WO2023037299A1 - Anti-inflammatory compositions comprising ginger and turmeric - Google Patents

Anti-inflammatory compositions comprising ginger and turmeric Download PDF

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Publication number
WO2023037299A1
WO2023037299A1 PCT/IB2022/058491 IB2022058491W WO2023037299A1 WO 2023037299 A1 WO2023037299 A1 WO 2023037299A1 IB 2022058491 W IB2022058491 W IB 2022058491W WO 2023037299 A1 WO2023037299 A1 WO 2023037299A1
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Prior art keywords
composition
ginger
extract
turmeric
various embodiments
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PCT/IB2022/058491
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French (fr)
Inventor
Vijay Jayasena
Madhuri Venigalla
David Leach
Gerald Muench
Xian Zhou
Chun Guang Li
Mitchell Low
Hans Wohlmuth
Ahmad Al-Khazaleh
Iftekhar Ahmed
Original Assignee
Integria Healthcare (Australia) Pty Limited
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Priority claimed from AU2021902926A external-priority patent/AU2021902926A0/en
Application filed by Integria Healthcare (Australia) Pty Limited filed Critical Integria Healthcare (Australia) Pty Limited
Publication of WO2023037299A1 publication Critical patent/WO2023037299A1/en

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    • 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
    • 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)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61K2236/30—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
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
    • 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
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • Anti-inflammatory compositions comprising ginger and turmeric
  • the invention relates to compositions comprising ginger and turmeric, or extracts thereof, wherein the composition elicits a synergistic anti-inflammatory effect.
  • the invention also relates to the use of such compositions in the treatment or prevention of inflammation, diseases or conditions associated with inflammation and/or to provide one or more health benefits to a subject.
  • diseases and conditions associated with the immune system and/or aberrant immune function together with the amelioration of symptoms associated with immune dysfunction remain significant medical and economic problems.
  • diseases and conditions include diseases and conditions associated with inflammation and chronic inflammation.
  • Ginger Zinc officinale Roscoe
  • turmeric Curcuma longa L.
  • the fresh or processed forms of ginger are used for gastrointestinal disorders, nausea, cold and pain in traditional medicines.
  • T cinnamonric is used extensively in diet for its flavour and colour, and as a medicinal plant for various indications such as sprains and swelling, and improving digestion.
  • Both ginger and turmeric have been investigated for their anti-inflammatory activities to prevent and manage several inflammatory diseases such as pain, diabetes, cardiovascular diseases, neurodegenerative diseases, cancer and respiratory disorders. However, little is known regarding the combined mechanisms of their activity and how the health benefits occur.
  • the invention provides a composition comprising turmeric and ginger, wherein the gingerturmeric dry weight or equivalent ratio is about 7:225 to about 63:25.
  • the composition comprises a gingerturmeric dry weight or equivalent ratio of about 7:225 to about 63:25, about 7:100 to about 28:25, about 3:25 to about 7:10, about 14:75 to about 63:25, about 7:25 to about 28:25, about 14:75 to about 28:25, about 21 :50 to about 7:10, about 49:75 to about 28:25, about 49:75 to about 7:10, about 7:10 to about 63:25 or about 7:10 to about 28:25.
  • the composition comprises a gingerturmeric dry weight or equivalent ratio of about 7:225, about 7:100, about 3:25, about 14:75, about 7:25, about 21 :50, about 49:75, about 7:10, about 28:25 and/or about 63:25. In various embodiments, the composition comprises a gingerturmeric dry weight or equivalent ratio of about 7:10.
  • the composition comprises turmeric and ginger, wherein the composition comprises less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% turmeric dry weight or equivalent, with respect to the ginger dry weight or equivalent, in the composition.
  • the composition comprises turmeric and ginger, wherein the composition comprises greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, or greater than 75% ginger dry weight or equivalent, with respect to the turmeric dry weight or equivalent, in the composition.
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 4:6 to about 9:1 , and wherein the turmeric extract has a drug extract ratio (DER) of 25:1 (25 parts dry turmeric extracted to yield 1 part final extract) and the ginger extract has a DER of about 7:1 (i.e. 7 parts dry ginger extracted to yield 1 part final extract), about 16:1 or about 12:1.
  • DER drug extract ratio
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1 , 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1 , 5:5 to 5:2, 6:4 to 9:1, 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9:1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9:1.
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1, 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1, 5:5 to 5:2, 6:4 to 9: 1 , 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9: 1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9: 1 and wherein the turmeric extract has a DER of 25:1, and the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1.
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 1:9, about 2:8, about 3:7, about 4:6, about 5:5, about 6:4, about 7:3, about 5:2, about 8:2 or about 9: 1 , and wherein the turmeric extract has a DER of about 25:1 and the ginger extract has a DER of about 7:1, about 16:1 or about 12:1.
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract has a DER of about 25:1 and the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1.
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 4:6 to about 9:1 , and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1 , and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
  • DCM dichloromethane
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1 , 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1 , 5:5 to 5:2, 6:4 to 9:1, 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9:1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9:1.
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1, 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1, 5:5 to 5:2, 6:4 to 9: 1 , 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9: 1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9: 1 and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
  • the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1
  • the ginger extract
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 1:9, about 2:8, about 3:7, about 4:6, about 5:5, about 6:4, about 7:3, about 5:2, about 8:2 or about 9: 1 , and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
  • DCM dichloromethane
  • the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1 , or is extracted with 100% ethanol and has a DER of about 12:1.
  • DCM dichloromethane
  • the invention provides a composition comprising: one or more of 6- gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s) and/or 10- shogaol (10s); and one or more of curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B).
  • the composition of the second aspect comprises ginger and turmeric, wherein the ginger comprises one or more of 6g, 8g, 10g, 6s, 8s and/or 10s, and the turmeric comprises one or more of C, D and/or B.
  • the composition of the second aspect comprises ginger and turmeric, and wherein the gingerturmeric dry weight or equivalent ratio is as defined in the first aspect.
  • the composition of the second aspect comprises a ginger extract and/or a turmeric extract.
  • the composition of the second aspect comprises a ginger extract as defined in the first aspect and/or a turmeric extract as defined in the first aspect, and/or a gingerturmeric dry weight or equivalent ratio as defined in the first aspect.
  • composition of any one of the above aspects comprises 6- shogaol (6s) and curcumin (C), wherein the 6s:C weight ratio is about 0.15:22.55 to about 0.34:3.76.
  • the composition of any one of the above aspects comprises a 6s:C weight ratio of about 0.15:22.55, about 0.19:18.79, about 0.22:15.04, about 0.26:11.28, about 0.27:10.74, about 0.30:7.52 or about 0.34:3.76. In various embodiments, the composition comprises a 6s:C weight ratio of about 0.27:10.74.
  • the composition of any one of the above aspects comprises a 6s:C weight ratio of about 0.27:10.74, about 0.49: 10.74 or about 0.35:10.74. In various embodiments, the composition comprises a 6s:C weight ratio of about 0.27-0.49:10.74. In various embodiments, the composition comprises a 6s:C weight ratio of about 0.27- 0.35:10.74.
  • the composition of any one of the above aspects comprises a 6s:C weight ratio of about 0.15:22.55 to about 0.19:18.79, about 0.15:22.55 to about 0.22:15.04, about 0.15:22.55 to about 0.26:11.28, about 0.15:22.55 to about 0.27:10.74, about 0.15:22.55 to about 0.30:7.52, about 0.15:22.55 to about 0.34:3.76, about 0.19:18.79 to about 0.22:15.04, about 0.19:18.79 to about 0.26:11.28, about 0.19:18.79 to about 0.27:10.74, about 0.19:18.79 to about 0.30:7.52, about 0.19:18.79 to about 0.34:3.76, about 0.22:15.04 to about 0.26:11.28, about 0.22:15.04 to about 0.27:10.74, about 0.22:15.04 to about 0.30:7.52, about 0.22:15.04 to about 0.34:3.76, about 0.26:11.28 to about 0.22:15.
  • the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 0.15:0.05:22.55 to about 0.34:0.10:3.76.
  • the 6s:10s:C weight ratio is about 0.15:0.05:22.55, about 0.19:0.06:18.79, about 0.22:0.07:15.04, about 0.26:0.08:11.28, about 0.27:0.08:10.74, about 0.30:0.09:7.52 or about 0.34:0.10:3.76.
  • the 6s:10s:C weight ratio is about 0.27:0.08:10.74.
  • the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 0.15:0.05:22.55 to about 0.19:0.06:18.79, about 0.15:0.05:22.55 to about 0.22:0.07:15.04, about 0.15:0.05:22.55 to about 0.26:0.08:11.28, about 0.15:0.05:22.55 to about 0.27:0.08:10.74, about 0.15:0.05:22.55 to about 0.30:0.09:7.52, about 0.15:0.05:22.55 to about 0.34:0.10:3.76, about 0.19:0.06:18.79 to about 0.22:0.07:15.04, about 0.19:0.06:18.79 to about 0.26:0.08:11.28, about 0.19:0.06:18.79 to about 0.27:0.08:10.74, about 0.19:0.06:18.79 to about 0.30:0.09:7.52, about 0.19:0.06:18.79 to about 0.34:
  • the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 0.15:0.03:0.05:22.55 to about 0.34:0.07:0.10:3.76.
  • the 6s:8s:10s:C weight ratio is about 0.15:0.03:0.05:22.55, about 0.19:0.04:0.06:18.79, about 0.22:0.05:0.07:15.04, about 0.26:0.05:0.08:11.28, about 0.27:0.06:0.08:10.74, about 0.30:0.06:0.09:7.52 or about 0.34:0.07:0.10:3.76.
  • the 6s:8s:10s:C weight ratio is about 0.27:0.06:0.08:10.74.
  • the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 0.15:0.03:0.05:22.55 to about 0.19:0.04:0.06:18.79, about 0.15:0.03:0.05:22.55 to about 0.22:0.05:0.07:15.04, about 0.15:0.03:0.05:22.55 to about 0.26:0.05:0.08:11.28, about 0.15:0.03:0.05:22.55 to about 0.27:0.06:0.08:10.74, about 0.15:0.03:0.05:22.55 to about 0.30:0.06:0.09:7.52, about 0.15:0.03:0.05:22.55 to about 0.34:0.07:0.10:3.76, about 0.19:0.04:0.06:18.79 to about 0.22:0.05:0.07:15.04, about 0.19:0.04:0.06:18.79 to about 0.26:0.05:0.08:11.28, about 0.19:0.04
  • composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 0.15:0.05:22.55:0.44:4.68 to about 0.34:0.10:3.76:0.07:0.78.
  • the 6s:10s:C:B:D weight ratio is about 0.15:0.05:22.55:0.44:4.68, about 0.19:0.06:18.79:0.37:3.90, about 0.22:0.07:15.04:0.30:3.12, about 0.26:0.08:11.28:0.22:2.34, about 0.27:0.08:10.74:0.21 :2.23, about 0.30:0.09:7.52:0.15:1.56 or about 0.34:0.10:3.76:0.07:0.78.
  • the 6s:10s:C:B:D weight ratio is about 0.27:0.08:10.74:0.21 :2.23.
  • the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 0.15:0.05:22.55:0.44:4.68 to about 0.19:0.06:18.79:0.37:3.90, about 0.15:0.05:22.55:0.44:4.68 to about 0.22:0.07:15.04:0.30:3.12, about 0.15:0.05:22.55:0.44:4.68 to about 0.26:0.08:11.28:0.22:2.34, about 0.15:0.05:22.55:0.44:4.68 to about 0.27:0.08:10.74:0.21 :2.23, about 0.15:0.05:22.55:0.44:4.68 to about 0.30:0.09:7.52:0.15:1.56, about 0.15:0.05:22.55:0.44:4.68 to about 0.34:0.10:3.76:0.07:0.78, about 0.19:0.06:18.79:0.37:3.90 to about 0.22:
  • the composition of any one of the above aspects comprises a 6s:C:B:D weight ratio of about 0.27:10.74:0.21 :2.23, about 0.49:10.74:0.21 :2.23 or about 0.35:10.74:0.21 :2.23. In various embodiments, the composition comprises a 6s:C:B:D weight ratio of about 0.27-0.49:10.74:0.21 :2.23. In various embodiments, the composition comprises a 6s:C:B:D weight ratio of about 0.27-0.35:10.74:0.21 :2.23.
  • composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.34:0.07:0.10:3.76:0.07:0.78.
  • the 6s:8s:10s:C:B:D weight ratio is about 0.15:0.03:0.05:22.55:0.44:4.68, about 0.19:0.04:0.06:18.79:0.37:3.90, about 0.22:0.05:0.07:15.04:0.30:3.12, about 0.26:0.05:0.08:11.28:0.22:2.34, about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.30:0.06:0.09:7.52:0.15:1.56 or about 0.34:0.07:0.10:3.76:0.07:0.78.
  • the 6s:8s:10s:C:B:D weight ratio is about 0.27:0.06:0.08:10.74:0.21 :2.23.
  • the composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.19:0.04:0.06:18.79:0.37:3.90, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.22:0.05:0.07:15.04:0.30:3.12, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.26:0.05:0.08:11.28:0.22:2.34, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.30:0.06:0.09:7.52:0.15:1.56, about 0.15:0.03:0.05:22.55:0.44:4.68
  • the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1.39:22.55 to about 3.13:3.76. In various embodiments, the 6g:C weight ratio is about 1.39:22.55, about 1.74:18.79, about 2.09:15.04, about 2.43:11.28, about 2.48:10.74, about 2.78:7.52 or about 3.13:3.76. In various embodiments, the 6g:C weight ratio is about 2.48:10.74.
  • the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1.39:22.55 to about 1.74:18.79, about 1.39:22.55 to about 2.09:15.04, about 1.39:22.55 to about 2.43:11.28, about 1.39:22.55 to about 2.48:10.74, about 1.39:22.55 to about 2.78:7.52, about 1.39:22.55 to about 3.13:3.76, about 1.74: 18.79 to about 2.09: 15.04, about 1 .74: 18.79 to about 2.43: 11 .28, about 1.74: 18.79 to about 2.48:10.74, about 1.74:18.79 to about 2.78:7.52, about 1.74:18.79 to about 3.13:3.76, about 2.09:15.04 to about 2.43:11.28, about 2.09:15.04 to about 2.48:10.74, about 2.09:15.04 to about 2.78:7.52, about 2.09:15.04 to about 3.13:3.76, about 2.43:23, about
  • the composition of any one of the above aspects comprises 6g, 6s and C, wherein the 6g:6s:C weight ratio is about 1.39:0.15:22.55 to about 3.13:0.34:3.76.
  • the 6g:6s:C weight ratio is about 1.39:0.15:22.55, about 1.74:0.19:18.79, about 2.09:0.22:15.04, about 2.43:0.26:11.28, about 2.48:0.27:10.74, about 2.78:0.30:7.52 or about 3.13:0.34:3.76.
  • the 6g:6s:C weight ratio is about 2.48:0.27:10.74.
  • the composition of any one of the above aspects comprises 6g, 6s and C, wherein 6g:6s:C weight ratio is about 1.39:0.15:22.55 to about 1.74:0.19:18.79, about 1.39:0.15:22.55 to about 2.09:0.22:15.04, about 1.39:0.15:22.55 to about 2.43:0.26:11.28, about 1.39:0.15:22.55 to about 2.48:0.27:10.74, about 1.39:0.15:22.55 to about 2.78:0.30:7.52, about 1.39:0.15:22.55 to about 3.13:0.34:3.76, about 1.74:0.19:18.79 to about 2.09:0.22:15.04, about 1.74:0.19:18.79 to about 2.43:0.26:11.28, about 1.74:0.19:18.79 to about 2.48:0.27:10.74, about 1.74:0.19:18.79 to about 2.78:0.30:7.52, about 1.74:0.19:18.79
  • the composition of any one of the above aspects comprises 6g, 10 and C, wherein the 6g:10:C weight ratio is about 1.39:0.39:22.55 to about 3.13:0.88:3.76.
  • the 6g:10:C weight ratio is about 1.39:0.39:22.55, about 1.74:0.49:18.79, about 2.09:0.59:15.04, about 2.43:0.69:11.28, about 2.48:0.70:10.74, about 2.78:0.78:7.52 or about 3.13:0.88:3.76.
  • the 6g:10:C weight ratio is about 2.48:0.70:10.74.
  • the composition of any one of the above aspects comprises 6g:10:C, wherein the 6g:10:C weight ratio is about 1.39:0.39:22.55 to about 1.74:0.49:18.79, about 1.39:0.39:22.55 to about 2.09:0.59:15.04, about 1.39:0.39:22.55 to about 2.43:0.69:11.28, about 1.39:0.39:22.55 to about 2.48:0.70:10.74, about 1.39:0.39:22.55 to about 2.78:0.78:7.52, about 1.39:0.39:22.55 to about 3.13:0.88:3.76, about 1.74:0.49:18.79 to about 2.09:0.59:15.04, about 1.74:0.49:18.79 to about 2.43:0.69:11.28, about 1.74:0.49:18.79 to about 2.48:0.70:10.74, about 1.74:0.49:18.79 to about 2.78:0.78:7.52, about 1.74:0.49:18.79 to about 1.48:
  • the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 1.39:0.21 :0.39:22.55 to about 3.13:0.47:0.88:3.76.
  • the 6g:8g:10:C weight ratio is about 1.39:0.21 :0.39:22.55, about 1.74:0.26:0.49:18.79, about 2.09:0.31 :0.59:15.04, about 2.43:0.37:0.69:11.28, about 2.48:0.37:0.70:10.74, about 2.78:0.42:0.78:7.52 or about 3.13:0.47:0.88:3.76.
