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WO2024171115A1 - Méthodes de préparation de zingérone, compositions contenant de la zingérone, et leurs utilisations - Google Patents

Méthodes de préparation de zingérone, compositions contenant de la zingérone, et leurs utilisations Download PDF

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Publication number
WO2024171115A1
WO2024171115A1 PCT/IB2024/051451 IB2024051451W WO2024171115A1 WO 2024171115 A1 WO2024171115 A1 WO 2024171115A1 IB 2024051451 W IB2024051451 W IB 2024051451W WO 2024171115 A1 WO2024171115 A1 WO 2024171115A1
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WIPO (PCT)
Prior art keywords
zingerone
inflammation
composition
arthritis
ginger
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Ceased
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PCT/IB2024/051451
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English (en)
Inventor
Cynthia HUNEFELD-GAIKEMA
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Evithe Ltd
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Evithe Ltd
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Priority to KR1020257030660A priority Critical patent/KR20250145108A/ko
Priority to AU2024221532A priority patent/AU2024221532A1/en
Priority to CN202480024232.2A priority patent/CN120957711A/zh
Publication of WO2024171115A1 publication Critical patent/WO2024171115A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/30—Other Organic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00—Processes
    • A23V2300/44—Supercritical state
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/10—Preparation or pretreatment of starting material
    • A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction

Definitions

  • the present disclosure relates to methods for preparing zingerone and compositions comprising zingerone.
  • beneficial compositions including pharmaceutical compositions and dietary compositions, and uses for these compositions.
  • Ginger Greek officinale'
  • rhizome is widely used as a spice and in traditional medicine. If consumed in reasonable quantities, ginger has few negative side effects. It is on the FDA's "generally recognized as safe” list.
  • the characteristic fragrance and flavour of ginger result from volatile oils that compose 1-3% of the weight of fresh ginger, primarily consisting of zingerone, shogaols, and gingerols with [6]-gingerol (l-[4'-hydroxy-3'- methoxypheny 1] - 5 -hydroxy- 3 -dec anone)
  • Zingerone also known as gingerone
  • Zingerone has been reported as being produced from gingerols during drying or heat treatment at temperatures of about 40 degrees Celsius as reported by Li et. al., 2016, “Chemical Characterisation and antioxidant activities comparison in fresh, dried, stir frying and carbonized ginger” Journal of Chromatography B Analyt. Technol. Biomed. Life Sci. 1011: 223-232.
  • Zingerone has a lower pungency and a spicy- sweet aroma.
  • Zingerone is also called vanillylacetone and is a crystalline solid that is reported to be sparingly soluble in water and soluble in ether.
  • Zingerone s water solubility value of 0.57 g/L and LogP value of 2.02 1.92 and logS of -2.5 is described on the FoodB compounds database; see https://foodb.ca/compounds/FDB010527. zingerone
  • Fresh ginger contains minimal zingerone, and it is known to be produced by cooking or drying of the ginger root, which causes dehydration of gingerol through the loss of a water molecule to produce zingerone and hexanal. See, e.g., Gopi et al. 2016, “Study on temperature dependent conversion of active components of ginger” Int. J. of Pharma Sciences 6(1): 1344-1347.
  • Shogaols are more pungent and have higher antioxidant activity and are not found in raw ginger, but are formed from gingerols during heating, storage or via acidity.
  • Shogaol is a dehydrated form of gingerol.
  • Zingerone was first isolated from the ginger root in 1917 by Hiroshi Nomura.
  • Nomura identified and later patented (US 1,263,796, issued April 23, 1918) a method for the synthesis of zingerone, in which vanillin and acetone are reacted under basic conditions to form dehydrozingerone. This compound was obtained in about 95% quantity.
  • This reaction was followed by catalytic hydrogenation of the intermediate compound in order to form zingerone, obtained in approximately 100% quantity.
  • Ginger compounds have been shown to be active against enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhoea. This type of diarrhoea is the leading cause of infant death in developing countries. It has been reported that Zingerone is likely the active constituent responsible for the antidiarrheal efficacy of ginger. The study concluded that ginger’s bioactive compounds significantly blocked the binding of enterotoxigenic Escherichia coli heat-labile enterotoxin to cellsurface receptor G Ml, resulting in the inhibition of fluid accumulation in the closed ileal loops of mice.
  • Zingerone has been shown to have an anti-inflammatory effect on liver inflammation in a peritonitis mouse model as reported by Kumar et al. 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): el06536.
  • Kumar et al. have also reported that zingerone can enhance the susceptibility of Pseudomonas aeruginosa cells to antibiotics. See Kumar et al., 2014, Life Sciences 117: 24-32. Kumar et al. concluded that zingerone was found to cause alterations in the cell surface properties of Pseudomonas aeruginosa thereby increasing the susceptibility of Pseudomonas aeruginosa cells to antibiotics.
  • this disclosure encompasses a method of producing zingerone by: (i) subjecting ginger root to an alkaline treatment in alkaline solution; (ii) subjecting juice obtained from ginger root to an alkaline treatment in an alkaline solution; or (iii) subjecting juice and marc obtained from ginger root to alkaline treatment in alkaline solution.
  • the ginger root is fresh.
  • the ginger root is dried at about 40 to about 70 degrees Celsius, or at about
  • the juice is obtained by macerating and/or pressing the ginger root.
  • the marc is obtained by juicing, macerating, and/or pressing the ginger root.
  • the ginger root is diced and subjected to the alkaline treatment.
  • the ginger root is diced, dried and subjected to the alkaline treatment.
  • the alkaline treatment is carried out at about 40 to about 70 degrees Celsius.
  • the alkaline treatment is carried out at about 50 to about 60 degrees Celsius. [0024] The alkaline treatment is carried out at about 55 to about 65 degrees Celsius.
  • the alkaline treatment is carried out at about 60 degrees Celsius.
  • the alkaline treatment is carried out for about 1-72 hours.
  • the alkaline treatment is carried out for about 1-48 hours.
  • the alkaline treatment is carried out for about 1-24 hours.
  • the alkaline treatment is carried out for about 1-30 hours, or about 1-20 hours, or about 1-10 hours, or about 1-5 hours.
  • the alkaline treatment is carried out for about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours.
  • the alkaline treatment is carried out for about 2 hours.
  • the alkaline treatment is carried out for about 1 hour.
  • KOH Potassium hydroxide
  • KOH potassium hydroxide
  • KOH KOH
  • v/v 6% KOH
  • v/v 5% to about 5.5% KOH
  • 1% to about 6% KOH (v/v) is used.
  • about 1.5% to about 5.5% KOH (v/v) is used.
  • about 2% to about 4% KOH (v/v) is used.
  • About 1.5% to about 3.5% KOH (v/v) is used.
  • the alkaline solution is neutralised with citric acid.
  • the alkaline solution is cooled during neutralisation to alleviate excess heat.
  • the alkaline solution is neutralised to obtain a pH of about 6.5 to about 7.5 or about 7.0 to about 7.3.
  • the neutralised solution is freeze dried.
  • the neutralised solution is heat dried. [0044] The neutralised solution is subjected to extraction of the zingerone.
  • the neutralised solution is dried and optionally subjected to extraction of the zingerone.
  • the drying is at about 50 degrees Celsius to at about 70 degrees Celsius.
  • the drying is at about 55 degrees Celsius to about 65 degrees Celsius.
  • the drying is at about 60 degrees Celsius.
  • the drying is for at least 24 hours.
  • the drying is for about 24 hours to about 28 hours.
  • the dried material is optionally milled.
  • the zingerone is optionally further extracted by one or more alcohol extraction steps.
  • the zingerone is optionally further extracted by one or more ethanol extraction steps.
  • the ethanol extraction is carried at about 35 degrees Celsius to at about 65 degrees Celsius.
  • the ethanol extraction is carried at about 45 degrees Celsius to about 55 degrees Celsius.
  • the ethanol extraction is carried at about 50 degrees Celsius.
  • the ethanol extraction is carried out for at least 7 days.
  • the ethanol extraction is carried out for 24 hours or less.
  • the ethanol extraction is carried out for at least 4 hours.
  • the ethanol extraction is carried out for about 4 to about 8 hours.
  • the ethanolic extract is optionally dried.
  • the zingerone is extracted using supercritical fluid extraction.
  • the zingerone is extracted by a supercritical fluid extraction followed by an alcohol extraction step.
  • the method produces a product, this being a composition that comprises zingerone.
  • the composition is free from or substantially free from aldehydes.
  • composition is a botanical extract.
  • the composition is an ethanolic extract.
  • the composition is a powder.
  • this disclosure encompasses a method of producing zingerone by subjecting ginger root extract to an alkaline treatment.
  • the ginger root extract is obtained by supercritical fluid extraction of the ginger root.
  • the ginger root extract is obtained by alcohol extraction of the ginger root.
  • the ginger root extract is obtained by juicing the ginger root.
  • the ginger root extract is obtained by juicing the ginger root to obtain a juice and a marc.
  • the juicing includes macerating and/or pressing the ginger root.
  • the alkaline treatment is carried out at about 30 to about 70 degrees Celsius.
  • the alkaline treatment is carried out at about 50 about 60 degrees Celsius.
  • the alkaline treatment is carried out at about 55 to about 65 degrees
  • the alkaline treatment is carried out at about 60 degrees Celsius.
  • the alkaline treatment is carried out for about 1-72 hours.
  • the alkaline treatment is carried out for about 1-48 hours.
  • the alkaline treatment is carried out for about 1-24 hours.
  • the alkaline treatment is carried out for about 1-30 hours, or about 1-20 hours, or about 1-10 hours, or about 1-5 hours.
  • the alkaline treatment is carried out for about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours.
  • the alkaline treatment is carried out for about 2 hours.
  • the alkaline treatment is carried out for about 1 hour.
  • Potassium hydroxide (KOH) is used.
  • KOH potassium hydroxide
  • KOH (v/v) About 0.1% to about 6% KOH (v/v) is used. About 0.5% to about 5.5% KOH (v/v) is used. About 1% to about 6% KOH (v/v) is used. About 1.5% to about 5.5% KOH (v/v) is used. About 2% to about 4% KOH (v/v) is used. About 1.5% KOH (v/v) to about 3.5% KOH (v/v) is used.
  • the alkaline solution is neutralised to obtain a pH of about 6.5 to about 7.5 or about 7.0 to about 7.3.
  • the dried material is optionally milled.
  • the dried material is optionally further extracted.
  • the zingerone is optionally further extracted by one or more alcohol extraction steps.
  • the zingerone is optionally further extracted by one or more ethanol extraction steps.
  • the ethanol extraction is carried out for at least 7 days.
  • the ethanol extraction is carried out for 24 hours or less.
  • the ethanol extraction is carried out for at least 4 hours.
  • the ethanol extraction is carried out for about 4 to about 8 hours.
  • the ethanolic extract is optionally dried.
  • the zingerone is optionally further extracted using supercritical fluid extraction.
  • the zingerone is optionally further extracted by a supercritical fluid extraction followed by an alcohol extraction step .
  • the method produces a product, this being a composition that comprises zingerone.
  • composition is free from or substantially free from aldehydes.
  • composition is a botanical extract.
  • the composition is an ethanolic extract.
  • the composition is a powder.
  • the method comprises (i) subjecting ginger root to an alkaline treatment in alkaline solution; or (ii) subjecting juice obtained from ginger root, and optionally marc obtained from ginger root, to an alkaline treatment in an alkaline solution, wherein the alkaline solution comprises about 1.5% to about 3.5% KOH (v/v), wherein the alkaline treatment is carried out for about 1 to about 2 hours, and wherein following alkaline treatment, the alkaline solution is neutralised to a pH of about 6.5 to about 7.5.
  • composition being a botanical extract comprising zingerone, the zingerone being prepared by a method of any one of the preceding aspects.
  • composition is free from or substantially free from aldehydes.
  • the composition is an ethanolic extract.
  • composition is a powder.
  • composition is formulated for administration as a pharmaceutical composition or a dietary composition.
  • composition is formulated as a dietary supplement.
  • composition comprising zingerone, the zingerone being prepared by a method of any one of the preceding aspects.
  • this disclosure encompasses a method of treating or preventing inflammation, comprising: administering to a subject a composition of any one of the preceding aspects, thereby treating or preventing the inflammation.
  • this disclosure encompasses the use of a composition of any one of the preceding aspects for preparing a medicament for treating or preventing inflammation.
  • a composition comprising zingerone for treating or preventing inflammation.
  • composition is obtained by a method of any one of the preceding aspects.
  • composition is obtained from a ginger root.
  • composition is obtained from fresh ginger root.
  • composition is obtained from dried ginger root.
  • composition is obtained from juice prepared from a ginger root.
  • the juice is prepared by macerating and/or pressing of the ginger root.
  • composition is obtained using an alkaline conversion step to convert the gingerol in the ginger root, or in the juice from ginger root, to zingerone.
  • composition is free from or substantially free from aldehydes.
  • composition is formulated as a powder.
  • composition is formulated as a tincture.
  • composition further comprises one or more anti-inflammatory agents.
  • composition further comprises one or more of: an analgesic compound, antipyretic compound, and psychotropic compound.
  • composition further comprises one or more of: a cannabinoid compound, mushroom compound, non-steroid anti-inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound.
  • NSAID non-steroid anti-inflammatory drug compound
  • the composition further comprises one or more of: acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone, cannabidiol, canna
  • the inflammation is acute or chronic inflammation.
  • the inflammation is an inflammatory disorder.
