brown resin with a characteristic ginger aroma. Figure 1 The resin obtained was very similar to that obtained in extraction A. 10 g of this resin (extract B) was taken and refrigerated under nitrogen purging for future possible bioassays. The extraction yield was 6.3%.

[0329] Gingerone and Aldehyde Analysis: Gingerone was quantified in the starting material (i.e., treated ginger) and the final two resins, as well as in samples from day 1, day 3, and day 7 of extraction B, by HPLC (note that the liquid sample from day 7 is equivalent to the final resin sample). The HPLC quantification method involved adding methanol and grinding the sample before analysis. The gingerone content in all fractions is shown in Table 6. The mass balance and yield of gingerone are shown in Table 7.

[0330] Table 6. Comparison of gingerone content in different fractions under different extraction conditions

[0331] Table 7. Mass balance and yield of gingerone

[0332] As indicated above, extract A was obtained by treatment at 40°C for 20 hours, while extract B was obtained by treatment at room temperature for 7 days. The results in Tables 6 and 7 indicate that higher levels of gingerone were obtained by treatment at room temperature for a longer period, although high yields could also be obtained by increasing the temperature to 40°C.

[0333] To determine the gingerone content in the raw material, it was first extracted into a suitable solvent. In one process, the extraction was performed with ethanol, resulting in a lower gingerone content (1.44 mg / g). In a second process, the extraction was performed with methanol, and the ginger was ground with the solvent in a mortar and pestle. This resulted in a higher gingerone content (3.1 mg / g). For reference, a Samoan laboratory reported a gingerone content of 1.86 mg / g for this material.

[0334] Based on this, the gingerone yield of extract A (i.e., the amount of gingerone in the extract relative to the amount of gingerone in the feed) was estimated to be 75%, and the gingerone yield of extract B was estimated to be 89%. When determining the gingerone content, the 6-gingerol peak in the HPLC analysis was consistently observed to be approximately 1 / 8 the area of ​​the gingerone peak. This indicates that the extraction had almost no effect on the ratio of gingerone to gingerol.

[0335] Samples of both final resins were dissolved in ethanol and examined by GC-MS for aldehyde analysis. Hexanal levels were found to be very low (too low to quantify). Hexanal is a byproduct of the reaction that forms gingerone, but it is volatile. The identity of the hexanal peak was confirmed by library matching of mass spectrometry data and separate injection of hexanal standards.

[0336] discuss

[0337] Each study described in this article effectively produced gingerone. Table 8 provides a comparison of the results from Example 2 and Example 3.

[0338] Table 8. Comparison of gingerone yields

[0339] Li et al. obtained similarity values ​​for fresh and dried ginger. 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. As described above, the progress of Example 2 far exceeds the standard reverse aldol reaction. As described above, Example 3 provides further progress using temperature and pH adjustments and extraction. As described herein, further progress is provided in Examples 5, 6, and 12.

[0340] Regarding Samoan ginger, it was noted that the ginger was not harvested at the required time (9 months in the ground), which affected the level of gingerol present in the ginger, and consequently the gingerone content in the final product. Therefore, additional benefits were expected. Regarding Fijian ginger, its gingerone content was significantly higher than that of Samoan ginger (10.2 : 1.44 = 7.08 x higher). This means that if the experimental conditions of Example 3 were applied, the total yield of Fijian ginger could be estimated at 310 mg / g. That is: 43.8 mg / g (the amount obtained from Samoan ginger in Example 3) x 7.08 (the higher starting content in Fijian ginger) = 310 mg / g.

[0341] Table 5 in Example 2 shows the yield of pH-treated ginger followed by CO2 extraction. 322 g of fresh ginger was found to provide 153 mg / g gingerone. In comparison, Example 3 used 785.9 g (2.4 times more product than used in Example 2) and provided 43.8 mg / g gingerone. However, this lower yield can be explained by the lower level (1.44 mg / g gingerone) in the starting material from Samoa.

[0342] Example 4: Processing method using juicing and alkali treatment

[0343] Overview: Ginger root was mechanically juiced, and the levels of 6-gingerol in the juice and residual solids were determined. Most of the 6-gingerol was present in the juice. Treatment of the juice with alkali showed that all 6-gingerol was efficiently converted to gingerone within 5–6 hours at 60°C.

[0344] Overview of juicing: Fresh ginger (500 g to 1000 g) is pretreated by mixing / soaking and pressing. The liquid fraction is retained, and the pulp is further washed with warm water (4 parts water to 1 part ginger) at 55-60°C for 10-15 minutes. This is done in a covered container. Then, pressing is performed again. The 6-gingerol content of each fraction is analyzed (a total of 5 analyses): 1) a fresh ginger sample taken immediately before treatment; 2) the liquid fraction after initial mixing / soaking; 3) the ginger pulp after initial mixing / soaking; 4) a second liquid fraction collected after further washing of the initial pulp; 5) the final ginger pulp after washing and pressing as described above. After the final pressing (i.e., samples 3 and 5 above), the moisture content of each batch of ginger pulp is read.

[0345] Overview of alkaline treatment: The liquid fraction was treated with an alkaline solution to convert it into gingerone. Samples were taken and analyzed for gingerone content at a series of time points. Samples were treated on a small scale with KOH (5% as previously stated). Treatment was performed at room temperature, 30°C, and 60°C using 1 ml of sample at 1 hour, 2 hours, 3 hours, and 5 hours. Additionally, one sample was treated at 60°C for 24 hours. Up to 15 samples were analyzed.

[0346] Juicing Method: Two fresh ginger root samples were obtained, one local (organic, from Evithe, Petone) and the second from Phil Rasmussen, Auckland. Both samples appeared to be plumper / juicier than ginger roots from a regular supermarket. Moisture content was determined by slicing each root into approximately 10 g pieces, freezing with liquid air, and then freeze-drying. 6-Gingerol content was determined by extraction of the roots with methanol. For this purpose, 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 min. HPLC analysis was then performed, with detection at 280 nm. The results are shown in Table 9.

[0347] Table 9. Moisture content of ginger

[0348] A sample from Auckland was selected for juicing. For this, 635 g of ginger root was processed using a household juicer. This included a rotary screw drive with a mesh juice filter and an adjustable solid nozzle (see [link to juicer]). Figure 4A and Figure 4B The juicer removed 516.3 g (81%) of liquid (juice 1, J1) and collected 101.5 g of solids (residue 1, M1). Some of the dried solids were taken for analysis, and 90.5 g was extracted with 360 ml of hot tap water. The mixture was allowed to stand for 15 minutes before being returned to the juicer. From this step, 350 g of juice (juice 2, J2) and 70.2 g of solids (residue 2, M2) were recovered.

[0349] Both liquids were refrigerated overnight. Both were cloudy with solid precipitate. The samples were shaken well before analysis or processing. The 6-gingerol content of the juice samples was analyzed 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.

[0350] The results are shown in Table 10 below. The total gingerol content in each material was obtained by multiplying the gingerol value by weight. The juice was found to contain 81.6% of the measured gingerol. The calculated total gingerol in the feed was lower than the recovery, indicating that some gingerol was extracted from the root. The percentage of gingerol is based on the total measured gingerol in the residue and juice (not the feed measurement).

[0351] Table 10. Weight and gingerol content

[0352] Alkali treatment: This work was performed on the first juice (J1) recovered during the juicing process (as above). Juice samples (after shaking to suspend all solids) were reacted with KOH at room temperature, 30°C, or 60°C for 1 h, 2 h, 3 h, 5 h, and 24 h at 60°C. Three concentrations of KOH were also tested: 0.5%, 1.0%, and 2.0%. A 2 N KOH solution (5.6 g KOH in 50 ml water) was prepared. To produce KOH concentrations of 0.5%, 1%, and 2% in each sample, 0.25 ml, 0.5 ml, or 1 ml of 2 N 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 completed by taking 200 µl from each sample, adding 200 µl of 1 N HCl, and then adding 500 µl of ethanol. After centrifugation, the samples were directly injected into the HPLC system. The peak areas of gingerone and gingerol were compared (see below).

[0353] These results indicate that treatment with 2% KOH can achieve complete conversion to gingerone within 5 hours. (See also...) Figure 5C Incubation at 60°C is particularly advantageous. See also Figure 5C The results are expressed as peak areas of gingerone (Z) and gingerol (G). It is noted that a solid was present in the KOH-treated sample, precipitated in the test tube. For analysis, the test tube was shaken, and a wide-mouth pipette was used for sampling to avoid clogging.

[0354] Example 5: Additional treatment method using juicing and alkali treatment

[0355] Overview: The objective is to produce ginger extracts that convert 6-gingerol to gingerone via a base-catalyzed reverse aldol reaction. The current process seeks to reduce processing time and water consumption. The process has been piloted on a scale of approximately 40 kg, with plans to further develop it on a scale of 200 kg.

[0356] In short, fresh ginger is received, treated with alkali, and then freeze-dried to produce treated ginger powder. This powder is 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.

[0357] Methodology: Imported fresh ginger underwent alkali pretreatment and drying. For this purpose, 36.67 kg of fresh ginger was pressed in a Vincent Corporation CP-4 screw press to produce two streams: ginger juice and pulp. The screw press was set to VSD speed of 50% and cone pressure of 2 bar. The pulp, after the initial pressing, was a second press to remove any remaining juice.

[0358] Combine the two juices. Heat the juice to 60°C and add KOH to a final concentration of 2% w / w. Maintain the alkalized juice at 60°C for 5 hours to convert gingerol to gingerone. Neutralize the juice to pH 7.2 by adding anhydrous citric acid. Freeze-dry and grind the juice now containing gingerone.

[0359] Samples were taken from: fresh ginger (ZINGO); two residues (GMARC and GMARC2); juice before the addition of KOH (GKOH0); juice after 2 hours, 3 hours and 5 hours of treatment (GKOH2, GKOH3, GKOH5); and the final dried extract (GPE).

[0360] In addition, the GMARC2 sample was extracted with hot water as follows: Water was added to GMARC2 at a ratio of 5:1 w:w. The mixture was heated to 60°C and maintained at this temperature for 15 minutes. Using the same settings as above, the extract was separated from the solids by screw pressing. Samples were taken from: extract (GMARC2 HWEX); residue (HW MARC). The total solids (LOD, 16 hours at 100°C) and gingerol or gingerone content of each sample were analyzed by HPLC.

[0361] Sample Analysis: Analyzed using HPLC with UV detection at 280 nm. Sample preparation was as follows: (1) Liquid samples (e.g., juice) were diluted 1:1 with ethanol and centrifuged. Liquid samples containing alkali were diluted 1:1:1 with 1 N HCl and ethanol; (2) Solid samples (e.g., raw ginger or ginger residue) were extracted by two extractions with ethanol (ultrasonic treatment, heating at 60°C for 20 minutes, vortexing and centrifugation) and the supernatants were combined. The solid extract (approximately 5 g) was typically prepared in 50 mL for analysis. Raw fresh ginger was coarsely chopped and then mixed with ethanol using an ULTRA-TURRAX® mixer. Quantification was performed by comparison with a standard curve prepared using gingerol. Gingerol was molecular weight corrected.

[0362] Pressing: 36.67 kg of raw Fijian ginger was received and pressed. The pressing was efficient, producing a large amount of light green juice and fibrous residue. 28.92 kg of juice was recovered from the first press. A second press was then performed on 6.6 kg of residue to recover an additional 2.18 kg of juice. The total juice yield was 31.1 kg, equivalent to 86% of the raw ginger feed by weight. The final recovered residue weight was 4.05 kg. At the end of an operation, there is typically 1-2 kg of residue remaining in the screw press. This results in a slight difference between the feed weight and the combined weight of residue and juice.

[0363] Hot water extraction: 3.62 kg of GMARC2 and 18.1 kg of water were heated to 60°C and extracted at 60°C for 15 minutes, followed by separation by screw pressing. 21.18 kg of the mixture was then pressed. Note that approximately 500 g of water was lost due to evaporation during extraction. 17.62 kg of extract was recovered and subjected to secondary sampling analysis. Additionally, 2.69 kg of residue was recovered.