  • the 6g:8g:10:C weight ratio is about 2.48:0.37:0.70:10.74.
  • the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 1.39:0.21 :0.39:22.55 to about 1.74:0.26:0.49:18.79, about 1.39:0.21 :0.39:22.55 to about 2.09:0.31 :0.59:15.04, about 1.39:0.21 :0.39:22.55 to about 2.43:0.37:0.69: 11.28, about 1.39:0.21 :0.39:22.55 to about 2.48:0.37:0.70:10.74, about 1.39:0.21 :0.39:22.55 to about 2.78:0.42:0.78:7.52, about 1.39:0.21 :0.39:22.55 to about 3.13:0.47:0.88:3.76, about 1.74:0.26:0.49:18.79 to about
  • composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 1.39:0.39:22.55:0.44:4.68 to about 3.13:0.88:3.76:0.07:0.78.
  • the 6g: 10:C:B:D weight ratio is about 1.39:0.39:22.55:0.44:4.68, to about 1.74:0.49:18.79:0.37:3.90, to about 2.09:0.59:15.04:0.30:3.12, to about 2.43:0.69:11.28:0.22:2.34, to about 2.48:0.70:10.74:0.21 :2.23, to about 2.78:0.78:7.52:0.15:1 .56, to about 3.13:0.88:3.76:0.07:0.78.
  • the 6g:10:C:B:D weight ratio is about 2.48:0.70:10.74:0.21 :2.23.
  • the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 1.39:0.39:22.55:0.44:4.68 to about 1.74:0.49:18.79:0.37:3.90, about 1.39:0.39:22.55:0.44:4.68 to about 2.09:0.59:15.04:0.30:3.12, about 1.39:0.39:22.55:0.44:4.68 to about 2.43:0.69:11.28:0.22:2.34, about 1.39:0.39:22.55:0.44:4.68 to about 2.48:0.70:10.74:0.21 :2.23, about 1.39:0.39:22.55:0.44:4.68 to about 2.78:0.78:7.52:0.15:1.56, about 1.39:0.39:22.55:0.44:4.68 to about 3.13:0.88:3.76:0.07:0.78, about 1.74:0.49:18.79:0.37:3.90 to about
  • composition of any one of the above aspects comprises 6g, 8g, 10, C, B and D, wherein the 6g:8g:10:C:B:D weight ratio is about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 3.13:0.47:0.88:3.76:0.07:0.78.
  • the 6g:8g:10:C:B:D weight ratio is about 1.39:0.21 :0.39:22.55:0.44:4.68, about 1.74:0.26:0.49:18.79:0.37:3.90, about 2.09:0.31 :0.59:15.04:0.30:3.12, about 2.43:0.37:0.69:11.28:0.22:2.34, about 2.48:0.37:0.70:10.74:0.21 :2.23, about 2.78:0.42:0.78:7.52:0.15:1.56 or about 3.13:0.47:0.88:3.76:0.07:0.78.
  • the 6g:8g:10:C:B:D weight ratio is about 2.48:0.37:0.70:10.74:0.21 :2.23.
  • the composition of any one of the above aspects comprises 6g, 8g, 10, C and D, wherein the 6g:8g:10:C:B:D weight ratio is about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 1.74:0.26:0.49:18.79:0.37:3.90, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.09:0.31 :0.59:15.04:0.30:3.12, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.43:0.37:0.69:11.28:0.22:2.34, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.48:0.37:0.70:10.74:0.21 :2.23, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.78:0.42:0.78:7.52:0.15:1 .56, about 1.39:0.21 :0.39:
  • the composition of any one of the above aspects comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78.
  • the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68, about 1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90, about 2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12, about 2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34, about 2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23, about 2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56 or about
  • the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23.
  • composition comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90, about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12, about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34, about 1.39:0.21 :0.39:0.15:0.03:0.05:0.255:0.44:4.68
  • the composition of any one of the above aspects comprises a 6g:8g:10:6s:C:B:D weight ratio of about 2.48:0.37 :0.70:0.27:10.74:0.21 :2.23, about 8:0.93:3.31:0.49:10.74:0.21:2.23 or about 7.1:0.74:2.69:0.35:10.74:0.21:2.23.ln various embodiments, the composition of any one of the above aspects comprises 6s and C, wherein the 6s:C weight ratio is about 1 :151 to about 1:11. In various embodiments, the 6s:C weight ratio is about 1:151 , about 1:101 , about 1:67, about 1:43, about 1:40, about 1:25 or about 1:11. In various embodiments, the 6s:C weight ratio is about 1:40.
  • the composition of any one of the above aspects comprises 6s and C, wherein the 6s:C weight ratio is about 1:151 to about 1:101, about 1 :151 to about 1 :67, about 1:151 to about 1:43, about 1:151 to about 1:40, about 1 :151 to about 1:25, about 1:151 to about 1:11, about 1:101 to about 1 :67, about 1:101 to about 1 :43, about 1:101 to about 1 :40, about 1:101 to about 1 :25, about 1:101 to about 1 :11, about 1 :67 to about 1 :43, about 1 :67 to about 1 :40, about 1 :67 to about 1 :25, about 1 :67 to about 1 :11, about 1 :43 to about 1 :40, about 1 :43 to about 1 :25, about 1 :43 to about 1 :11, about 1 :43 to about 1 :40, about 1 :43 to about 1 :25, about 1 :43 to about 1 :11, about 1 :40
  • the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 5:1:723 to about 5:1:54.
  • the 6s: 10s:C weight ratio is about 5: 1 :723, about 5: 1 :482, about 5: 1 :321 , about 5:1:207, about 5:1:193, about 5:1 :120 or about 5:1:54.
  • the 6s:10s:C weight ratio is about 5:1 :193.
  • the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 5:1:723 to about 5:1:482, about 5:1:723 to about 5:1:321, about 5:1:723 to about 5:1:207, about 5:1 :723 to about 5:1 :193, about 5:1 :723 to about 5:1:120, about 5:1:723 to about 5:1:54, about 5:1 :482 to about 5:1 :321, about 5: 1 :482 to about 5: 1 :207, about 5: 1 :482 to about 5:1 :193, about 5: 1 :482 to about 5:1 :120, about 5:1 :482 to about 5:1:54, about 5:1 :321 to about 5:1:207, about 5:1 :321 to about 5:1:193, about 5:1:321 to about 5:1:207
  • the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 5:1:1:723 to about 5:1:1:54.
  • the 6s:8s: 10s:C weight ratio is about 5:1 :1 :723, about 5:1 :1 :482, about 5:1:1:321 , about 5:1 :1:207, about 5:1:1:193, about 5:1:1:120 or about 5:1 :1:54.
  • the 6s:8s:10s:C weight ratio is about 5:1 :1:193.
  • the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 5:1:1:723 to about 5:1:1:482, about 5:1:1:723 to about 5:1:1:321, about 5:1:1:723 to about 5:1:1:207, about 5:1:1:723 to about 5:1:1:193, about 5:1:1:723 to about 5:1:1:120, about 5:1:1:723 to about 5:1:1:54, about 5:1:1:482 to about 5:1:1:321, about 5:1:1:482 to about 5:1:1:207, about 5:1:1:482 to about 5:1:1:193, about 5:1:1:482 to about 5:1:1:120, about 5:1:1:482 to about 5:1:1:54, about 5:1:1:1
  • the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 5:1:723:14:150 to about 5:1:54:1:11.
  • the 6s:10s:C:B:D weight ratio is about 5:1:723:14:150, about 5:1:482:9:100, about 5:1:321:6:67, about 5:1:207:4:43, about 5:1:193:4:40, about 5:1:120:2:25 or about 5:1:54:1:11.
  • the 6s:10s:C:B:D weight ratio is about 5:1:193:4:40.
  • the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 5:1:723:14:150 to about 5:1:482:9:100, about 5:1:723:14:150 to about 5:1:321:6:67, about 5:1:723:14:150 to about 5:1:207:4:43, about 5:1:723:14:150 to about 5:1:193:4:40, about 5:1:723:14:150 to about 5: 1 : 120:2:25, about 5: 1 :723: 14: 150 to about 5: 1 :54: 1 : 11 , about 5: 1 :482:9: 100 to about 5:1:321:6:67, about 5:1:482:9:100 to about 5:1:207:4:43, about 5:1:482:9:100 to about 5:1:1:1:
  • composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 5:1:1:723:14:150 to about 5:1:1:54:1:11.
  • the 6s:8s:10s:C:B:D weight ratio is about 5:1:1 :723: 14: 150, about 5:1:1 :482:9: 100, about 5: 1 : 1 :321 :6:67, about 5:1:1 :207:4:43, about 5:1 :1:193:4:40, about 5:1:1 :120:2:25 or about 5:1:1:54:1:11.
  • the 6s:8s:10s:C:B:D weight ratio is about 5:1:1:193:4:40.
  • the composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 5:1:1:723:14:150 to about 5:1:1:482:9:100, about 5:1 :1:723:14:150 to about 5:1:1:321:6:67, about 5:1 :1:723:14:150 to about 5:1:1:207:4:43, about 5:1 :1:723:14:150 to about 5:1:1:193:4:40, about 5:1:1:723:14:150 to about 5:1:1 :120:2:25, about 5:1:1:723:14:150 to about
  • the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1 :16 to about 1:1. In various embodiments, the 6g:C weight ratio is about 1:16, about 1 :11, about 1:7, about 1 :5, about 1 :4, about 1:3 or about 1:1. In various embodiments, the 6g:C weight ratio is about 1 :4.
  • the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1 :16 to about 1:11 , about 1:16 to about 1:7, about 1:16 to about 1:5, about 1:16 to about 1:4, about 1:16 to about 1:3, about 1:16 to about 1:1, about 1:11 to about 1:7, about 1:11 to about 1:5, about 1:11 to about 1:4, about 1:11 to about 1:3, about 1 :11 to about 1:1, about 1 :7 to about 1:5, about 1:7 to about 1:4, about 1 :7 to about 1 :3, about 1 :7 to about 1:1, about 1 :5 to about 1 :4, about 1 :5 to about 1 :3, about 1 :5 to about 1:1, about 1 :4 to about 1 :3, about 1 :4 to about 1 : 1 or about 1 :3 to about 1:1.
  • the 6g:C weight ratio is about 1 :16 to about 1:11 , about 1:16 to about 1:7, about 1:16 to
  • the composition comprises 6g, 6s and C, wherein the 6g:6s:C weight ratio is about 9:1:151 to about 9:1:11. In various embodiments, the 6g:6s:C weight ratio is about 9:1:151, about 9:1 :101, about 9:1:67, about 9:1 :43, about 9:1:40, about 9:1:25 or about 9:1 :11. In various embodiments, the 6g:6s:C weight ratio is about 9:1:40.
  • the composition of any one of the above aspects comprises 6g, 6s and C, wherein the 6g:6s:C weight ratio is about 9:1:151 to about 9:1:101, about 9:1:151 to about 9:1:67, about 9:1:151 to about 9:1:43, about 9:1:151 to about 9:1:40, about 9:1:151 to about 9:1:25, about 9:1:151 to about 9:1:11, about 9:1:101 to about 9:1:67, about 9:1:101 to about 9:1:43, about 9:1:101 to about 9:1:40, about 9:1:101 to about 9:1:25, about 9:1:101 to about 9: 1 : 11 , about 9: 1 :67 to about 9: 1 :43, about 9: 1 :67 to about 9: 1 :40, about 9: 1 :67 to about 9: 1 :25, about 9: 1 :67 to about 9:1:11,
  • the composition of any one of the above aspects comprises 6g, 10 and C, wherein the 6g:10:C weight ratio is about 4:1:57 to about 4:1:4.
  • the 6g: 10:C weight ratio is about 4: 1 :57, about 4: 1 :38, about 4: 1 :26, about 4:1:16, about 4:1:15, about 4:1:10 or about 4:1:4.
  • the 6g:10:C weight ratio is about 4:1:15.
  • the composition of any one of the above aspects comprises 6g, 10 and C, wherein the 6g:10:C weight ratio is about 4:1:57 to about 4:1:38, about 4:1:57 to about 4:1:26, about 4:1:57 to about 4:1:16, about 4:1:57 to about 4:1:15, about 4:1:57 to about 4: 1 : 10, about 4: 1 :57 to about 4: 1 :4, about 4: 1 :38 to about 4: 1 :26, about 4: 1 :38 to about 4:1:16, about 4:1:38 to about 4:1:15, about 4:1:38 to about 4:1:10, about 4:1:38 to about 4:1:4, about 4: 1 :26 to about 4:1:16, about 4: 1 :26 to about 4:1:15, about 4: 1 :26 to about 4:1:10, about 4: 1 :26 to about 4:1:4, about 4: 1 : 16 to about 4:1:15, about 4: 1 : 16 to about 4:1:10, about 4:1:16 to about 4:1:4, about 4:1:15 to about 4:1:10,
  • the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 7:1:2:108 to about 7:1:2:8.
  • the 6g:8g:10:C weight ratio is about 7:1:2:108, about 7:1:2:72, about 7:1:2:48, about 7:1:2:31, about 7:1:2:29, about 7:1:2:18 or about 7: 1:2:8.
  • the 6g:8g:10:C weight ratio is about 7:1:2:29.
  • the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 7:1:2:108 to about 7:1:2:72, about 7:1:2:108 to about 7:1:2:48, about 7:1:2:108 to about 7:1:2:31, about 7:1:2:108 to about 7:1:2:29, about 7:1:2:108 to about 7:1:2:18, about 7:1:2:108 to about 7: 1 :2:8, about 7:1:2:72 to about 7:1 :2:48, about 7:1 :2:72 to about 7: 1 :2:31 , about 7: 1 :2:72 to about 7:1 :2:29, about 7:1:2:72 to about 7:1:2:18, about 7:1:2:72 to about 7: 1 :2:8, about 7:1:2:48 to about 7:1:2
  • the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 45:13:723:14:150 to about 45:13:54:1 :11.
  • the 6g:10:C:B:D weight ratio is about 45:13:723:14:150, about 45:13:482:9:100, about 45:13:321 :6:67, about 45:13:207:4:43, about 45:13:193:4:40, about 45:13:120:2:25 or about 45:13:54:1 :11.
  • the 6g:10:C:B:D weight ratio is about 45:13:193:4:40.
  • the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 45:13:723:14:150 to about 45:13:482:9:100, about 45:13:723:14:150 to about 45:13:321 :6:67, about 45:13:723:14:150 to about 45:13:207:4:43, about 45:13:723:14:150 to about 45:13:193:4:40, about 45: 13:723: 14: 150 to about 45: 13: 120:2:25, about 45: 13:723: 14: 150 to about 45: 13:54: 1 : 11 , about 45:13:482:9:100 to about 45:13:321 :6:67, about 45:13:482:9:100 to about 45:13:207:4:43, about 45:13:482:9:100 to about 45:13:193:4:
  • composition of any one of the above aspects comprises 6g, 8g, 10, C, B and D, wherein the 6g:8g:10:C:B:D weight ratio is about 45:7:13:723:14:150 to about 45:7:13:54:1 :11.
  • the 6g:8g:10:C:B:D weight ratio is about 45:7:13:723:14:150, about 45:7:13:482:9:100, about 45:7:13:321 :6:67, about 45:7: 13:207:4:43, about 45:7: 13: 193:4:40, about 45:7: 13: 120:2:25 or about 45:7: 13:54: 1 : 11 .
  • the 6g:8g:10:C:B:D weight ratio is about 45:7:13:193:4:40.
  • the composition of any one of the above aspects comprises 6g, 8g, 10, C, B and D, wherein the 6g:8g:10:C:B:D weight ratio is about 45:7:13:723:14:150 to about 45:7:13:482:9:100, about 45:7:13:723:14:150 to about 45:7:13:321 :6:67, about 45:7:13:723:14:150 to about 45:7:13:207:4:43, about 45:7:13:723:14:150 to about 45:7:13:193:4:40, about 45:7:13:723:14:150 to about 45:7:13:120:2:25, about 45:7: 13:723: 14: 150 to about 45:7: 13:54: 1 : 11 , about 45:7: 13:482:9: 100 to about 45:7: 13:321 :6:67, about 45:7: 13:482:9
  • the composition of any one of the above aspects comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :723:14:150 to about 45:7:13:5:1 :1 :54:1 :11.
  • the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :723:14:150, about 45:7:13:5:1 :1 :482:9:100, about 45:7:13:5:1 :1 :321 :6:67, about 45:7:13:5:1 :1 :207:4:43, about 45:7:13:5:1 :1 :193:4:40, about 45:7:13:5:1 :1 :120:2:25 or about 45:7:13:5:1 :1 :54:1 :11.
  • the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :193:4:40.
  • the composition of any one of the above aspects comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :723:14:150 to about 45:7:13:5:1 :1 :482:9:100, about 45:7:13:5:1 :1 :723:14:150 to about 45:7: 13:5: 1 : 1 :321 :6:67, about 45:7: 13:5: 1 : 1 :723: 14: 150 to about 45:7:13:5:1 :1 :207:4:43, about 45:7:13:5:1 :1 :723:14:150 to about 45:7:13:5:1 :1 :193:4:40, about 45:7: 13:5: 1 : 1 : 1 :
  • composition of any one of the above aspects comprises, consists essentially of, or consists of ginger and turmeric.
  • composition of any one of the above aspects comprises ginger from the rhizome of Zingiber officinale Roscoe.
  • the ginger is fresh or dried.
  • composition of any one of the above aspects comprises a ginger extract derived from the rhizome of Zingiber officinale Roscoe.