  • the inflammation is inflammation of one or more of: an immune disorder; an arthritic disorder; an infection; a cardiac, circulatory, or pulmonary disorder; a neurological disorder; and a neoplastic disorder.
  • the inflammation is an inflammation affecting one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen, lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.
  • the inflammation is inflammation of one or more of: Alzheimer’s disease, early stage Alzheimer’s disease, ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerated colitis), Crohn's disease, dementia, early stage dementia, depression, diabetes, fibromyalgia, gout, infection (e.g., microbial infection), immune mediated inflammatory disease, inflammatory bowel disease (IBD), interstitial cystitis, multiple sclerosis (MS), polymyalgia psoriasis, scleroderma, and Sjogren’s syndrome, and systemic lupus erythematosus (SLE; lupus).
  • colitis e.g., ulcerated colitis
  • Crohn's disease dementia
  • early stage dementia dementia
  • depression depression
  • diabetes fibromyalgia
  • gout infection
  • infection e.g., microbial infection
  • immune mediated inflammatory disease e.g., inflammatory bowel disease (IBD
  • the inflammation is inflammation of one or more of: rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (JIA), lupus arthritis, osteoarthritis, polymyalgia rheumatica, psoriatic arthritis, reactive arthritis, scleroderma arthritis, Sjogren’s syndrome arthritis.
  • JIA juvenile idiopathic arthritis
  • osteoarthritis polymyalgia rheumatica
  • psoriatic arthritis reactive arthritis
  • scleroderma arthritis Sjogren’s syndrome arthritis.
  • the inflammation is inflammation of one or more of: atherosclerosis, coronary artery disease, pulmonary artery hypertension, hypoxia-induced pulmonary hypertension, pneumonia, acute respiratory distress syndrome, coronavirus respiratory disorder, and cytokine storm syndrome.
  • the inflammation is inflammation of one or more of: breast cancer, leukaemia, multiple myeloma, myelodysplastic syndrome, pancreatic cancer, and prostate cancer.
  • composition [00145] In various aspects for the composition:
  • composition is formulated for topical administration, or oral administration.
  • composition is formulated as a solid, semi-solid, or liquid.
  • composition is formulated as a solution, tincture, gel, jelly, gummy, powder, tablet, or capsule.
  • composition is provided in a sachet.
  • the composition comprises a dose of about 10 mg to about 3000 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 1500 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 1000 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 500 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 300 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 150 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 100 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 75 mg of zingerone.
  • the composition comprises a dose of about 10 mg to about 50 mg of zingerone.
  • composition is formulated for co-administration with one or more antiinflammatory agents.
  • composition is formulated for co-administration with one or more of: an analgesic compound, antipyretic compound, and psychotropic compound.
  • the composition is formulated for co-administration with one or more of: a cannabinoid compound, mushroom compound, non-steroid anti-inflammatory drug compound (NSAID), opioid compound, salicylate compound, and steroid compound.
  • NSAID non-steroid anti-inflammatory drug compound
  • the composition is formulated for co-administration with one or more of: acetaminophen, aspirin, celecoxib, diclofenac, difhmisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, eptazocinem, betamet
  • composition of a preceding aspect for preparing a medicament for treating or preventing inflammation in a subject.
  • Also encompassed is a method of treating or preventing inflammation in a subject comprising administering to the subject a composition of a preceding aspect.
  • Figure 1 Photograph depicting fresh ginger root.
  • Figure 2 HPLC UV chromatogram traces (280 nm) for alkaline treated ginger.
  • Figure 3 Schematic showing processing comparison.
  • Figure 4A Photograph depicting juicing machine and raw ginger prior to juicing.
  • Figure 4B Photograph depicting juicing of raw ginger in progress.
  • Figure 5A Ginger juice treated with KOH (0.5%) and analysed by HPLC. Peak areas shown for zingerone (Z) and gingerol (G).
  • Figure 5B Ginger juice treated with KOH (1%) and analysed by HPLC. Peak areas shown for zingerone (Z) and gingerol (G).
  • Figure 5C Ginger juice treated with KOH (2%) and analysed by HPLC. Peak areas shown for zingerone (Z) and gingerol (G).
  • Figure 6A Ginger marc produced by pressing.
  • Figure 6B Ginger juice produced by pressing.
  • Figure 7 Schematic showing ethanolic extraction process and evaporation.
  • Figure 8A GCMS TIC analysis for ethanolic extract.
  • Figure 8B Comparison of ethanolic extract with hexanal standard. Shown are 2-7 minute regions of chromatogram.
  • Figure 9 Dose response curve for cytotoxicity assay. Disclosed botanical extract assessed.
  • Figure 10 Dose response curve for nitric oxide assay. Disclosed botanical extract assessed.
  • Figure 11 Dose response curve for IL-6 assay. Disclosed botanical extract assessed.
  • Figure 12 Dose response curve for cytotoxicity, NO, and IL-6 assays. Disclosed botanical extract assessed.
  • Figure 13 Cell viability of RAW264.7 cells was measured with the WST- 1 assay. Comparative study for disclosed botanical extract and commercially sourced zingerone.
  • Figure 14 Interleukin (IL)-6 produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). Comparative study for disclosed botanical extract and commercially sourced zingerone.
  • Figure 15 Interleukin (IL)-10 produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). Comparative study for disclosed botanical extract and commercially sourced zingerone.
  • Figure 16 Tumour necrosis factor (TNF)-a produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS). Comparative study for disclosed botanical extract and commercially sourced zingerone.
  • TNF Tumour necrosis factor
  • Figures 17A-17B Both testing methods showed efficacy of dexamethasone as a positive control.
  • Figure 17A shows results for Method 1 analysis.
  • Figure 17B shows results for Method 2 analysis.
  • Figures 18A-18B Both testing methods performed similarly for MTT scoring.
  • Figure 18A shows results for Method 1 analysis.
  • Figure 18B shows results for Method 2 analysis.
  • Figures 19A-19D Disclosed botanical extract showed a cytotoxic effect at 150, 100, 75 and 50 pM. Data is presented as the mean of three biological replicates ⁇ SEM. Non-linear regression was computed using GraphPad Prism 9.0.
  • Figure 19A shows results for synthetic zingerone.
  • Figure 19B shows results for acetyl zingerone.
  • Figure 19C shows results for ferulic acid.
  • Figure 19D shows results for disclosed botanical extract.
  • Figure 20 Disclosed botanical extract produces a dose dependent inhibition of IL-6 production from stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ⁇ SEM.
  • Figure 21 Treatment with the disclosed botanical extract at 25 pM produces significantly less IL-6 compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 pM. Data is presented as the mean of three biological replicates ⁇ SEM. Repeated Measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P ⁇ 0.05, ***: P ⁇ 0.001
  • Figure 22 Treatment with the disclosed botanical extract at 25 pM significantly lowered IL-6 levels compared to vehicle control. Data is presented as the mean of three biological replicates ⁇ SEM. Repeated Measures Two-Way ANOVA with a Sidak correction for multiple comparisons was computed in GraphPad Prism 9.0. *:
  • Figures 23A-23D No observable effect on IL-6 production from unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ⁇ SEM.
  • Figure 23A shows results for synthetic zingerone.
  • Figure 23B shows results for acetyl zingerone.
  • Figure 23C shows results for ferulic acid.
  • Figure 23D shows results for disclosed botanical extract.
  • Figure 24 Disclosed botanical extract reduces TNF (TNF-a) production by stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ⁇ SEM.
  • Figure 25 Treatment with the disclosed botanical extract at 25 pM produces significantly less IL-6 compared to treatment with acetyl zingerone at 150 pM. Data is presented as the mean of three biological replicates ⁇ SEM. Repeated Measures One-Way ANOVA with a Turkey Correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P ⁇ 0.05.
  • FIG. 26 Reduction of TNF production by stimulated RAW264.7 cells. Ferulic acid at 150 pM produces a significant reduction of TNF compared to vehicle. Data is presented as the mean of three biological replicates ⁇ SEM. Repeated Measures Two- Way ANOVA with a Sidak correction for multiple comparisons was computed in GraphPad Prism 9.0. *: P ⁇ 0.05, **: P ⁇ 0.01.
  • Figures 27A-27D No observable effect on TNF production from unstimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ⁇ SEM.
  • Figure 27A shows results for synthetic zingerone.
  • Figure 27B shows results for acetyl zingerone.
  • Figure 27C shows results for ferulic acid.
  • Figure 27D shows results for disclosed botanical extract.
  • Figure 28 Disclosed botanical extract reduces NO levels in stimulated RAW264.7 cells. Data is presented as the mean of three biological replicates ⁇ SEM.
  • Figure 29 Treatment with the disclosed botanical extract at 25 pM produces significantly lower NO levels compared to treatment with synthetic zingerone, acetyl zingerone, or ferulic acid at 150 pM. Data is presented as the mean of three biological replicates ⁇ SEM. Repeated Measures One-Way ANOVA with a Turkey correction for multiple comparisons was computed in GraphPad Prism9.0.
  • Figure 30 Treatment with synthetic zingerone at 150 pM produces a significantly higher levels of NO compared to treatment with vehicle. Data is presented as the mean of three biological replicates ⁇ SEM. Repeated Measures Two-Way ANOVA with a Sidak correction for multiple comparisons was computed in GraphPad Prism 9.0.
  • Figure 31 Flow chart for a large scale ginger juicing method.
  • Figure 32 Flow chart for a large scale zingerone extraction method with ginger juice as starting material.
  • Figure 33 Flow chart for a large scale zingerone extraction method with ginger marc as starting material.
  • Figures 34A-34B Stability of zingerone extract obtained by large scale production. Testing was carried out over two months.
  • Figure 34A shows zingerone content at 5°C and 40°C.
  • Figure 34B shows pH levels at 5°C and 40°C.
  • each range that is specified includes all possible combinations of numerical values between the lowest value and the highest value enumerated (e.g., 1, 1.1, 2, 3, 3.3, 4, 5.5, 6, 7, 8.9, 9 and 10) and also any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.9), and, therefore, all subranges of all ranges expressly disclosed herein are hereby expressly disclosed.
  • the numeric values provided in parentheses here are only examples of what is specifically intended and all possible combinations of numerical value between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure in a similar manner.
  • the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element can be taken to mean one element or more than one element.
  • the term “about” is used to indicate that a value includes the standard deviation of error for the method being employed to determine the value, for example, levels of compounds or dosage levels, as described in detail herein. In particular, the term “about” encompasses up to a 10% deviation (positive and negative) in the stated value or range.
  • the term “comprising”, as used herein, may refer to the presence of zingerone or a zingerone extract in a composition.
  • the zingerone or zingerone extract may be at least 1%, at least 2%, at least 4%, at least 5%, at least 10%, at least 12%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by weight of the composition (% w/w).
  • the zingerone or zingerone extract may be at least 1%, at least 2%, at least 4%, at least 5%, at least 10%, at least 12%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by volume of the composition (% w/v or % v/v).
  • the term “substantially free” in relation to aldehydes refers to a product having negligible aldehyde levels.
  • the product may be, for example, a composition as described herein, or may be, for example, a product produced by a method as described herein.
  • aldehyde levels may be less than 20 ppm, less than 15 ppm, less than 10 ppm, less than 7.5 ppm, less than 5 ppm, less than 2 ppm, less than 1.5 ppm, less than 1 ppm, less than 0.75 ppm, less than 0.5 ppm, less than 0.2 ppm, less than 0.1 ppm, less than 0.05 ppm, less than 0.005 ppm, or less than 0.0005 ppm.
  • the term “alkaline treatment” as used herein means the exposure of a sample (e.g., ginger, ginger juice, ginger marc, or any combination thereof) to an aqueous solution containing alkali having a pH greater than 7.
  • solutions comprising, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide and any combination thereof.
  • the alkaline treatment can occur at a range of temperatures as described herein and that the alkaline solution may be heated prior to or during exposure to the sample comprising ginger or an extract from ginger.
  • the alkaline solution will have a chemically effective amount of alkali present to convert at least some gingerol present in the sample to zingerone. Particular methodologies are described in detail herein.
  • an “extract” of this disclosure will refer to a botanical extract (also referred to as a “botanical drug”). More specifically, an “extract” refers to a composition where one or more liquid, solid, or chemical constituents of a plant or plant part has been isolated or concentrated. For example, a liquid, solid, or semi- solid extract may be obtained. An extract may be obtained by one or more of: juicing, pressing, macerating, mashing, milling, or other standard processes. Solvent-based extraction is also included. Solid extracts are specifically noted, for example, powders obtained from drying or evaporation. As specific exemplifications, an extract may be prepared as a dry form, or may be prepared in the form of a solution.
  • a “zingerone extract” refers to an extract comprising zingerone, as prepared/produced from ginger root (i.e., ginger rhizome, which can also be referred to as “ginger”). Particular extracts and their production methods are described in detail herein.
  • composition encompasses a product comprising one or more active components (e.g., combinations as set out herein), and one or more suitable excipients comprising other ingredients. These may be physiologically acceptable excipients. Encompassed is any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the active components.
  • the composition may comprise any suitable solvate or salt of each compound.
  • an extract of this disclosure can be prepared as a composition suitable for administration to a subject, or suitable for formulation for administration to a subject.
  • Various exemplary compositions are described in detail herein.
  • a “pharmaceutical composition” refers to a composition administered to a subject, for example, to treat or prevent inflammation.
  • a “dietary composition” refers to a composition to be ingested by a subject, for example, to alleviate or prevent inflammation.
  • “administration of’ or “administering” refers to the providing the disclosed extract or a composition formulated from the disclosed extract to a subject.