[0364] Alkali treatment: For this purpose, 1.236 kg of 50% KOH solution was added to 31.1 kg of ginger juice to achieve a target KOH concentration of 2%. The pH after adding KOH was 12.18. After adding KOH, 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 adding citric acid was 7.23.

[0365] Freeze-drying: The treated juice was transferred to a freeze dryer tray and frozen overnight, then transferred to a Cuddon FD80 freeze dryer. A total of 28.51 kg of juice was loaded onto the tray and dried. Approximately 3 kg of juice was lost before freeze-drying due to manual handling. After drying, 2.86 kg of dried extract was collected, representing a total drying of 10% of the juice mass. This was ground and sampled a second time. After grinding, secondary sampling, and handling the loss, a total of 2.19 kg was stored in foil bags until further processing. Approximately 1.6 kg of this was sent for ethanol extraction (see Example 6).

[0366] The mass balance summary is as follows.

[0367] Juicing

[0368] Alkali treatment

[0369] freeze-drying

[0370] The levels of gingerol and gingerone were measured as described above. The results are shown in Table 11 below.

[0371] Table 11. Levels of gingerol and gingerone

[0372] This table shows the gingerol content at different stages of pretreatment and processing. Based on these measurements, a total of 19.1 g of 6-gingerol was found in 36.67 kg of raw ginger feed. The gingerone content in the dried ginger extract (GPE) was 5.7 mg / g. Therefore, the total gingerone content (before loss and milling) in 2.86 kg of dried powder was 16.30 g. The gingerol concentration in the pre-conversion juice (GKOH-0) was 6.9 mg / g (on a dry basis). When converted back to a wet basis, using a solids concentration of 6.67% (before adding KOH and anhydrous citric acid to increase the total solids content to approximately 10%), the total gingerol in the juice was 14.32 g. The GKOH-2, -3, and -5 values ​​for 6-gingerol are pending confirmation.

[0373] The mass balance between gingerol in the juice and gingerone in the final powder extract is not precisely consistent. This may be attributed to variations in measurements. From the overall mass balance calculations, the amount of residue appears to be increased. When the residue was extracted with hot water, the extract contained 0.22 mg / g of 6-gingerol, corresponding to a total of 5.7 g of 6-gingerol in the extract, approximately 25% of the 6-gingerol in the feed. For water extraction, this requires a total of 18 kg of water. This, in turn, increases the mass of KOH and citric acid and increases the drying load by 58%. Therefore, in some cases, water extraction may need to be omitted.

[0374] Notably, these experiments demonstrate that almost complete recovery of 6-gingerol (in the form of gingerone) was achieved in the final product. Samples GKOH2, GKOH3, and GKOH5 collected during the conversion reaction showed that the conversion from 6-gingerol to gingerone occurred during the first two hours of treatment, and the gingerone levels in samples collected after two hours of treatment did not increase significantly. Considering that the conversion appears to be complete after two hours, this incubation time (or even a shorter incubation time) would be sufficient.

[0375] The conclusion is that the processing method, including pressing followed by KOH treatment in the juice phase, is an effective production method. This represents a significant advancement beyond the standard alkaline-catalyzed reverse aldol reaction. Further experiments will use more than 200 kg of ginger as feedstock. The 6-gingerol level in this batch is 1.1 mg / g, which is expected to be reflected in the corresponding gingerone level.

[0376] Example 6: Ethanol Extraction Process and Analysis

[0377] Overview: Fresh ginger was received, treated with alkali, and then freeze-dried to produce treated ginger powder (see Example 5 above). The powder was extracted with ethanol at room temperature for 3 days, and samples were taken at 24, 48, and 72 hours to evaluate the extraction process.

[0378] Methodology: The treated ginger produced as described above (Example 5) was refrigerated until use. Approximately half of the received ginger was extracted with XNS food-grade ethanol at room temperature. The treated ginger and ethanol (using a ginger-to-ethanol weight ratio of 1:5) were placed together in a 10 L glass container equipped with a top stainless steel stirrer. The mixture was stirred for 72 hours at a speed sufficient to prevent solids from settling at the bottom.

[0379] After 24 hours, the stirrer was turned off, and the solids were allowed to settle for 10 minutes. Then, 50 ml of sample was removed from the top. After another 24 hours, a second sample was removed using the same procedure. After a total of 72 hours, stirring was stopped, and the mixture was filtered under vacuum using filter paper. A sample was taken from the filter cake, and a final tincture sample representing 72 hours was taken from the filtrate. The remaining filtrate was labeled ZINGOEE. Approximately 300 ml of the ZINGOEE sample was taken and stored refrigerated in a glass bottle. In a further step, all ZINGOEE was evaporated to produce a total resin volume of 43.5 g, with a value of 88.9 mg / g gingerone.

[0380] The remaining ethanol tincture was evaporated under vacuum using a Buchi R220SE rotary evaporator operating at 50 mbar and 40°C until a volume reduction of approximately 31-fold was achieved. The resulting concentrated extract (ZINGOCE) was then analyzed, and after confirming the gingerone content, it was diluted with food-grade ethanol. A small sample of this concentrated extract was used to produce a standardized tincture containing approximately 12 mg of gingerone per gram. Two separate samples of this standardized tincture were sent to SCU (Australia) for analysis, while a third sample remained on-site for gingerone analysis.

[0381] Results: Methods and results are as follows Figure 7As shown. For these experiments, 801.5 g of the received treated ginger was used for ethanol extraction together with 4007.3 g of food-grade ethanol. After stirring at room temperature (16–20 °C) for 72 hours, the mixture was filtered to obtain 3556.7 g of a clear, brown aromatic ethanol tincture [ZINGOEE], and 1033.4 g of filter cake (i.e., waste ginger solid). Approximately 140 g of ethanol was lost due to evaporation during extraction. The total weight of the ethanol tincture produced was 3631.7 g, including samples collected at 24 and 48 hours.

[0382] Gingerone content in the raw material (i.e., treated ginger, GPE) and samples extracted with ethanol at room temperature for 24, 48, and 72 hours was quantified by HPLC. Waste ginger solids (i.e., filter cake generated during filtration) were also analyzed. The gingerone content in all fractions is shown in Table 12. HPLC results showed minimal differences between the three extraction times, indicating that 24 hours was sufficient for extraction.

[0383] Table 12. Gingerone content of the samples (mg / g)

[0384] The filtrate [ZINGOEE] had a gingerone concentration of 1.1 mg / g, meaning there were 3.88 g of gingerone in the liquid, or 85% of the initial gingerone, indicating reasonable recovery. The filter cake had a gingerone concentration of 0.61 mg / g, meaning there were 0.63 g of gingerone in the filter cake. However, it was noted that the filter cake still contained a certain amount of ethanol solution. In further processing, the filter cake could be washed with clean ethanol to flush out as much extract as possible.

[0385] The gingerone mass balance of the ethanol extraction process was 98.5%. This is based on 3.88 g in the extract, plus 0.63 g in the filter cake, divided by 4.58 g in the feed. Room temperature extraction was determined to provide favorable gingerone recovery. Increasing the extraction temperature may result in higher gingerone recovery, but room temperature extraction is clearly effective.

[0386] After removing approximately 300 ml of ZINGOEE sample, the remaining extract (3249 g) was evaporated under vacuum to produce 104.9 g of concentrated extract [ZINGOCE] containing 33 mg / g gingerone (3.46 g of gingerone). The total solids content of this concentrated extract was 37.1% (measured by loss during drying at 110°C), indicating that the final oleoresin weight achievable if all ethanol were removed would be 38.9 g. Extrapolating this figure to the total amount of ZINGOEE produced, the extraction yield of this method is approximately 5.4%. A standardized tincture containing 12 mg / g gingerone was prepared by mixing 25.5 g of ZINGOCE with 44.5 g of food-grade ethanol. A sample of this tincture was sent for further analysis.

[0387] Furthermore, further analysis showed that no aldehydes were present in the final product. (See also...) Figure 8A and Figure 8B To conduct these assessments, the presence of aldehydes in the treated ginger ethanol extract samples was analyzed using GCMS. If present, it was expected to be (primarily) hexanal derived from 6-gingerol. The powdered formulations (e.g., the pre-extracted powders from Examples 4 and 5 above) were also expected to be aldehyde-free. The next step would involve evaporating the ethanol tincture to produce a thick ethanol paste.

[0388] Conclusion: The proposed method, involving screw pressing, followed by KOH treatment of the juice phase, neutralization with citric acid, and then freeze-drying, proved highly effective, achieving almost complete recovery of 6-gingerol from the juice phase. Furthermore, after treatment with 2% KOH at 60°C for 5 hours, all 6-gingerol was converted to gingerone. The pretreated dried powder currently contains 5.32 mg / g gingerone, at least twice the concentration of previous manufacturing techniques.

[0389] Ethanol extraction at room temperature for 24 hours is an optional step to achieve a gingerone recovery rate of at least 85%. Washing the solids with fresh ethanol after extraction can further improve the recovery rate. In these methods, evaporation of the ethanol extract [ZINGOEE] achieves a significant volume reduction, followed by recombination of the concentrated extract [ZINGOCE] with fresh ethanol to produce a standardized tincture containing a target dose of 12 mg / g gingerone.

[0390] For the extraction process, the overall Z mass balance (in / out) was 98.5%. The gingerone content in the filtrate (ZINGOEE) was 1.1 mg / g, and the gingerone content in the concentrated extract (ZINGOCE) was 33 mg / g. This method produced 104.9 g of ZINGOCE at 33 mg / g, leaving 3.46 g of gingerone. This value is lower than the 3.88 g in ZINGOCE because approximately 400 g of ZINGOCE was removed before evaporation (for testing, plus approximately 300 mL of retained sample). Taking this into account, the calculation results are in excellent agreement. The total gingerone mass balance before evaporation was calculated to be 98.5% (=(0.63 + 3.88) / 4.58).

[0391] The results obtained in this work indicate that 100 kg of fresh ginger with a 6-gingerol content of 0.5 mg / g will produce 3.6 kg of a standardized tincture with a gingerone content of 12 mg / g. This demonstrates that higher 6-gingerol levels should result in higher gingerone yields.

[0392] Example 7: Anti-inflammatory activity of gingerone composition

[0393] Overview: These studies were conducted to determine the anti-inflammatory activity of the disclosed plant extracts (ethanol extract (tincture); see Example 6) normalized to gingerone content. Nitric oxide (NO) and IL-6 levels were assessed.

[0394] Methodology: The anti-inflammatory activity of different test compounds / extracts and controls on lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells, in the presence or absence of RAW264.7 cells incubated in standard cell medium (DMEM, 5% fetal bovine serum), was determined. The production of inflammatory mediators (including NO and IL-6) was measured using a method established by commercial 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 using three replicates (maximum concentration 40 µM gingerone) (n=9) with relevant internal controls. Furthermore, cytotoxicity of each sample was determined by the MTT assay (tetrazolium dye MTT, chemically named 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Assay parameters for each assay are summarized in Table 13.

[0395] Table 13. Anti-inflammatory assay and positive control

[0396] The tincture from Example 6 was used in these studies. For NO and IL-6 assays, cultured RAW264.7 cells were counted and placed in 96-well plates (0.8 x 10⁻⁶). 5 Cells were placed in wells and incubated for 48 hours. The medium was then aspirated and replaced with fresh medium, followed by the addition of the test compound. The compound was incubated for 1 hour before the addition of the stimulant. The plate was then incubated for 18 hours, and the supernatant of the target medium was analyzed to determine the remaining cell viability using the MTT assay.

[0397] Positive controls were selected based on their widespread use in similar assays and included N-(3-(aminomethyl)benzyl)acetamide (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 common cytokine inhibitor). Data are presented as mean and standard error of mean (sem) (n=9).

[0398] The dose-response curve was fitted using Graph Pad Prism. The 95% confidence interval (CI) was calculated using the entire dataset, and the IC was obtained. 50 The estimated error in the value. The IC50 concentration was not reached at the test concentration. 50 In the case of IC 50 95% of CI requires graphical extrapolation to calculate IC. 50 In IC 50 Where estimates have been obtained through graphical extrapolation, unless otherwise stated, "approximately (~)" is used to indicate that they are estimates and have not been experimentally determined. Graphical extrapolation yields wider 95% confidence intervals.