  • the ginger extract is derived from dried or fresh ginger.
  • the ginger extract is an extract of dried ginger and has a drug extract ratio of about 7:1 (7 parts dry ginger extracted to yield 1 part final extract), about 16:1 or about 12:1.
  • the ginger extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting ginger with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication, liquid carbon dioxide and/or other organic solvents.
  • the ginger extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting ginger with 90 % ethanol, 100% ethanol or DCM.
  • the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
  • DCM dichloromethane
  • the composition of any one of the above aspects comprises an artificial ginger composition comprising isolated, pure and/or synthetic 6g, 8g, 10g, 6s, 8s and/or 10s.
  • the 6g, 8g, 10g, 6s, 8s and/or 10s are synthetic, the 6g, 8g, 10g, 6s, 8s and/or 10s are isolated or purified from ginger, or the 6g, 8g, 10g, 6s, 8s and/or 10s are a combination of purified, isolated and/or synthetic.
  • the composition of any one of the above aspects comprises turmeric from the rhizomes Curcuma longa L. In various embodiments, the turmeric fresh or dried. In various embodiments, the composition of any one of the above aspects comprises a turmeric extract derived from the rhizome of Curcuma longa L. In various embodiments, the turmeric extract is derived from fresh or dried turmeric. In various embodiments, the turmeric extract is an extract of dried turmeric and has a drug extract ratio (DER) of about 25:1 (25 parts dry turmeric extracted to yield 1 part final extract).
  • DER drug extract ratio
  • the turmeric extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting turmeric with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol or warm water by sonication, liquid carbon dioxide, other organic solvents and/or liquid carbon dioxide.
  • the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1
  • the composition comprises a turmeric extract with a drug extract ratio of 25:1 and the ginger extract has a drug extract ratio of about 7:1, about 16:1 or about 12:1.
  • the composition of any one of the above aspects comprises pure, isolated and/or synthetic curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B).
  • the C, D and/or B are synthetic, the C, D and/or B are isolated and/or purified from turmeric, or the C, D and/or B are a combination of purified, isolated and/or synthetic.
  • the composition comprises C, D and B; C and D; C and B; or C.
  • the composition of any one of the above aspects comprises ginger and turmeric, wherein the turmeric is a turmeric extract and the ginger is a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract has a drug to extract ratio (DER) of about 25:1 and wherein the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1.
  • DER drug to extract ratio
  • the composition of any one of the above aspects comprises ginger and turmeric, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
  • the present invention provides a pharmaceutical composition comprising the composition of any one of the above aspects.
  • the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.
  • the present invention provides a method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition and/or pharmaceutical composition of any one of the above aspects.
  • the present invention provides a method of treating a disease or condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition and/or pharmaceutical composition of any one of the above aspects.
  • the present invention provides a method of conferring one or more health benefits to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition and/or pharmaceutical composition of any one of the above aspects.
  • the present invention provides a use of a composition and/or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for reducing inflammation.
  • the present invention provides a use of a composition and/or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a disease or condition associated with inflammation.
  • the present invention provides a use of a composition and/or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for conferring one or more health benefits.
  • Figure 1 shows an isobologram of G-T combinations (1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 5:2, 8:2, 9:1 , w/w) at IC50 on NO (A), IL-6 (B) and TNF (C) assays (n ⁇ 3).
  • Figure 2 shows Cl-Fa curves of G-T 5:2 on NO (A), IL-6 (B) and TNF (C) assays (n ⁇ 3) in RAW264.7 cells.
  • B Quantification of iNOS positive and iNOS negative counting from the immunofluorescent staining. #p ⁇ 0.05 of iNOS positive compared with blank. *p ⁇ 0.05 of iNOS positive compared with LPS. ***p ⁇ 0.001 of iNOS positive compared with LPS.
  • Figure 4 shows isobolograms (A) of G-T combinations (1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 5:2, 8:2, 9:1 , w/w) at IC50 and Cl-Fa curve (B) of G-T 5:2 on TNF (A) assay in THP-1 cells (n ⁇ 3).
  • Figure 5 shows the activation of Nrf2/H0-1 pathway by G-T 5:2.
  • the maximum activation of Nrf2 by G-T 5:2 was significantly higher than that of G (p ⁇ 0.05) and higher than T at 25 pg/mL.
  • B The Cl- Fa curve of G-T 5:2 on Nrf2 activation showed that there was strong synergy at all concentration levels of G-T 5:2 in activating Nrf2.
  • Figure 6 shows the effect of 10 pg/mL of G, T, GT ( Figures 6A and 6C), and 5 pg/mL of S, C or SC ( Figures 6B and 6D) on the TLR4-TRAF6-MAPK pathway in LPS- stimulated RAW264.7 cells.
  • Cells were pre-treated with G, T, GT, S, C, SC 1 h prior to LPS (100 ng/mL) for various optimal time points.
  • Protein expression levels of phosphorylated p38 (p-p38), JNK (p-JNK), CJUN (p-CJUN), and TLR4 and TRAF-6 were analysed by western blot using whole protein extract.
  • B- actin was used as internal control.
  • B Quantification of % of nuclei positive p65 of staining in unstimulated, LPS stimulated macrophages with and without various treatments. Bar represents 10 pm. Data points represent mean ⁇ standard deviation from analysis of 9 separate images. #### p ⁇ 0.0001 LPS vs.
  • FIG. 10 shows dose-response curves of T/artificial turmeric (AT: mixed curcuminoids equivalent to their content in T) on NO assay (A) and cell viability (B), G-T/G- AT 5:2 on NO assay (C) and G-AT 5:2 (D) on NO production and cell viability.
  • AT mixed curcuminoids equivalent to their content in T
  • B G-T/G- AT 5:2 on NO assay
  • D G-AT 5:2
  • Figure 11 shows S, C and SC inhibited LPS activated iNOS expression in the RAW264.7 cells by immunofluorescent staining.
  • B Quantification of iNOS positive and negative counting. #p ⁇ 0.05 LPS vs. blank. ****p ⁇ 0.0001 of iNOS positive compared with LPS.
  • Figure 14 shows human TH P-1 cells (differentiated by 50 nM PM A) were treated media
  • B Data points represent mean ⁇ standard deviation from analysis of 3 separate images. *** p ⁇ 0.001 compared with LPS. # p ⁇ 0.01 LPS vs. blank.
  • Figure 18 shows a simplified diagram of G-T 5:2 actions on the neuroinflammation triculture system.
  • Figure 19 shows effect of 10 pg/mL of G, T, GT, and 5 pg/mL of S, C or SC combinations on the mmu-microRNA(miR)-155-5p expression in LPS and IFN-y-stimulated RAW264.7ells.
  • Cells were cultured in T25 cell flasks and were pre-treated with G, T, GT, S, C, SC 1 h prior to LPS (1 pg/mL) for 24h. The samples were then subjected to real-time PCR after RNA collection and cDNA synthesis.
  • the mmu-miR-155-5p fold changes compared to untreated cells (B) were expressed as the mean ⁇ SEM, # p ⁇ 0.05 vs. blank control (B); *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 vs. LPS-stimulated cells.
  • Figure 20 shows dose-response curves of NO production (% relative to control) in concentrations (0.3125 -10 pg /ml) of ginger solvent extracts in RAW264.7 cells tested by Griess assay.
  • Figure 21 shows individual and dual response curves of G with T in inhibiting NO.
  • A. Dose-response curves of A. G(DCM), T and G(DCM)-T.
  • B. G(ETOH), T and G(ETOH)-T.
  • Figure 22 shows the inhibitory effects of G(DCM)-T and G(ETOH)-T (5:2) on NO production (%) versus concentration (pg/ml) as shown in A and D, respectively.
  • C and F Isobologram curves for G(DCM)-T and G(ETOH)-T (5:2) on NO.
  • B and E Cl values verses NO inhibition effect (Fa) by ‘Calcusync’ software.
  • Dotted line is the reference line, where Cl value equals 1 ; solid line represents Cl values at different Fa
  • weight ratio refers to w/w, such that a weight ratio of 2:1 could equivalently be expressed as 2:1 (w/w).
  • a “subject” may be human or a non-human animal.
  • a "subject" to which administration is contemplated includes, but is not limited to, humans (i.e. , a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals, including primates, commercially relevant mammals (such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys).
  • the animal is a mammal.
  • the animal may be a male or female at any stage of development.
  • the animal may be a transgenic animal or genetically engineered animal.
  • the subject is non-human animal.
  • a "patient” as used herein will typically refer to a human subject in need of treatment of a disease.
  • condition As used herein, the terms “condition”, “disease,” and “disorder” are used interchangeably.
  • treatment As used herein, the terms “treatment,” “treat,” and “treating” or variations thereof, is to be considered in its broadest context. It may refer to reducing, ameliorating, reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or condition described herein. As used herein, a “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or the delay, prevention, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured.
  • a composition useful in treatment as contemplated herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, provide improvement to a patient or subject's quality of life, or delay, prevent, or inhibit the onset of a disease, disorder, or condition.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • preparation is intended to include the formulation of the one or more active ingredients with encapsulating materials as a carrier which may provide a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it.
  • a carrier which may provide a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it.
  • sachets are included. Tablets, powders, sachets, and capsules can be used as solid dosage forms suitable for oral administration. If desired for reasons of convenience or patient acceptance, pharmaceutical tablets may be provided in chewable form, using techniques well known in the art.
  • administer refers to any method which, in sound medical practice, delivers the composition to a subject in such a manner as to provide a therapeutic effect, and may include implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a composition as herein described in or on a subject.
  • terapéuticaally effective with reference to an amount or dosage of a composition or medicament refers to an amount of a composition that is sufficient to effectively prevent, ameliorate or eliminate inflammation in a subject.
  • the term should not be seen as limiting. It may refer to an amount of a dosage of a composition or medicament that optimises the antiinflammatory effects on a subject depending on desired application.
  • an "effective amount” or a "therapeutically effective amount” of a composition, or of an active agent or ingredient or pharmaceutically active agent or ingredient refer to an amount of the composition or pharmaceutically active agent sufficient to have a therapeutic effect upon administration.
  • a therapeutically effective amount of the composition or pharmaceutically active agent may, will, or is expected to elicit a response, such as an anti-inflammatory response, and/or provide a health benefit or therapeutic effect, and/or cause a relief of symptoms, and/or effect a treatment.
  • Effective amounts of the composition or pharmaceutically active agent will vary with the particular condition or conditions being treated or the health benefit to be achieved, the severity of the condition, the duration of the treatment, the specific components of the composition being used, and like factors.
  • enhancing the biological activity, function, health, or condition of a cell or an organism refers to the process of augmenting, fortifying, strengthening, or improving.
  • an “artificial extract” in the context of the invention refers to a composition which comprises one or more of a purified, isolated or synthetic bioactive compound of a natural plant (e.g. ginger or turmeric).
  • dry weight or equivalent refers to the quantity of extract extracted from a given quantity of dry weight raw material (dry herb).
  • dry weight or equivalent refers to the quantity of extract extracted from a given quantity of dry weight raw material (dry herb).
  • the bioactive compounds are 6-gingerol, 8-gingerol, 10-gingerol, 6shogaol, 8-shogaol, 10- shogaol, curcumin, desmethoxycurcumin and/or bisdemethoxycurcumin.
  • the phrase “drug extract ratio” or “DER” refers a ratio of dry raw material to final extract quantity. Where the final extract is a dry extract, the DER refers to a w/w ratio. Where the final extract is a liquid extract, the DER will generally be understood to refer to a w/v ratio.
  • the present invention relates to anti-inflammatory compositions comprising turmeric and ginger, and the use of such compositions to provide one or more health benefits and/or one or more anti-inflammatory responses in an animal subject.
  • compositions comprising turmeric and ginger, wherein the composition elicits a synergistic anti-inflammatory effect.
  • a composition comprising at least one turmeric extract and at least one ginger extract, wherein the composition elicits a synergistic antiinflammatory effect.
  • Ginger and turmeric comprise a variety of bioactive compounds, including gingerols shogaols and curcuminoids.
  • ginger and turmeric compounds 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s), 10-shogaol (10s); and one or more of curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B) , particularly 10s, 6s and C, and more particularly 6s and C, are considered by the inventors, without wishing to be bound by theory, to be at least part responsible for some of the synergistic anti-inflammatory activity associated with certain combinations of turmeric and ginger, or extracts thereof.
  • the ginger in the composition described herein is fresh or dried ginger.
  • the ginger is a ginger extract, such as an extract derived from ginger or an artificial ginger extract.
  • the ginger extract is derived from dried or fresh ginger.
  • Ginger comprises bioactive compounds such as shogaols (6-, 8- and 10-shogaol) and gingerols (6-, 8- and 10-gingerol).
  • the ginger or ginger extract in the composition described herein comprises 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s) and/or 10-shogaol (10s).
  • the ginger or ginger extract in the composition described herein comprises: at least two compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least three compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least four compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least five compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; or 6g, 8g, 10g, 6s, 8s and 10s.
  • Ginger and ginger extracts can be prepared using techniques well known in the art.
  • the ginger or ginger extract is derived from the whole or any part of the rhizome of Zingiber officinale Roscoe.
  • Zingiber officinale Roscoe in the composition described herein, or for the preparation of extracts in the composition described herein, may be gathered from the wild and/or cultivated.
  • the ginger in the composition described herein is raw ginger or an extract derived from ginger.
  • the ginger extract is a ginger extract.
  • Suitable ginger, ginger extracts or raw materials suitable for making such extracts for use in the composition described herein are also commercially available. For example, from commercial suppliers of medicinal plants and spices, and/or extracts thereof. For example, ginger tincture, powder, tablets, liquid drops and capsules. Those skilled in the art would appreciate that other suppliers of suitable raw materials or extracts can be selected using the disclosure and examples presented herein.
  • extracts from Zingiber officinale Roscoe for use in the composition described herein are well known in the art, including solvent extraction methods.
  • solvent extraction comprising extracting ginger with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication, and/or extraction comprising other organic solvents.
  • solvent extraction comprising extracting ginger with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication, and/or extraction comprising other organic solvents.
  • DCM dichloromethane
  • EA ethyl acetate
  • alcohol aqueous alcohol
  • ethanol ethanol
  • methanol warm water by sonication
  • extraction comprising other organic solvents.
  • supercritical fluid extraction using liquid carbon dioxide as the solvent.
  • the extraction may
  • the ginger extract may be obtained by a solvent extraction method comprising extracting ginger with 90 % ethanol, 100% ethanol or DCM.
  • the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1 , or is extracted with 100% ethanol and has a DER of about 12:1.
  • ginger extracts include (but are not limited to): ginger extracts with a drug to extract ratio (DER) of 1:2 (Liquid) obtained by extraction with90% aqueous ethanol, DER of 1:2 (Liquid) obtained by extraction with 60-70% aqueous ethanol or a DER of 30:1 (Liquid Cone.) obtained by extraction with 100% carbon dioxide.
  • the ginger extract has a drug to extract ratio (DER) of 1:2 (Liquid) obtained by extraction with 90% aqueous ethanol, a DER of 1:2 (Liquid) obtained by extraction with 60- 70% aqueous ethanol or a DER of 30:1 (Liquid Cone.) obtained by extraction with 100% carbon dioxide
  • the ginger extract is an artificial ginger extract.
  • the artificial ginger extract is a composition comprising one or more bioactive compounds of ginger.
  • the artificial ginger extract is a composition comprising one or more purified, isolated and/or synthetic bioactive compounds 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol and 10-shogaol.
  • the artificial ginger extract comprises isolated, pure and/or synthetic 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s) and/or 10- shogaol (10s).
  • the artificial ginger extract comprises: at least two compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least three compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least four compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least five compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; or 6g, 8g, 10g, 6s, 8s and 10s.
  • the bioactive compounds 6-gingerol, 8-gingerol, 10-gingerol, 6- shogaol, 8-shogaol and/or 10-shogaol in the artificial ginger extract are isolated or purified from ginger, using techniques well known in the art.
  • bioactive compounds 6-gingerol, 8-gingerol, 10-gingerol, 6- shogaol, 8-shogaol and/or 10-shogaol are synthetic compounds manufactured using techniques well known in the art.
  • composition described herein comprises an artificial ginger extract with a combination of purified, isolated and/or synthetic 6g, 8g, 10g, 6s, 8s and/or 10s.
  • Curcuminoids are the major bioactive components in turmeric and include curcumin, desmethoxycurcumin and bisdemethoxycurcumin. Of these three, the most prevalent in turmeric is curcumin.
  • the turmeric or turmeric extract in the composition described herein comprises one or more of curcumin (C), desmethoxycurcumin (D) and bisdemethoxycurcumin (B).
  • the turmeric comprises C, D and B; C and D; C and B; or C.
  • the turmeric in the composition described herein is from the rhizomes of Curcuma longa.
  • the turmeric is fresh or dried turmeric.
  • the turmeric extract is an extract of dried turmeric.
  • the turmeric extract is a semi-refined turmeric extract from the rhizomes of Curcuma longa.
  • Turmeric and turmeric extracts in the compositions described herein can be prepared using techniques well known in the art.
  • Curcuma longa L. in the composition described herein, or for the preparation of turmeric extracts in the composition described herein, may be gathered from the wild and/or cultivated.
  • Suitable turmeric, turmeric extracts or raw materials suitable for making such extracts for use in the composition described herein are commercially available, for example from Integria Healthcare (Australia) Pty Ltd (Eight Mile Plains, QLD, Australia) or from other commercial suppliers of medicinal plants and spices, and/or extracts thereof. Those skilled in the art would appreciate that other suppliers of suitable raw materials or extracts can be selected using the disclosure and examples presented herein.