  • the disclosed extract and disclosed composition can 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 subject.
  • one or more oral formulations may be used, or one or more topical formulations may be used, or one or more oral formulation may be used in conjunction with one or more topical formulations.
  • routes of administration and formulations for administration are provided herein.
  • an “anti-inflammatory agent” refers to a constituent that mitigates one or more symptoms of inflammation. Included amongst these are pharmaceutical agents, phytochemical agents, plant components, plant extracts, and essential oils, as well as tisanes and other infusions, along with various other constituents, to assist in the reduction of inflammation. Such may be utilised in combination with the compositions and extracts of this disclosure, and may be used to assist in regulating an immune response.
  • Co-administration refers to the combined use of active components, for example, for therapy or for cosmetic enhancement, and includes the administration of co-formulations (i.e., combination formulations), as well as the simultaneous, sequential, or separate administration of distinct formulations.
  • co-formulations i.e., combination formulations
  • in conjunction refers to the combined use of one or more active components and a device/procedure. This can include use of the active component(s) preceding use of the device/procedure, simultaneously with the device/procedure, and/or following use of the device/procedure.
  • inflammation includes any degree of inflamed tissue in a subject that persists for any period of time. Particular exemplifications include acute inflammation or chronic inflammation. The inflammation may be associated with pain. The inflammation may be that of the joint, skin, lung, heart, circulatory system, digestive tract, genitourinary tract, etc. These and other types of inflammation are encompassed herein.
  • a “symptom” of inflammation includes one or more of: pain, heat, redness, swelling, and loss of function. Also noted as markers of inflammation are the production of proinflammatory cytokines (e.g., IL-6, TNF-a) and/or proinflammatory small molecules (e.g., NO), as well as the activation and/or accumulation of immune cells.
  • proinflammatory cytokines e.g., IL-6, TNF-a
  • proinflammatory small molecules e.g., NO
  • a “subject” may be a human or non-human animal, particularly a mammal, including cattle, sheep, goats, pigs, horses, and other livestock, including, as well, dogs, cats, and other domesticated pets.
  • the subject is a human being.
  • Preventing refers to halting or delaying the onset or progression of inflammation or a disorder involving inflammation.
  • a preventative measure may result in the stoppage or delay of development of the inflammation or the disorder, or its symptom(s), a prevention of progression of the inflammation or the disorder, or its symptom(s), or a lessening of the developed inflammation or disorder, or it symptom(s), if such happen to arise.
  • a preventative measure may also act in supporting, maintaining, and/or protecting of a bodily system. It should be understood that the term “treating or preventing” does not exclude the possibility of obtaining both treatment and prevention of the disorder.
  • a “therapeutic” effect or “therapeutic” method may include treatment, or prevention, or both.
  • Treating refers to ameliorating or resolving inflammation or a disorder involving inflammation.
  • a treatment will result in the reduction, e.g., amelioration or resolution, of the inflammation or the disorder, or one or more symptoms of the inflammation or the disorder.
  • Resolution in the context of a treatment includes partial or complete reversal of the inflammation or the disorder, or its symptom(s). Partial or complete healing is encompassed by this, e.g., an improvement in one or more relevant health parameters.
  • a treatment can include a lessening in the expression of the inflammation or the disorder, or its symptom(s).
  • a treatment may also suppress existing inflammation/inflammatory disorder or its symptom(s), or put existing inflammation/inflammatory disorder or its symptom(s) into remission.
  • the healing of wounds and rashes is specifically noted.
  • “Alleviation” refers to ameliorating inflammation or a disorder involving inflammation.
  • An alleviation will result in the reduction, e.g., amelioration, of the inflammation or the disorder, or one or more symptoms of the inflammation or the disorder.
  • Healing is specifically encompassed, e.g., an improvement in one or more relevant health parameters.
  • An alleviation includes a lessening in the expression of the inflammation or the disorder, or its symptom(s).
  • the term “effective amount” refers to a sufficient quantity of the active component(s), in a suitable composition, and in a suitable dosage form to treat or prevent a noted disorder, or at least one symptom thereof.
  • the “effective amount” will vary depending on the component(s) used, the type of therapy, and the species, age, weight, heath, etc, of the subject to be treated.
  • Combination refers to combined use of two or more components (e.g., two or more active components). Usage may be by co-formulated components (i.e., combination formulations), or by simultaneous, sequential, or separate use of components (e.g., via different formulations, same formulations, or co-formulations). These and other specific combinations are encompassed in this disclosure.
  • zingerone compositions prepared from ginger root in accordance with the disclosed methods have significant anti-inflammatory activity, which exceed the activity of commercially available zingerone compositions. Therefore, the present disclosure relates generally to a zingerone composition prepared from ginger root, and methods of preparation of such.
  • this disclosure provides a method of producing zingerone from ginger root by subjecting the ginger root to an alkaline treatment.
  • the alkaline treatment may include incubation in an alkaline solution as described herein.
  • the ginger root may be fresh ginger root.
  • the disclosed preparation methods produce a highly efficacious botanical extract.
  • the ginger root may be harvested less than 48 hours before processing, less than 24 hours before processing, or less than 12 hours before processing, or less than 6 hours before processing, or less than 3 hours before processing.
  • fresh ginger may have a moisture content of about 80% to about 95%, about 81% to about 95%, or about 82% to about 95%, or about 83% to about 95%, or about 85% to about 95% on a wet basis.
  • the ginger root may be dried prior to treatment.
  • the ginger root may be dried at about 40° to about 70°C, or at about 55° to about 65°C, or at about 60°C. Drying may be carried out for about 1-72 hours, or about 1-48 hours, 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.
  • the ginger root selected for use in the disclosed methods may have a minim level of gingerol, e.g., 6-gingerol.
  • the ginger root e.g., fresh ginger root
  • the ginger root may have about 0.3 to about 10 mg/g, or about 0.3 to about 9 mg/g, or about 0.3 to about 8 mg/g, or about 0.3 to about 7 mg/g, or about 0.3 to about 6 mg/g, or about 0.4 to about 5 mg/g of 6-gingerol.
  • the ginger root may have at least 1 mg/g, at least 2 mg/g, at least 3 mg/g, at least 4 mg/g, or at least 5 mg/g of 6-gingerol.
  • the method comprises subjecting juice and/or marc from the ginger root to an alkaline treatment.
  • the ginger juice and/or ginger marc may be obtained by macerating and/or pressing.
  • the macerating may comprise homogenising with a blender, food processor, or similar machinery.
  • machine or hand presses may be utilised. Screw pressing is specifically noted.
  • the solid material remaining after juicing (ginger marc) may be re-juiced to obtain ginger juice. This may be repeated as needed.
  • the various juice samples and marc samples may be combined before alkaline treatment, e.g., juice sample A + juice sample B, or marc sample A + marc sample B, or juice sample A, B + marc sample A, B.
  • the ginger marc may be subjected to hot water treatment to obtain a diluted juice.
  • water may be added to the marc at a ratio of about 6 to about 1 (—6:1), or about 5 to about 1 (—5:1), or about 4 about 1 (—4:1), or about 3 to about 1 (—3:1) by weight.
  • the water may be, for example, at about 40°C to about 80°C, or at about 50°C to about 70°C, or at about 55°C to about 65°C, or at about 60°C.
  • the incubation time in the water may be about 5 minutes to about 60 minutes, or about 10 minutes to about 30 minutes, or about 15 minutes to about 20 minutes, or about 15 minutes.
  • the diluted juice samples may then be subjected to alkaline treatment.
  • the diluted juice samples may be combined with other juice samples before alkaline treatment.
  • potassium hydroxide may be used in the alkaline treatment.
  • a solid form of KOH may be used, e.g., KOH pellets.
  • the solid form of KOH may be at about 100% starting concentration, or at about 90% to about 100% starting concentration.
  • a liquid form of KOH may be used.
  • the liquid form of KOH may be at about 50% starting concentration, or at about 40% to about 60%, or at about 45% to about 55%.
  • the concentration of KOH used in the treatment mixture may be, for example, about 0.1% to about 6%, or about 0.5% to about 5.5%, or about 1% to about 6%, or about 1.5% to about 5.5%, or about 2% to about 4%, or about 1.5% to about 3.5%, or about 2% (v/v).
  • calcium hydroxide Ca(OH)2 may be used in the alkaline treatment.
  • a liquid form of Ca(OH)2 may be used in the alkaline solution.
  • the concentration of Ca(OH)2 used in the treatment mixture may be, for example, about 0.5% to about 4%, about 1.5% to about 3.5%, about 1% to about 2%, or about 3.0% (v/v).
  • Liquid forms may include, for example, stock solutions of about 25% to about 65%, or about 30% to about 60%, or about 35% to about 55%, or about 45% to about 55%, or about 50%.
  • the alkaline treatment may achieve a pH level for the treatment solution of about pH 9 to about pH 14, or about pH 9.5 to about pH 13.5, or about pH 10 to about pH 13.5, or about pH 10.5 to about pH 13.5, or about pH 11.5 to about pH 13.5, or about pH 12.5 to about pH 13.5, or a pH of at least 13.
  • the alkaline treatment may be carried out for a sufficient time and at a sufficiently elevated temperature to obtain desired levels of zingerone.
  • the alkaline treatment may be carried out for about 1-72 hours, or about 1-48 hours, or about 1-24 hours.
  • Further examples include treatment for about 1 to about 30 hours, or about 1 to about 20 hours, or about 1 to about 10 hours, or about 1 to about 15 hours, or about 1 to about 7 hours, or about 1 to about 6 hours, or about 1 to about 5 hours, or about 1 to about 4 hours, or about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours, or at least 1 hour, or about 2 hours, or about 1 hour.
  • the alkaline treatment may be carried out at about 40° to about 70°C, or about 50° to about 60°C, or about 55° to about 65 °C, or about 60°C. It will be understood that lower temperatures can allow for longer treatment periods.
  • alkaline treatments performed at room temperature can carried out for about 3 days to about 9 days, or for about 5 days to about 9 days, or for about 5 days to about 7 days.
  • the treatment mixture may be further processed, for example, by one or more of: neutralisation, extraction, and drying.
  • neutralisation citric acid or other acid composition may be utilised.
  • neutralisation may achieve a pH of about 6.4 to about 7.4, or about 6.5 to about 7.5, or about 6.6 to about 7.6, or about 6.9 to about 7.4, or about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.
  • about 10 to about 700 g/L citric acid may be utilised, or about 10 to about 600 g/L, or about 10 to about 500 g/L, or about 10 to about 400 g/L, or about 10 to about 300 g/L, or about 10 to about 200 g/L, or about 10 to about 100 g/L, or about 10 to about 50 g/L, or about 10 to about 40 g/L, or about 10 to about 30 g/L, or about 10 to about 20 g/L, or about 15 to about 16 g/L citric acid may be utilised.
  • zingerone extraction may be achieved by one or more alcohol extractions, e.g., one or more ethanol extractions.
  • an alcohol extraction e.g., ethanol extraction
  • alcohol extraction may be carried out for 24 hours or less, for example, for at least 4 hours, or for about 4 to about 12 hours, or for about 4 to about 8 hours.
  • the alcohol extraction may be carried out, for example, at about 35 °C to about 65°C, or at about 45°C to about 55°C, or at least 50°C, or at about 50°C.
  • One or more drying steps may be used before and/or after extraction (e.g., alcohol extraction.
  • extraction e.g., alcohol extraction.
  • freeze drying or heat drying may be utilised.
  • the amount of ethanol in the extract composition may be reduced by air drying, rotary evaporation, lyophilisation, or other techniques.
  • certain drying methods e.g., rotary drying
  • the temperature and pressure can be increased to remove residual liquid(s).
  • drying can be carried out under vacuum, for example, at 50 mBar or less, 45 mBar or less, or 40 mBar or less.
  • drying can be carried out at about 35°C to about 50°C, or at about 35°C to about 45°C, or at least 30°C, or at least 35°C, or at least 40°C, or at about 40°C.
  • drying may be carried out, e.g., for at least 18 hours, or at least 24 hours, or at least 28 hours, or at least 30 hours, or about 18 to about 28 hours, or about 24 to about 28 hours.
  • the drying may be carried out, for example, at about 45°C to about 75°C, or at about 55°C to about 65°C, or no more than 60°C, or at about 60°C. Other alternatives for drying are described herein.
  • the ginger root may be washed or sterilised.
  • the plant component e.g., fruit or seed
  • the plant component may be passed through an assembly having one or more roller brushes for removing any adhering foreign matter.
  • Conventional washing techniques may then be employed. For example, it is possible to use a series of spray nozzles to wash the components. Wash additives aiding cleansing or reducing the microbial count on the plant components may be employed according to local regulations and requirements.
  • the plant components may be washed by a chlorine wash and/or an ozone impregnated water wash followed by a fresh water rinse.
  • a liquid or semi-solid zingerone composition may be prepared from ginger root.
  • zingerone components may be extracted by chemical means (e.g., solvent-based extraction).
  • Solvent-based extraction may utilise one or more of: water, methanol, ethanol, or 2-propanol, extraction.
  • Supercritical fluid extraction for example CO2 extraction may also be used to extract zingerone.
  • emulsions, pastes, suspensions, and syrups are also be used.
  • the ginger root may be heated for several hours, strained, and reduced to a thick, concentrated form.
  • the paste can be spread on a flat sheet, or transferred to a packaging, for example, a bag, tube, jar, bottle, or other container.
  • the paste may be transferred aseptically. It may be desired to prepare the paste from mature plant components.
  • the paste may be a smooth preparation.