[0399] Results: The tincture from Example 6 was analyzed to determine its anti-inflammatory activity. Anti-inflammatory activity was assessed using key inflammatory mediators (NO and IL-6). Cytotoxicity of each sample was also determined using the MTT assay.

[0400] Cytotoxicity: The effect of the compound on cell viability was determined using MTT assay. Potency was monitored to avoid false positives because dead cells do not produce inflammatory mediators. Cytotoxicity was determined spectrophotometrically because mitochondrial dehydrogenases present in living cells cleave the tetrazolium ring of MTT, producing purple MTT formazan. All doses were tested, up to 40 µM gingerone. Results are as follows: Figure 9 As shown. According to the cytotoxicity assay, the disclosed tincture has an IC50 value of [missing information]. 50 It was determined to be 40 µM gingerone.

[0401] NO assay: NO, a free radical metabolite, has been shown to have many physiological functions, acting as both a signaling molecule and a toxic agent in inflammation (Coleman, 2001). Inhibition of iNOS and reduction in NO levels secreted by immune cells may be contributing factors to its anti-inflammatory activity. Therefore, the disclosed tinctures were assayed to determine whether they exhibited inhibition of the inflammatory signaling molecule NO. The dose-dependent effects of the tinctures on NO are shown in... Figure 10 According to NO determination, the IC50 of the disclosed tincture is... 50 It was determined to be 9.2 µM gingerone.

[0402] IL-6 assay: IL-6 is considered a pro-inflammatory cytokine. IL-6 is secreted by T cells and macrophages, stimulating immune responses. IL-6 is responsible for increasing the production of neutrophils in the bone marrow. It supports B cell growth and antagonizes T cell differentiation into regulatory T cells. It can cross the blood-brain barrier and initiate PGE2 synthesis 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 disclosed tincture on IL-6 is shown in... Figure 11 According to IL-6 determination, the IC50 of the tincture is... 50 It was measured to be 4.6 µM gingerone.

[0403] from Figure 10 , Figure 11 and Figure 12 As can be seen, the disclosed tincture inhibits NO and IL-6 in a dose-dependent manner. The effective inhibition levels of NO and IL-6 are significantly lower than those required for cytotoxicity, validating the observed anti-inflammatory effect of the tincture.

[0404] Example 8: Comparative anti-inflammatory test of gingerone compositions

[0405] Overview: These studies were conducted to determine the anti-inflammatory bioactivity of the disclosed plant extract (ethanol extract (tincture); see Example 6) in RAW264.7 macrophages compared with commercially available gingerone.

[0406] Methodology: The extract (ethanol extract (tincture); Rx7 / 22 / 161) was prepared as described in Example 6. The extract was normalized to contain 33 mg gingerone per mL. Commercially available gingerone powder (Vigon #500938) was obtained and freshly dissolved in ethanol to 33 mg / mL on the day of use. The ethanol-concentrated sample was further diluted in cell medium before addition to cell cultures. An ethanol solvent control was included in cell culture experiments to rule out any potential solvent effects.

[0407] RAW264.7 cells in growth medium (DMEM, 10% FBS, PSN, 2 mM L-glutamine) were thawed at a concentration of 0.8 x 10⁻⁶ cells / mL. 5 Cells / wells were placed in 96-well tissue culture plates and incubated at 37°C / 5% CO2 under humid conditions for 48 hours. Waste medium was then aspirated and replaced with medium containing 5% FBS. Treatment was added to obtain a final concentration of 0.625–40 µM gingerone (or an equivalent dilution of the ethanol solvent control). Cells were incubated with the treatment for 1 hour before adding 50 ng / mL lipopolysaccharide (LPS; from *E. coli* O111:B4).

[0408] Before collecting the conditioned medium, cells were co-incubated with the treatment and LPS for 18 hours, centrifuged, and the cell-free supernatant was collected. Cell viability was determined by WST-1 assay. Controls included unstimulated cells (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 samples).

[0409] For the cytotoxicity assay, an equal volume of a 1:5 mixture of WST-1 media was added to each 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 to remove background). Hydrogen peroxide was included as a positive control for cytotoxicity. Results were normalized to the LPS-stimulated control and expressed as a percentage of cell viability. Sample concentrations were considered cytotoxic if cell viability was below 80% of the unstimulated control.

[0410] For IL-6 assays, interleukin-6 (IL-6) was analyzed in cell-free conditioned media collected after treatment and LPS stimulation using a bead-based multiplex assay panel (Legendplex MU Th1 / Th2 8-plex panel; BioLegend #741054), and measurements were performed using a Cytex Aurora Spectral 3 laser flow cytometer. As a follow-up to the IL-6 assays, assays were performed to measure IL-10 and TNF.

[0411] Statistical analysis was performed using Minitab 18.0. Paired Student's t-tests were performed to obtain statistical differences between treatments at each time point. Data are presented as mean ± standard error of the mean (SEM). Each experiment included three replicate wells, and three separate cell experiments were conducted.

[0412] Results: Cell viability was measured after incubation with the compound / extract. In this assay, WST-1 reacts with mitochondrial enzymes to form a colored dye, which can be measured by absorbance. Therefore, WST-1 is a measure of cellular metabolism, and a decrease in the WST-1 reaction rate may indicate cell death. Hydrogen peroxide (H2O2) was used as a positive control to induce cell death and ensure the validity of the assay.

[0413] The results of cytotoxicity were shown in Figure 13 The figure depicts cell viability levels after 18 hours of treatment with 50 ng / mL lipopolysaccharide (LPS), LPS with the disclosed plant extract or gingerone (0.6 to 40 µM), and an equivalent ethanol control. Data are mean ± SEM (n=3) from three independent experiments. Assay validation was determined using hydrogen peroxide (H2O2).

[0414] In the left figure, a clear dose-dependent cytotoxic response was observed in cell samples exposed to H2O2, confirming the assay was performed as expected. Untreated controls with WST-1 values ​​< 80% (dashed lines) were considered cytotoxic. Cytotoxicity was assessed in RAW264.7 cells 18 hours after exposure to the published plant extract or commercially available gingerone. Gingerone concentrations ranged from 0.3 µM to 40 µM. No significant cell death was detected by WST-1 assay in RAW264.7 cells exposed to the ethanol extract or commercially available gingerone at the tested concentrations. Figure 13 In other words, no data point was below 80% of the WST-1 response in the untreated LPS-stimulated control. This validates the results of the cytokine assay (see results below).

[0415] A cell model for IL-6 assessment explored the pro-inflammatory response in a mouse macrophage line (RAW264.7) upon exposure to bacterial endotoxin (lipopolysaccharide; LPS). Mechanistically, LPS interacts with a membrane-bound TLR4 receptor, triggering an intracellular signal transduction cascade to activate NF-κB, a transcription factor that regulates the expression of many pro-inflammatory cytokines, including IL-6.

[0416] The results of the IL-6 measurement are shown in Figure 14This figure depicts IL-6 levels in RAW264.7 cells after 18 hours of treatment with lipopolysaccharide (LPS), LPS + 10 µg / mL dexamethasone (Dex), LPS + a publicly available plant extract or commercially available gingerone (0.6 µM to 40 µM). Data are mean ± SEM from three independent experiments (n=3). An asterisk indicates significantly lower IL-6 production at the corresponding gingerone concentration (p < 0.05). No error bar indicates treatment at 20,000 pg / mL.

[0417] It was found that exposing RAW264.7 cells to LPS for 18 hours significantly induced IL-6 secretion to concentrations above 20,000 pg / mL, exceeding the upper limit of accuracy for assays. Figure 2 Following co-treatment of RAW264.7 cells with equivalent ethanol concentrations present in the disclosed plant extracts and commercially available gingerone dilutions, IL-6 secretion remained above the upper limit of assay. Co-treatment of RAW264.7 cells with LPS + the disclosed plant extracts or LPS + commercially available gingerone in the concentration range of 0.6 µM to 10 µM also exceeded the upper limit of accuracy, making it difficult to assess the anti-inflammatory bioactivity of these samples at these concentrations.

[0418] Notably, co-treatment of RAW264.7 macrophages with LPS and the disclosed plant extract or the highest concentrations of commercially available gingerone assessed (20 µM and 40 µM) resulted in a significant decrease in IL-6, within the limits of assay accuracy. Specifically, co-treatment of RAW264.7 with the disclosed plant extract at 20 µM and 40 µM gingerone resulted in significantly (p < 0.05) lower IL-6 production compared to co-treatment with commercially available gingerone. These findings suggest that the disclosed plant extract is more effective than commercially available gingerone in reducing IL-6 production at these concentrations.

[0419] A specific decrease in IL-10 was also observed. Figure 15IL-10 levels in RAW264.7 cells were depicted after 18 hours of treatment with lipopolysaccharide (LPS), LPS + 10 µg / mL dexamethasone (Dex), and LPS + a publicly disclosed plant extract, or LPS + commercially available gingerone (1.25 µM to 40 µM). Data are mean ± SEM (n=3) from three independent experiments. Carrot shape indicates significantly lower IL-10 production compared to the corresponding ethanol control (p < 0.05). Asterisk indicates significantly lower IL-10 production at the corresponding gingerone concentration (p < 0.05). Treatments at 15,000 pg / mL had no error bar indication at 215,000 pg / mL and were above the limit of accuracy of the assay.

[0420] The results showed that RAW264.7 macrophages exposed to LPS for 18 hours induced a significant increase in IL-10, which could be attenuated by co-treatment with dexamethasone (a known immunosuppressant). Co-treatment with the disclosed plant extract containing 10 µM and 20 µM equivalent gingerone (instead of commercially available gingerone) significantly attenuated LPS-induced IL-10 secretion in RAW264.7 (p < 0.05). Furthermore, IL-10 secretion in RAW264.7 cells co-treated with the disclosed plant extract at 20 µM and 40 µM gingerone was significantly lower than that at these concentrations of commercially available gingerone (p < 0.05).

[0421] The results for TNF are shown in Figure 16 This figure depicts TNF levels in RAW264.7 cells after 18 hours of treatment with lipopolysaccharide (LPS), LPS + 10 µg / mL dexamethasone (Dex), and LPS + a publicly disclosed plant extract, or LPS + commercially available gingerone (1.25 µM to 40 µM). Data are mean ± SEM from three independent experiments (n=3). An asterisk indicates significantly lower IL-6 production at the corresponding gingerone concentration (p < 0.05). Treatments at 15,000 pg / mL had no error bar indication at 215,000 pg / mL and were above the limit of accuracy of the assay.

[0422] The results showed that RAW264.7 macrophages exposed to LPS for 18 hours induced a significant increase in TNF, which could be attenuated by co-treatment with dexamethasone (a known immunosuppressant). Co-treatment of cells with the disclosed plant extracts or pure gingerone compounds had no significant effect on attenuating LPS-induced TNF secretion (p > 0.05).

[0423] Overall, the disclosed plant extract (containing naturally occurring gingerone) exhibits significant and superior anti-inflammatory activity compared to commercially available gingerone, particularly regarding IL-6 and IL-10 levels. Future assays will be performed at generally lower inducible levels for more accurate comparisons.

[0424] Further review of these studies revealed an error by the researchers recording the concentrations of gingerone in the published plant extracts (tinctures). Although indicated as 33 mg / mL (0.170 M) in these studies, the actual gingerone concentration in the tinctures was 33 mg / g (0.134 M). Consequently, the gingerone concentrations in the tincture dilutions were incorrectly labeled as 0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM. Based on the correct starting concentration of 33 mg / g, the correct gingerone concentrations in the tincture dilutions can be calculated as 0.493 µM, 0.986 µM, 1.97 µM, 3.95 µM, 7.89 µM, 15.8 µM, and 31.6 µM, respectively.