  • turmeric extracts from Curcuma longa L. for use in the composition described herein are well known in the art, including for example solvent extraction and sonication.
  • the turmeric extract is a semi-refined turmeric extract.
  • the turmeric extract is obtained by extraction with ethyl acetate (EA), 100% hexane, dichloromethane (DCM), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication and/or extraction comprising other organic solvents.
  • EA ethyl acetate
  • DCM dichloromethane
  • alcohol aqueous alcohol
  • ethanol ethanol
  • methanol warm water by sonication and/or extraction comprising other organic solvents.
  • Another example includes extracting turmeric with liquid carbon dioxide.
  • the turmeric extract may be an extract of dried turmeric with a DER of 25:1 (25 parts dry turmeric extracted to yield 1 part final extract).
  • the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1.
  • suitable turmeric extracts include (but are not limited to) turmeric extracts with a drug extract ratio (DER) of 1:1 (Liquid) obtained by extraction with69% aqueous ethanol, a DER of 25:1 (Dry Cone.) obtained by extraction with99% ethyl acetate or a DER of 55:1 (Dry Cone.) obtained by extraction with93% aqueous ethanol.
  • DER drug extract ratio
  • the turmeric in the composition described herein is an extract selected from a turmeric extract with a drug extract ratio (DER) of 1 :1 (Liquid) obtained by extraction with 69% aqueous ethanol, a DER of 25:1 (Dry Cone.) obtained by extraction with 99% ethyl acetate or a DER of 55:1 (Dry Cone.) obtained by extraction with 93% aqueous ethanol.
  • DER drug extract ratio
  • the turmeric extract is an artificial turmeric extract.
  • the artificial turmeric extract is a composition comprising one or more of the bioactive compounds of turmeric.
  • the artificial turmeric extract is a composition comprising one or more of the bioactive compounds curcumin (C), desmethoxycurcumin (D) and bisdemethoxycurcumin (B).
  • the artificial turmeric composition comprises, consists essentially of or consists of C, D and B; C and D; C and B; or C.
  • the C, D and/or B are synthetic.
  • the C, D and/or B are isolated and/or purified from turmeric.
  • the composition of the invention comprises an artificial turmeric extract with a combination of purified, isolated and/or synthetic C, D and/or B.
  • the bioactive compounds C, D and/or B in the artificial turmeric composition are isolated from turmeric by methods that are well known to a person skilled in the art.
  • bioactive compounds C, D and/or B are synthetic.
  • the composition described herein comprises, consists essentially of, or consists of the bioactive ingredients: a) ginger, an extract derived from ginger and/or an artificial ginger extract; and b) turmeric, an extract derived from turmeric and/or an artificial turmeric extract.
  • the composition described herein comprises, consists essentially of, or consists of the bioactive compounds: a) 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol and/or 10-shogaol; and b) curcumin, desmethoxycurcumin and/or bisdemethoxycurcumin.
  • synergistic effect such as a synergistic effect mediated by a composition described herein
  • a synergistic effect is an effect that is of greater magnitude than the sum of the effects elicited by the individual active constituents alone.
  • synergism such as that embodied in a synergistic response, can be assessed and determined in a number of different ways, including by methods described and exemplified herein, such as isobologram and combination index (Cl) models based on the Loewe Additivity principle well known in the art and described and exemplified herein.
  • a synergistic effect can be a lowered effective dosage compared to the effective dosage of the individual active constituents alone, a reduction in cytotoxicity compared to the cytotoxicity of the individual constitutions alone, or an enhanced anti-inflammatory activity compared to the antiinflammatory activity of the individual constitutions alone.
  • the synergistic effect mediated by the composition described herein is an enhanced anti-inflammatory activity.
  • the composition described herein is an anti-inflammatory composition.
  • the anti-inflammatory effect is activation of the Nrf2/H0-1 antiinflammatory pathway, or inhibition of the MAPK/JNK, TLR4-TRAF6-MAPK and/or IKK/NFKB inflammatory pathways.
  • the anti-inflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators. For example, pro- and/or secondary-inflammatory mediators.
  • the antiinflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more of IL-6, nitric oxide (NO), TNF, I L-1 a, I L-1 b, eotaxin, interferon-y (IFN-y), IL- 12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1, MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155.
  • the antiinflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more of the above inflammatory mediators by one or more immune cells, including one or more mammalian immune cells.
  • immune cells including one or more mammalian immune cells.
  • pro- and/or secondary-inflammatory mediators for example, pro- and/or secondary-inflammatory mediators.
  • the anti-inflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more of IL-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL- 13, G-CSF, GM-CSF, KC, MCP-1, MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL- 10, 1 L-17 and/or miRNA-155-5p by one or more immune cells, including one or more mammalian immune cells.
  • the one or more immune cells are macrophages or monocytes.
  • the anti-inflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more proinflammatory chemokines.
  • the chemokines are associated with the acute inflammatory phase.
  • the one or more chemokines are selected from CXCL5, IL-8 (CXCL8), CXCL10, CXCL11 , MIG, and/or MIP-3a.
  • composition described herein elicits a synergistic activation of the Nrf2/H0-1 anti-inflammatory pathway, or inhibition of the MAPK/JNK, TLR4- TRAF6-MAPK and/or IKK/NFKB inflammatory pathways.
  • composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators.
  • composition described herein elicits a synergistic decrease in pro- and/or secondary-inflammatory mediators.
  • composition described herein elicits a synergistic decrease in any one or more of IL-6, NO, TNF, IL-1 a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1, MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p.
  • the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators by one or more immune cells, including one or more mammalian immune cells.
  • the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of pro- and/or secondary-inflammatory mediators by one or more immune cells, including one or more mammalian immune cells.
  • the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of IL-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p by one or more immune cells, including one or more mammalian immune cells.
  • the one or more immune cells are macrophages or monocytes
  • the synergistic effect such as a synergistic effect mediated by a composition described herein, is an enhanced anti-neuroinflammatory activity.
  • the invention provides a method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition as described herein.
  • the invention provides a method of treating a disease or condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition as described herein.
  • the inflammation, or the disease or condition associated with inflammation is associated with an increase in expression of IL-6, NO, TNF, IL-1 a, I L-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p.
  • the inflammation or the disease or condition associated with inflammation is associated with activation of MAPK/JNK, TLR4-TRAF6-MAPK and/or I KK/NFKB inflammatory pathways.
  • the inflammation, or disease or condition associated with inflammation is associated with an increase in the expression and/or elevated expression of one or more proinflammatory chemokines.
  • the chemokines are associated with the acute inflammatory phase.
  • the one or more chemokines are selected from CXCL5, IL-8 (CXCL8), CXCL10, CXCL11 , MIG, and/or MIP-3a.
  • the inflammation, or the disease or condition associated with inflammation is associated with a cytokine storm.
  • the cytokine storm is an LPS and IFN-y induced cytokine storm.
  • the cytokine storm is induced by a viral infection.
  • the inflammation is chronic inflammation.
  • the chronic inflammation is neuroinflammation.
  • the disease or condition associated with inflammation is associated with chronic inflammation.
  • the disease or condition associated with chronic inflammation is selected from the group consisting of: cardiovascular disease, cancer, diabetes, metabolic syndrome, autoimmune diseases, depression, declining cognitive function, Alzheimer’s and other dementias, and age-related macular degeneration.
  • the disease or condition associated with inflammation is associated with neuroinflammation.
  • the disease or condition associated with inflammation is selected from the group consisting of: atherosclerosis, pain, depression, hyperlipidemia, atherogenesis, blood clots, hypertension, asthma, wounds, aging, hypoglycaemia, arthritis, cytokine storm, sepsis, ulcers.
  • the inflammation is reduced by, or the disease or condition associated with inflammation treated by, a synergistic activation of the Nrf2/H0-1 antiinflammatory pathway, or inhibition of the MAPK/JNK, TLR4-TRAF6-MAPK and/or I KK/NFKB inflammatory pathways; a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators; a synergistic decrease in pro- and/or secondary-inflammatory mediator; a synergistic decrease in any one or more of interleukin (IL)-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17 and/
  • IL
  • the invention provides a method of conferring one or more health benefits to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition as described herein.
  • the one or more health benefits is selected from the group comprising: supporting healthy immune function, improving acute inflammation or pain, improving chronic inflammation or decreasing susceptibility to diseases linked to chronic inflammation.
  • the one or more health benefits is selected from the group comprising: a decrease in inflammatory mediators, one or more anti-atherosclerotic effects, one or more immunological benefits, one or more analgesic effects, one or more antidementia effects, one or more anti-depressant effects, one or more cognitive benefits, one or more chemopreventive effects, one or more anti-cancer effects, one or more hypolipidemic effects, one or more anti-atherogenic effects, one or more anti-platelet and/or anti-thrombotic effects, one or more anti-diabetic effects, one or more anti-hypertensive effects, one or more anti-asthma effects, one or more wound-healing effects, one or more anti-aging effects, one or more hypoglycemic effects, one or more anti-arthritis effects, one or more anti-cytokine storm effects, one or more anti-sepsis effects and one or more anti-ulcer effects, wherein one or more of the above potential therapeutic
  • the subject is a mammal.
  • the mammal may be of either sex and may be at any stage of development.
  • the subject is a human.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal such as a dog or cat.
  • the subject is a livestock animal such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate.
  • the animal is a genetically engineered animal.
  • the animal is a transgenic animal.
  • composition as described in any one or more of the embodiments described herein is a pharmaceutical composition.
  • the composition further comprises one or more pharmaceutically acceptable carriers.
  • the specific dose level selected for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the condition undergoing therapy.
  • Dosage levels of the active ingredients in the compositions may be varied so as to provide an amount of the active ingredient which is effective to achieve the desired therapeutic effect for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the composition is administered to deliver about 50 to 50,000 mg per day to an adult human subject. In another example, the composition is administered to deliver about 500 to 2000 mg per day of turmeric or about 500 to 2000 mg per day of curcumin to an adult human subject, and/or about 500 to 3000 mg per day of ginger to an adult human subject.
  • the composition is formulated for topical application.
  • the topical composition is formulated as a cream, ointment, liniment, spray or plaster.
  • the composition is formulated for oral administration.
  • the oral composition is a liquid, tablet, capsule, powder or wafer.
  • compositions described herein comprise one or more pharmaceutically acceptable carriers, excipients, or diluents, as required.
  • the composition is a liquid composition.
  • Liquid compositions include solutions, suspensions, and emulsions.
  • the liquid composition comprises a solvent, for example an organic solvent or aqueous organic solvent.
  • the liquid composition comprises a solvent, for example ethanol, methanol or ethyl acetate.
  • Solutions can be prepared by combining the active ingredient(s) with a suitable solvent, such as an organic solvent or aqueous organic solvent and optionally adding suitable colorants, flavors, stabilizers and thickening agents as desired.
  • a suitable solvent such as an organic solvent or aqueous organic solvent
  • the composition is a solid preparation or dosage form, such as a powder, tablet, dispersible granule, capsule, sachet, or suppository.
  • a carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents. It can also be an encapsulating material.
  • the carrier In powders, the carrier will typically be a finely divided solid which is in admixture with the active ingredients. In tablets, the actives are mixed with a carrier having the necessary binding properties in suitable proportions and compacted to the shape and size desired.
  • Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, silicas including colloidal silicas, glycollates, low melting point wax, cocoa butter, and the like.
  • the composition described herein is formulated to increase the oral bioavailability of the 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, curcumin, desmethoxycurcumin or bisdemethoxycurcumin, or combinations thereof, present in the composition.
  • Methods of increasing bioavailability are well known to a person skilled in the art (see for example Stohs SJ et al., Molecules. 2020;25(6):1397).
  • compositions described herein comprise a carrier selected from lipids (such as liposomes, phytosomes), cyclodextrin, whey protein, soy lecithin or soluble fibre (such as galactomannans from Fenugreek, also known as Trigonella foenum-graecum), micro-particle technology and nano-particle technology.
  • lipids such as liposomes, phytosomes
  • cyclodextrin such as whey protein, soy lecithin or soluble fibre (such as galactomannans from Fenugreek, also known as Trigonella foenum-graecum)
  • micro-particle technology such as galactomannans from Fenugreek, also known as Trigonella foenum-graecum
  • the compositions described herein are formulated for administration by a delivery system selected from micelles, liposomes, phospholipid complexes, microemulsions, nanoemulsions, emulsions, solid lipid nano
  • Solid dosage forms are in certain embodiments formulated in single or divided dosage forms (such as multiple tablets). Those skilled in the art will appreciate that multiple tablets may be required to deliver certain doses and achieve the required patient compliance.
  • the ginger and turmeric are administered as a single formulation or administered as a separate ginger formulation and a separate turmeric formulation. In various embodiments of the method or use described herein, the ginger and turmeric are administered simultaneously, sequentially, or separately in separate dosage forms.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.
  • the examples describe the analysis and preparation of various extracts and compositions, and analyses of the anti-inflammatory effects of such extracts and compositions.
  • Crude material of ginger and semi-refined powder of turmeric (LOT NO. E17E041) was obtained from Integria Healthcare (Australia) Pty Ltd, Eight Mile Plains, OLD, Australia. Crude material of ginger (100 g) was ground and extracted in 90% ethanol (1 L) by sonication. The supernatant was decanted and filtered. The collected supernatant was then dried by rotary evaporation. This ginger extract had a drug extract ratio of 7:1 (7 parts dry ginger was extracted to yield 1 part final extract).
  • the semi-refined powder of turmeric was dissolved in ethyl acetate and mixed by sonication. The supernatant was filtered and collected. This turmeric extract had a drug extract ratio of 25:1 (25 parts dry turmeric was extracted to yield 1 part final extract).
  • the filtered extracts of ginger (G) and turmeric (T) were dried and stored at -20°C until further use.
  • the chemical profile of ginger and turmeric was analysed by high-performance liquid chromatography with established methods ( Lee, S., Khoo, C., Halstead, C. W., Huynh, T., & Bensoussan, A., 2007. Liquid chromatographic determination of 6-, 8-, 10- gingerol, and 6-shogaol in ginger (Zingiber officinale) as the raw herb and dried aqueous extract. Journal of AOAC international. 90, 5, 1219-1226; and ; XXII, U., & XVII, N., 1990. The United States Pharmacopeia.
  • Murine RAW264.7 macrophages cells were cultured at 37°C in Dulbecco's Modified Eagle Medium (DM EM, Lonza, Victoria, Australia) supplemented with 5% foetal bovine serum (FBS; Life Technologies, Victoria, Australia), and 1% penicillin-streptomycin (Life Technologies, Victoria, Australia) in a humidified atmosphere containing 5% CO2 and 95% air.
  • Human monocytic THP-1 cells were maintained in culture medium (RPMI 1640, Lonza) containing 10% of FBS and 1 % penicillin-streptomycin.
  • THP-1 cells monocytes were differentiated into macrophages by 24 h incubation with 100 nM phorbol 12-myristate 13- acetate (PMA, Sigma, Australia).
  • nitric oxide (NO) production stimulated by LPS and murine interferon-gamma (IFN-y; Sigma, Australia) in RAW264.7 cells was measured by its stable metabolite nitrite based on the Griess reaction ( Zhou, X., Razmovski-Naumovski, V., Chang, D., Li, C., Kam, A., Low, M., et al., 2016. Synergistic effects of danshen (salvia miltiorrhiza radix et rhizoma) and sanqi (notoginseng radix et rhizoma) combination in inhibiting inflammation mediators in RAW264. 7 cells. BioMed Research International. 2016.).
  • the cell supernatants from RAW264.7 cells treated with various samples were collected 24 hours after stimulation by LPS and IFN-y.
  • the stored supernatants from RAW264.7 cells were analysed for TNF and IL-6 synthesis using commercial ELISA kits (Lonza, Australia) according to the manufacturer’s instructions.
  • the absorbance was measured at 410 nm ( Zhou, X., 2016. Synergistic behaviour of Salvia and Notoginseng species in vascular diseases.).
  • THP-1 cells were primed with PMA (100 nM, Sigma, Australia) and were co-incubated with various samples for 2 hours before the stimulation with LPS (1 pg/mL) for another 24 hours to enable the measurement of TNF using commercial ELISA kits (Lonza, Australia).
  • LPS 1 pg/mL
  • cytotoxicity of tested samples was controlled by methyl thiazolyl tetrazolium (MTT) and Alamar Blue assay. After the media was withdrawn from cells, MTT (0.12 mg/mL, Thermo Fisher Scientific, Australia) was added to the cells and incubated for 4 hours. The supernatant was then discarded and replaced with 100 pL dimethyl sulfoxide (DMSO, Sigma, Australia) and the optical density was measured using a microplate reader (BMG l_ABTECH FLUOstar OPTIMA, Mount Eliza, Victoria, Australia) at 510 nm.
  • MTT methyl thiazolyl tetrazolium
  • Alamar Blue assay After the media was withdrawn from cells, MTT (0.12 mg/mL, Thermo Fisher Scientific, Australia) was added to the cells and incubated for 4 hours. The supernatant was then discarded and replaced with 100 pL dimethyl sulfoxide (DMSO, Sigma, Australia) and the optical density was measured using
  • resazurin 0.01 mg/mL of resazurin (Sigma, Australia) was added to the cells after the media was withdrawn.