  • a pressing assembly may be adapted to perform a pulping or comminution process. Such process can be relatively mild and gentle (soft pulping) compared to conventional fruit pulping techniques. With soft pulping, no significant disintegration or lysis of cells is utilised.
  • the press belts may be multiple loops rotated about a series of pulleys. The distance separating the press belts may decrease in the direction of travel of the plant component. In this way, increased force may be exerted upon the plant component as it travels along the length of the pressing assembly.
  • a pressing assembly or mechanical press may be used to obtain juice from the ginger root, as described herein.
  • mechanical maceration may be used to obtain juice.
  • commercial juicing equipment may be utilised.
  • the ginger root component (e.g., zingerone or zingerone extract) may be processed by a freezing step. This may be followed by or used in conjunction with a drying or evaporation step. In an alternative embodiment, the component is dried or evaporated, and then processed to a powder without an intervening freezing step. Methods involving air drying or heat-assisted drying (e.g., oven drying) may be used. Drying may be obtained, for example, by one or more of: sun or solar drying, hot air drying, batch drying, rotary drying, tunnel drying, belt drying, fluidised bed drying, impingement drying, puff drying, drum drying, spray drying, vacuum drying, freeze drying, or osmotic drying.
  • air drying or heat-assisted drying e.g., oven drying
  • Exemplary temperatures for drying include about 50°C-70°C, about 55°C-65°C, or at least 50°C, at least 55°C, at least 60°, or at least 65°C.
  • Evaporation may be obtained, for example, by one or more of: pan evaporation, batch evaporation, tube evaporation, rising film evaporation, falling film evaporation, rising-falling film evaporation, or agitated film evaporation.
  • Combinations of various drying and evaporation methods may be used. For example, filtering followed by freeze drying may be used.
  • the ginger root component e.g., zingerone or zingerone extract
  • freezing may be carried out within 24 or 48 hours, as needed.
  • Standard freezing methodologies may be utilised.
  • Blast freezing is particularly desirable for use with the present disclosure.
  • the component may be frozen in standard sized pales, which are used to collect the frozen product after processing.
  • the component for example, can be stored frozen (e.g., at -18°C) until it is required.
  • the component may then be freeze dried, i.e., lyophilised. Freeze drying techniques are widely used.
  • the freeze-drying cycle may be up to 48 hours.
  • the process may be carried out to such that water formation is avoided, and the moisture content is minimised during processing.
  • freeze drying/lyophilising does not exclude the use of higher temperatures (i.e., higher than freezing temperatures).
  • higher temperatures may be used for removing residual moisture during the secondary drying phase for lyophilisation/freeze drying procedures.
  • the resulting dried or evaporated component from ginger root may then be milled into a powder, which can then be utilised as appropriate. Standard milling methods may be utilised. Standard mesh sizes may be used to produce the powder, for example, US 20, US 23, US 30, US 35, US 40, US 45, or US 50 mesh sizes may be used.
  • the sieve size for the powder may range from 1.0 to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or may be about 1.0 mm, about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47 mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm.
  • the composition may be prepared as a pharmaceutical composition.
  • the composition can also be prepared as a dietary composition, for example, a functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement).
  • the composition may be prepared in liquid or solid form, or semi-solid form.
  • Various formulations are encompassed in this disclosure.
  • it may be desirable to formulate the composition into a powder.
  • the powder may be provided in free flowing form or as a solid cake.
  • the composition may be provided as a powder for forming a suspension, powder for forming a solution, bulk granules, or bulk powder.
  • the powder may be prepared as tablets or capsules, or other formulations, as described in detail herein.
  • 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.
  • the solid may be used as such (e.g., with milling, sieving, or other processing), or may be re-suspended to obtain a liquid or semi-solid form for use as a pharmaceutical composition or dietary composition, as described herein.
  • zingerone compositions prepared from ginger root in accordance with the methods disclosed herein have significant anti-inflammatory and immunomodulating properties that are useful for reducing cellular inflammation markers, reducing tissue damage, antioxidant action, as well as for treating or preventing inflammation and inflammatory disorders in a subject.
  • the composition of the present disclosure may be prepared as one or more formulations, including pharmaceutical compositions and dietary compositions.
  • the percentage of zingerone or zingerone extract in the composition may be about 0.01% to about 30%, or about 1% to about 30%, or about 1% to about 15%, or about 1% to about 10%, or about 1% to about 9%, or about 1% to about 8%, or about 0.1% to about 7%, or about 0.1% to about 6%, or about 0.1% to about 5%, or about 0.1% to about 4%, or about 0.1% to about 4%, or about 0.1% to about 3%, or a percentage of at least about 1%, at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, or at least about 50%, or a percentage of about 6.25%, about 12.5%, or about 25%, these percentage
  • a solid composition may include about 0.5 to about 300 mg/g zingerone, about 1 to about 150 mg/g zingerone, about 1 to about 100 mg/g zingerone, or about 1 to about 80 mg/g, or about 1 to about 60 mg/g, or about 1 to about 50 mg/g, or about 1 to about 40 mg/g, or about 1 to about 20 mg/g, about 1 to about 15 mg/g, or about 1 to about 10 mg/g, or about 10 to about 60 mg/g zingerone, or about 10 to about 50 mg/g zingerone, or about 10 to about 40 mg/g zingerone, or about 10 to about 30 mg/g zingerone, or about 10 to about 20 mg/g zingerone, or about 10 to about 15 mg/g zingerone, or at least about 50 mg/g zingerone, or at least about 40 mg/g zingerone, or at least about 10 mg/g zingerone (w/w).
  • a liquid or semi-solid composition may include about 0.5 to about 300 mg/ml zingerone, about 1 to about 150 mg/ml zingerone, about 1 to about 100 mg/ml zingerone, or about 1 to about 80 mg/ml, or about 1 to about 60 mg/ml, about 1 to about 50 mg/ml, or about 1 to about 40 mg/ml, or about 1 to about 20 mg/ml, or about 1 to about 15 mg/ml, or about 1 to about 10 mg/ml, or about 10 to about 60 mg/ml zingerone, or about 10 to about 50 mg/ml zingerone, or about 10 to about 40 mg/ml zingerone, or about 10 to about 30 mg/ml zingerone, or about 10 to about 20 mg/ml zingerone, or about 10 to about 15 mg/ml zingerone, or at least about 50 mg/ml zingerone, or at least about 40 mg/
  • topical compositions may be prepared, for example: for use on hands (e.g., hand creams), pre-operative tissue (e.g., surgical preparations for skin), mucous membranes (e.g., treatments for bladder, urethral, or vaginal inflammation, or cleansing of these cavities prior to medical procedures), wounds or burns (e.g., ointments, bandages, or dressings), mouth or throat (e.g., mouthwashes or lozenges), or eye (e.g., eye drops or ointments).
  • hands e.g., hand creams
  • pre-operative tissue e.g., surgical preparations for skin
  • mucous membranes e.g., treatments for bladder, urethral, or vaginal inflammation, or cleansing of these cavities prior to medical procedures
  • wounds or burns e.g., ointments, bandages, or dressings
  • mouth or throat e.g., mouthwashes or lozenges
  • eye e.g.
  • topical compositions may include one or more of: diluents (e.g., ethanol or other alcohol), emollients (e.g., PEG-45, palm kernel glycerides, or isopropyl myristate), humectants (e.g., glycerine or methylpropanediol), carriers (e.g., one or more oils), occlusive agents (e.g., mineral oil or dimethicone) other conditioning agents (e.g., behentrimonium methosulfate or polyquaternium-7), and surfactants (e.g., mild surfactants (e.g., amphoacetate, isethionate, sulfosuccinate, in particular, sodium lauroamphoacetate, sodium cocoyl isethionate, disodium oleoamido sulfosuccinate, sodium lauryl sulfate, sodium C
  • diluents
  • oils include olive oil, coconut oil (e.g., coconut-derived MCT oil), palm oil (e.g., palm kernel- derived MCT oil), any other MCT oil (medium-chain triglyceride oil), and any combination thereof.
  • Other possible carriers include lecithin (e.g., liquid form) and propylene glycol. Any combination of the carriers set out herein is also noted.
  • compositions may be prepared for various routes of administration, including oral formulas. Also included are compositions prepared for other routes of enteral or parenteral administration.
  • Enteral formulations include but are not limited to: oral, rectal, sublingual, sublabial, and buccal preparations.
  • Parenteral formulations include but are not limited to: nasal, intraocular, vaginal, intralesional, transdermal, and transmucosal preparations. Standard methods are available for formulating pharmaceutical compositions. See, e.g., Remington: Essentials of Pharmaceutics, 2013, Pharmaceutical Press, London.
  • composition of this disclosure may be prepared as a powder, or in any other suitable dosage form.
  • Topical formulations may be prepared, for example, as aerosols, balms, creams, dressings, drops, emulsions, films, foams, gels, jellies, liquids, lotions, masks, oils, ointments, pastes, powders, salves, soaps, sprays, suspensions, solutions, tinctures, and vapours.
  • Further topical formulations include bandages, dressings, patches, pads, sponges, strips, tapes, and others noted herein.
  • the composition may be formulated as a semi-solid or liquid composition, for example, for oral administration (e.g., taken directly by mouth or via inclusion in capsules or in other forms), or for enteral or parenteral administration (e.g., taken by injection, feeding tube, or in other forms).
  • the composition may be formulated as a powder to be encapsulated, tableted, or added to or incorporated in other products.
  • Oral formulations may be prepared, for example, as draughts, drops, elixirs, emulsions, liquids, linctuses, solutions, sprays, suspensions, syrups, tonics, or, as films, gels, jellies, gummies, lozenges, nuggets, pastes, purees, pomaces, powders, pills, or strips.
  • oral formulations may be prepared as a tablets or as capsules, for example, with liquid, semi-solid, or solid contents.
  • Oral formulations may be provided in sachet form, for example, a powder sachet, or a gel or jelly sachet.
  • oral formulations comprising thin strips, or comprising solids in a capsule to mix with food or drink.
  • the oral formulation may be provided as shooters or shots (to be consumed by mouth), for example, liquid shots, gel or jelly shots, paste shots, or powder shots.
  • composition formulas which include the powder of the present disclosure mixed with other beneficial agents, e.g., one or more anti-inflammatory agents.
  • beneficial agents e.g., one or more anti-inflammatory agents.
  • Other formulas are also possible, as described herein.
  • the dissolution time for an oral formulation can be modified for a rapid effect or for sustained release.
  • Oral formulations may also contain a mixture of slow and fast release particles to produce rapid and sustained absorption in the same dose.
  • Special coatings can be used with oral formulations such as tablets and capsules to impart resistance to stomach acids.
  • Oral formulations can also be coated with sugar, varnish, or wax to improve taste.
  • tablets may be prepared as rapid dissolve tablets and capsules may be prepared as extended release capsules.
  • the tablets may be scored tablets, chewable tablets, effervescent tablets, orally disintegrating tablets, or tablets for forming a suspension.
  • the capsules may be gel capsules, for example, and may include solid, semisolid, or liquid contents. This includes gel capsules made by single piece gel encapsulation and two piece gel encapsulation. Hard shell capsules and soft shell capsules are specially noted. Non-gelatine capsules are also noted, as well as caplets.
  • formulations will be suitable for topical or other applications.
  • Particular formulations of interest are: eye formulas (e.g., drops, ointments), ear formulas (e.g., drops, ointments), nasal or airway formulas (e.g., drops, sprays, insufflation compositions, inhalation compositions, nebulisation compositions), skin formulas (e.g., soaps, sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, patches, tapes, bandages, dressings, sponges, vapours) throat or mouth formulas (e.g., drops, lozenges, mouthwashes, toothpastes, sprays, pastes, gels, jellies, gummies), mucous membrane formulas (e.g., sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, dressings, sponges).
  • eye formulas e.g., drops, oin
  • Solid, semi-solid, and liquid compositions can combine zingerone or a zingerone extract with one more compounds to ensure a stable and active composition.
  • an oral formulation such as a tablet or capsule, may include: about 5 to about 50% w/w zingerone or zingerone extract; up to about 80% w/w of one or more fillers, lubricants, glidants, or binders; and up to about 10% w/w of compounds to ensure easy disintegration, disaggregation, and dissolution of the tablet in the stomach or the intestine.
  • an extract of this disclosure may be mixed with one or more carrier substances such as glycerol, glyceryl esters, hydrogenated oils, polyethylene glycols, poloxamers, etc, and included in a capsule (e.g., gel capsule).
  • carrier substances such as glycerol, glyceryl esters, hydrogenated oils, polyethylene glycols, poloxamers, etc.
  • the composition may contain various excipients, for example, one or more: solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, thickening agents, viscosity enhancer s/reducers, emollients, surfactants, chelating agents, adjuvants, anti-adherents, anti-caking agents, binders, coatings, disintegrants, lubricants, glidants, flow agents, sorbents, flavours, flavour masking agents, colours, sweeteners, or preservatives.
  • solubilizers for example, one or more: solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, thickening agents, viscosity enhancer s/reducers, emollients, surfactants, chelating agents, adjuvants, anti-adherents, anti-caking agents, binders, coatings, disintegrants, lubricants, glidants, flow
  • the composition may include less than 1% of a preservative, for example, about 0.005% to about 0.5%, or about 0.05% to about 0.15%, or may include about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, or about 0.2% of a preservative, these percentages being representative of w/v values or w/w values.
  • Useful preservatives include but are not limited to sorbic acid, sodium sorbate, potassium sorbate, citric acid, ascorbic acid, malic acid, tartaric acid, propionic acid, and benzoic acid, for example, in the form of its sodium salt, e.g., sodium benzoate.