[0425] This means that the results for the activities of IL-6, IL-10, and TNF in the published plant extracts were all underestimated, as the concentrations of gingerone used from commercial sources (0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM) were significantly higher than those of the published plant extracts (0.493 µM, 0.986 µM, 1.97 µM, 3.95 µM, 7.89 µM, 15.8 µM, and 31.6 µM). In any case, these initial results are very positive, and replication studies are currently being conducted using correct calculations.

[0426] Table 14. Overview of the results of IL-6 inhibition

[0427] The MTT assay was used to identify the cytotoxic effects of compound treatments on RAW264.7 cells. No cytotoxic effects were observed with any of the compounds tested. Under Method 1 conditions, the MTT absorbance was significantly lower than that under unstimulated Method 2 conditions and under both stimulated and unstimulated Method 2 conditions. Figure 18A Method 1 stimulation; Figure 18B (Method 2 stimulation). This decrease is expected because the mechanism of IFN-γ involves inhibiting cell proliferation, resulting in fewer cells present compared to (-)IFN-γ conditions. Specifically, fewer cells mean fewer mitochondria metabolizing MTT into formazan, resulting in lower absorbance.

[0428] Conclusion: Due to its IL-6 inhibitory activity, 100 nM dexamethasone was shown to be an effective control. Method 2 was chosen for the remaining experiments because it demonstrated compatibility with the other data obtained.

[0429] Example 9: Comparison of methods for assessing anti-inflammatory activity and survival rate

[0430] Overview: These studies were conducted to: (1) identify the most effective positive control; and (2) compare two different methods for assessing inflammatory activity. The experiments produced the following outputs: survival rate as measured by MTT; and IL-6 production as measured by ELISA.

[0431] The evaluation method is summarized as follows:

[0432] Methodology: Dexamethasone and synthetic gingerone were obtained from Sigma-Aldrich. For these studies, dexamethasone (100 nM) was used as a positive control; anhydrous ethanol was used as a mediator control; and synthetic gingerone (20 µM) was used for the test treatment. The tests included: (1) IL-6 ELISA: measuring IL-6 produced by stimulated / unstimulated RAW264.7 cells; (2) MTT assay: identifying potential cytotoxicity by measuring the metabolism of MTT to formazan.

[0433] On day 0, remove the flask used for Method 2 conditions from the 37°C / 5% CO2 incubator. Pour out the growth medium. Next, add 10 mL of CTCM and wash the cells. Pour out the growth medium again. Then, add 10 mL of CTCM and collect the cells using a rubber spatula. Transfer the resulting cell suspension to 50 mL Falcon centrifuge tubes. Centrifuge at 400 x g for 5 minutes. Count the cells after applying a 1:10 trypan blue dilution. Then, dilute the sample to 1 x 10⁻⁶. 6 Cells / mL. Then, seed or add cells at 80 µL / well (80,000 cells / well). For stimulated Method 2 samples, add 120 µL of medium. Repeat this step for unstimulated Method 2 samples. Return cells to a 37°C / 5% CO2 incubator and incubate for 48 hours.

[0434] On Day 1, prepare the drug diluent (see below). Remove the flask used for Method 1 conditions from the 37°C / 5% CO2 incubator. Pour out the growth medium. Next, add 10 mL of CTCM and wash the cells. Pour out the growth medium again. Then, add 10 mL of CTCM and collect the cells using a rubber spatula. Transfer the resulting cell suspension to 50 mL Falcon centrifuge tubes. Centrifuge at 400 x g for 5 minutes. Count the cells after applying a 1:10 trypan blue dilution. Then, dilute the sample to 1 x 10⁻⁶. 6 Cells / mL. Seed cells at 50,000 cells / well (50 µL). For stimulated Method 1 samples, add 50 µL of IFN-γ (final concentration 20 U / mL). For unstimulated samples, seed cells and bring the well volume to 200 µL.

[0435] On Day 2, prepare LPS and treatment conditions for Method 2 samples. For stimulated Method 2 samples, add 50 µL of LPS (final concentration 20 ng / mL) and 50 µL of treatment. For unstimulated Method 2 samples, centrifuge these samples and remove 50 µL of CTCM from the treatment wells. Add 50 µL of treatment to these wells. For stimulated Method 1 samples, centrifuge these samples and remove 100 µL of medium from the wells. Add 50 µL of LPS (final concentration 50 ng / mL) and 50 µL of treatment to these wells. For unstimulated Method 1 samples, centrifuge these samples and remove 50 µL of medium from the wells. Add 50 µL of treatment to these wells. Begin the IL-6 ELISA capture setup.

[0436] On day 3, wash the ELISA plate and begin blocking. Collect the supernatant (170 µL) from each sample. Next, add 50 µL of warm CTCM to the wells. Then, add 20 µL of 5 mg / mL MTT solution. Return the plate to a 37°C / 5% CO2 incubator for 45 minutes. While incubating with MTT, wash away the ELISA capture and add blocking solution. After 45 minutes, add 10 µL of MTT dissolving agent. Continue the ELISA procedure. Perform cell counting on four fields. The average cell number per field is as follows: (48+58+67+62) / 4 = 58.75 cells in 10 µL. This is then calculated as 58.75. 10 10⁴ = 5,875,000 cells / mL. Then, this is calculated as 5,875,000 cells / mL in 10 mL. 10 = 58,750,000 cells. Therefore, to produce 1 x 10⁻⁶ cells... 6Cells / mL solution, prepare up to 58.75 mL of cell suspension.

[0437] To prepare the diluents: (1) The IFN-γ stock solution was 5,000,000 U / 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 wells: 20 ng / mL). For Method 2, a 200 ng / mL solution was required (final concentration in wells: 50 ng / mL); (3) 2 µL of gingerone was added to 198 µL of CTCM to prepare a synthetic gingerone stock solution. This yielded a 1544.6401 µM stock solution. 25.89 µL of this stock solution was added to 474.11 µL of CTCM. This yielded an 80 µM stock solution. The 80 µM stock solution (50 µL) (together with LPS) was added to the sample to achieve a final concentration of 20 µM (0.013% ethanol).

[0438] To prepare the drug diluent: (1) 2 µL of dexamethasone was added to 998 µL of CTCM to prepare a dexamethasone stock solution. This yielded a 5096 nM stock solution. 78.49 µL of the 5096 nM stock solution was added to 921.51 µL of CTCM. This yielded a 400 nM stock solution. 50 µL of the 400 nM stock solution (together with LPS) was added to the sample to achieve a final concentration of 100 nM (0.003% ethanol); (2) 2 µL of ibuprofen was added to 579.704 µL of CTCM to prepare an ibuprofen stock solution. This yielded a 1000 µM ibuprofen stock solution. 400 µL of the 1000 µM ibuprofen stock solution was added to 600 µL of CTCM to prepare a 400 µM stock solution. (2) Add 400 µM stock solution (50 µL) (together with LPS; 0.03% ethanol) to the sample; (3) Add 2 µL of risperidone to 248 µL of CTCM to prepare a risperidone stock solution. This yields a 584.67 µM stock solution. Add 342.1 µL of the 584.67 µM stock solution to 157.9 µL of CTCM. This yields a 400 µM stock solution. Add 400 µM stock solution (50 µL) (together with LPS) to the sample to achieve a final concentration of 100 µM (0.14% ethanol).

[0439] For ELISA measurement of IL-6: Reagents were obtained from BD Biosciences. Capture: Purified NA / LE rat anti-mouse IL-6; catalog number 554398, clone MP5-20F3; Standard: Recombinant mouse IL-6; catalog number 554582; Detection: Biotinylated rat anti-mouse IL-6 antibody; catalog number 554402, clone MP5-32C11; SA-HRP: Streptomycin HRP; catalog number 554066; TMB: BD OptEIA™ TMB substrate kit; catalog number 555214. When standards were included, two complete ELISA plates (each with 96 wells) were used. A total volume of 11 mL was prepared for 50 µL / well sample and a total volume of 22 mL was prepared for 100 µL / well sample. For capture, overnight incubation was performed at 4°C. For capture antibody, the stock solution was 1000 µg / mL, with a desired final concentration of 1 µg / mL. Perform the following calculation: 1000 µg / mL Vi = 1 µg / mL 11,000 μL. Based on this calculation, the equivalent of 11 µL of rat anti-mouse IL-6 Vi was added to 11 mL of pH 9.0 capture buffer. The buffer was added to the plate at 50 µL / well. Four washes were performed after capture.

[0440] For blocking, incubation was performed at room temperature for 2 hours. For this, 100 µL of 10% FCS was added to each well. This is calculated as: 10% FCS in 50 mL 1X PBS = 5 mL FCS. Three washes were performed after blocking. The plate was incubated at room temperature for 2 hours. The stimulated sample was diluted to 10 µL in 100 µL of 5% FCS. 5% FCS is equivalent to adding 2.5 mL of FCS to 50 mL of 1X PBS. Added at 50 µL / well. Standards were prepared as follows: 16 µL was added to 784 µL of 5% FCS. The maximum concentration was 4 ng / mL. Serial dilutions of 100 µL were performed to 7.8125 pg / mL. Samples with 0 pg / mL were also included. Serial dilutions were produced in duplicate. Four washes were performed after standard preparation.

[0441] For the initial detection step, incubation was performed at room temperature for 1 hour. The stock solution was 500 µg / mL, and the desired final concentration was 0.5 µg / mL. The following calculations were performed: 500 µg / mL Vi = 0.5 µg / mL 11,000 µL. Based on this calculation, the equivalent of 11 µL of biotinylated rat anti-mouse IL-6 Vi was added to 11 mL of 5% FCS / PBS. 10 µL was added to each well. Six washes were performed after this initial assay step. Incubation with streptavidin-horseradish peroxidase (SA-HRP) was performed at room temperature for 1 hour. For this purpose, a 1:2000 dilution of SA-HRP was prepared. The calculation was as follows: 11,000 / 2000 = 5.5 µL. Based on this calculation, 5.5 µL of SA-HRP was added to 11 mL of 5% FCS / PBS. 50 µL was added to each well. Eight washes were performed after the SA-HRP step.

[0442] For the final assay step, incubation with TMB (3,3',5,5'-tetramethylbenzidine) was performed. TMB was prepared by separately adding 11 mL of TMB-A and 11 mL of TMB-B to 15 mL Falcon centrifuge tubes covered with foil. After washing following SA-HRP incubation, the TMB-A and TMB-B samples were combined and added to the plate at 100 µL / well. Color development was allowed. Color development was stopped by adding 100 µL of H2SO4 to the wells. The plate was then read using a plate reader.

[0443] Results: Interleukin-6 (IL-6) was produced in response to LPS in both experimental settings, with method 2 producing higher levels. Figure 17A Method 1 stimulation; Figure 17B Method 2 stimulation). The unstimulated conditions in both settings showed no significant change under either treatment. Dexamethasone (100 nM) was the only control compound capable of inhibiting IL-6 in either setting. In the Method 1 setting, dexamethasone showed greater inhibition of IL-6 (29.32% of stimulated / untreated conditions and 33.42% of the medium) compared to the Method 2 setting (64.03% of stimulated / untreated conditions and 63.62% of the medium). An overview of IL-6 levels is presented in Table 14 below.

[0444] Example 10: Analysis of anti-inflammatory activity and cell viability in comparative tests

[0445] Overview: The methodology as indicated in the previous embodiments was used to evaluate various biomarkers, including: (1) survival: identifying concentrations with cytotoxic effects, measured by the MTT test; (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.

[0446] Some data have been presented as the logarithm of the test concentration to facilitate data visualization, and nonlinear regression has been performed where appropriate. The test concentrations and their corresponding logarithms are listed below. 10 value.

[0447] Table 15. Test concentration and calculated log 10 concentration

[0448] Methodology: Anti-inflammatory activity and survival rate were evaluated as follows.