  • the cells with resazurin were incubated at 37°C for 2 h, and the viability of the cells was measured for fluorescence intensity (Ex: 530 nm and Em: 590 nm) in a microplate reader ( Liu, J., Chen, X., Ward, T., Pegram, M., & Shen, K., 2016., Tumor Biology. 37, 7, 9825-9835. 10.1007/s13277-015-4650-1).
  • the multi-bioplex assay was conducted using the Bio-Plex 200 system with a commercial kit (Bio-plex Pro Mouse Cytokine Grp I Panel 23-Plex from BIO-RAD, catalogue number. #M60009RDPD) according to the instructions from the supplier ( Kim, Y. J., & Park, W., 2016.
  • the 23 pro-inflammatory cytokines included I L-1 a, I L-1 b, eotaxin, IL- 6, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, TNF, IL-5, IL-9, IL-13, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM- CSF), monocyte chemoattractant protein-1 (MCP-1), macrophage Inflammatory Protein-1 (MIP)-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17.
  • the up-regulation after the stimulation of LPS and IFN-y was analysed by unpaired t-test using GraphPad P
  • THP-1 cells were seeded in a 24 well plate overnight, and incubated with S (2.5 pg/mL), C (2.5 pg/mL) and SC (2.5 pg/mL) for 1h followed by the stimulation with LPS (1 pg/mL) and human recombinant IFN-y (50 ng/mL). After 24h, the cell supernatant was collected and the total protein amount was determined by the Pierce BCA Protein Assay (Thermo Fisher Scientific, Australia). The cell viability was controlled by the Alamar blue assay. The equal amount of total protein collected from each sample was applied to the human XL Cytokine Array Kit (R&D, Australia) based on the manufacturers’ protocol.
  • the Cl-Fa curve suggested the correlation between the Cl values and the effective levels on a certain biological target (i.e., the suppressive effect on NO) (Zhou, X., Razmovski-Naumovski, V., Kam, A., Chang, D., Li, C., Bensoussan, A., et al., 2017., Medicines. 4, 4, 85, and Zhou, X., Razmovski-Naumovski, V., Kam, A., Chang, D., Li, C. G., Chan, K., et al., 2019., BMC complementary and alternative medicine. 19, 1 , 50.).
  • Intracellular inducible nitric oxide synthase (iNOS) expression and nuclear factor kappa B (NF- KB) nuclear translocation were assessed using fluorescence microscopy.
  • RAW264.7 cells were plated in the 8 well glass chamber at 20,000 cells/well, incubated overnight, and then co-incubated with samples of interest or vehicle (0.1 % DMSO) in DM EM serum-free medium for 1 hour prior to LPS (50 ng/mL) for 30 minutes. Cells were then washed with ice-cold phosphate-buffered saline (PBS) and fixed using 2% paraformaldehyde for 30 minutes at room temperature. Triton X 100 (0.1%) was used to permeabilize the cells for 20 minutes.
  • PBS ice-cold phosphate-buffered saline
  • CTCF corrected total cell fluorescence
  • MCF-7 AREc32 cells (transfected with Nrf2) were cultured and seeded at a density of 1.0 x 10 6 cells/mL in 96 well plates. After 48 hours’ incubation, the cells were activated with tert- Butylhydroquinone (tBHQ, Sigma, Australia) as a positive control, samples of interest, or medium only (negative control). Cells were then incubated with Alamar Blue (0.01 mg/mL resazurin) to examine the cell viability.
  • tBHQ tert- Butylhydroquinone
  • the Alamar Blue solution was replaced with a triton lysis buffer (tris HCI: 1.705%, tris base: 0.508%, 5M NaCI: 1.5%, 1M MgCI2: 0.3%, Triton X 100 pure liquid: 0.75%) for 30 minutes.
  • the cells were then transferred to a new white micro-tire plate and mixed with luciferin buffer (D-luciferin 30 mg/mL: 0.525%, DTT 1 M: 3%, coenzyme A 10 mM: 1.5%, ATP 100 mM: 0.45%) for 100 pL per well.
  • Nrf2 The absorbance was measured within 30 minutes at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
  • the activation of Nrf2 was calculated by the fold increase compared to a negative control (cells with medium only). Cell viability was presented as a percentage relative to the negative control (%).
  • RAW264.7 cells were grown for 24 h in sterile petri-dish (94 x 16 mm) until confluent. Cells were then treated with various samples for 1 h before being activated with LPS (1 pg/mL). After incubation for various time points, cell lysates were harvested and subjected to protein estimation by the Bradford protein assay kit (Thermo Fisher Scientific, Australia). Lysates were separated by SDS-PAGE electrophoresis and electro-transferred to a PVDF membrane.
  • PBST PBS with 1 % tween 20
  • the membranes were incubated with anti-phospho-p38, anti- p38, anti-phospho JNK, anti-JNK, anti-phospho-cJUN, anti-cJUN, anti-HO-1 , anti- -actin, anti- TLR4, and anti-TRAF-6 (1 :1000, all from Cell Signalling Technology, USA) overnight at 4°C.
  • immunoreactive bands were detected by incubating with secondary antibody conjugated with anti-mouse/anti-rabbit horseradish peroxidase (Cell Signalling Technology, USA) for 1 h. Immunoreactive bands were visualized by an ECL kit (Thermo Fisher Scientific, Australia). Specific bands were analysed and the intensity was quantified using Image J software.
  • the tri-cultured neuroinflammation model was established using a transwell polycarbonate membrane cell culture inserts with a 6-well plate (Corning®Costar®Transwell® Cell culture inserts, Sigma, Australia) according to our previous published protocol (Zheng, Y.F., & Zhou X., 2020. A novel tri-culture model for neuroinflammation. Journal of Neurochemistry. 156:249-261). Firstly, the insert membrane of the transwell was placed upside down and coated with 1% polylysine (Sigma, Australia).
  • the transwell was then placed in a 6-well plate with 1500 pL DMEM containing 10% FBS in each well. After 24 h of incubation, the transwell was combined with the 6-well plate containing N2A cells to construct a tri-culture model and the medium was replaced with DMEM-F12 (Lonza, Australia) with 10% FBS.
  • the medium in the lower compartment was replaced with serum-free DMEM-F12, and in the upper compartment was replaced with either 1000 pL of serum-free DMEM-F12 (normal control), 1 ug/mL LPS or G-T 5:2 at various concentrations for another 24 h before bioassays.
  • the supernatant in the lower compartment was collected and used for the measurement of NO expression.
  • the transwell was then placed in a fresh 6- well plate, and the upper and lower compartments were filled with 0.2 mL of Evans blue [0.25% Evans blue, (Sigma, Australia)] and 0.5 mL PBS, respectively.
  • RNA was obtained from the RAW264.7 cells and harvested by the mirVana RNA isolation kit (Thermo Fisher Scientific, Australia). The quantity and purity of total RNA was determined by nanodrop spectrophotometer. The reverse transcription (RT) of approximately 10 ng of total RNA was performed using the TaqManTM MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Australia) to synthesise cDNA. Then the qPCR was performed after the TaqManTM MicroRNA Assay using TaqManTM Fast Advanced Master Mix (Thermo Fisher Scientific, Australia). The primer sequences for murine microRNA-155-5p (mmu-miR-155-5p) and internal controls using U6 were also purchased from Thermo Fisher Scientific (Australia). The calculation of the relative fold gene expression of samples with qPCR was followed the delta-delta Ct method (also known as the 2-AACt method).
  • G-T 5:2 also showed a synergistic effect with a Cl value of 0.61 and IC50 value of 5.83 pg/mL.
  • the ratio of 5:2 is within the range of 7:3 to 8:2.
  • Figure 2A demonstrated a synergistic NO inhibitory activity of G-T 5:2 (Cl ⁇ 1 ) at the inhibition level ranged from 0-80%.
  • most cells expressed iNOS green fluorescence (Figure 3A, p ⁇ 0.0001 vs. vehicle control).
  • G and T (3.13-25 pg/mL) inhibited IL-6 in a dose-dependent manner with IC50 values of 32.91 and 16.10 pg/mL, respectively.
  • IC50 values 32.91 and 16.10 pg/mL, respectively.
  • Table 1 B all the tested combinational extracts of G and T dose-dependently inhibited IL-6 expression with IC50 values ranging from 9.07 to 29.87 pg/mL.
  • G-T (3:7, 4:6, 5:5, 6:4 and 5:2, w/w) with certain ratios exhibited stronger IL-6 inhibitory effects than their individual counterparts, evidenced by lower IC50 values (9.07 -13.98 pg/mL) than that of G (32.91 pg/mL) or T (16.10 pg/mL) alone.
  • G-T (3:7 to 9:1) showed synergy with Cl values from 0.43 to 0.70.
  • Figure 2B demonstrates synergy of G-T 5:2 in inhibiting IL-6 expressions (Cl ⁇ 1) when Fraction affected (Fa) was in the range of 0.25 - 0.97 (25%-97% inhibitory level).
  • G-T 5:2 Although the IC50 value of G-T 5:2 was higher than that of T (21.07 pg/mL vs. 14.63 pg/mL), G-T 5:2 showed synergy at all tested concentrations with Cl values of 0.39, 0.28 and 0.20 at 25, 50 and 75% inhibitory levels (Figure 2C). 1.1.4 G-T combinations showed lower cytotoxicity and higher therapeutic index against inflammation in RAW264.7 cells
  • G and T showed cytotoxicity on RAW264.7 cells when the concentration was higher than 50 and 25 pg/mL with LC50 values of 104.3 and 83.90 pg/mL, respectively.
  • G-T combinations (6:4, 8:2, 9:1 and 5:2) showed lower cytotoxicity with higher LC50 values ranging from 104.8 to 115.8 pg/mL (Table 1A, 1 B and/or 1C).
  • G-T 5:2 also showed a lower cytotoxicity level than G and T with LC50 estimated to be 115.8 pg/mL.
  • G-T combinations that showed the highest therapeutic index in NO, IL-6 and TNF were 6:4, 5:2 and 5:5, respectively.
  • the therapeutic index of G-T 5:2 throughout the three assays consistently showed relatively high values of therapeutic index (19.86, 12.76 and 5.33, respectively).
  • Table 1A Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS and IFN-y-induced NO in RAW264.7 cells (n>3).
  • Table 1B Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS and IFN-y-induced IL-6 in RAW264.7 cells (n>3).
  • Table 1C Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS and IFN-y-induced TNF assays in RAW264.7 cells (n>3).
  • G-T combinations The inhibitory effect of G-T combinations on TNF was investigated in human monocytic THP- 1 cells. As shown in Table 2, G (1 .63 - 25 pg/mL) did not show any significant TNF inhibitory activity, whereas T (1.63 - 25 pg/mL) dose-dependently inhibited TNF in response to LPS with IC50 of 8.54 pg/mL. G-T combinations showed a TNF inhibitory effect in a dose-dependent manner with IC50 ranging from 4.79 to 57.13 pg/mL. The potency for G-T combinations 1 :9, 2:8, 3:7, 4:6, 5:5, 6:4 were stronger (with lower IC50 values) than the single contributor of G and T.
  • G-T combinations showed synergistic interaction in inhibiting TNF expression at IC50 level (Figure 4A).
  • G-T combinations (3:7, 4:6, 5:5, 6:4, 7:3, 5:2, 8:2, 9:1) showed lower cytotoxicity than T with LC50 ranging from 52.03 - 118.90 pg/mL.
  • the therapeutic index values for most G-T combinations (6:4 to 1 :9) were higher than G (no effect) and T (5.53).
  • G-T 6:4 was calculated to have the highest therapeutic index of 10.70, followed by G-T 5:2 at 8.85.
  • G-T 5:2 (1.63 - 25 pg/mL) showed a comparable effect as T with a slightly lower IC50 value of 8.32 pg/mL. However, the cytotoxicity of G-T 5:2 was much lower than that of T (73.61 pg/mL vs. 47.24 pg/mL) which led to a superior therapeutic index (8.85 vs. 5.53). As analysed by Cl model, G-T 5:2 showed a strong synergy within the tested concentration (Figure 4B), with Cl values of 0.25, 0.32 and 0.41 at 25%, 50% and 75% inhibitory level, respectively.
  • Anti- IL-2 (36) 5.9910.40 69.3815.02 11.59** inflammatory IL-3 (18) 1.6710.05 6.0610.08 3.64** mediators IL-4 (39) 4.6510.00 7.5710.24 1.63*
  • G did not demonstrate significant inhibitory effects on most cytokines, with prominent inhibitory effects on GM-CSF (73), KC (57) and MCP-1 only with IC50 values ranging from 5.97 to 16.69 pg/mL.
  • T showed potent activities on most of the cytokines (excluding MIP-1b) with the IC50 values ranged from 2.15 to 7.26 pg/mL.
  • G-T 5:2 showed dose-dependent inhibitory activities on most of the tested mediators (excluding G-CSF) with IC50 ranging from 3.84 to 13.62 pg/mL.
  • IC50 ranging from 3.84 to 13.62 pg/mL.
  • the doses required to reach 50% of G and T in the combined extract to reduce cytokines were generally lower than that of individual extracts.
  • Nrf2/H0-1 mediated signalling pathway is the main cellular defence mechanism against oxidative stress and inflammation.
  • the effect of G-T 5:2 on Nrf2 pathway was investigated using the Nrf2 luciferase assay in MCF AREc32 cells.
  • a prototypical and potent Nrf2 activator tert-Butylhydroquinone (tBHQ)
  • tBHQ tert-Butylhydroquinone
  • Extracts of G, T and G-T 5:2 were tested in the Nrf2 luciferase assay and the comparison of their induction on Nrf2 at 25 and 12.5 pg/mL are shown in Figure 5A.
  • G-T 5:2 produced a higher maximum-fold induction of Nrf2 (11.57 ⁇ 3.30) compared to that of G or T (2.04 ⁇ 0.22 and 10.11 ⁇ 1.60, respectively).
  • Turkey non-parametric analysis showed that the maximum-fold activation of G-T 5:2 was significantly higher than that of G (p ⁇ 0.001), but comparable to that of T and tBHQ (at 4.16 pg/mL). Strong synergistic interaction was suggested from the changes of Cl and isobologram models (Figure 5B). Cl values were consistently lower than 1 at all tested concentrations with Cl values at 0.13, 0.06 and 0.03 at Fa 0.25, 0.5 and 0.75, respectively.
  • FIG. 5C cells with medium only (Blank) did not show significant expression of HO-1, however, the co-incubation of G-T 5:2, G and T triggered expression of HO-1.
  • Figure 5D showed the quantitative analysis of the HO-1 upregulation by G, T and G-T 5:2.
  • the activated expression of HO-1 by G-T 5:2 was 1.92 ⁇ 0.37 times that of blank control (p ⁇ 0.05).
  • G and T also showed an increasing trend of HO-1 expressions, however, did not reach statistical significance, with 1.46 ⁇ 0.32 and 1.03 ⁇ 0.19 fold, respectively.
  • the synergistic anti-inflammatory activity of G-T 5:2 maybe at least partially associated with the activation of the Nrf2-H0-1 pathway.
  • TLR-4 The Toll-like receptor-4 (TLR-4), a key pattern recognition receptor, recognizes and binds LPS which contacts down-stream tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), then activates mitogen-activated protein kinases (MAPKs) and nuclear transcription factor-kappa B (NF-KB) signalling pathways.
  • TNFR tumor necrosis factor receptor
  • TRAF6 mitogen-activated protein kinases
  • NF-KB nuclear transcription factor-kappa B
  • Figure 7 shows the three trials and data analysis of the individual and combined effects of G- T in regulating the key protein kinase and expressions in the MAPK/JNK/CJUN pathways.
  • DAPI blue was used to stain the cell nucleus and fluor 594 red fluorescence represents p65 expression within the cell.
  • p65 was observed almost exclusively in the cytoplasm outside the nucleus ( Figure 9A, blank).
  • LPS 100 ng/mL
  • a dramatic increase of p65 was seen in the nuclei as indicated by the overlap of the p65- fluor 594 red fluorescence with the DAPI blue staining in the nucleus.
  • Curcumin (C), desmethoxycurcumin (D) and bisdemethoxycurcumin (B) also dose-dependently inhibited NO and IL-6 with IC50 values that ranged from 3.69 to 8.30 pg/mL.
  • IC50 values ranged from 3.69 to 8.30 pg/mL.
  • the summary of IC50 values of individual compounds is shown in Supplementary material 3.
  • pair-wise combinations of single compounds in G and T either combined by their content in G-T 5:2 or by IC50 values on NO and IL-6 assays generally exhibited antagonistic effects (Supplementary material 4 and 5).
  • pair-wise combinations of G with curcuminoids (G-C, G-D, G-B), T with gingerols (T-6g, T-8g, T-10g) or shogaols (T-6s, T-8s, T-10s) in 5:2 also displayed antagonistic effects (Supplementary material 6).
  • T An artificial T (AT, constructed by C, B and D based on their content in T) was tested in the NO assay together with G at 5:2, and the dose-response curves and IC50 values were comparable to that of original T and G-T 5:2 ( Figure 10). Since C is the dominant compound that accounted for approximately 60% of the total weight of T, the compounds combination was limited to 2 gingerols -C, and 2 shogaols - C for the synergistic investigation.
  • Combinations of 6s8s-C, 6s10s-C and 8s10s-C (0.1 -25 pg/mL) based on their content in G- T 5:2 were tested on NO assay and the results were compared with combinations of 6g8g-C, 6g10g-C, and 8g10g-C 5:2 (0.4 -15 pg/mL).
  • the inventors further demonstrated the effect of SC in inhibiting iNOS on RAW264.7 cells using fluorescent staining.
  • S, C and SC at 2.5 pg/mL all significantly inhibited iNOS expression with p ⁇ 0.0001.