  • Other useful excipients include but are not limited to: stearin, magnesium stearate, and stearic acid; saccharides and their derivatives, e.g., disaccharides: sucrose, lactose; polysaccharides and their derivatives, e.g., starches, cellulose or modified cellulose such as microcrystalline cellulose and cellulose ethers such as hydroxypropyl cellulose; sugar alcohols such as isomalt, xylitol, sorbitol and maltitol; proteins such as gelatin; polysaccharides such as pectin; gums and other thickeners, e.g., acacia gum, gellan gum, guar gum, locust bean gum, xanthan gum, agar, arrowroot carrageenan, gelatine, glycerine, kudzu, lecithin, starch; synthetic polymers such as polyvinylpyrrolidone, polyethylene glycol; fatty acids and their derivative
  • a wide array of delivery systems may be utlised, for example, nanoparticle delivery systems such as polymeric nanoparticles (e.g., PEG, PLGA, PLA, chitosan, etc), lipid-based nanoparticles (e.g., liposomes, micellar nanoparticles, phytosomes etc), nanocrystals/nanoshells, and inorganic nanoparticles (e.g., metal nanoparticles, dendrimers, etc).
  • polymeric nanoparticles e.g., PEG, PLGA, PLA, chitosan, etc
  • lipid-based nanoparticles e.g., liposomes, micellar nanoparticles, phytosomes etc
  • nanocrystals/nanoshells e.g., metal nanoparticles, dendrimers, etc.
  • phytosomes which may include lecithin, may be used to increase absorption of the zingerone or zingerone extract, both top
  • Liquid compositions may be stored, for example, in vials, bags, ampoules, cartridges, or prefilled syringes. The composition may also be transferred from a vial to a larger container and mixed with other materials. Dried or evaporated compositions may be stored, for example, in vials, cartridges, dual chamber syringes, or prefilled mixing systems. Before administration, a dry-form composition may be reconstituted as a liquid before being administered.
  • Exemplary unit dosages of the composition include: about 0.1 mg to about 1000 mg zingerone or zingerone extract, about 1 mg to about 500 mg zingerone or zingerone extract, about 1 mg to about 250 mg zingerone or zingerone extract, about 1 mg to about 200 mg zingerone or zingerone extract, about 1 mg to about 100 mg zingerone or zingerone extract, about 1 mg to about 50 mg zingerone or zingerone extract, or about 1 mg to about 25 mg zingerone or zingerone extract.
  • the dosage may be formulated for administration once per week, twice per week, three times per week, every other day, once per day, twice per day, or three times per day, or more as needed.
  • the dosage may be adjusted for paediatric, geriatric, overweight, underweight, or other patients, where required. Dosage modification can be made in accordance with standard methods. It is to be appreciated therefore that a wide range of unit dose forms may be envisioned and prepared.
  • the disclosed compositions can be used to treat or prevent inflammation and various health conditions associated with inflammation.
  • the disclosed compositions may be utilised to reduce proinflammatory cytokine or proinflammatory small molecule levels in a subject.
  • the proinflammatory cytokine may be an interleukin cytokine such as IL-6 and/or IL- 10.
  • the proinflammatory cytokine may be a tumour necrosis factor such as TNF.
  • the proinflammatory small molecule may be nitric oxide.
  • the composition may comprise zingerone or a zingerone extract, as produced by the methods set out herein.
  • the composition of the present disclosure may also be prepared as one or more pharmaceutical forms.
  • the composition can be prepared as one or more dietary forms, for example, a functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement).
  • the disclosed compositions may be used to target one or more inflammatory disorders.
  • the inflammation is an inflammation affecting one or more of: a joint, skin, eye, ear, nose, mouth, throat, oesophagus, kidney, bladder, liver, spleen, lung, heart, brain, circulatory system, digestive system, endocrine system, genitourinary system, lymphatic system, nervous system, and skeletal system.
  • disorders of interest include but are not limited to: immune system disorders (e.g., autoimmune disorders) such as Alzheimer’s disease (e.g., early stage Alzheimer’s disease), ankylosing spondylitis, arthritis, asthma, colitis (e.g., ulcerated colitis), Crohn's disease, dementia (e.g., early stage dementia), depression, disease, diabetes, fibromyalgia, gout, immune mediated inflammatory disease, infection (e.g., microbial infection), inflammatory bowel disease (IBD), interstitial cystitis, multiple sclerosis (MS), polymyalgia psoriasis, scleroderma, and Sjogren’s syndrome, and systemic lupus erythematosus (SLE; lupus); arthritic disorders such as rheumatoid arthritis, ankylosing spondylitis arthritis, fibromyalgia arthritis, gout arthritis, juvenile idiopathic arthritis (J
  • disorders of interest include musculoskeletal disorders (e.g., disorders of the bones, cartilage, digits, joints, limbs, muscles, tendons, etc), and back disorders (e.g., conditions of the spine or soft tissue of the back).
  • inflammatory disorders associated with infections e.g., gingival inflammation.
  • anti-inflammatory therapies for the skin may be used for one or more of: blisters, dermatitis, eczema, hives, lesions, papules, plaques, psoriasis, rashes, rosacea, ulcers, and wounds.
  • Wounds may be acute or chronic (e.g., non-healing or recurring wounds). Surgical wounds and scarring are specifically noted.
  • the disclosed compositions may be used together with one or more anti-inflammatory agents.
  • the composition may be prepared as a combined formulation with one or more anti-inflammatory agents.
  • the composition may be utilised as a separate formulation along with one or more antiinflammatory agents.
  • two or more actives e.g., a zingerone composition and an anti-inflammatory agent
  • a composition as described herein may be used in conjunction with various medical or non-medical procedures. Use of the composition may be carried out prior to, during, or after the procedure(s), or any combination thereof.
  • anti-inflammatory agents may include one or more constituents obtained from plants, for example, one or more plant compounds, concoctions, extractions, and/or oils. These include constituents from: manuka (e.g., L. scoparium), houhere (e.g., Hoheria angustifolia, Hoheria glabrata, Hoheria lyallii, Hoheria populnea, Hoheria sexstylosa), horopito (e.g., Pseudowintera colorata), kawakawa (e.g., Piper excelsum), koromiko (e.g., Hebe stricta, Hebe salicifolia, or Hebe elliptical), poroporo (e.g., Solarium aviculare), pukatea (e.g., Laurelia novae -zelandiae), and others.
  • manuka e.g., L. scoparium
  • houhere e.g.,
  • Extracts from manuka e.g., manuka oil
  • kawakawa e.g., leaf extraction
  • constituents from Psilocybe spp. such as P. azurescens, P. semilanceata, and P. cyanescens, as well as those from Cannabis spp., such as C. sativa.
  • 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.
  • chamomile e.g., Roman chamomile
  • clary sage clove
  • copaiba eucalyptus
  • fennel e.g., fennel
  • frankincense e.g., frankincense
  • hops lavender
  • marjoram e.g., sweet marjoram
  • patchouli peppermint, rose, rosemary, tea tree, thyme, and turmeric.
  • honey e.g., manuka honey
  • arnica e.g., arnica oil, cream, or gel
  • activated charcoal e.g., for various skin formulas
  • yarrow e.g., for various skin formulas
  • comfrey e.g., for ointments or creams
  • anti-inflammatory agents may include one or more drug compounds. Included amongst these are analgesic agents, antipyretic agents, and psychotropic agents. Exemplifications include acetaminophen, aspirin, celecoxib, diclofenac, diflunisal, etodolac, etoricoxib, felbinac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, lidocaine, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, tenoxicam, and others.
  • NS AID compounds and salicylate compounds are specifically noted.
  • opioid compounds e.g., KOR inhibitors
  • steroid compounds e.g., corticosteroids
  • Exemplifications include butorphanol, nalbuphine, levorphanol, levallorphan, pentazocine, phenazocine, and eptazocinem. Further exemplifications include betamethasone, cortisone, deflazacort, dexamethasone, ethamethasoneb, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone. Cannabinoid compounds and mushroom compounds are also noted. Exemplifications include cannabidiol, cannabigerol, and tetrahydrocannabinol. Further exemplifications include psilocybin and psilocin.
  • anti-inflammatory agents may be adapted and utilised in accordance with this disclosure. Any combination of anti-inflammatory agents can be utilised. Any medical devices and procedures may also be used in conjunction with the disclosed compositions and extracts.
  • compositions of the present disclosure are useful as anti-inflammatory formulations.
  • compositions can be used in methods to reduce or delay inflammation in certain tissue.
  • This tissue includes: a joint, skin, ear, eye, nose, mouth, gums, throat, digestive, cardiac, circulatory, pulmonary, vaginal, and urinary tract tissues, and others noted herein.
  • the compositions may be applied, for example, to bums, to lessen the chance of inflammation, or to the skin before surgery, to combat inflammation on the skin around the operation site.
  • the compositions may be used as hand cleansers (e.g., soaps or hand sanitisers), to be applied with or without water.
  • the compositions may be used for minor skin irritations, cuts, or grazes.
  • compositions may be used as mouthwashes or gargles, for example, to combat inflammation from mouth sores or gingival inflammation.
  • Compositions may also be utilised as lozenges and throat sprays, for example, to relieve a sore throat.
  • Eye drops or ointments may be used to combat inflammation of the eye, including the eye lid.
  • compositions of the present disclosure also find use as formulations, which can be used in methods for treating or preventing inflammations and inflammatory disorders, as described herein.
  • the inflammation may affect one or more physiological components, including one or more parts of: the articular system, circulatory system, respiratory system, digestive system, renal system, excretory system, reproductive system, integumentary system, nervous system, lymphatic system, endocrine system, muscular system, skeletal system, and sensory system.
  • compositions including parenteral (e.g., topical) and enteral (e.g., oral) administration, as described herein.
  • Enteral administration may be by duodenal tubing or gastric tubing, including nasogastric tubing, or other standard means.
  • Oral administration may be by tablets, capsules, sachets, drops, elixirs, linctuses, solutions, emulsions, suspensions, draughts, purees, pastes, pomaces, syrups, gels, jellies, gummies, tonics, or various other means.
  • Topical administration may be by drops, sprays, ointments, soaps, pads, sponges, dressings, bandages, or various other means. Standard modes of administration may be utilised by a skilled person.
  • the compositions disclosed herein are not limited to a particular form for administration.
  • the compositions may be administered at a dose of about 1 to about 3000 mg zingerone or zingerone extract, or about 100 to about 3000 mg zingerone or zingerone extract, or about 100 to about 2500 mg zingerone or zingerone extract, or about 100 to about 2000 mg zingerone or zingerone extract, or about 1 to about 1800 mg zingerone or zingerone extract, or about 100 to about 1600 mg zingerone or zingerone extract, or about 100 to about 1400 mg zingerone or zingerone extract, or about 100 to about 1200 mg zingerone or zingerone extract, or about 100 to about 1000 mg zingerone or zingerone extract.
  • Additional exemplifications include about 10 to about 300 mg dose of zingerone or zingerone extract, or about 10 to about 200 mg dose of zingerone or zingerone extract, or about 10 to about 150 mg dose of zingerone or zingerone extract, or about 10 to about 100 mg dose of zingerone or zingerone extract, or about 10 to about 80 mg dose of zingerone or zingerone extract, or about 10 to about 60 mg dose of zingerone or zingerone extract, or about 10 to about 55 mg dose of zingerone or zingerone extract, or about 10 to about 50 mg dose of zingerone or zingerone extract, or about 10 to about 40 mg dose of zingerone or zingerone extract.
  • These ranges of dosages are particularly useful for a ginger component (e.g., zingerone or zingerone extract) that is dried and milled to a powder.
  • EFSA European Food Safety Authority
  • NOAEE No Observed Adverse Effects
  • exemplary dosages may be determined, for example, for an average 70 kg human subject.
  • Such exemplary dosages may include, for example, about 1 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 45 mg/kg, or about 5 mg/kg to about 40 mg/kg, or about 5 mg/kg to about 45 mg/kg, or about 5 mg/kg to about 35 mg/kg, or about 5 mg/kg to about 30 mg/kg, or about 5 mg/kg to about 25 mg/kg, or about 5 mg/kg to about 20 mg/kg, or about 5 mg/kg to about 15 mg/kg for zingerone or zingerone extract.
  • Additional exemplary dosages may include, for example, about 0.1 mg/kg to about 20 mg/kg, or about 0.1 mg/kg to about 15 mg/kg, or about 0.1 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 8 mg/kg, or about 0.5 mg/kg to about 6 mg/kg, or about 0.1 mg/kg to about 4 mg/kg, or about 0.1 mg/kg to about 2 mg/kg, or about 0.1 mg/kg to about 1 mg/kg, for zingerone or zingerone extract.
  • the dosages as indicated herein may be administered once per week, every other day, once per day, twice per day, three times per day, or less or more, as needed. Administration may be made with food, or before a meal.
  • the appropriate dosage and dosage form will be readily determined by skilled persons.
  • Extract 1 and Extract 2 * The dry weight of Extract 1 and Extract 2 was estimated by drying a 5 ml portion of each extract.
  • the 400 g fresh ginger root supplied was shown to contain 260 mg of 6- gingerol (i.e. 0.65 mg/g). This would mean the theoretical maximum yield of zingerone would be in the order of 171 mg from the treated material (loss of weight due to lower molecular weight of zingerone vs 6-gingerol). About 50% of the 6-gingerol was unconverted. It is envisioned that further studies could be used to increase the alkaline conversion of 6-gingerol to zingerone.