[0449] On day 0, remove the dedicated T75 flask from the incubator for self-replication. In a laminar flow hood, gently tap the flask to detach any unattached cells. Pour out the growth medium and replace it with 10 mL of CTCM. Gently shake the flask to wash the cells. Pour out the wash medium and replace it with 10 mL of CTCM. Use a rubber spatula to detach the cells. Transfer the suspension to 50 mL Falcon centrifuge tubes. Centrifuge the cells at 400 x g for 5 minutes. Resuspend the cells in 10 mL of CTCM. Count the cells after applying a 1:10 trypan blue dilution. Dilute the cells to 1 x 10⁻⁶. 6 Cells / mL. Seed cells at 80 µL / well. To do this, add 120 µL to bring the volume to 200 µL. Place the plate back in the incubator. Continue seeding cells as instructed. In this way, the plate is dedicated to both stimulated and unstimulated samples. Let the plate stand for 48 hours before performing any treatment.

[0450] Round 1 counting (stimulated / unstimulated): Average cell count in four fields of view = 54 -> x 10 x10 4 This means there are 5,400,000 cells in 1 mL, which is equivalent to 54,000,000 cells in 10 mL. For dilution, add 44 mL of CTCM to achieve 1 million cells / mL.

[0451] Second round of counting (after stimulation): Average cell count in four visual fields = 15.7 -> x 10 x 10 4 This means there are 1,570,000 cells per mL, which is equivalent to 15,700,000 cells per 10 mL. For dilution, add 5.70 mL of CTCM to achieve 1 million cells / mL.

[0452] Second round of counting (unstimulated): Average cell count in four fields of view = 25.75 -> x 10 x 10 4This means there are 2,575,000 cells per mL, which is equivalent to 25,750,000 cells per 10 mL. For dilution, add 15.75 mL of CTCM to achieve 1 million cells / mL.

[0453] Round 3 counting (after stimulation): Average cell count in four visual fields = 25 x 10 x 10 4 This means there are 2,500,000 cells in 1 mL, which is equivalent to 25,000,000 cells in 10 mL. For dilution, add 15 mL of CTCM to achieve 1 million cells / mL.

[0454] Round 3 counting (unstimulated): Average cell count in four fields of view = 32 -> x 10 x 10 4 This means there are 3,200,000 cells in 1 mL, which is equivalent to 32,000,000 cells in 10 mL. For dilution, add 22 mL of CTCM to achieve 1 million cells / mL.

[0455] On Day 1, preparation was performed. Synthetic gingerone, acetyl gingerone, and ferulic acid were used in a stock solution of 360,416.0231 µM. Acetyl gingerone was obtained from Sytheon Ltd. Synthetic gingerone and ferulic acid were obtained from Sigma-Aldrich. To produce the required dose, 1199.4 µL of CTCM was added to 2 µL aliquots of each stock solution. This produced a 600 µM test solution for each compound. When added to cells, the concentration was 150 µM to 50 µL in 200 µL. The disclosed plant extract was used in a stock solution of 134,000 µM. To produce the required dose, 6 µL of the extract was added together with 1334 µL of LTCCM. This produced a 600 µM test solution. See the overview table below.

[0456]

[0457] On day 2, stimulation and treatment began. The plates were removed from the incubator. The plates were centrifuged at 400 xg for 5 minutes. The medium (150 µl) was removed and replaced with 50 µL of CTCM. 50 µl of test solution for each treatment was added to the appropriate wells. The plates were returned to the incubator for 1 hour. LPS solution was prepared. For this, 2 µL of aliquots (1,000,000 ng / mL) were added to 9,998 µL of CTCM to obtain a 200 ng / mL solution (final concentration 50 ng / mL). LPS solution was added at 50 µL / well. For the unstimulated condition, treatment was added at 50 µL / well, followed by 50 µL of CTCM. The plates were returned to the incubator for 18 hours. All ELISA plates were plated. MTT reagent was prepared. This was done by mixing 50 mg MTT into 10 mL of 1X PBS. This yielded a 5 mg / mL reagent solution, which could be used for 6 plates.

[0458] On day 3, supernatant collection, MTT assay, and ELISA were performed. For supernatant collection, the plate was centrifuged at 400 xg for 5 minutes. A multichannel pipette was set to 170 µL to remove the supernatant. The supernatant was transferred to labeled UB-96WP containers. For the MTT assay, CTCM was preheated in a 37°C water bath. After collecting the supernatant, 50 µL of warm 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 color development, 100 µL of MTT dissolving solution was added. Incubation was performed overnight.

[0459] ELISA reagents: from BD Biosciences. Capture: purified NA / LE rat anti-mouse IL-6; catalog number 554398, clone MP5-20F3; Standard: recombinant mouse IL-6; catalog number 554582; Detection: biotinylated rat anti-mouse IL-6 antibody; catalog number 554402, clone MP5-32C11; SA-HRP: streptavidin HRP; catalog number 554066; TMB: BD OptEIA™ TMB substrate kit; catalog number 555214. Solutions included: (1) ELISA capture buffer pH 6: 14.196 Na2HPO4 in 1 L mq H2O. Adjusted pH with HCl. (2) ELISA capture buffer pH 9: 14.196 Na2HPO4 in 1 L mq H2O. Adjusted pH with NaOH. (3) ELISA washing solution: 1 mL Tween® 20, 200 mL 10X PBS and 1800 mL mqH2O. (4) 5% FCS in PBS: 2.5 mL FCS in 47.5 mL 1X PBS. (5) 10% FCS in PBS: 5 mL FCS in 45 mL 1X PBS. (6) 0.18 M H2SO4: 9.78 mL concentrated H2SO4 in 1 L mL H2O.

[0460] ELISA Assay: Spread ELISA plates with capture antibodies suspended in ELISA capture buffer, 50 µL / well. Adjust the pH based on the cytokines being tested (see overview table below). Incubate the spread ELISA plates overnight at 4°C. The next day, remove the plates from the refrigerator. Wash the plates four times with ELISA wash buffer. Next, add 100 µL / well blocking solution to the plates. Incubate the plates at room temperature for 2 hours. Wash the plates three times. Dilute the supernatant according to the cytokines being tested. Add the test samples along with the cytokine standard curve, quantifying at 50 µL / well (see overview table below). Incubate the plates at room temperature for 2 hours, or overnight at 4°C. For serial ELISA, transfer the supernatant to another plate before washing. Perform four washes.

[0461] For the assay, suspend the biotinylated detection antibody in FCS and PBS and add it to the plate at 50 µL / well. Incubate the plate at room temperature for 1 hour. Wash the plate six times. Suspend the streptavidin-horseradish peroxidase in FCS and PBS and add it to the plate at 50 µL / well. Incubate the plate at room temperature in the dark for 1 hour. Place the required volumes of TMB-A and TMB-B in separate Falcon centrifuge tubes. Store these tubes at room temperature in the dark during SA-HRP incubation. Wash the plate eight times. Add the TMB solution to the plate at 100 µL / well. Allow the color to develop. To stop the reaction, add 0.18 M H2SO4 solution at 100 µL / well. See the table below. Read the plate at 450 nm using a PerkinElmer EnSpire® microplate reader.

[0462]

[0463] MTT assay: The day before the assay, dissolve MTT powder in 1X DPBS to a concentration of 5 mg / mL. 20 µL / well is required per plate. For 96 wells, this requires 1920 µL of MTT solution. The concentration is 5 mg MTT per mL of solution. This equates to 10 mg MTT per 96-well plate. On the day of assay, warm the CTCM in a 37°C water bath. While warming the medium, centrifuge the cell culture (RAW264.7) at 400 xg for 5 minutes. Remove the supernatant using a multichannel pipette set to 180 µL and transfer it to labeled U-bottom 96-well plates. Store the supernatant at -20°C until analysis. Add 60 µL of warmed CTCM to the remaining cells to bring the volume to 80 µL / well. For this, add 20 µL of MTT solution. Incubate the plate at 37°C / 5% CO2 for 45 minutes. After 45 minutes, add 50 µL of MTT dissolution solution. This includes 10% SDS (w / v), 0.01 MHCl, with the pH adjusted to 4.0 using sodium hydroxide; or 10% SDS (w / v), 45% DMF, with the pH adjusted to 4.0 using acetic acid. Cover the plate with aluminum foil and let it stand overnight. After overnight incubation, read the plate at 580 nm using a PerkinElmer EnSpire® microplate reader.

[0464] NO determination: The Griess reaction protocol was used. NO production was measured via the NaNO2 product in a 96-well format. Reagents included: (1) Greiss solution A (50 mL): 1% (w / v) sulfanilamide (500 mg), 2.5% phosphoric acid, stored at 40°C 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 protected from light; (3) 2.5% phosphoric acid (100 mL): 2.94 mL 85% phosphoric acid, 97.6 mL ddH2O. For plate standards, the first and second columns (horizontal) of each plate included a 1:1 dilution of NaNO2 (500 µM to 0 µM). In columns A and B (bottom), 50 µl of the medium was added to each well. The first well in each column received 95 µl. Add 5 µl of 10 mM NaNO2 to the first well. Take 50 µl from each well and dilute along the columns, excluding the last well in each column (baseline). For the test sample, remove 170 µl of supernatant from the well without disturbing the cell monolayer at the bottom of each well. From this 170 µl sample, transfer 50 µl of sample (triple) to a flat-bottomed 96-well plate. For the Greiss reaction, mix equal volumes of Greiss solution A and Greiss solution B to the desired volume (approximately 5 mL per 96-well plate). Add the mixed Greiss solution A+B to each well (50 µl per well). Remove any air bubbles using a blower. Read the absorbance at 570 nm.

[0465] Results—Survival: The MTT assay was used to identify any decrease in metabolism, which can indicate the cytotoxic effect of any compound. The only compound found to have a cytotoxic effect was the disclosed plant extract at 50 µM and above. Data summary is provided in Figures 19A to 19D , as well as in Tables 16 and 17 below.

[0466] Table 16. IC50 of MTT analysis 50 calculate

[0467] Table 17. Closest to IC 50 concentration

[0468] Results: IL-6: IL-6 is a common pro-inflammatory cytokine produced by macrophages upon LPS stimulation. IL-6 is involved in the early transmission of innate immune responses. The only substantial inhibition of IL-6 production in stimulated RAW264.7 macrophages was from the disclosed plant extract (…). Figure 20 The most potent IL-6 inhibition was observed in the 25 µM plant extract, reducing IL-6 production to 41% of the anhydrous ethanol control. Figure 20 Significant inhibition of IL-6 production was also observed in 6.25 µM and 12.5 µM plant extracts. Figure 20 Surprisingly, the 25 µM plant extract significantly inhibited IL-6 more than the 150 µM synthetic gingerone. Figure 20 and Figure 21 A summary of the dose-dependent inhibition of the disclosed plant extracts is listed in Table 18 below. Potential inhibition was observed upon treatment with 150 µM synthetic gingerone or acetyl gingerone. This treatment with synthetic gingerone or acetyl gingerone reduced IL-6 to 64% and 69% of the anhydrous ethanol control, respectively. These results are summarized in Table 18 below. Ferulic acid did not affect IL-6 production at any of the concentrations tested. Concentrations of the disclosed plant extracts associated with cytotoxicity are omitted to avoid obfuscation of data.

[0469] Table 18. Overview of the results of IL-6 inhibition

[0470] To determine whether RAW264.7 cells treated with the disclosed plant extract had significantly lower IL-6 levels compared to cells treated with synthetic gingerone, acetyl gingerone, or ferulic acid, a one-way ANOVA was used. IL-6 levels were significantly lower in cells treated with 25 µM of the disclosed plant extract compared to treatment with 150 µM of synthetic gingerone, acetyl gingerone, or ferulic acid. Figure 21 Two-way ANOVA was also performed to determine whether the IL-6 levels observed under the compound treatment conditions differed significantly from those under the mediator treatment conditions. Treatment with 25 µM of the disclosed plant extract or 150 µM of synthetic gingerone / acetyl gingerone resulted in significantly lower IL-6 levels compared to the corresponding mediator treatments. Figure 22 ).

[0471] Under unstimulated conditions ( Figures 23A to 23D The levels of IL-6 were extremely low relative to those under stimulated conditions, which was expected. No trend was observed due to the low levels of cytokines produced under unstimulated conditions. This provides confidence that the test group did not induce IL-6 production. Since IL-6 levels under unstimulated conditions are low and typically 0 pg / mL, this data was not normalized to the anhydrous ethanol control.