  • the most prominent activity was seen in SC with almost no iNOS positive cells observed.
  • the combination of 6s10sC 5:2 was selected and tested in LPS induced-TNF assay in RAW264.7 and THP-1 cells.
  • the results of SC were compared with that of individual components and G-T extracts to see whether it represented a higher level of bioactivity.
  • the concentrations of 6s, 10s and C in G-T 5:2 at 50 mg/mL were 0.27, 0.08 and 10.74 pg/mL, respectively.
  • the ratio in SC was 0.27:0.08:10.74, and the concentrations in 10 pg/mL were 0.24, 0.07 and 9.68 pg/mL, respectively.
  • the SC mixture at 10 pg/mL was then series diluted with DMSO before being subjected to the cellular assays.
  • SC showed the highest TNF inhibitory effect with IC50 value of 7.03 pg/mL compared to 6s10s (S) or C.
  • S (0-0.45 pg/mL) failed to exhibit any significant activity whereas C (0-18.53 pg/mL) exhibited dose-dependent activity with IC50 of 8.69 pg/mL.
  • a low amount of S (0.23 pg/mL) mixed with C (6.80 pg/mL) did not dilute the effect of C, but reached the same level of C when it was used at 8.69 pg/mL.
  • Both isobologram and Cl showed strong synergy of SC, with Cl value consistently lower than 1 ( Figure 12A and B).
  • IL-6 production in RAW264.7 cells reached approximately 195.414 ng/mL, detected by a commercial IL-6 ELISA kit used according to the manufacturer’s instructions.
  • IC50 values of compounds were all lower than that of the extracts (IC50 values of G, T and G-T 5:2 were 24.75, 5.16, 3.63 pg/mL).
  • cytokines operate in a large network in the immune system, where the action of one cytokine is regulated by the presence or absence of other cytokines.
  • the anti-cytokine effect of SC was demonstrated in a broader range using human XL Cytokine Array Kit (R&D, Australia). In this cytokine array assay, differences of up to 102 cytokines between samples can be determined simultaneously.
  • THP-1 cells were seeded in a 24 well-plate overnight, and incubated with RPMI media, S (2.5 pg/mL), C (2.5 pg/mL) and SC (2.5 pg/mL) for 1 hour followed by stimulation with LPS (1 pg/mL) and human IFN-y (50 ng/mL). After 24h, the cell supernatant was collected and the total protein amount was determined by the Pierce BCA Protein Assay (ThermoFisher, Australia). Then equal amounts of total protein from each sample was applied to the cytokine array kit to conduct the assay based on the manufacturers’ protocol.
  • Typical cytokine images in cells stimulated with LPS and IFN-y only, pre-treated with S, C and SC before the stimulation are shown in Figure 14A.
  • Cytokines detected with LPS and IFN-y stimulation are listed in Figure 14B.
  • the average expression levels of spot density for each cytokine are shown in Figure 14C.
  • CXCL family contains chemokines for the recruitment of the first line of innate immune effector cells to sites of infection and inflammation which contribute to elimination of pathogens, but it may also contribute significantly to disease-associated processes, including tissue injury.
  • IL-8 is a classical inflammatory cytokine (chemokine) that stimulates neutrophil chemotaxis during bacterial exposure to help eliminate a pathogen. These actions have a relatively rapid onset in the initial stage of inflammation.
  • CXCL10 and CXCL11 are mainly induced by IFN-y and their actions have been associated with the migration of Th1-polarized effector T cells to inflamed sites during the adaptive immune response.
  • SC demonstrated greater inhibitory effects in inhibiting a range of chemokines under the acute inflammatory phase, and the effect was greater than S or C alone.
  • the inhibitory effects covered chemokines and main cytokines (IL-6 and TNF).
  • IL-6 and TNF main cytokines
  • Previous studies have demonstrated the potential use of C against cytokine storm. So far, there has not been any study on the possible inhibitory effect of shogaol or ginger in inhibiting CXCL11 .
  • SC is a better candidate for a broad anti-inflammatory and anticytokines action and has great potential against LPS and IFN-y induced cytokine storm.
  • Nrf2 luciferase assay for the evaluation of Nrf2 activation.
  • tBHQ was used as positive control and Nrf2 activation by treatments was compared to that of a negative control (cell with medium only).
  • the results show that SC, S and C activated Nrf2 in a dose-dependent manner.
  • the maximum-induction of Nrf2 induced by SC at 16.40 pg/mL was 13.27 ⁇ 1.54 times which was markedly higher than that of the maximum induction of Nrf2 by S and C at 6.17 ⁇ 1.00 and 6.65 ⁇ 0.36 times, respectively (p ⁇ 0.05).
  • the maximum induction of Nrf2 by SC was comparable to that of tBHQ.
  • DAPI blue was used to stain the cell nucleus and fluor 594 red fluorescence represents p65 expression within the cell.
  • p65 was observed almost exclusively in the cytoplasm outside the nucleus ( Figure 16A, blank).
  • LPS 100 ng/mL
  • a dramatic increase of p65 was seen in the nuclei as indicated by the overlap of the p65-fluor 594 red fluorescence with the DAPI blue staining in the nucleus.
  • the inhibitory effect of p65 translocation was compared among S, C and SC at 5 pg/mL. As shown in Figure 16A and B, S did not show significant inhibition in p65 translocation, whereas C showed an inhibitory effect of p65 translocation to some extent. SC showed the most potent inhibitory activity in p65 translocation with a prominent amount of p65 expressed outside the nucleus. The statistical analysis showed that the nuclear p65 localization was significantly reduced by SC (p ⁇ 0.01 vs. vehicle + LPS), and the reduction was greater than that of S and C at 5 pg/mL (p ⁇ 0.5).
  • G-T significantly inhibited the phosphorylation of p38, JNK and CJUN in the LPS-stimulated MyD88 dependent signalling pathway.
  • SC key contributing compounds
  • G-T 5:2 (10-20 pg/mL) treatment markedly attenuated the increase of NO (p ⁇ 0.001) in the tri-culture system.
  • the integrity of MVEC cells in the tri-culture system was damaged after LPS exposure for 24h at 1 pg/mL as evidenced by significant increased leakage (p ⁇ 0.05), but it was restored by G-T 5:2 treatment (10-20 pg/mL) significantly (p ⁇ 0.001).
  • G-T 5:2 (10-20 pg/mL) protected neuron survival by restoring the cell viability impaired by cytokine release in the tri-culture system (p ⁇ 0.05).
  • miRs Small endogenous RNA molecules known as microRNAs (miRs or miRNAs) have been shown to play an important role in regulating the inflammatory response by specifically binding to the 3’IITR of target miRNAs.
  • miR-155 has been implicated in the control of the inflammatory response, which creates a negative feedback loop in LPS-induced signalling pathways.
  • G-T and SC the combined effect of G-T and SC on the production miR-155 by using miR- 155 qPCR, and any crosstalk at the miRNA level that contributes to the consequent synergistic anti-cytokine effects was investigated.
  • MiR-155 is rapidly activated by NFKB which then induces the IKK signalosome complex and amplifies the signal of the downstream inflammatory response and the excessive release of the proinflammatory cytokines (Mahesh G and Biswas R., Journal of Interferon and Cytokine research. 2019. 39 (6): 321-330).
  • the synergistic decrease in microRNA 155-5p of SC composition was likely to be attributed to the supressed NFKB pathway and led to further supressed downstream inflammatory response.
  • Ginger raw material fresh ginger root
  • a laboratory sieve 500 microns, 200 mm diameter, ENDECOTTS, UK.
  • the dried powder 50g was weighed and then dissolved in 500 ml of 100% ethanol or DCM.
  • the solution was first shaken manually to ensure the powder is evenly spread in the solvent, then subjected to sonication (Thermoline Scientific, Power Sonic420, Korea) at room temperature for 30 minutes on maximum power to maximise the solubility in the solvents.
  • the solution was then filtered using a funnel with a filter paper (Whatman, circle filter paper 125mm Grade 4: 20-25 pm, Sigma-Aldrich, Germany), then the filtered solution was collected into a fresh 500ml round bottom flask.
  • the flask was weighed before and after the evaporation to calculate the extraction yield.
  • the filtered solution in the flask was then evaporated using a rotary evaporator (Buchi R-215 Rotavapor System including Buchi B-491 Heating Bath, BUCHI, Germany) under the vacuum (Buchi V-850 vacuum controller, BUCHI, Germany) at 40 °C.
  • the extract was then subjected to freeze-dry using a freeze drier (ChristTM LDplus freeze-dryer alpha 1-4 Id plus, Germany) overnight.
  • the dried extract was dried using nitrogen gas.
  • the dried extract was collected and resuspended in methanol and stored in a -20 °C freezer for the subsequent analysis or stored into a -80 °C freezer for further use.
  • the collected extract was then labelled as DCM (G(DCM)) or EtOH (G(ETOH)) extracts.
  • Turmeric semi-refined granule powder (extracted by ethyl acetate) was provided by Integria Healthcare, Australia, which contains curcumin, demethxycurcumin, and bisdemethoxycurcumin only.
  • the turmeric granule (50g) was re-suspended in ethyl acetate (500 ml) and sonicated for 30 min.
  • the solution was filtered and concentrated by the rotary evaporator following the same procedure as for ginger.
  • the extract was stored under -20°C for subsequent analysis or -80°C until further use.
  • This turmeric extract had a drug extract ratio of 25:1 (25 parts dry turmeric was extracted to yield 1 part final extract).
  • Ginger extracts were dissolved in methanol (HPLC grade, Honeywell B&J ACS/HPLC, Korea) to a final concentration of 1 mg/ml and filtered using syringe filter 4.5 um (Sigma-Aldrich, Sydney, Australia) before subjecting to HPLC analysis.
  • the reference standards of 6-gingerol (6g), 8- gingerol (8g), 10-gingerol (10), 6-shogaol (6s), 8-shogaol (8s), and 10-shogaol (10s) were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China; purity > 98%).
  • the reference compounds' standard stock solution was prepared in methanol at 0.8 mg/ml and stored at - 80 °C until further use.
  • a mixed standard solution containing all six compounds was prepared with each standard concentration at 0.167 mg/ml. It was serially diluted in a 1 :2 ratio to form a standard calibration curve.
  • the mixed standards were prepared at concentrations that produced comparable peak sizes.
  • HPLC-PDA High-performance liquid chromatography-photodiode array detection
  • the HPLC-PDA analysis was performed on the Shimadzu UFLC system (Shimadzu, Australia), comprising an LC-30AD pump, SIL-30ACHT autosampler, SPD-M20A PDA detector and DGU-20A5 inline solvent degasser. The system was controlled using Class-VP 7.4SP4 software.
  • HPLC analysis of the extracts was performed using an Alltech Alltima (Alltech Australia) reverse phase C18 column (46 x 150 mm I.D., 5 pm).
  • the HPLC-PDA analysis of ginger was a modification method from Jung et al. (2019).
  • Solution A (Acetonitrile, dilute phosphoric acid (0.1 %), and methanol (55:44:1) was used as mobile phase in isocratic flow at rate 1 ml/min for 60 min followed by washing method involving solution B (100% Acetonitrile) in between each run as shown in Table 7.
  • the column temperature was kept at 30 °C throughout the analysis.
  • the samples were kept at 4 °C.
  • the injection volume was 25 pl.
  • the PDA UV 200-500 nm
  • UV 280 nm was used to quantify the gingerols and shogaols peaks.
  • the amounts of each analyte were calculated using the corresponding Regression equations.
  • the relative standard deviation (RSD) was used as a repeatability metric.
  • the intra-day precision was assessed by analysing four concentrations of each marker compound three times within a day, and the inter-day reproducibility was assessed over three days (Chan et al., 2004).
  • the RAW264.7 cell line (provided from the NICM Health Research Institute) was preserved in Dulbecco's Modified Eagle Medium (DMEM) (Lonza, Australia) having 4.5 g/l D-glucose and supplemented with 2 mM I- GlutaMax, 100 U penstrep, and 5% FBS (foetal bovine serum) French origin (Bovogen Biologicals, Keilor East, Australia). Then it was incubated at 37°C, 5% CO2 in 95% air in a vented flask T75 cm 2 (Sigma-Aldrich, Sydney, Australia). The cells were sub-cultured every 3-4 days to sustain a controllable number.
  • DMEM Dulbecco's Modified Eagle Medium
  • the viable and total cell number was analysed by the Vi-CELL XR cell viability analyser (Beckman Coulter, Australia). The cells with a passage under 30 were used for the bioassays.
  • Raw264.7 cells were diluted to (23.52 x 10 6 , 24 x 10 6 and 29.28 x 10 6 cells/ml), respectively, in complete DMEM.
  • the cells (23.52x10 A 4/well, 100 pl, 24x10 A 4/well, 100 pl and 29.28x10 A 4/well 100 pl) were seeded in 96 well plates and incubated for 48 h at 37°C (5% CO2) to allow attachment of the cells and reach consistent confluence.
  • the ginger solvents were prepared by weighing (10 mg) of each extract, then were dissolved in 1 ml of DMSO in 1.5 ml centrifuge tube. After the solvent was added, the solutions were vortexed for > 10 min or until dissolved.
  • (50 ml) of each ginger extract solution were mixed with (20 ml) of turmeric extract (5:2) ratio, before preparing the drug 96 well plate for dilution.
  • Drugs 96 well plate was prepared to dilute ginger solution prior adding into cells, the first row of the plate was filled with 198 ml of fresh DMEM serum-free mixed with 2 ml of each extract solutions that were prepared in DMSO to produce a final volume of 200 ml in the first row of wells. After filling the rest of the wells with 100 ml of fresh DMEM serum-free, the drugs solution was mixed in each well by multichannel pipette and diluted in (1 :2) ratio to produce six serial concentrations (10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/ml), before adding to the cell plates. After seeding the cells for 48 h, the old media was discarded and replaced with another (170 pl) of fresh DMEM serum-free media, the prepared DMSO with ginger solvent’s and turmeric extract solutions were added (20 pl) into each well.
  • LPS lipopolysaccharides
  • the Griess method is a quantitative colourimetric method for the measurement of nitrites.
  • Sodium nitrite was used as a standard to quantify NO release in this model.
  • sulfanilic acid sulphanilamide dissolved in 5% phosphoric acid
  • nitrite stimulated from the cells
  • acid solution a diazonium salt
  • the concentration of NO in the cell culture medium can be measured by the Griess reagent proportional to the quantity of nitrite (the end product of NO).
  • the cell supernatant (100 pl) was collected 16h after the activation with LPS and IFN- and transferred to a new 96 well plate.
  • the Griess reagent was then added to the supernatant with 50 pl of 1% sulfanilamide in 2.5% phosphoric acid and 50 pl of 0.1% N-(1 naphthyl) ethylenediamine dihydrochloride.
  • the Griess reagent with the presence of NO produces a violet colour immediately, and the colour density measured under 545 nm absorbance is proportional to the production of NO.
  • the amount of nitrite was normalised to the control wells, leading to non-stimulated cells providing 0 % NO production and LPS stimulated cells that provide 100 % with NO production.
  • MTT colourimetric assay was used to measure the viability of RAW264.7 cells.
  • MTT is a powdered yellow tetrazole. As the MTT solution is applied to the cells, it reaches the living cells and progresses into the mitochondria, reducing the NAD(P)H-dependent cellular oxidoreductase enzymes that are only present in viable cells (Liu et al., 1997). It then transforms into an insoluble dark purple formazan complex (Liu et a!., 1997). As a result, the purple colour density, which represents the amount of insoluble formazan produced in viable cells, can be used to indicate cell viability.
  • the insoluble formazan is usually solubilized with organic solvents such as isopropanol and dimethyl sulfoxide (DMSO), and this colour measurement can be detected using a microplate reader at a wavelength of 510-590 nm.
  • organic solvents such as isopropanol and dimethyl sulfoxide (DMSO)
  • DMSO dimethyl sulfoxide
  • MTT solution 50 pl of 0.12 mg/ml in complete medium
  • MTT solution 50 pl of 0.12 mg/ml in complete medium
  • 100 pl of DMSO was added to dissolve the formazan crystals.
  • the plate was then shaken for approximately 3 minutes before the absorbance was measured at 545 nm using the FlUOstar omega microplate reader (BMG LABTECH, The Microplate Reader Company, Australia).
  • Figure 21 shows Individual and dual response curves of G with T in inhibiting NO.
  • A. Doseresponse curves of A. G(DCM), T and G(DCM)-T.
  • B. G(ETOH), T and G(ETOH)-T.
  • Figure 22 shows the inhibitory effects of G(DCM)-T and G(ETOH)-T (5:2) on NO production (%) versus concentration (pg/ml) as shown in A and D, respectively.
  • C and F Isobologram curves for G(DCM)-T and G(ETOH)-T (5:2) on NO.
  • B and E Cl values verses NO inhibition effect (Fa) by ‘Calcusync’ software.
  • Dotted line is the reference line, where Cl value equals 1; solid line represents Cl values at different Fa
  • Table 15 represent G-T combinations of synergistic effects at different concentrations level.
  • the Cl values of G(ETOH)-T were below 1 at all Fa levels, which showed synergy when Fa was at 0.25, 0.5, 0.75 and 0.95 respectively, the Cl values of G(DCM)-T at Fa level 0.25 and 0.5 were below 1 , which shows synergy effect.
  • 6-shogaol (6s) Y 79816x + 74.868 1.00 0.21 0.64 0.16 0.85
  • Curcumin (C) Y 67047x - 1803.5 0.99 8.88 26.67 0.10 1.27
  • Supplementary material Content of bioactive compounds in ginger and turmeric extracts quantified by HPLC-PDA (n>3) and their content in G-T 5:2 dried extract in the cell-based assays.