  • drying the extract fully would give a concentration of approximately 16 mg/g of solid extract. Higher concentrations would be expected if starting with a higher initial content of 6-gingerol and/or with more complete conversion. A theoretical 25 mg dose, for example, could then potentially be achieved by formulation of the dried extract directly into an oil or glycerol carrier to give the required zingerone dose in one or two 500 mg capsules.
  • Drying Samples of the fresh ginger material imported from Fiji were sliced into 2-5 mm slices and placed on a single layer on perforated oven trays. Drying was performed in a forced convection. Drying was carried out at a moderate temperature (60°C) with the goal of eliminating moisture without conversion of gingerol. This process was considered finished when the moisture content of the ginger reached 7%. The dried ginger obtained was stored refrigerated until it was used in the extraction trials.
  • Catalysed conversion Small scale, preliminary trials were performed by treating around 1.6 g of fresh chopped Fijian sourced ginger with aqueous solutions of 0.5% KOH (pH 14), 1% Ca(OH)2 (pH 11.6) and 1% sodium carbonate (pH 10.5). The volume: weight ratio of reagent added:ginger was around 3:1. The samples were then shaken and placed in a fumehood at room temperature, or ovens at 37°C or 60°C, overnight, before being analysed. It is to be appreciated that suitable water baths could be used to keep the samples at the desired temperature for the desired period of time.
  • Table 2-1 Composition of treated and freeze-dried ginger
  • the CO2 extract contained 153 mg/g of zingerone (15.3%) and 103 mg/g (10.3%) 6-gingerol, with a zingerone/gingerol ratio of approximately 1.5.
  • the CCT+cthanol extract contained 91 mg/g of zingerone and 52 mg/g of gingerol (zingerone/gingerol ratio of approximately 1.75).
  • the marc or residual ginger post extraction was also analysed, and found to contain 5.4 mg/g of zingerone. When the mass of feed, extracts and marc is taken into account (Table 4), the zingerone mass balance can be calculated at 90.6%.
  • the zingerone extraction yield (i.e., grams of zingerone extracted per 100 g of zingerone in the feed) was only 37% when calculated based on the extracts. However, 54% of the initial zingerone present in the feed remains unextracted in the marc, so the zingerone extraction yield can also be calculated as 46% when based on the marc results. This accounts for the “missing” zingerone. Since the proportion of unextracted zingerone is significant, the extraction process could be further improved to reduce this.
  • the extraction yield of gingerol is higher than that of zingerone (82% in the untreated sample and 71% in the treated sample), since it is more soluble in CO2.
  • Example 1 The samples were centrifuged at 2000 rpm to separate the water from the oleoresin. After centrifugation the bulk of the water was then removed by pipette. The resultant resin was then removed. Absolute alcohol was added to one of the resin samples (5 ml ethanol in approximately 3 grams resin) to produce a tincture (Sample 1). The other sample was kept in resin form (Sample 2). Sample 1 and Sample 2 were analysed for zingerone content. The concentration of zingerone in the tincture (Sample 1) was calculated at 23 mg/g of tincture while that of the treated resin (Sample 2) was 52 mg/g.
  • the water is able to extract some of the zingerone from the resin it may also be possible to produce a high yield of zingerone after treatment by drying down the total neutralised alkaline treatment product. Further process optimisation is to be undertaken to improve yields given the finding that after alkaline treatment zingerone appears to be more soluble in water than previously reported.
  • Extraction A The treated ginger produced by SROS (Scientific Research Organisation of Samoa) was supplied in two separate plastic bags. The contents of both bags were combined, frozen at -80°C and subsequently milled using a knife mill (Wiley) with a 2 mm mesh attached. The milled material (782.3 g) was then placed in a round bottom flask along with food grade ethanol with a ratio of approximately 5:1 by weight. The flask was then placed in a water bath at 40°C and 5 rpm stirring overnight (total extraction time 20 hours).
  • Extraction B The ginger was frozen and milled as described above. The milled material (785.9 g) was placed in a bucket along with food grade ethanol with a ratio of approximately 5: 1 by weight. The ginger was macerated in ethanol at room temperature (22-29°C) over 7 days. Samples were taken on days 1, 3 and 7. After 7 days, the mixture was filtered under vacuum and the ethanol was removed by rotary evaporation under vacuum to produce 49.3 g of highly viscous, dark brown resin with a characteristic ginger aroma (Figure 1), very similar to the one obtained in Extraction A. 10 g of this resin (Extract B) were taken out and stored refrigerated and under nitrogen flush for possible future bioassays. Extraction yield was 6.3%.
  • Zingerone and aldehyde analysis Quantification of zingerone was carried out by HPLC in the starting material (i.e., treated ginger) and the final two resins, as well as in the samples on days 1, 3 and 7 for Extraction B (note that the liquid sample from day 7 is equivalent to the final resin sample).
  • the HPLC quantification method included methanol addition and grinding the sample before analysis. Zingerone content for all fractions is shown in Table 6. Zingerone mass balance and yield are shown in Table 7.
  • Extract A was obtained by treatment at 40 degrees, duration 20 hrs, while Extract B was obtained by treatment at room temperature, duration 7 days.
  • Tables 6 and 7 show that higher levels of zingerone were obtained by longer treatment at room temperature, although high yields were also obtained by a temperature increase to 40°C.
  • the zingerone yield (i.e., the amount of zingerone in the extract relative to the zingerone in the feed) was estimated at 75% for Extraction A and 89% for Extraction B.
  • the 6-gingerol peak seen in the HPLC analysis when determining zingerone content was consistently observed at around l/8th the peak area of zingerone. This suggests that extraction had little effect on the zingerone to gingerol ratio.
  • Example 2 shows the output of the pH treated ginger followed by CO2 extraction. It was found that 322 grams of fresh ginger provides 153 mg/g zingerone. In comparison to this, Example 3 utilises 785.9 grams (2.4 times more compared to amount of product used in Example 2) and provides 43.8 mg/g zingerone. However, this lower yield can be explained by the lower levels in the starting material from Samoa (1.44 mg/g zingerone).
  • Example 4 Processing methods using juicing and alkaline treatment
  • Ginger root was mechanically juiced and the levels of 6- gingerol were determined for juice and remnant solids. The majority of the 6-gingerol was present in the juice. Treatment of the juice with alkali showed that effectively all of the 6- gingerol was converted to zingerone in 5-6 hrs at 60°C.
  • Alkaline treatment This work was performed on the first juice (JI) recovered from the juicing process (above). Samples of juice (after shaking to suspend all solids) were allowed to react with KOH at RT, 30 or 60°C for 1, 2, 3, 5, and also at 60°C for 24 hours. Three concentrations of KOH were also trialled, 0.5, 1.0 and 2.0%. A solution of 2 N KOH was prepared (5.6 g KOH in 50 ml water). To generate 0.5%, 1% and 2% KOH concentrations in each sample, 0.25, 0.5 or 1 ml of the 2 N KOH was added to 5.5 ml of juice and shaken.
  • Example 5 Additional processing methods using juicing and alkaline treatment
  • the two juices were combined.
  • the juice was heated to 60°C and KOH was added to a final concentration of 2% w/w.
  • the alkalised juice was held at 60°C for 5 hours to convert gingerol to zingerone.
  • the juice was neutralised to a pH of 7.2 by addition of anhydrous citric acid.
  • GMARC2 HWEX the extract
  • HW MARC the marc
  • Sample analysis Analysis was performed using HPLC with UV detection at 280 nm.
  • Sample preparation was as follows: (1) liquid samples such as juice were diluted 1 : 1 with ethanol and centrifuged. In-process liquid samples with alkali present were diluted with 1 N HC1 and ethanol 1 : 1 : 1 (2) solid samples such as raw ginger or ginger marc were extracted by double extraction with ethanol (ultrasonication, heat at 60°C for 20 minutes, vortexed and centrifuged) and combining supernatants. Solid extracts (from approximately 5 g) were generally made up to 50 mL for analysis. The raw fresh ginger was roughly chopped and then blended with ethanol using an ULTRA-TURRAX® type mixer. Quantification was performed by comparison to a standard curve prepared using zingerone. A molecular weight correction was made for gingerol.
  • Hot water extraction 3.62 kg of GMARC2 and 18.1 kg of water were heated to 60°C and then separated by screw pressing after 15 minutes of extraction at 60°C. Next, 21.18 kg of mixture was pressed. Noting that approximately 500 g of water was lost as evaporation during the extraction. From this, 17.62 kg of extract was recovered and subsampled for analysis. In addition, 2.69 kg of marc was recovered.
  • Freeze drying The treated juice was transferred to freeze dryer trays and frozen overnight before transferring to the Cuddon FD80 freeze dryer. A total of 28.51 kg of juice was loaded onto trays and dried. Approximately 3 kg of juice was lost prior to freezing and drying as a result of manual handling. After drying, 2.86 kg of dried extract was collected- a total of 10% of the mass of juice dried. This was ground and subsampled. After grinding and subsampling and handling losses a total of 2.19 kg was packed into foil bags for storage until further processing. Approximately 1.6 kg of this was sent for ethanol extraction (see Example 6).
  • This table shows the gingerol content of different fractions during pretreatment and the treatment process. From these measurements, there was a total of 19.1 g of 6-gingerol in the 36.67 kg of raw ginger feed.
  • the dried ginger extract (GPE) had a zingerone content of 5.7 mg/g. Therefore, the total zingerone in the 2.86 kg of dried powder (before losses and milling losses), was 16.30 g.
  • the concentration of gingerol in the juice before conversion (GKOH-0) was 6.9 mg/g on a dry basis.
  • Results The process and results are set out in Figure 7. For these experiments, 801.5 g of the received treated ginger was used in the ethanol extraction, along with 4007.3 g of food grade ethanol. After 72 hours at room temperature (16-20°C) under stirring, the mixture was filtered and 3556.7 g of a clear brown, aromatic ethanol tincture was obtained [ZINGOEE], as well as 1033.4 g of cake (i.e., spent ginger solids). Approximately 140 g of ethanol was lost due to evaporation during the extraction. The total weight of ethanolic tincture produced, including the samples taken at 24 and 48 hours, was 3631.7 g.
  • the zingerone mass balance for the ethanol extraction process is 98.5%. This is based on 3.88 g in the extract, plus 0.63 g in the cake, divided by 4.58 g in the feed. It was determined that room temperature extraction provided advantageous recovery of zingerone. It is possible that increasing the extraction temperature could lead to higher zingerone recovery, but room temperature extraction is clearly effective.
  • the ethanol extraction at room temperature for 24 hours is an optional step achieving at least 85% recovery of zingerone.
  • the recovery could be further increased by washing the solids with fresh ethanol after extraction.
  • the ethanolic extract [ZINGOEE] was evaporated to achieve a significant volume reduction, followed by reconstitution of the concentrated extract [ZINGOCE] with fresh ethanol in order to produce a standardised tincture containing the target dose of 12 mg/g zingerone.
  • the overall Z mass balance (out/in) was 98.5%.
  • the zingerone content in the filtrate (ZINGOEE) was 1.1 mg/g, and the zingerone content in the concentrated extract (ZINGOCE) was 33 mg/g.
  • the method produced 104.9 g of ZINGOCE at 33 mg/g, leaving 3.46 g zingerone. This number is lower than the 3.88 g in ZINGOCE because -400 g of ZINGOCE was removed before evaporation (for testing, plus -300 mL retention sample). Taking this into account, the calculations fit very well.
  • Example 7 Anti-inflammatory activity for zingerone composition
  • LPS lipopolysaccharide
  • DMEM standard cell culture media
  • DMEM foetal bovine serum 5%
  • DMEM standard cell culture media
  • MTT assay tetrazolium dye MTT, which is chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
  • Example 6 The tincture from Example 6 was utilised for these studies.
  • NO and IL-6 assays the cultured RAW264.7 cells were counted and plated (0.8 x 10 5 cells/well) in 96 well plates and incubated for 48 hours. The medium was then aspirated and replaced with fresh medium followed by the addition of the test compounds. The compounds were incubated for 1 hour prior to the addition of the stimulant. The plates were then incubated for 18 hours and the supernatant analysed for the mediator of interest, the remaining cell viability was determined by MTT.
  • the positive controls were selected based on their widespread use in similar assays, including N-(3-(aminomethyl)benzyl)acetamidine (1400W), a slow, tight binding inhibitor of inducible nitric-oxide synthase (iNOS) (Garvey et al., 1997, J Biol Chem 272(8):4959-63) and dexamethasone, a commonly used cytokine inhibitor.
  • the dose response curves were fit using Graph Pad Prism.
  • the 95% confidence intervals (CI) were calculated using the whole data set and gave an estimate error in the IC50 value.
  • Results The tincture from Example 6 was assayed to determine antiinflammatory activity. The anti-inflammatory activity was assessed using key mediators of inflammation: NO, and IL-6. The cytotoxicity of each sample was also determined by MTT assay.
  • Cytotoxicity The effect of the compounds on the viability of the cells was determined by MTT. Potency was monitored to avoid false positives, as dead cells do not produce inflammatory mediators. Cytotoxicity was determined spectrophotometrically as mitochondrial dehydrogenase present in viable cells cleaves the tetrazolium ring of MTT to yield a purple MTT formazan. All doses were tested up to 40 pM zingerone. The results are shown in Figure 9. From the cytotoxicity assay, the EC50 for the disclosed tincture was determined as 40 pM zingerone.