[0472] Results—TNF: TNF is also a pro-inflammatory cytokine produced by macrophages upon LPS stimulation. Maximum inhibition of TNF was observed in stimulated RAW264.7 cells after treatment with 150 µM ferulic acid. This treatment reduced TNF to 61% of the level in the anhydrous ethanol control. Figure 24 At a concentration of 50 µM, the inhibition of ferulic acid treatment gradually decreased to the level of the anhydrous ethanol control. 150 µM synthetic gingerone showed a significant moderate inhibitory effect on TNF production, reducing TNF levels to approximately 80% of the anhydrous ethanol control. The disclosed plant extracts also showed moderate inhibitory effects on TNF, reducing TNF levels to approximately 80% of the anhydrous ethanol control at 25 µM, 12.5 µM, and 6.25 µM. Acetyl gingerone had no effect on TNF production. Concentrations of the disclosed plant extracts related to cytotoxicity are omitted to avoid data confounding. Results are summarized in Table 19 below.

[0473] Table 19. Overview of the results of TNF inhibition

[0474] No significant difference was observed when treated with the disclosed plant extract at 25 µM compared to treatment with 150 µM synthetic gingerone or ferulic acid. Figure 25 A statistically significant difference was observed between treatment with 25 µM of the disclosed plant extract and treatment with 150 µM acetylshogaol. A two-way ANOVA was also performed to determine whether TNF levels under the compound treatment conditions were statistically significant compared to those under the medium treatment conditions. Based on this analysis, treatment with 150 µM ferulic acid showed significantly lower TNF levels compared to medium treatment. Figure 26 ).

[0475] Under unstimulated conditions ( Figures 27A to 27D In the unstimulated condition, TNF levels were much lower and generally not higher than the ELISA background reading. No trend toward TNF production was observed under unstimulated conditions. This provides confidence that the test group would not induce TNF production. Since TNF levels were low under unstimulated conditions and typically 0 pg / mL, this data was not normalized to the anhydrous ethanol control.

[0476] Results—NO: Nitric oxide (NO) is a small molecule released by macrophages during the inflammatory response and can kill invading pathogens. Only the disclosed plant extracts showed inhibitory activity against NO, reducing it to 77% of the anhydrous ethanol control. Figure 28When compared with anhydrous ethanol, synthetic gingerone caused an increase in NO at higher test concentrations (50 µM, 75 µM, 100 µM, and 150 µM). 150 µM acetylgingone also caused a slight increase in NO levels. Ferulic acid did not alter NO production. Concentrations of the disclosed plant extracts associated with cytotoxicity have been omitted to avoid obfuscation of data. Results are summarized in Table 20 below.

[0477] Table 20. Overview of the results of NO production

[0478] It is worth noting that since nitric oxide measurements can only be performed under stimulated conditions, a large amount of supernatant is required.

[0479] To determine whether RAW 264.7 cells treated with 25 µM of the disclosed plant extract had significantly lower NO levels compared to other treatments, a one-way ANOVA was used. Treatment with the disclosed plant extract at 25 µM resulted in significantly lower NO levels compared to treatment with 150 µM of synthetic gingerone, acetylshogaol, and ferulic acid. Figure 29 To determine whether the observed NO changes were significantly different from those of the medium, a two-way ANOVA was used. Treatment with the disclosed plant extract at 25 µM did not produce significantly lower NO levels compared to treatment with the medium. However, treatment with 150 µM synthetic gingerone produced significantly higher NO levels than treatment with the medium. Figure 30 ).

[0480] Conclusion: Cytotoxicity was observed in the disclosed plant extract at a concentration of approximately 50 µM. The disclosed extract showed maximum inhibition of IL-6 from stimulated RAW264.7 cells. Treatment with 25 µM of the disclosed plant extract reduced IL-6 levels to less than 50% of the anhydrous ethanol control. This inhibition of IL-6 by the disclosed extract was statistically significant compared to the medium control and compared to treatment with synthetic gingerone, acetyl gingerone, or ferulic acid. Treatment with the disclosed plant extract also moderately inhibited TNF from stimulated RAW264.7 cells. Furthermore, 25 µM of the disclosed plant extract inhibited NO production from stimulated RAW264.7 cells. This inhibition of NO by the disclosed extract was statistically significant compared to treatment with synthetic gingerone, acetyl gingerone, or ferulic acid.

[0481] Other tested compounds produced mixed results, and in most cases, their efficacy was generally low. Treatment with 150 µM synthetic gingerone or acetyl gingerone moderately inhibited IL-6. These reductions were statistically significant when compared to treatment with the mediated compound. Treatment with 150 µM synthetic gingerone moderately reduced TNF levels. 150 µM ferulic acid also inhibited TNF production, reducing its levels to approximately 60% of the anhydrous ethanol control. This reduction was considered statistically significant when compared to treatment with the mediated compound. Ferulic acid had no effect on NO production. Both synthetic gingerone and acetyl gingerone increased NO production. The increase in NO caused by synthetic gingerone was considered statistically significant when compared to treatment with the mediated compound.

[0482] In summary, the disclosed plant extracts were observed to be significantly superior to the commercially available compounds tested. When compared to commercially available gingerone and acetyl gingerone, the plant extracts exhibited potent inhibition of pro-inflammatory cytokine expression and the production of pro-inflammatory small molecules. These results demonstrate the superior therapeutic efficacy of plant extracts in blocking / interfering with inflammatory pathways.

[0483] It should be recognized that the excellent inhibitory activity of the plant extracts is particularly surprising, especially considering that the concentrations of the plant extracts used in the comparative tests (e.g., 25 µM) are significantly lower than those of commercially available compounds (e.g., 100 µM or 150 µM).

[0484] Example 11: Preliminary tests in animals

[0485] Overview: Study of gingerone administration in an animal model of encephalitis (EAE).

[0486] Materials and Methods: Six groups of female C57BL / 6J mice (9 to 11 weeks old, n=5 per group), along with a healthy control group, were administered the mediator control, gingerone (10 mg / kg), or other test compounds via oral gavage. EAE induction was initiated on day 1. The test compounds were administered daily from day 5 to day 30.

[0487] Results: EAE was not successfully induced in this study (data not shown). However, body weight was monitored in mice as an indicator of safety and tolerability. During the treatment period, the average body weight increased by 0.3 g, with an average body weight of 19.7 g at the start of treatment and 20.0 g at the end of treatment. No significant changes in body weight were observed with gingerone treatment, indicating that the dose used was safe and tolerable.

[0488] Example 12: Large-scale production process

[0489] Overview: Large-scale production processes have been implemented. Each run allows for a maximum of 700 ± 50 kg of ginger rhizomes, as outlined below.

[0490] Step 1: Preliminary juicing of ginger rhizomes.

[0491] Description: A total of 1360 kg of ginger was transported to Phytex's Synergy Foodgroup manufacturing facility in Brookvale, Sydney. The material was stored under refrigeration (4°C ± 3°C) overnight. During processing, the material was transferred back to refrigeration to minimize the risk of degradation. Ginger juice was collected in 200 L blue polyethylene drums and transported back to the Phytex plant. Solid residue was collected in separate 200 L blue polyethylene drums (see, for example,...). Figure 31 Clean the equipment and juicing area, and maintain / calibrate the equipment. Use cleaning labels or cleaning records as needed.

[0492] Operation Details A: Divide the ginger rhizomes into two equal portions, approximately 700 kg each. Store the second 700 kg frozen until use the following week. Process the first 700 kg through a belt juicer (Voran EBP500). Collect all juice from the juicer into a 200 L blue HDPE bucket (supplied by Phytex) on a wooden tray. Place all solids / pulp from the juicer into a separate 200 L blue HDPE bucket (supplied by Phytex). Process the solids / pulp a second time through a belt juicer (Voran EBP500) to further remove liquid from the solids. Combine the liquid from the second press with the original 200 L blue HDPE bucket containing the liquid from the first press.

[0493] Run B Details: Every other week, begin the second 700 kg juice extraction. Pass the second 700 kg through a belt juicer (Voran EBP500). Place all juice from the juicer into a 200 L blue HDPE tank (supplied by Phytex) on a wooden pallet. Collect all solids / pulp from the juicer into a separate 200 L blue HDPE tank (supplied by Phytex). Pass the pulp a second time through the belt juicer (Voran EBP500) to further remove liquid from the pulp. Combine the liquid from the second press with the original 200 L blue HDPE tank containing the liquid from the first press.

[0494] The total volume of ginger juice and solid residue is transferred for process sampling and preparation for ultrafiltration (UF). The mass balance and yield percentage are calculated and recorded. At the end of step 1, approximately 600 L ± 50 L of ginger juice and 200 kg + 50 kg of ginger residue / solid are obtained from each batch of 700 kg + 50 kg ginger rhizomes.

[0495] Step 2 (i): Alkali treatment of ginger juice and ginger residue. See, for example, Figure 32 The flowchart in the document.

[0496] Introduction: Run A and Run B are performed with two equal KOH treatments. The maximum capacity of the extraction vessel is 400 L, therefore each run (A or B) is divided into two parts (Run A1 / A2 and Run B1 / B2). Check the equipment maintenance / calibration and cleanliness status before proceeding with the treatment. Use cleaning labels or cleaning records as needed.

[0497] Ginger Juice Processing (300 L per run): Measure the volume of ginger juice obtained from multiple 200 L HDPE barrels. Record the net weight of all HDPE barrels. Use a 1 m plastic mixing paddle to mix the remaining ginger juice / settleable solids in each 200 L HDPE barrel to ensure solids are resuspended. Attach a clamp to the pour cap (cap with a 25 mm polyethylene valve) of each 200 L HDPE barrel. Use a barrel lifter to pour all solids fractions into the extraction vessel to combine with the UF concentrate. Insert the wall-mounted agitator purple propeller into the extraction vessel attached with 2 x 100 mm stainless steel propellers. Set the propeller height to the appropriate level to mix the juice volumes. Attach the wall-mounted agitator to the utility plate and begin stirring at 50 Hz in a clockwise direction. Scoop out the settleable sludge from the bottom of each barrel and transfer it to the extraction vessel (dissolved by heating and alkali treatment). Rinse each barrel with 1 L of water to remove all residual ginger juice and add it to the extraction vessel.

[0498] Add potassium hydroxide to the ginger juice: Begin stirring and add 2.0% w / w KOH. This is based on the total volume of juice obtained from the ginger rhizome (raw material). Record the volume of the ginger juice and the initial pH level before adding KOH. Zero the blue HDPE bucket on a 150 kg scale. The alkali treatment includes weighing the total amount of ginger juice. Calibrate the pH electrode with pH 7.0 and pH 4.0 buffer solutions, and check the calibration with pH 2.0 buffer solution. In the extraction vessel, add 50% KOH to the ginger juice extract at the following ratio: KOH weight (kg) = ginger juice weight (kg) x 0.03 kg KOH (50%). Adding at this ratio will raise the pH to 13.0 + 1.0. All pH readings are adjusted with automatic temperature compensation (Mettler ToledopH meter). When the pH range is reached, continue stirring for another 15 minutes (minimum). Record the final pH and the volume of KOH added.

[0499] Alkali treatment procedure for ginger juice: Insert a stainless steel steam coil into the extraction vessel and connect it to the steam inlet on the utility wall using a dedicated steam hose. Open the steam valve on the wall to allow steam to heat the coil. Calibrate the pH electrode using pH 7.0 and pH 4.0 buffer solutions, and check the calibration using a pH 2.0 buffer solution. Heat the ginger juice (pH > 13) to 60°C ± 2°C and stir for 60 minutes. Adjust the steam flow rate to ensure the alkaline liquid is maintained within the temperature range (60°C ± 2°C). Take a sample from the stirred alkaline ginger juice (20 mL) to monitor the conversion of 6-gingerol to gingerone. Record the volume of the alkaline ginger juice.