  • # 10 pg/mL refers to a working concentration of G-T 5:2 in the cell-based assays (western blot, p65 transcription, etc.)
  • *10 pg/mL was the highest concentration when subjecting to the cells for the bioassays, and followed by five two-fold serial dilutions.
  • G or T was prepared in 50 mg/mL, and the single compounds (B, C, D, 6g, 8g, 10g, 6s, 8s and 10s) were prepared in the concentration equivalent to their content in G or T at 50 mg/mL. Then they were mixed by taking 50 pL from G or its derived compounds (6g, 8g, 10g, 6s, 8s and 10s) with 20 pL from T or its derived compounds (C, B or D). The pair-wise mixture was then diluted by 1:1000 followed by two-fold serial dilutions before subjecting to cells.
  • Extract and Ratio of the Composition NO assay IL-6 assay compounds components in in the mixture combination the mixture at highest by their based on their working content in G- concentrations concentration T 5:2 (mg/mL) in G/T when at 50 mg/mL subjecting to cells (pg/mL)
  • Supplementary material 7 Inhibitory effects of mixed gingerols or shogaols with curcumin (content equivalent to G-T 5:2) on LPS stimulated NO expressions in RAW264.7 cells (n>3).

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Abstract

Provided herein are compositions comprising ginger and turmeric, or extracts thereof, wherein the composition elicits a synergistic anti-inflammatory effect. Also provided is the use of such compositions in the treatment or prevention of inflammation, diseases or conditions associated with inflammation and/or to provide one or more health benefits to a subject.

Description

Anti-inflammatory compositions comprising ginger and turmeric
RELATED APPLICATIONS
This application derives priority from Australian patent application number 2021902926, incorporated herein by reference.
FIELD OF THE INVENTION
The invention relates to compositions comprising ginger and turmeric, or extracts thereof, wherein the composition elicits a synergistic anti-inflammatory effect. The invention also relates to the use of such compositions in the treatment or prevention of inflammation, diseases or conditions associated with inflammation and/or to provide one or more health benefits to a subject.
BACKGROUND OF THE INVENTION
The treatment and/or prevention of diseases and conditions associated with the immune system and/or aberrant immune function, together with the amelioration of symptoms associated with immune dysfunction remain significant medical and economic problems. Such diseases and conditions include diseases and conditions associated with inflammation and chronic inflammation.
Ginger (Zingiber officinale Roscoe) and turmeric (Curcuma longa L.) are known dietary plants that are extensively used in diet, tea, supplements and natural products. They are both considered as functional foods and medicinal plants due to a variety of health benefits. The fresh or processed forms of ginger are used for gastrointestinal disorders, nausea, cold and pain in traditional medicines. T urmeric is used extensively in diet for its flavour and colour, and as a medicinal plant for various indications such as sprains and swelling, and improving digestion. Both ginger and turmeric have been investigated for their anti-inflammatory activities to prevent and manage several inflammatory diseases such as pain, diabetes, cardiovascular diseases, neurodegenerative diseases, cancer and respiratory disorders. However, little is known regarding the combined mechanisms of their activity and how the health benefits occur. A previous study has demonstrated enhanced activities of the mixture of ginger and turmeric extracts in a selected ratio of 1 :1 in reducing systematic inflammation in vivo (Ramadan, G., & El-Menshawy, O., 2013., International Journal of Rheumatic Diseases. 16, 2, 219-229). The anti-inflammatory effects and potential for therapeutic benefits are generating a renewed interest in the scientific body of research, and there remains a need for effective compositions derived from naturally occurring extracts, particularly those which are capable of eliciting an anti-inflammatory response.
It is an object of the invention to provide one or more anti-inflammatory compositions and/or uses of said compositions, that overcome or ameliorate at least one of the disadvantages of the prior art, or at least provide the public with a useful choice.
In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
BRIEF DESCRIPTION OF THE INVENTION
In a first aspect, the invention provides a composition comprising turmeric and ginger, wherein the gingerturmeric dry weight or equivalent ratio is about 7:225 to about 63:25.
In various embodiments, the composition comprises a gingerturmeric dry weight or equivalent ratio of about 7:225 to about 63:25, about 7:100 to about 28:25, about 3:25 to about 7:10, about 14:75 to about 63:25, about 7:25 to about 28:25, about 14:75 to about 28:25, about 21 :50 to about 7:10, about 49:75 to about 28:25, about 49:75 to about 7:10, about 7:10 to about 63:25 or about 7:10 to about 28:25.
In various embodiments, the composition comprises a gingerturmeric dry weight or equivalent ratio of about 7:225, about 7:100, about 3:25, about 14:75, about 7:25, about 21 :50, about 49:75, about 7:10, about 28:25 and/or about 63:25. In various embodiments, the composition comprises a gingerturmeric dry weight or equivalent ratio of about 7:10.
In various embodiments, the composition comprises turmeric and ginger, wherein the composition comprises less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, or less than 25% turmeric dry weight or equivalent, with respect to the ginger dry weight or equivalent, in the composition. In various embodiments, the composition comprises turmeric and ginger, wherein the composition comprises greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, or greater than 75% ginger dry weight or equivalent, with respect to the turmeric dry weight or equivalent, in the composition.
In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 4:6 to about 9:1 , and wherein the turmeric extract has a drug extract ratio (DER) of 25:1 (25 parts dry turmeric extracted to yield 1 part final extract) and the ginger extract has a DER of about 7:1 (i.e. 7 parts dry ginger extracted to yield 1 part final extract), about 16:1 or about 12:1.
In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1 , 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1 , 5:5 to 5:2, 6:4 to 9:1, 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9:1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9:1. In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1, 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1, 5:5 to 5:2, 6:4 to 9: 1 , 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9: 1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9: 1 and wherein the turmeric extract has a DER of 25:1, and the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1.
In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 1:9, about 2:8, about 3:7, about 4:6, about 5:5, about 6:4, about 7:3, about 5:2, about 8:2 or about 9: 1 , and wherein the turmeric extract has a DER of about 25:1 and the ginger extract has a DER of about 7:1, about 16:1 or about 12:1. In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract has a DER of about 25:1 and the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1.
In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 4:6 to about 9:1 , and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1 , and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1 , 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1 , 5:5 to 5:2, 6:4 to 9:1, 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9:1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9:1. In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is from 4:6 to 9:1, 4:6 to 8:2, 4:6 to 5:2, 5:5 to 9:1, 5:5 to 5:2, 6:4 to 9: 1 , 6:4 to 8:2, 6:4 to 5:2, 7:3 to 9: 1 , 7:3 to 8:2, 7:2 to 5:2, 5:2 to 8:2, or 5:2 to 9: 1 and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 1:9, about 2:8, about 3:7, about 4:6, about 5:5, about 6:4, about 7:3, about 5:2, about 8:2 or about 9: 1 , and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1. In various embodiments, the invention provides a composition comprising a turmeric extract and a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1 , or is extracted with 100% ethanol and has a DER of about 12:1.
In a second aspect, the invention provides a composition comprising: one or more of 6- gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s) and/or 10- shogaol (10s); and one or more of curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B).
In various embodiments, the composition of the second aspect comprises ginger and turmeric, wherein the ginger comprises one or more of 6g, 8g, 10g, 6s, 8s and/or 10s, and the turmeric comprises one or more of C, D and/or B. In various embodiments, the composition of the second aspect comprises ginger and turmeric, and wherein the gingerturmeric dry weight or equivalent ratio is as defined in the first aspect.
In various embodiments, the composition of the second aspect comprises a ginger extract and/or a turmeric extract. In various embodiments, the composition of the second aspect comprises a ginger extract as defined in the first aspect and/or a turmeric extract as defined in the first aspect, and/or a gingerturmeric dry weight or equivalent ratio as defined in the first aspect.
In various embodiments, the composition of any one of the above aspects comprises 6- shogaol (6s) and curcumin (C), wherein the 6s:C weight ratio is about 0.15:22.55 to about 0.34:3.76.
In various embodiments, the composition of any one of the above aspects comprises a 6s:C weight ratio of about 0.15:22.55, about 0.19:18.79, about 0.22:15.04, about 0.26:11.28, about 0.27:10.74, about 0.30:7.52 or about 0.34:3.76. In various embodiments, the composition comprises a 6s:C weight ratio of about 0.27:10.74.
In various embodiments, the composition of any one of the above aspects comprises a 6s:C weight ratio of about 0.27:10.74, about 0.49: 10.74 or about 0.35:10.74. In various embodiments, the composition comprises a 6s:C weight ratio of about 0.27-0.49:10.74. In various embodiments, the composition comprises a 6s:C weight ratio of about 0.27- 0.35:10.74.
In various embodiments, the composition of any one of the above aspects comprises a 6s:C weight ratio of about 0.15:22.55 to about 0.19:18.79, about 0.15:22.55 to about 0.22:15.04, about 0.15:22.55 to about 0.26:11.28, about 0.15:22.55 to about 0.27:10.74, about 0.15:22.55 to about 0.30:7.52, about 0.15:22.55 to about 0.34:3.76, about 0.19:18.79 to about 0.22:15.04, about 0.19:18.79 to about 0.26:11.28, about 0.19:18.79 to about 0.27:10.74, about 0.19:18.79 to about 0.30:7.52, about 0.19:18.79 to about 0.34:3.76, about 0.22:15.04 to about 0.26:11.28, about 0.22:15.04 to about 0.27:10.74, about 0.22:15.04 to about 0.30:7.52, about 0.22:15.04 to about 0.34:3.76, about 0.26:11.28 to about 0.27:10.74, about 0.26:11.28 to about 0.30:7.52, about 0.26:11.28 to about 0.34:3.76, about 0.27:10.74 to about 0.30:7.52, about 0.27:10.74 to about 0.34:3.76 or about 0.30:7.52 to about 0.34:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 0.15:0.05:22.55 to about 0.34:0.10:3.76. In various embodiments, the 6s:10s:C weight ratio is about 0.15:0.05:22.55, about 0.19:0.06:18.79, about 0.22:0.07:15.04, about 0.26:0.08:11.28, about 0.27:0.08:10.74, about 0.30:0.09:7.52 or about 0.34:0.10:3.76. In various embodiments, the 6s:10s:C weight ratio is about 0.27:0.08:10.74.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 0.15:0.05:22.55 to about 0.19:0.06:18.79, about 0.15:0.05:22.55 to about 0.22:0.07:15.04, about 0.15:0.05:22.55 to about 0.26:0.08:11.28, about 0.15:0.05:22.55 to about 0.27:0.08:10.74, about 0.15:0.05:22.55 to about 0.30:0.09:7.52, about 0.15:0.05:22.55 to about 0.34:0.10:3.76, about 0.19:0.06:18.79 to about 0.22:0.07:15.04, about 0.19:0.06:18.79 to about 0.26:0.08:11.28, about 0.19:0.06:18.79 to about 0.27:0.08:10.74, about 0.19:0.06:18.79 to about 0.30:0.09:7.52, about 0.19:0.06:18.79 to about 0.34:0.10:3.76, about 0.22:0.07: 15.04 to about 0.26:0.08:11.28, about 0.22:0.07:15.04 to about 0.27:0.08:10.74, about 0.22:0.07:15.04 to about 0.30:0.09:7.52, about 0.22:0.07:15.04 to about 0.34:0.10:3.76, about 0.26:0.08:11.28 to about 0.27:0.08:10.74, about 0.26:0.08:11.28 to about 0.30:0.09:7.52, about 0.26:0.08:11.28 to about 0.34:0.10:3.76, about 0.27:0.08:10.74 to about 0.30:0.09:7.52, about 0.27:0.08:10.74 to about 0.34:0.10:3.76 or about 0.30:0.09:7.52 to about 0.34:0.10:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 0.15:0.03:0.05:22.55 to about 0.34:0.07:0.10:3.76. In various embodiments, the 6s:8s:10s:C weight ratio is about 0.15:0.03:0.05:22.55, about 0.19:0.04:0.06:18.79, about 0.22:0.05:0.07:15.04, about 0.26:0.05:0.08:11.28, about 0.27:0.06:0.08:10.74, about 0.30:0.06:0.09:7.52 or about 0.34:0.07:0.10:3.76. In various embodiments, the 6s:8s:10s:C weight ratio is about 0.27:0.06:0.08:10.74.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 0.15:0.03:0.05:22.55 to about 0.19:0.04:0.06:18.79, about 0.15:0.03:0.05:22.55 to about 0.22:0.05:0.07:15.04, about 0.15:0.03:0.05:22.55 to about 0.26:0.05:0.08:11.28, about 0.15:0.03:0.05:22.55 to about 0.27:0.06:0.08:10.74, about 0.15:0.03:0.05:22.55 to about 0.30:0.06:0.09:7.52, about 0.15:0.03:0.05:22.55 to about 0.34:0.07:0.10:3.76, about 0.19:0.04:0.06:18.79 to about 0.22:0.05:0.07:15.04, about 0.19:0.04:0.06:18.79 to about 0.26:0.05:0.08:11.28, about 0.19:0.04:0.06:18.79 to about 0.27:0.06:0.08:10.74, about 0.19:0.04:0.06:18.79 to about 0.30:0.06:0.09:7.52, about 0.19:0.04:0.06:18.79 to about 0.34:0.07:0.10:3.76, about 0.22:0.05:0.07:15.04 to about 0.26:0.05:0.08:11.28, about 0.22:0.05:0.07:15.04 to about 0.27:0.06:0.08:10.74, about 0.22:0.05:0.07:15.04 to about 0.30:0.06:0.09:7.52, about 0.22:0.05:0.07:15.04 to about 0.34:0.07:0.10:3.76, about 0.26:0.05:0.08:11.28 to about 0.27:0.06:0.08:10.74, about 0.26:0.05:0.08:11.28 to about 0.30:0.06:0.09:7.52, about 0.26:0.05:0.08:11.28 to about 0.34:0.07:0.10:3.76, about 0.27:0.06:0.08:10.74 to about 0.30:0.06:0.09:7.52, about 0.27:0.06:0.08:10.74 to about 0.34:0.07:0.10:3.76 or about 0.30:0.06:0.09:7.52 to about 0.34:0.07:0.10:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 0.15:0.05:22.55:0.44:4.68 to about 0.34:0.10:3.76:0.07:0.78. In various embodiments, the 6s:10s:C:B:D weight ratio is about 0.15:0.05:22.55:0.44:4.68, about 0.19:0.06:18.79:0.37:3.90, about 0.22:0.07:15.04:0.30:3.12, about 0.26:0.08:11.28:0.22:2.34, about 0.27:0.08:10.74:0.21 :2.23, about 0.30:0.09:7.52:0.15:1.56 or about 0.34:0.10:3.76:0.07:0.78. In various embodiments, the 6s:10s:C:B:D weight ratio is about 0.27:0.08:10.74:0.21 :2.23.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 0.15:0.05:22.55:0.44:4.68 to about 0.19:0.06:18.79:0.37:3.90, about 0.15:0.05:22.55:0.44:4.68 to about 0.22:0.07:15.04:0.30:3.12, about 0.15:0.05:22.55:0.44:4.68 to about 0.26:0.08:11.28:0.22:2.34, about 0.15:0.05:22.55:0.44:4.68 to about 0.27:0.08:10.74:0.21 :2.23, about 0.15:0.05:22.55:0.44:4.68 to about 0.30:0.09:7.52:0.15:1.56, about 0.15:0.05:22.55:0.44:4.68 to about 0.34:0.10:3.76:0.07:0.78, about 0.19:0.06:18.79:0.37:3.90 to about 0.22:0.07:15.04:0.30:3.12, about 0.19:0.06:18.79:0.37:3.90 to about 0.26:0.08:11.28:0.22:2.34, about 0.19:0.06:18.79:0.37:3.90 to about 0.27:0.08:10.74:0.21 :2.23, about 0.19:0.06:18.79:0.37:3.90 to about 0.30:0.09:7.52:0.15:1.56, about 0.19:0.06:18.79:0.37:3.90 to about 0.34:0.10:3.76:0.07:0.78, about 0.22:0.07:15.04:0.30:3.12 to about 0.26:0.08:11.28:0.22:2.34, about 0.22:0.07:15.04:0.30:3.12 to about 0.27:0.08:10.74:0.21 :2.23, about 0.22:0.07:15.04:0.30:3.12 to about 0.30:0.09:7.52:0.15:1.56, about 0.22:0.07:15.04:0.30:3.12 to about 0.34:0.10:3.76:0.07:0.78, about 0.26:0.08:11.28:0.22:2.34 to about 0.27:0.08:10.74:0.21 :2.23, about 0.26:0.08:11.28:0.22:2.34 to about 0.30:0.09:7.52:0.15:1.56, about 0.26:0.08:11.28:0.22:2.34 to about 0.34:0.10:3.76:0.07:0.78, about 0.27:0.08:10.74:0.21 :2.23 to about 0.30:0.09:7.52:0.15:1.56, about 0.27:0.08:10.74:0.21 :2.23 to about 0.34:0.10:3.76:0.07:0.78 or about 0.30:0.09:7.52:0.15:1.56 to about 0.34:0.10:3.76:0.07:0.78.