  • NO assay NO is a radical metabolite, which has been shown to have numerous physiological functions both as a signalling molecule and as a toxic agent in inflammation (Coleman, 2001). The inhibition of iNOS and reduction in NO levels secreted by immune cells may be a contributing factor to anti-inflammatory activity. The disclosed tincture was therefore assayed to determine if they showed inhibition on the inflammatory signalling molecule NO. The dose dependent effects of the tincture on NO are shown in Figure 10. From the NO assay, the IC50 for the disclosed tincture was determined as 9.2 pM zingerone.
  • IL-6 assay IL-6 is considered a pro-inflammatory cytokine. IL-6 is secreted by T cells and macrophages, which stimulates an immune response. IL-6 is responsible for the increased production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to the differentiation of T cells into regulatory T cells. It can cross the blood-brain barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature set point (Banks, Kastin, & Gutierrez, 1994). The inhibition of IL-6 release by immune cells points to anti-inflammatory activity. The dose dependent effects of the disclosed tincture on IL-6 are shown in Figure 11. From the IL-6 assay, the IC50 for the tincture was determined as 4.6 pM zingerone.
  • RAW264.7 cells in growth media were plated at 0.8 x 10 5 cells/well in 96-well tissue culture treated plates and incubated at 37°C / 5% CO2 in a humidified environment for 48 hours. The spent media was then aspirated and replaced with media containing 5% FBS. Treatments were added to give final concentrations of 0.625 - 40 pM zingerone (or equivalent dilutions for ethanol solvent control). The cells were incubated with the treatments for 1 hour prior to the addition of 50 ng/mL lipopolysaccharide (LPS; from Escherichia coli O111:B4).
  • LPS lipopolysaccharide
  • the cells were co-incubated with treatments and LPS for 18 hours before conditioned media was harvested, centrifuged and cell-free supernatants collected. The viability of the remaining cells was determined by WST-1 assay. Controls included were unstimulated (cells with no LPS), LPS (cells exposed to 50 ng/mL LPS), Dex (cells exposed to 1 or 10 pg/mL dexamethasone before addition of 50 ng/mL LPS), and media (media containing no cells or samples).
  • cytotoxicity assay For the cytotoxicity assay, an equal volume of a 1:5 mixture of WST- Emedia was added to each cell well and incubated for 10 minutes. Immediately prior to measurement of absorbance, an equal volume of DPBS was added and absorbance read at 440 nm (and 620 nm for removal of background). Hydrogen peroxide was included as a cytotoxic positive control. Results were normalised to LPS-stimulated control and expressed as percentage cell viability. Sample concentrations where cell viability is below 80% of the unstimulated control were deemed cytotoxic.
  • IL-6 Interleukin 6
  • IL-6 Interleukin 6
  • a beadbased multiplex assay panel Legendplex MU Thl/Th2 8-plex panel; BioLegend #741054
  • Cytex Aurora Spectral 3 laser flow cytometer As a follow-up to the IL-6 assay, assays were performed to measure IL- 10 and TNF.
  • Results The viability of the cells after incubation with the compounds/extracts was measured.
  • WST-1 reacts with a mitochondrial enzyme to form a coloured dye which can be measured by absorbance.
  • WST-1 is a measure of cellular metabolism, and a reduction in the WST-1 reaction rate can be indicative of cellular death.
  • Hydrogen peroxide (H2O2) is used as a positive control to induce cell death and ensure the validity of the assay.
  • the cell model used for IL-6 assessments explores the pro-inflammatory response by a mouse macrophage cell line (RAW264.7) when exposed to a bacterial endotoxin (lipopolysaccharide; LPS).
  • LPS lipopolysaccharide
  • NF-KB a transcription factor that regulates the expression of many proinflammatory cytokines, including IL-6.
  • Results for IL-6 measurements are shown in Figure 14.
  • This figure depicts IL-6 levels produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS), LPS + 10 pg/mL dexamethasone (Dex) and LPS + disclosed botanical extract or commercially sourced zingerone (0.6 to 40 pM) for 18 hours.
  • the asterisk denotes significantly lower IL-6 production from zingerone at the corresponding concentration (p ⁇ 0.05).
  • Treatments with no error bars at 20,000 pg/mL indicate values >20,000 pg/mL.
  • FIG. 15 depicts IL- 10 levels produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS), LPS + 10 pg/mL dexamethasone (Dex) and LPS + disclosed botanical extract or LPS + commercially sourced zingerone (1.25 to 40 pM) for 18 hours.
  • Carrot denotes significantly lower IL- 10 production compared with corresponding ethanol control (p ⁇ 0.05).
  • Asterisk denotes significantly lower IL- 10 production from zingerone at the corresponding concentration (p ⁇ 0.05).
  • Treatments with no error bars at 15,000 pg/mL indicate values 215,000 pg/mL and are above the assays limit of accuracy.
  • Results for TNF are shown in Figure 16.
  • This figure depicts TNF levels produced by RAW264.7 cells after treatment with lipopolysaccharide (LPS), LPS + 10 pg/mL dexamethasone (Dex) and LPS + disclosed botanical extract or LPS + commercially sourced zingerone (1.25 to 40 pM) for 18 hours.
  • Asterisks denotes significantly lower IL-6 production from zingerone at the corresponding concentration (p ⁇ 0.05).
  • Treatments with no error bars at 15,000 pg/mL indicate values 215,000 pg/mL and are above the assays limit of accuracy.
  • the zingerone concentrations for the tincture dilutions have been incorrectly noted as 0.625, 1.25, 2.5, 5, 10, 20, and 40 pM. Based on the correct starting concentration of 33 mg/g, the zingerone concentrations for the tincture dilutions can be correctly calculated as 0.493, 0.986, 1.97, 3.95, 7.89, 15.8, and 31.6 pM, respectively.
  • the MTT assay was used identify cytotoxic effects of compound treatment on the RAW264.7 cells. No cytotoxic effect was identified for any compound tested.
  • Method 1 conditions there was a notable drop in the MTT absorbance relative to the unstimulated Method 2 conditions, and both stimulated and unstimulated Method 2 conditions (Figure 18A: Method 1 stimulation; Figure 18B: Method 2 stimulation).
  • This drop is expected as IFN-y’s mechanism involves stopping cellular proliferation, resulting in less cells present compared to (-) IFN-y conditions. In particular, fewer cells means fewer mitochondria to metabolise the MTT to formazan, resulting in lower absorbance.
  • Dexamethasone and synthetic zingerone were obtained from Sigma-Aldrich. For these studies, dexamethasone (100 nM) was used as the positive control); absolute ethanol was used as the vehicle control; and synthetic zingerone (20 pM) was used for test treatments. The testing included: (1) IL-6 ELISA - measures production of IL-6 from stimulated/unstimulated RAW264.7 cells; (2) MTT assay - identifies potential cytotoxicity by measuring metabolism of MTT to formazan.
  • Cells were counted after applying a 1 in 10 trypan blue dilution. Samples were then diluted to 1 x 10 6 cells/mL. Cells were plated at 50,000/well (50 pL). For stimulated Method 1 samples, 50 pL IFN-y was added (final concentration 20 U/mL). For unstimulated samples, cells were plated and well volume was made up to 200 pL.
  • IFN-y stock solution was 5,000,000 U/mL. This was diluted to 24,000 U/mL in CTCM; (2) LPS stock solution was 1,000,000 ng/mL.
  • a 80 ng/mL solution was required (final concentration 20 ng/mL in the wells).
  • a 200 ng/mL solution was required (final concentration 50 ng/mL in the wells);
  • Synthetic zingerone stock solution was prepared by adding 2 pL of zingerone into 198 pL CTCM . This gave a 1544.6401 pM stock. From this stock, 25.89 pL was added to 474.11 pL CTCM. This gave an 80 pM stock. The 80 pM stock (50 pL) was added to samples (along with LPS) to achieve a final concentration 20 pM (0.013% ethanol).
  • Dexamethasone stock solution was prepared by introducing 2 pL of dexamethasone into 998 pL CTCM . This gave a 5096 nM stock solution. From the 5096 nM stock solution, 78.49 pL was added to 921.51 pL CTCM. This gave a 400 nM stock solution. The 400 nM stock solution (50 pL) was added to samples (along with LPS) to achieve a final concentration of 100 nM (0.003% ethanol); (2) Ibuprofen stock solution was prepared by introducing 2 pL ibuprofen into 579.704 pL CTCM.
  • IL-6 Reagents from BD Biosciences. Capture: purified NA/LE rat anti-Mouse IL-6; Cat. No. 554398, clone MP5-20F3; Standard: recombinant mouse IL-6; Cat. No. 554582; Detection: biotin rat anti-Mouse IL-6 antibody; Cat. No. 554402, clone MP5-32C11; SA-HRP: Streptavidin HRP; Cat. No. 554066; TMB: BD OptEIATM TMB substrate reagent set; Cat. No. 555214. Two full ELISA plates (each with 96 wells) were utilised when including standards.
  • a total of 11 mL volume was prepared for 50 pL/well samples and a total of 22 mL volume was prepared for 100 pL/well samples.
  • TMB 3, 3', 5, 5'- tetramethylbenzidine
  • the TMB was prepared by introducing 11 mL TMB-A and 11 mL TMB-B into separate 15 mL falcon tubes covered in tinfoil. After the washing following SA-HRP incubation, the TMB-A and TMB-B samples were combined and this was introduced to the plates at 100 pL/well. Colour was allowed to develop. Colour development was stopped by adding 100 pL H2SO4 to wells. The plates were read on a plate reader.
  • Example 10 Anti-inflammatory activity and cell viability analysis in comparative testing
  • a dedicated T75 flask was retrieved for replicate from incubator.
  • the flask was gently tapped to detach any non-adherent cells.
  • the growth media was decanted and replaced with 10 mL CTCM.
  • the flask was gently swayed to wash the cells.
  • the wash media was decanted and replaced with 10 mL CTCM.
  • Using a rubber scraper cells were detached.
  • the suspension was transferred to a 50 mL Falcon tube.
  • the cells were centrifuged at 400 x g for 5 minutes.
  • the cells were resuspended in 10 mL CTCM. Cells were counted after applying a 1 in 10 trypan blue dilution.
  • Cells were diluted to 1 x 10 6 cells/mL. Cells were plated at 80 pL/well. To this, we added 120 pL to bring the volume to 200 pL. The plate was returned to the incubator. Cells were continued to be plated as indicated. In this way, plates were dedicated to stimulated and unstimulated samples. Plates were left for 48 hours before any treatment occurred.
  • the plates were returned to the incubator for 18 hours. All ELISA plates were coated.
  • the MTT reagent was prepared. This was done by mixing 50 mg MTT into 10 mL IX PBS. This produced a 5 mg/mL reagent solution for use with 6 plates.
  • ELISA reagents From BD Biosciences. Capture: purified NA/LE rat anti-Mouse IL-6; Cat. No. 554398, clone MP5-20F3; Standard: recombinant mouse IL-6; Cat. No. 554582; Detection: biotin rat anti-Mouse IL-6 antibody; Cat. No. 554402, clone MP5-32C11; SA-HRP: Streptavidin HRP; Cat. No. 554066; TMB: BD OptEIATM TMB substrate reagent set; Cat. No. 555214. Solutions included: (1) ELISA capture buffer pH 6: 14.196 Na2HPO4 in 1 L mqH20.
  • ELISA assays ELISA plates were coated with a capture antibody suspended in an ELISA capture buffer at 50 pL/well. The pH was adjusted based on the cytokine being tested (see summary tables, below). The coated ELISA plates were incubated at 4°C. overnight. The following day, the plates were retrieved from the refrigerator. The plates were washed four times in ELISA wash buffer. Next, 100 pL/well blocking solution was added to the plates. The plates were incubated at room temperature for 2 hours. The plates were washed three times. The supernatant was diluted depending on the cytokine being tested.
  • test sample was added along with a standard curve of the cytokine to quantify at 50 pL/well (see summary tables, below).
  • the plates were incubated for 2 hours at room temperature or overnight at 4°C.
  • the supernatant was transferred to another plate before washing. Washing was carried out four times.
  • a biotinylated detection antibody was suspended in a solution of FCS and PBS and added to the plates at 50 pL/well. The plates were incubated for 1 hour at room temperature. The plates were washed six times. Streptavidin -horseradish peroxidase was suspended in a solution of FCS and PBS. This was introduced to the plates at 50 pL/well. The plate was incubated at room temperature, in darkness, for 1 hour. The required volumes of TMB-A and TMB-B were placed into separate falcon tubes. These were stored at room temperature, in darkness, during the SA-HRP incubation. The plates were washed eight times.
  • TMB solution was introduced to the plates at 100 pL/well. Colour was allowed to develop. To stop the reaction, 0.18 M H2SO4 solution was added at 100 pL/well. See tables, below. Plates were read using a PerkinElmer EnSpire® plate reader at 450 nm.
  • MTT assays On the day preceding the assay, MTT powder was dissolved in IX dPBS to a concentration of 5 mg/mL. Each plate required 20 pL/well. For 96 wells, this required 1920 pL MTT solution to be prepared. The concentration was 5 mg of MTT per mL of solution. This equated to 10 mg MTT per 96 well plate.
  • CTCM On the day of the assay, CTCM was warmed in a 37°C water bath. While media was warming, cell cultures (RAW264.7) were centrifuged at 400 x g for 5 minutes. Using a multichannel pipette set to 180 pL, supernatant was removed and transferred to a labelled U-bottom 96 well plate.
  • the supernatant was stored at -20 °C until analysis.
  • 60 pL warmed CTCM was added to bring the volume to 80 pL/well.