[0500] Neutralization procedure for ginger juice: Stir the alkaline ginger juice for another 5 minutes, then neutralize the solution with >98.0% citric acid. Connect the SS vortex tube in the extraction vessel to cooling water and circulate the cooling water. Adding citric acid (>98%) to the alkaline juice indicates a strong acid-base reaction, which is exothermic and generates heat. Apply cooling water to mitigate excess heat in the solution. Prepare the citric acid before use. This is based on a starting weight of 1340 kg of raw material. Weigh the citric acid granules (18.0 kg) into a 20 L purple polyethylene tank. Prepare an additional citric acid aqueous solution (50% w / v) by dissolving 1.0 kg of citric acid granules in 1.0 L of pure water in the 20 L purple polyethylene tank. Dissolve using a stainless steel hand stirrer. While stirring the alkaline ginger juice in the extraction vessel, add the citric acid granules. This is done by adding 10 kg at a time and waiting for the granules to dissolve. Monitor the pH of the solution during this procedure. Continue to slowly add citric acid using 500 ml spoons each time, waiting 30 seconds before adding the next spoonful. After adding and dissolving all citric acid (18.0 kg), obtain the pH reading. Then add 50% aqueous citric acid solution until a pH range of 7.0–7.3 is obtained. Once this desired range is reached, continue stirring for another 15 minutes. All pH adjustments were performed with automatic temperature compensation (Mettler Toledo pH meter). The resulting solution was then labeled “gingerone liquid.” Record the final pH, the volume of added citric acid (>98%), and the volume of gingerone liquid. A sample was removed from the gingerone liquid (20 mL) to monitor the conversion of 6-gingerol to gingerone.

[0501] Step 2 (ii): Alkali treatment of ginger residue. For example, see, Figure 33 The flowchart in the document.

[0502] Processing ginger residue / solids (50 kg per run): Insert the wall-mounted agitator into the extraction vessel equipped with three large 150 mm propellers. Start stirring at 50 Hz. Add 50 kg of total solid residue from a 200 L HDPE tank to the extraction vessel. Ensure that the solids are continuously drawn into the liquid and that the solution moves freely with the agitator. If the solution becomes too viscous, add 20 L of water.

[0503] Add potassium hydroxide to the ginger residue / solid: Add 2% w / w KOH. This is based on the weight of the total volume of juice extract obtained from the ginger rhizome raw material. Zero the blue HDPE bucket on a 150 kg scale. The total amount of residue is weighed during alkali treatment. Calibrate the pH electrode with pH 7.0 and pH 4.0 buffer solutions, and check the calibration with pH 2.0 buffer solution. In the extraction vessel, add 50% KOH to the ginger juice extract at the following ratio: KOH weight (kg) = ginger residue weight (kg) x 0.03 kg KOH (50%). The calculation takes into account the weight of the ginger residue (50 kg) and the added water (20 kg). Adding at this ratio will raise the pH to 13.0 ± 1.0. When the pH range is reached, continue stirring for another 15 minutes (minimum). Record the final pH and the volume of KOH added.

[0504] Alkali treatment procedure for ginger residue / solid: Insert the SS vapor cyclone tube into the extraction vessel and connect it to the steam inlet on the utility wall using a dedicated steam hose. Open the steam valve on the wall to allow steam to heat the cyclone tube. Calibrate the pH electrode using pH 7.0 and pH 4.0 buffer solutions, and check the calibration using pH 2.0 buffer solution. Heat the ginger residue (pH > 13) to 60°C ± 2°C and stir for 60 minutes. Adjust the steam flow rate to ensure the alkaline liquid is maintained within the temperature range (60°C ± 2°C). Take a sample from the stirred alkaline ginger residue (20 mL) to monitor the conversion of 6-gingerol to gingerone. Record the volume of the alkaline ginger residue.

[0505] Neutralization of Ginger Residue / Solid: Stir the alkaline ginger residue for another 5 minutes, then neutralize the solution with >98.0% citric acid. Connect the SS vortex tube in the extraction vessel to cooling water and circulate the cooling water. This is to mitigate excessive heat from the exothermic reaction (acid + base). Prepare the citric acid as described above before use. While stirring the alkaline ginger residue in the extraction vessel, add citric acid granules. This is done by adding 5 kg and waiting for the granules to dissolve. Monitor the pH of the solution during this procedure. Continue to slowly add citric acid using 500 ml spoons each time, waiting 30 seconds before adding the next spoonful. After adding and dissolving all the citric acid (18 kg, total), obtain the pH reading. Then add 50% aqueous citric acid solution until a pH range of 7.0–7.3 is obtained. When this desired range is reached, continue stirring for another 15 minutes. All pH reading adjustments are performed with automatic temperature compensation (Mettler Toledo pH meter). The resulting solution is then labeled “Ginger Residue”. Record the final pH, the volume of added citric acid (>98%), and the volume of gingerone residue. Remove a sample from the gingerone residue (20 mL) to monitor the conversion of 6-gingerol to gingerone. Calculate the mass balance and yield percentage.

[0506] Step 3: Gingerone powder is produced by drying and grinding.

[0507] Oven Drying: Verify equipment maintenance / calibration and cleanliness. Set the drying unit using a calibrated COMARS digital recorder with a stainless steel probe. Record the time taken to remove each barrel of gingerone concentrate from the freezer for drying. Record the Brix% and theoretical gingerone content. All trays in the oven have Teflon pads for drying. Mix the barrels using a polypropylene paddle, then remove a certain volume of liquid using a 4 L jug. Transfer this to a 2 L jug and place it on each tray. Next, pour 3 L onto the Teflon pad on each tray. Load the trays onto each rack, ensuring a gap (not less than 30 mm) between trays for airflow and to allow the drying material to break up. Set the oven temperature to 60°C. After 24 hours, inspect and scrape each tray, scraping off any softer portions of the Teflon pads as needed. The bottom of the product is placed on an inverted tray. Gently break up the product using a plastic scraper to promote drying. Load the trays back into the oven and dry for another 4 hours (total 28 hours). When fully dried (24 or 28 hours), manually crush the product using a stainless steel scraper and collect it in 100-micron polyethylene bags. Weigh and record the weight of each bag, then place it in a 100 L square movable bowl. Weigh and record the weight of the square movable bowl. Then, prepare the product for grinding.

[0508] Grinding: Before grinding, check that the stainless steel hammer mill is clean and assemble a clean 1 mm mesh sieve (“fine gingerone mesh sieve”). Assemble the cover with a 3 mm rubber gasket seal and tighten it securely with a thumbscrew to lock the cover in place. Run the hammer mill for 10 seconds to ensure proper rotation and clearance. Assemble a 100-micron polyethylene bag into the outlet of the grinding chamber and secure it with an adjustable clamp. Start the top vacuum. Slowly transfer the coarse, dried gingerone material into the feed chute using a polyethylene scoop. Pour two scoops into the chute each time, opening the inlet cover every few seconds. All material in the chute passes through the stainless steel sieve and is collected in the polyethylene bag collector. Weigh all bags and calculate to determine the mass balance and yield percentage. Remove a representative sample (20 g) from each bag and assess the moisture percentage determination. Place all 20 kg bags into an additional 100-micron polyethylene bag. Evacuate any remaining air gaps before performing a double seal using a pulse heat sealer. Store the bags in a refrigerator. Perform quality analysis on the final product (gingerone powder). See Table 21 below. The quality indicators were found to be excellent.

[0509] Table 21. Quality assessment of gingerone extract (powder)

[0510] Step 4: Ethanol extraction of gingerone powder.

[0511] Introduction: As outlined below, up to 75 kg of dry powder can be extracted in a single extraction step. Verify the equipment maintenance / calibration and cleanliness (including the extraction vessel). Attach a 40 mm stainless steel filter to the outlet at the bottom of the extraction vessel. Hand-tighten the filter to the bottom 25 mm thread to filter out solids after extraction agitation. Assemble a dedicated stainless steel heating vortex tube inside the extraction vessel for later steam connection in this step. Attach a glass condenser vortex tube to the lid of the extraction tank and connect the inlet / outlet to the cooling water inlet / outlet on the wall. Circulate the cooling water through the glass condenser vortex tube.

[0512] Single Extraction: For extraction, add 95% ethanol to a clean, capped extraction vessel. Use the following addition rate: 95% ethanol volume = gingerone powder amount (kg) x 5 kg. Insert the purple WMS shaft through the bearing cap in the extraction vessel cap. Start stirring at 200 to 300 rpm to promote complete mixing of the ethanol solution. Slowly add 75 kg of dried gingerone powder (from step 3) to the extraction vessel through the 100 mm inlet / outlet hole on the vessel cap. Continue stirring during this addition. After all powder has been added, close the inlet / outlet hole and seal the cap. Record all initial temperatures before heating. Next, connect the steam supply to the stainless steel rotary tube in the extraction vessel. Monitor the temperature of the ethanol solution while stirring and maintain it at a minimum of 50°C for at least 4 hours. Turn off the stirrer and allow the solution to stand for 20 minutes. Record the final temperature before filtering the ethanol extract. Remove the sample from the ethanol extract (20 mL) and test for gingerone.

[0513] Filtration of residual solids in the ethanol extract: Drain the contents of the ethanol extract into two clean 200L HDPE buckets. Continue stirring while draining. Let the ethanol extract stand for 30 minutes to allow any solids to settle. A hopper plate filter is fitted with a Z1 pad (5 microns). Assemble and compact using a stainless steel plunger tool. Use a bucket lifter to pour the upper layer of the ethanol extract through the hopper plate filter. Pour the poured solution into a 55-micron pre-filter and collect it in a bucket. Collect the filtered ethanol extract from the hopper plate filter into two clean blue HDPE buckets. When the hopper plate filter is full of solids, apply a full vacuum to the apparatus for 10 minutes until all liquid is collected. After each vacuum filtration, empty the hopper plate filter and place it in a clean polyethylene bucket. Repeat this process until all ethanol extract has been filtered through the Z1 pad. Record the volume of 95% ethanol wash. Remove a sample from the combined filtered ethanol extract (20 mL) and test for gingerone. Store the two containers containing the filtered ethanol extract in a refrigerator.

[0514] Rotary Evaporation of Ethanol Extract: Remove the filtered ethanol extract from three 15 L barrels from the refrigerator and allow it to equilibrate to room temperature. Prepare, dry, and weigh the Heidolph round-bottom flasks. Set up the Heidolph 20 L rotary evaporator and add 1 L of fresh 95% ethanol to the RBF. Connect the cooling water inlet / outlet lines to the appropriate valves on the wall and adjust the valves to change the flow rate as needed. Connect the rotary evaporator to the Heidolph vacuum pump. Connect the white 4 mm HDPE tubing, immersed in 95% ethanol, to the inlet tap of the rotary evaporator. Begin circulating the cooling water through the glass condenser. Remove residual water during the evaporation phase by running at a minimum of 50 mBar and 40°C. Therefore, set the vacuum and begin slowly rotating the round-bottom flasks (approximately 60 rpm). As each barrel is emptied, transfer the inlet line to the next barrel until all three barrels (total 45 L) have been drawn into the rotary evaporator and collected in the condenser flask. Observe the pressure, color, etc.

[0515] Standardization of the ethanol extract: For each rotary evaporator run (45 L of filtered ethanol extract from three barrels), concentrate the volume to the 4 L mark on the outside of the RBF. This mark approximately represents the standard concentration of 4 L (12.5% ​​mg / ml) of 45 L of ethanol extract. At this point, remove a 10 mL sample of the concentrated tincture and test for gingerone. If the gingerone concentration is greater than 12.5 mg / ml, add 1 L of the combined filtered ethanol extract from step 4 and continue evaporation until the correct concentration is achieved. If the gingerone concentration is less than 12.5 mg / ml, continue evaporation until the correct concentration is achieved. Rinse twelve 4 L amber glass bottles with fresh 95% ethanol and drain for 30 minutes. When the standardization is 12.5 mg / ml, remove the RBF from the rotary evaporator and place it in a 15 L barrel. Pour the standardized gingerone tincture into the rinsed and drained bottles, leaving 30 mm of the bottom thread. Cover the filled vials with polyethylene caps with Teflon seals and tighten by hand. Cover the neck and cap with sealing film. Place the sealed vials in a 100-micron polyethylene bag and double-heat seal. Place these sealed vials in a box and refrigerate the box. Perform quality analysis on the final product (ethanol extract). See Table 22 below. Excellent quality indicators were found. The stability of the final product was assessed and confirmed after 2 months of storage under accelerated conditions (40°C ± 2°C / 75% ± 5% RH) and independently after 2 months of storage under cooled conditions (5°C). Extremely high stability was found. See Tables 23 and 24 below. Some changes were observed at the 1-month time point due to the small sample amount tested under accelerated conditions (less than 2 g) (Table 23). It should be understood that larger sample amounts during storage can enhance product stability by reducing headspace in the container. Good stability is expected over at least 6 months of storage.