  • 20 pL MTT solution was added.
  • the plate was returned to the 37°C/5% CO2 incubator for 45 minutes. After 45 minutes, 50 pL MTT solubilizing solution was added. This included 10% SDS (w/v), 0.01 M HC1 - pH adjusted to 4.0 with sodium hydroxide; or 10% SDS (w/v), 45% DMF - pH adjusted to 4.0 with acetic acid.
  • the plate was covered with tinfoil and left overnight. After overnight incubation, the plate was read using a PerkinElmer EnSpire® plate reader at 580 nm.
  • NO assays A Griess reaction protocol was utilised. NO production was measured via NaNO2 product in a 96 well format.
  • Reagents included: (1) Greiss solution A (50 mL): 1% (w/v) sulphanilamide (500 mg), 2.5% phosphoric acid, stored at 40°C and protected from light; (2) Greiss solution B (50 mL): 0.1% (w/v) N-(l- naphthyl)ethylenediamine (50 mg), 2.5% phosphoric acid, stored at 40°C and protected from light; (3) 2.5% phosphoric acid (100 mL): 2.94 mL 85% phosphoric acid, 97.6 mL ddfLO.
  • rows 1 and 2 (across) of each plate included 1:1 dilutions of NaNO2 from 500 pM to 0 pM.
  • rows A and B (down) 50 pl of culture media was added to each well. The first well in each row received 95 pl. To the first wells, 5 pl of 10 mM NaNO2 was added. From these wells, 50 pL was taken and dilutions made down the row excluding the last well of each row (base line).
  • 170 pl supernatant was removed from the wells without disturbing the cellular monolayer on the bottom of each well. From this 170 pl sample, 50 pl samples (in triplicate) were transferred to a flat- bottomed 96 well plate.
  • Greiss reaction For the Greiss reaction, equal volumes of Greiss solution A and Greiss solution B were mixed to the desired volume (about 5 mL total per 96 well plate). The mixed Greis solution A+B was added to each well (50 pl per well). Any bubbles were removed using a hair dryer. Absorbance was read at 570 nm.
  • Results - viability The MTT assay was used to identify any drops in metabolism which may be indicative of a cytotoxic effect of any of the compounds. The only compound found to have a cytotoxic effect was the disclosed botanical extract at and above 50 pM. The data is summarised in Figures 19A-19D and Tables 16 and 17, below.
  • IL-6 is a common proinflammatory cytokine produced by macrophages upon stimulation with LPS. IL-6 is involved in propagating the early innate immune response. The only substantial inhibition of IL-6 production from stimulated RAW264.7 macrophages was from the disclosed botanical extract ( Figure 20). The most potent IL-6 inhibition was seen with the botanical extract at 25 pM, reducing the IL-6 production to 41% of the absolute ethanol control ( Figure 20). Considerable inhibition of IL-6 production was also seen with the botanical extract at 6.25 pM and 12.5 pM ( Figure 20).
  • TNF is also a proinflammatory cytokine produced by macrophages when stimulated with LPS.
  • the greatest inhibition of TNF from stimulated RAW264.7 cells was observed after treatment with ferulic acid at 150 pM.
  • This treatment reduced TNF to 61% of the absolute ethanol control ( Figure 24).
  • concentrations of 50 pM the inhibition from ferulic acid treatment tapered off to the level of the absolute ethanol control.
  • Synthetic zingerone had an apparently modest inhibitory effect on TNF production at 150 pM, reducing the level of TNF to around 80% of the absolute ethanol control.
  • the disclosed botanical extract also had a modest inhibitory effect on TNF, reducing the level of TNF to around 80% of the absolute ethanol control at 25, 12.5 and 6.25 pM. acetyl zingerone had no effect on TNF production. Concentrations of the disclosed botanical extract associated with cytotoxicity were omitted to avoid confounding data. Results are summarised in Table 19, below.
  • Nitric oxide (NO) is a small molecule released by macrophages during the inflammatory response which is capable of killing invading pathogens. Only the disclosed botanical extract had an inhibitory effect on NO, reducing it to 77% of the absolute ethanol control ( Figure 28). Synthetic zingerone caused elevations at the higher range of concentrations tested (50, 75, 100 and 150 pM) when compared to the absolute ethanol control. Acetyl zingerone at 150 pM also caused a slight elevation in NO levels. Ferulic acid did not appear to alter NO production. Concentrations of the disclosed botanical extract associated with cytotoxicity were omitted to avoid confounding data. Results are summarised in Table 20, below.
  • the superior inhibitory activity of the botanical extract is particularly surprising, especially given that the comparative testing utilised the botanical extract at significantly lower concentrations (e.g., 25 pM) than the commercially available compounds (e.g., 100 pM or 150 pM).
  • mice EAE was not successfully induced in this study (data not shown). However, body weights of the mice were monitored as an indication of safety and tolerability. On average, body weight increased by 0.3 g over the treatment period, with an average weight of 19.7 g at the beginning of the treatment period and an average weight of 20.0 g at the end of the treatment period. No significant change in body weight was observed for the zingerone treatment, which indicated the dosage used was safe and tolerable.
  • Step 1 Initial juicing of ginger rhizome.
  • the first 700 kg batch was passed through the belt press juicer (Voran EBP500). All juice obtained from the juicer was collected into 200 L blue HDPE drums (provided by Phytex) on a wooden pallet. All solid/marc obtained from the juicer was placed into a separate 200 L blue HDPE drum (provided by Phytex).
  • the solid marc was passed through the belt press juicer (Voran EBP500) a second time to remove further liquid from the solid marc. The liquid from the second press was combined with the original 200 L blue HDPE drums containing the first-pressed liquid.
  • the second 700 kg batch was passed through the belt press juicer (Voran EBP500). All juice obtained from the juicer was placed into 200 L blue HDPE drums (provided by Phytex) on a wooden pallet. All solid/marc obtained from the juicer was collected into a separate 200 L blue HDPE drum (provided by Phytex).
  • the solid marc was passed through the belt press juicer (Voran EBP500) a second time to remove further liquid from the solid marc. The liquid from the second press was combined with the original 200 L blue HDPE drums containing the first-pressed liquid.
  • Run A and Run B were performed in two equal treatments with KOH.
  • the extraction vessel had maximum capacity of 400 L, so each run (A or B) was performed in two parts (run A1/A2 and run B 1/B2).
  • Equipment maintenance/calibration and cleaning status was checked before proceeding with treatment. Clean tags or cleaning logs were used as needed.
  • Processing of ginger juice 300 L per run: The volume of ginger juice obtained in the multiple 200 L HDPE drums was measured. Net weights were recorded for all HDPE drums. The ginger juice/settleable solids remaining in each 200 L HDPE drum were mixed using a l m plastic mixing paddle to ensure solids were resuspended.
  • a clamp was attached to the decanting lid (lid with 25 mm poly valve) to each 200 L HDPE drum. Using the drum lifter all solid fractions were poured into the extraction vessel to combine with the UF concentrate. A wall mounted stirrer purple propeller was inserted into the extraction vessel with 2 x 100 mm stainless steel propellers attached. Propeller heights were set to the appropriate height with to mix the juice volume. The wall mounted stirrer was connected on utilities board and stirring was commenced in a clockwise direction at 50 Hz. The settleable sludge from the bottom of each drum was scooped out and transferred to the extraction vessel (to be dissolved by the heat and alkaline treatment). Each drum was hosed with I L water to remove all residual ginger juice, and this was added to the extraction vessel.
  • Extra citric acid aqueous solution (50% w/v) was prepared by dissolving 1.0 kg citric acid granules into 1.0 L purified water in a 20 L purple polythene bucket. This was dissolved with a stainless steel hand stirrer. Whilst alkaline ginger juice was stirring in the extraction vessel, citric acid granules were added. This was done by adding 10 kg and waiting for the granules to dissolve. The pH of the solution was monitored during this process. The slow addition of citric acid was continued using 500 ml scoops at a time and waiting 30 seconds before the adding the next scoop. Once all of the citric acid was added and dissolved (18.0 kg), a pH reading was obtained.
  • KOH potassium hydroxide
  • Step 3 Production of zingerone powder by drying and milling.
  • Oven drying Equipment maintenance/calibration and cleaning status were confirmed. The drying over was set up with calibrated COMARK digital logger with stainless steel probe. Time was recorded for each drum of zingerone concentrate to be taken from the cold room to prepare for drying. The Brix% and theoretical zingerone content were noted. The oven was loaded with all trays having Teflon mats for drying. The drum was mixed using a polypropylene paddle before using a 4 L jug to remove a volume of liquid. This was subsequently transferred to a 2 L jug to allow placement into each tray. Next, 3 L was poured onto the Teflon mat for each tray.
  • the trays were loaded onto each shelf to ensure a gap between the trays (not less than 30 mm) and allow for airflow and to allow breaking up of dried materials.
  • the oven temperature was set at 60°C. After 24 hours, each tray was inspected and scraped to release the softer portions off the Teflon mat as needed. The underside of the product was positioned on the top of the flipped tray. The product was gently broken up using the plastic spatula to facilitate drying. The trays were loaded back into the oven and dried for a further 4 hours (28 hours total). When sufficient dried (at 24 or 28 hours), the product was crushed manually with a stainless steel spatula and collected in 100 micron poly bag. Each bag was weighed and recorded and placed into a 100 L square mobile tub. The square mobile tub was weighed and recorded. The product was then ready for milling.
  • Milling Prior to milling, the stainless steel hammer mill was checked as clean and fit with a clean 1 mm mesh screen (“fine zingerone screen”). The lid was fitted with a 3 mm rubber mat seal and the thumb screw was firmly tightened to lock the lid in place. The hammer mill was run for 10 seconds to ensure correct rotation and clearances. A 100 micron poly bag was fitted to the outlet of the mill chamber and tightened with an adjustable clamp. The overhead dust extractor was activated. Slowly, the coarse dry zingerone material was transferred into the feed chute using the poly scoop. Two scoops at a time were emptied into the chute and the inlet flap was opened every few seconds. All the material in the chute was passed through the stainless steel screen and collected in a poly bag collector.
  • Step 4 Ethanol extraction of zingerone powder.
  • vol 95% ethanol amt. zingerone powder (kg) x 5 kg.
  • the purple WMS shaft was inserted through the bearing gland in the lid of the extraction vessel. Stirring was commenced at 200-300 rpm to facilitate complete mixing of the ethanol solution.
  • 75 kg of dry zingerone powder (from Step 3) was added to the extraction vessel through the 100 mm access port on the lid of the vessel. Stirring was continued during this addition. Once all the powder was added, the access port was closed and the lid was sealed. All initial temperatures were recorded before heating. Then, the steam supply was connected to the stainless steel coil in the extraction vessel. Whilst stirring, the temperature of the ethanolic solution was monitored and maintained at a minimum of 50°C for at least 4 hours. The stirrer was turned off and the solution was allowed to stand for 20 minutes. The final temperature was recorded prior to filtration of the ethanolic extract. A sample was removed from the ethanolic extract (20 mL) and tested for zingerone.
  • Rotary evaporation of the ethanolic extract Three 15 L buckets of filtered ethanolic extract were removed from the cold room and allowed to equilibrate to room temperature. A Heidolph round bottom flask was prepared, dried, and weighed. The Heidolph 20 L rotary evaporator was set up with 1 L fresh 95% ethanol added to the RBF. The cooling water inlet/outlet lines was connected to appropriate valves on the wall and the valve was adjusted to alter flow rate as required. The rotary evaporator was connected to the Heidolph vacuum pump. A white 4 mm HDPE tube, submerged in 95% ethanol, was connected to the inlet tap on the rotary evaporator.
  • Circulation of cooling water was commenced through the glass condenser. Residual water was removed during the evaporation stage by running at a minimum 50 mBar and 40°C. Accordingly, the vacuum was set and slow rotation of the round bottom flask was commenced (approximately 60 rpm). As each bucket was emptied, the inlet tube was transferred to the next bucket until all three buckets (45 L total) had been bled into the rotary evaporator and collected in the condenser flask. Observations were made for pressure, colour, etc.
  • zingerone concentration of zingerone was less than 12.5 mg/ml, evaporation was continued until the correct concentration was achieved. Twelve amber 4 L glass bottles were rinsed with fresh 95% ethanol and left to drain for 30 minutes. When standardised to 12.5 mg/ml, the RBF was removed from the rotary evaporator and placed into a 15 L bucket. The standardised zingerone tincture was decanted into the rinsed and drained bottles, leaving 30 mm from the bottom thread on the bottle. The filled bottles were capped with poly lids having Teflon seals and hand tightened. Parafilm was wrapped around the bottle neck and lid. Sealed bottles were placed into 100 micron poly bags and double heat sealed. These were placed in a box and the box was put into the cold room.

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Abstract

Sont divulguées des méthodes de préparation de zingérone à partir de gingembre ainsi que des compositions obtenues à l'aide de ces méthodes, et des méthodes d'utilisation de ces compositions. Sont en outre divulguées précisément des compositions bénéfiques et des méthodes les utilisant.
PCT/IB2024/051451 2023-02-15 2024-02-15 Méthodes de préparation de zingérone, compositions contenant de la zingérone, et leurs utilisations Ceased WO2024171115A1 (fr)

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AU2024221532A AU2024221532A1 (en) 2023-02-15 2024-02-15 Methods of preparation of zingerone, compositions comprising zingerone, and uses therefor
CN202480024232.2A CN120957711A (zh) 2023-02-15 2024-02-15 制备姜油酮的方法、包含姜油酮的组合物和其用途

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