[0516] Table 22. Quality assessment of gingerol extract (tincture)

[0517] Table 23. Stability assessment of gingerone extract (tincture)

[0518] Table 24. Stability assessment of gingerone extract (tincture)

[0519] Those skilled in the art can utilize the disclosure and teachings herein to generate other implementations and variations without excessive experimentation. All such implementations and variations are considered part of this disclosure.

[0520] Therefore, those skilled in the art will readily understand from this disclosure that, based on such related embodiments, subsequent modifications, substitutions, and / or variations can be made to implement substantially the same functions or achieve substantially the same results as the embodiments described herein. Therefore, this disclosure is intended to cover within its scope any modifications, substitutions, and variations to the processes, manufactures, material compositions, compounds, means, methods, and / or steps disclosed herein.

[0521] The description herein may include subject matter that is beyond the scope of the claimed invention. This subject matter is included to aid in understanding the invention.

[0522] In this specification, references have been made to external sources of information, including patent specifications and other documents, generally for the purpose of providing context for discussing the features of this disclosure. Unless otherwise stated, such references to sources of information in any jurisdiction should not be construed as an admission that such sources are prior art or constitute part of common general knowledge in the art.

Claims

1. Methods for producing gingerone include: (i) subject ginger roots to alkaline treatment in an alkaline solution; or (ii) subject the juice and / or residue obtained from ginger root to alkaline treatment in an alkaline solution; This produces gingerone.

2. The method according to claim 1, wherein the ginger root is fresh.

3. The method according to claim 1 or 2, wherein: (a) Chop the ginger root mentioned in (i); (b) Chop and dry the ginger root described in (i); or (c) The juice and / or residue of (ii) obtained by soaking and / or pressing ginger root.

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

5. The method according to claim 4, wherein the alkaline solution comprises: (a) Approximately 1% to approximately 6% KOH (v / v); (b) Approximately 1.5% to approximately 5.5% KOH (v / v); (c) Approximately 2% to approximately 4% KOH (v / v); (d) Approximately 1.5% to approximately 3.5% KOH (v / v); or (e) Approximately 2% KOH (v / v).

6. The method according to any one of claims 1 to 3, wherein calcium hydroxide (Ca(OH)2) is used in the alkaline solution.

7. The method according to claim 6, wherein the alkaline solution comprises: (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).

8. The method according to any one of claims 1 to 7, wherein: (a) The alkaline treatment is carried out at 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 approximately 60 degrees Celsius.

9. The method according to any one of claims 1 to 8, wherein: (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 alkali 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.

10. The method according to any one of claims 1 to 9, further comprising the following additional steps: (a) Neutralize the alkaline solution; (b) Neutralize the alkaline solution and then dry it; or (c) Neutralize the alkaline solution, optionally dry it, and then perform one or more additional extraction steps.

11. The method of claim 10, wherein the one or more additional extraction steps comprise: (a) One or more ethanol extraction steps; (b) Supercritical fluid extraction; or (c) Supercritical fluid extraction, followed by one or more ethanol extraction steps.

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

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

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

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

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

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

18. The composition according to any one of claims 14 to 17, wherein it is formulated as a solution, tincture, gel, jelly, gummies, powder, tablets or capsules.

19. The composition according to any one of claims 14 to 18, wherein it is formulated to comprise: (a) Gingerone in doses of approximately 10 mg to approximately 1500 mg; (b) Gingerone in doses of approximately 10 mg to approximately 1000 mg; (c) Gingerone in doses of approximately 10 mg to approximately 150 mg; (d) Gingerone in doses of approximately 10 mg to approximately 100 mg; or (e) Gingerone in doses of approximately 10 mg to approximately 50 mg.

20. The composition according to any one of claims 14 to 19, wherein: (a) It is formulated for use in combination with other anti-inflammatory agents; (b) Formulated for use in conjunction with one or more of the following: analgesic compounds, antipyretic compounds, and psychotropic compounds; (c) Formulated for use in conjunction with one or more of the following: cannabinoid compounds, fungal compounds, nonsteroidal anti-inflammatory drug compounds (NSAIDs), opioid compounds, salicylates, and steroid compounds; (d) Contains one or more of the following: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodoxacin, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxapazine, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levonorphanol, levonorphanone, pentazocine, phenazocine, etazoline, betamethasone, cortisone, dexamethasone, dexamethasone, ethammethasone, hydrocortisone, methylprednisolone, prednisolone, prednisolone, triamcinolone, cannabidiol, cannabinol, tetrahydrocannabinol, psilocybin, and dephosphorylated psilocybin.

21. The composition according to any one of claims 14 to 20, used for treating or preventing inflammation.

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

23. The composition according to claim 22, wherein: (a) The inflammation described is an inflammatory condition; (b) The inflammation requires regulation; (c) The inflammation described is associated with immune disorders; (d) The inflammation described is related to arthritis symptoms; (e) The inflammation described is related to infection; (f) The inflammation described is associated with heart disease, circulatory disorders, or lung diseases; (g) The inflammation is related to a neurological disorder; and / or (h) The inflammation mentioned is related to hyperplastic conditions.

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

25. The composition according to any one of claims 21 to 24, wherein the inflammation is associated with one or more of the following: 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 diseases, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma, Sjögren's syndrome, and systemic lupus erythematosus.

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

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

28. The composition according to any one of claims 21 to 24, wherein the inflammation is associated with one or more of the following: vesicles, dermatitis, eczema, urticaria, lesions, papules, plaques, psoriasis, rash, rosacea, ulcers, and wounds.

29. Use of the composition according to claim 14 for the preparation of a medicament for the treatment or prevention of inflammation in an individual.

30. The use according to claim 29, wherein the drug provides to reduce or slow the progression of the inflammation.

31. The use according to claim 29 or 30, wherein: (a) The drug is formulated as a solid, semi-solid, or liquid; and / or (b) The drug is formulated for topical or oral administration.

32. The use according to any one of claims 29 to 31, wherein the drug is formulated as a solution, gel, jelly, gummies, powder, tablets or capsules.

33. The use according to claim 32, wherein the drug is formulated to comprise: (a) Gingerone in doses of approximately 10 mg to approximately 1500 mg; (b) Gingerone in doses of approximately 10 mg to approximately 1000 mg; (c) Gingerone in doses of approximately 10 mg to approximately 150 mg; (d) Gingerone in doses of approximately 10 mg to approximately 100 mg; or (e) Gingerone in doses of approximately 10 mg to approximately 50 mg.

34. The use according to any one of claims 29 to 33, wherein: (a) The composition is formulated for co-administration with other anti-inflammatory agents; (b) The composition is formulated for co-administration with one or more of the following: analgesic compounds, antipyretic compounds, and psychotropic compounds; (c) The composition is formulated for co-administration with one or more of the following: cannabinoid compounds, fungal compounds, nonsteroidal anti-inflammatory drug compounds (NSAIDs), opioid compounds, salicylates, and steroid compounds; (d) The drug comprises one or more of the following: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etoricoxib, etoricoxib, biphenylacetic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxapazine, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levonorphanol, levonorphanone, pentazocine, phenazocine, etanertin, betamethasone, cortisone, defcoccal, dexamethasone, ethammethasone, hydrocortisone, methylprednisolone, prednisolone, prednisolone, triamcinolone, cannabidiol, cannabinol, tetrahydrocannabinol, psilocybin, and dephosphated psilocybin.

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

36. The use according to claim 35, wherein: (a) The inflammation described is an inflammatory condition; (b) The inflammation requires regulation; (c) The inflammation described is associated with immune disorders; (d) The inflammation described is related to arthritis symptoms; (e) The inflammation described is related to infection; (f) The inflammation described is associated with heart disease, circulatory disorders, or lung diseases; (g) The inflammation is related to a neurological disorder; and / or (h) The inflammation mentioned is related to hyperplastic conditions.

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

38. The use according to any one of claims 35 to 37, wherein the inflammation is associated with one or more of the following: 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 diseases, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma, Sjögren's syndrome, and systemic lupus erythematosus.

39. The use according to any one of claims 35 to 37, wherein the inflammation is associated with one or more of the following: 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 Sjögren's syndrome arthritis.

40. The use according to any one of claims 35 to 37, wherein the inflammation is associated with one or more of the following: atherosclerosis, coronary artery disease, pulmonary hypertension, hypoxic pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory distress, and cytokine storm syndrome; or originates from one or more of the following: breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

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

42. A method for treating or preventing inflammation, the method comprising administering to an individual the composition according to claim 15, thereby treating or preventing said inflammation.

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

44. The method according to claim 42 or 43, wherein: (a) The composition is applied as a solid, semi-solid, or liquid; and / or (b) The composition is administered by topical or oral application.

45. The method according to any one of claims 42 to 44, wherein the composition is administered as a solution, gel, jelly, gummies, powder, tablets or capsules.

46. ​​The method of claim 45, wherein the composition is applied as follows: (a) Gingerone in doses of approximately 1 mg to approximately 5000 mg; (b) Gingerone in doses of approximately 1 mg to approximately 1500 mg; (c) Gingerone in doses of approximately 5 mg to approximately 500 mg; (d) Gingerone in doses of approximately 1 mg to approximately 15 mg; or (e) Gingerone in doses of about 1 mg to about 10 mg.

47. The method according to any one of claims 42 to 46, wherein: (a) The composition is administered in combination with another anti-inflammatory agent; (b) The composition is administered in combination with one or more of the following: analgesic compounds, antipyretic compounds, and psychotropic compounds; (c) The composition may be used in combination with one or more of the following: cannabinoid compounds, fungal compounds, nonsteroidal anti-inflammatory drug compounds (NSAIDs), opioid compounds, salicylates, and steroid compounds; (d) The composition is administered in combination with one or more of the following: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etoricoxib, etoricoxib, biphenylacetic acid, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxapazine, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levonorphanol, levonorphanone, pentazocine, phenazocine, etanertin, betamethasone, cortisone, dexamethasone, dexamethasone, ethammethasone, hydrocortisone, methylprednisolone, prednisolone, prednisolone, triamcinolone, cannabidiol, cannabinol, tetrahydrocannabinol, psilocybin, and dephosphated psilocybin.

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

49. The method according to claim 48, wherein: (a) The inflammation described is an inflammatory condition; (b) The inflammation requires regulation; (c) The inflammation described is associated with immune disorders; (d) The inflammation described is related to arthritis symptoms; (e) The inflammation described is related to infection; (f) The inflammation described is associated with heart disease, circulatory disorders, or lung diseases; (g) The inflammation is related to a neurological disorder; and / or (h) The inflammation mentioned is related to hyperplastic conditions.

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

51. The method according to any one of claims 48 to 50, wherein the inflammation is associated with one or more of the following: 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 diseases, inflammatory bowel disease, interstitial cystitis, multiple sclerosis, polymyalgia psoriasis, scleroderma, Sjögren's syndrome, and systemic lupus erythematosus.

52. The method according to any one of claims 48 to 50, wherein the inflammation is associated with one or more of the following: 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 Sjögren's syndrome arthritis.

53. The method according to any one of claims 48 to 50, wherein the inflammation is associated with one or more of the following: atherosclerosis, coronary artery disease, pulmonary hypertension, hypoxic pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory distress, and cytokine storm syndrome; or originates from one or more of the following: breast cancer, leukemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.

54. The method according to any one of claims 48 to 50, wherein the inflammation is associated with one or more of the following: vesicles, dermatitis, eczema, urticaria, lesions, papules, plaques, psoriasis, rashes, rosacea, ulcers, and wounds.

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