In various embodiments, the composition of any one of the above aspects comprises a 6s:C:B:D weight ratio of about 0.27:10.74:0.21 :2.23, about 0.49:10.74:0.21 :2.23 or about 0.35:10.74:0.21 :2.23. In various embodiments, the composition comprises a 6s:C:B:D weight ratio of about 0.27-0.49:10.74:0.21 :2.23. In various embodiments, the composition comprises a 6s:C:B:D weight ratio of about 0.27-0.35:10.74:0.21 :2.23.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.34:0.07:0.10:3.76:0.07:0.78. In various embodiments, the 6s:8s:10s:C:B:D weight ratio is about 0.15:0.03:0.05:22.55:0.44:4.68, about 0.19:0.04:0.06:18.79:0.37:3.90, about 0.22:0.05:0.07:15.04:0.30:3.12, about 0.26:0.05:0.08:11.28:0.22:2.34, about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.30:0.06:0.09:7.52:0.15:1.56 or about 0.34:0.07:0.10:3.76:0.07:0.78. In various embodiments, the 6s:8s:10s:C:B:D weight ratio is about 0.27:0.06:0.08:10.74:0.21 :2.23.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.19:0.04:0.06:18.79:0.37:3.90, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.22:0.05:0.07:15.04:0.30:3.12, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.26:0.05:0.08:11.28:0.22:2.34, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.30:0.06:0.09:7.52:0.15:1.56, about 0.15:0.03:0.05:22.55:0.44:4.68 to about 0.34:0.07:0.10:3.76:0.07:0.78, about 0.19:0.04:0.06:18.79:0.37:3.90 to about 0.22:0.05:0.07:15.04:0.30:3.12, about 0.19:0.04:0.06:18.79:0.37:3.90 to about 0.26:0.05:0.08:11.28:0.22:2.34, about 0.19:0.04:0.06:18.79:0.37:3.90 to about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.19:0.04:0.06:18.79:0.37:3.90 to about 0.30:0.06:0.09:7.52:0.15:1.56, about 0.19:0.04:0.06:18.79:0.37:3.90 to about 0.34:0.07:0.10:3.76:0.07:0.78, about 0.22:0.05:0.07:15.04:0.30:3.12 to about 0.26:0.05:0.08:11.28:0.22:2.34, about 0.22:0.05:0.07:15.04:0.30:3.12 to about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.22:0.05:0.07:15.04:0.30:3.12 to about 0.30:0.06:0.09:7.52:0.15:1.56, about 0.22:0.05:0.07:15.04:0.30:3.12 to about 0.34:0.07:0.10:3.76:0.07:0.78, about 0.26:0.05:0.08:11.28:0.22:2.34 to about 0.27:0.06:0.08:10.74:0.21 :2.23, about 0.26:0.05:0.08:11.28:0.22:2.34 to about 0.30:0.06:0.09:7.52:0.15:1.56, about 0.26:0.05:0.08:11.28:0.22:2.34 to about 0.34:0.07:0.10:3.76:0.07:0.78, about 0.27:0.06:0.08:10.74:0.21 :2.23 to about 0.30:0.06:0.09:7.52:0.15:1.56, about 0.27:0.06:0.08:10.74:0.21 :2.23 to about 0.34:0.07:0.10:3.76:0.07:0.78 or about 0.30:0.06:0.09:7.52:7.52:7.52 to about 0.34:0.07:0.10:3.76:0.07:0.78.
In various embodiments, the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1.39:22.55 to about 3.13:3.76. In various embodiments, the 6g:C weight ratio is about 1.39:22.55, about 1.74:18.79, about 2.09:15.04, about 2.43:11.28, about 2.48:10.74, about 2.78:7.52 or about 3.13:3.76. In various embodiments, the 6g:C weight ratio is about 2.48:10.74.
In various embodiments, the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1.39:22.55 to about 1.74:18.79, about 1.39:22.55 to about 2.09:15.04, about 1.39:22.55 to about 2.43:11.28, about 1.39:22.55 to about 2.48:10.74, about 1.39:22.55 to about 2.78:7.52, about 1.39:22.55 to about 3.13:3.76, about 1.74: 18.79 to about 2.09: 15.04, about 1 .74: 18.79 to about 2.43: 11 .28, about 1.74: 18.79 to about 2.48:10.74, about 1.74:18.79 to about 2.78:7.52, about 1.74:18.79 to about 3.13:3.76, about 2.09:15.04 to about 2.43:11.28, about 2.09:15.04 to about 2.48:10.74, about 2.09:15.04 to about 2.78:7.52, about 2.09:15.04 to about 3.13:3.76, about 2.43:11.28 to about 2.48:10.74, about 2.43:11.28 to about 2.78:7.52, about 2.43:11.28 to about 3.13:3.76, about 2.48:10.74 to about 2.78:7.52, about 2.48:10.74 to about 3.13:3.76 or about 2.78:7.52 to about 3.13:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6g, 6s and C, wherein the 6g:6s:C weight ratio is about 1.39:0.15:22.55 to about 3.13:0.34:3.76. In various embodiments, the 6g:6s:C weight ratio is about 1.39:0.15:22.55, about 1.74:0.19:18.79, about 2.09:0.22:15.04, about 2.43:0.26:11.28, about 2.48:0.27:10.74, about 2.78:0.30:7.52 or about 3.13:0.34:3.76. In various embodiments, the 6g:6s:C weight ratio is about 2.48:0.27:10.74.
In various embodiments, the composition of any one of the above aspects comprises 6g, 6s and C, wherein 6g:6s:C weight ratio is about 1.39:0.15:22.55 to about 1.74:0.19:18.79, about 1.39:0.15:22.55 to about 2.09:0.22:15.04, about 1.39:0.15:22.55 to about 2.43:0.26:11.28, about 1.39:0.15:22.55 to about 2.48:0.27:10.74, about 1.39:0.15:22.55 to about 2.78:0.30:7.52, about 1.39:0.15:22.55 to about 3.13:0.34:3.76, about 1.74:0.19:18.79 to about 2.09:0.22:15.04, about 1.74:0.19:18.79 to about 2.43:0.26:11.28, about 1.74:0.19:18.79 to about 2.48:0.27:10.74, about 1.74:0.19:18.79 to about 2.78:0.30:7.52, about 1.74:0.19:18.79 to about 3.13:0.34:3.76, about 2.09:0.22: 15.04 to about 2.43:0.26:11.28, about 2.09:0.22:15.04 to about 2.48:0.27:10.74, about 2.09:0.22:15.04 to about 2.78:0.30:7.52, about 2.09:0.22: 15.04 to about 3.13:0.34:3.76, about 2.43:0.26: 11 .28 to about 2.48:0.27:10.74, about 2.43:0.26:11.28 to about 2.78:0.30:7.52, about 2.43:0.26:11.28 to about 3.13:0.34:3.76, about 2.48:0.27:10.74 to about 2.78:0.30:7.52, about 2.48:0.27:10.74 to about 3.13:0.34:3.76 or about 2.78:0.30:7.52 to about 3.13:0.34:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10 and C, wherein the 6g:10:C weight ratio is about 1.39:0.39:22.55 to about 3.13:0.88:3.76. In various embodiments, the 6g:10:C weight ratio is about 1.39:0.39:22.55, about 1.74:0.49:18.79, about 2.09:0.59:15.04, about 2.43:0.69:11.28, about 2.48:0.70:10.74, about 2.78:0.78:7.52 or about 3.13:0.88:3.76. In various embodiments, the 6g:10:C weight ratio is about 2.48:0.70:10.74.
In various embodiments, the composition of any one of the above aspects comprises 6g:10:C, wherein the 6g:10:C weight ratio is about 1.39:0.39:22.55 to about 1.74:0.49:18.79, about 1.39:0.39:22.55 to about 2.09:0.59:15.04, about 1.39:0.39:22.55 to about 2.43:0.69:11.28, about 1.39:0.39:22.55 to about 2.48:0.70:10.74, about 1.39:0.39:22.55 to about 2.78:0.78:7.52, about 1.39:0.39:22.55 to about 3.13:0.88:3.76, about 1.74:0.49:18.79 to about 2.09:0.59:15.04, about 1.74:0.49:18.79 to about 2.43:0.69:11.28, about 1.74:0.49:18.79 to about 2.48:0.70:10.74, about 1.74:0.49:18.79 to about 2.78:0.78:7.52, about 1.74:0.49:18.79 to about 3.13:0.88:3.76, about 2.09:0.59: 15.04 to about 2.43:0.69:11.28, about 2.09:0.59:15.04 to about 2.48:0.70:10.74, about 2.09:0.59:15.04 to about 2.78:0.78:7.52, about 2.09:0.59: 15.04 to about 3.13:0.88:3.76, about 2.43:0.69: 11 .28 to about 2.48:0.70:10.74, about 2.43:0.69:11.28 to about 2.78:0.78:7.52, about 2.43:0.69:11.28 to about 3.13:0.88:3.76, about 2.48:0.70:10.74 to about 2.78:0.78:7.52, about 2.48:0.70:10.74 to about 3.13:0.88:3.76 or about 2.78:0.78:7.52 to about 3.13:0.88:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 1.39:0.21 :0.39:22.55 to about 3.13:0.47:0.88:3.76. In various embodiments, the 6g:8g:10:C weight ratio is about 1.39:0.21 :0.39:22.55, about 1.74:0.26:0.49:18.79, about 2.09:0.31 :0.59:15.04, about 2.43:0.37:0.69:11.28, about 2.48:0.37:0.70:10.74, about 2.78:0.42:0.78:7.52 or about 3.13:0.47:0.88:3.76. In various embodiments, the 6g:8g:10:C weight ratio is about 2.48:0.37:0.70:10.74. In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 1.39:0.21 :0.39:22.55 to about 1.74:0.26:0.49:18.79, about 1.39:0.21 :0.39:22.55 to about 2.09:0.31 :0.59:15.04, about 1.39:0.21 :0.39:22.55 to about 2.43:0.37:0.69: 11.28, about 1.39:0.21 :0.39:22.55 to about 2.48:0.37:0.70:10.74, about 1.39:0.21 :0.39:22.55 to about 2.78:0.42:0.78:7.52, about 1.39:0.21 :0.39:22.55 to about 3.13:0.47:0.88:3.76, about 1.74:0.26:0.49:18.79 to about 2.09:0.31 :0.59:15.04, about 1.74:0.26:0.49:18.79 to about 2.43:0.37:0.69:11.28, about 1.74:0.26:0.49: 18.79 to about 2.48:0.37:0.70: 10.74, about 1.74:0.26:0.49: 18.79 to about 2.78:0.42:0.78:7.52, about 1.74:0.26:0.49:18.79 to about 3.13:0.47:0.88:3.76, about 2.09:0.31 :0.59:15.04 to about 2.43:0.37:0.69:11.28, about 2.09:0.31 :0.59:15.04 to about 2.48:0.37:0.70:10.74, about 2.09:0.31 :0.59:15.04 to about 2.78:0.42:0.78:7.52, about 2.09:0.31 :0.59:15.04 to about 3.13:0.47:0.88:3.76, about 2.43:0.37:0.69:11.28 to about 2.48:0.37:0.70:10.74, about 2.43:0.37:0.69:11.28 to about 2.78:0.42:0.78:7.52, about 2.43:0.37:0.69:11.28 to about 3.13:0.47:0.88:3.76, about 2.48:0.37:0.70:10.74 to about 2.78:0.42:0.78:7.52, about 2.48:0.37:0.70:10.74 to about 3.13:0.47:0.88:3.76 or about 2.78:0.42:0.78:7.52 to about 3.13:0.47:0.88:3.76.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 1.39:0.39:22.55:0.44:4.68 to about 3.13:0.88:3.76:0.07:0.78. In various embodiments, the 6g: 10:C:B:D weight ratio is about 1.39:0.39:22.55:0.44:4.68, to about 1.74:0.49:18.79:0.37:3.90, to about 2.09:0.59:15.04:0.30:3.12, to about 2.43:0.69:11.28:0.22:2.34, to about 2.48:0.70:10.74:0.21 :2.23, to about 2.78:0.78:7.52:0.15:1 .56, to about 3.13:0.88:3.76:0.07:0.78. In various embodiments, the 6g:10:C:B:D weight ratio is about 2.48:0.70:10.74:0.21 :2.23.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 1.39:0.39:22.55:0.44:4.68 to about 1.74:0.49:18.79:0.37:3.90, about 1.39:0.39:22.55:0.44:4.68 to about 2.09:0.59:15.04:0.30:3.12, about 1.39:0.39:22.55:0.44:4.68 to about 2.43:0.69:11.28:0.22:2.34, about 1.39:0.39:22.55:0.44:4.68 to about 2.48:0.70:10.74:0.21 :2.23, about 1.39:0.39:22.55:0.44:4.68 to about 2.78:0.78:7.52:0.15:1.56, about 1.39:0.39:22.55:0.44:4.68 to about 3.13:0.88:3.76:0.07:0.78, about 1.74:0.49:18.79:0.37:3.90 to about 2.09:0.59:15.04:0.30:3.12, about 1.74:0.49:18.79:0.37:3.90 to about 2.43:0.69:11.28:0.22:2.34, about 1.74:0.49:18.79:0.37:3.90 to about 2.48:0.70:10.74:0.21 :2.23, about 1.74:0.49:18.79:0.37:3.90 to about 2.78:0.78:7.52:0.15:1 .56, about 1.74:0.49:18.79:0.37:3.90 to about 3.13:0.88:3.76:0.07:0.78, about 2.09:0.59:15.04:0.30:3.12 to about 2.43:0.69:11.28:0.22:2.34, about 2.09:0.59:15.04:0.30:3.12 to about 2.48:0.70:10.74:0.21 :2.23, about 2.09:0.59:15.04:0.30:3.12 to about 2.78:0.78:7.52:0.15:1.56, about 2.09:0.59:15.04:0.30:3.12 to about 3.13:0.88:3.76:0.07:0.78, about 2.43:0.69:11.28:0.22:2.34 to about 2.48:0.70:10.74:0.21 :2.23, about 2.43:0.69:11.28:0.22:2.34 to about 2.78:0.78:7.52:0.15:1 .56, about 2.43:0.69:11.28:0.22:2.34 to about 3.13:0.88:3.76:0.07:0.78, about 2.48:0.70:10.74:0.21 :2.23 to about 2.78:0.78:7.52:0.15:1 .56, about 2.48:0.70:10.74:0.21 :2.23 to about 3.13:0.88:3.76:0.07:0.78 or about 2.78:0.78:7.52:0.15:1.56 to about 3.13:0.88:3.76:0.07:0.78.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, C, B and D, wherein the 6g:8g:10:C:B:D weight ratio is about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 3.13:0.47:0.88:3.76:0.07:0.78. In various embodiments, the 6g:8g:10:C:B:D weight ratio is about 1.39:0.21 :0.39:22.55:0.44:4.68, about 1.74:0.26:0.49:18.79:0.37:3.90, about 2.09:0.31 :0.59:15.04:0.30:3.12, about 2.43:0.37:0.69:11.28:0.22:2.34, about 2.48:0.37:0.70:10.74:0.21 :2.23, about 2.78:0.42:0.78:7.52:0.15:1.56 or about 3.13:0.47:0.88:3.76:0.07:0.78. In various embodiments, the 6g:8g:10:C:B:D weight ratio is about 2.48:0.37:0.70:10.74:0.21 :2.23.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, C and D, wherein the 6g:8g:10:C:B:D weight ratio is about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 1.74:0.26:0.49:18.79:0.37:3.90, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.09:0.31 :0.59:15.04:0.30:3.12, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.43:0.37:0.69:11.28:0.22:2.34, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.48:0.37:0.70:10.74:0.21 :2.23, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 2.78:0.42:0.78:7.52:0.15:1 .56, about 1.39:0.21 :0.39:22.55:0.44:4.68 to about 3.13:0.47:0.88:3.76:0.07:0.78, about 1.74:0.26:0.49:18.79:0.37:3.90 to about 2.09:0.31 :0.59:15.04:0.30:3.12, about 1.74:0.26:0.49:18.79:0.37:3.90 to about 2.43:0.37:0.69:11.28:0.22:2.34, about 1.74:0.26:0.49:18.79:0.37:3.90 to about 2.48:0.37:0.70:10.74:0.21 :2.23, about 1.74:0.26:0.49:18.79:0.37:3.90 to about 2.78:0.42:0.78:7.52:0.15:1 .56, about 1.74:0.26:0.49:18.79:0.37:3.90 to about 3.13:0.47:0.88:3.76:0.07:0.78, about 2.09:0.31 :0.59:15.04:0.30:3.12 to about 2.43:0.37:0.69:11.28:0.22:2.34, about 2.09:0.31 :0.59:15.04:0.30:3.12 to about 2.48:0.37:0.70:10.74:0.21 :2.23, about 2.09:0.31 :0.59:15.04:0.30:3.12 to about 2.78:0.42:0.78:7.52:0.15:1.56, about 2.09:0.31 :0.59:15.04:0.30:3.12 to about 3.13:0.47:0.88:3.76:0.07:0.78, about 2.43:0.37:0.69:11.28:0.22:2.34 to about 2.48:0.37:0.70:10.74:0.21 :2.23, about 2.43:0.37:0.69:11.28:0.22:2.34 to about 2.78:0.42:0.78:7.52:0.15:1 .56, about 2.43:0.37:0.69:11.28:0.22:2.34 to about 3.13:0.47:0.88:3.76:0.07:0.78, about 2.48:0.37:0.70:10.74:0.21 :2.23 to about 2.78:0.42:0.78:7.52:0.15:1 .56, about 2.48:0.37:0.70:10.74:0.21 :2.23 to about 3.13:0.47:0.88:3.76:0.07:0.78 or about 2.78:0.42:0.78:7.52:0.15:1.56 to about 3.13:0.47:0.88:3.76:0.07:0.78.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78. In various embodiments, the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68, about 1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90, about 2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12, about 2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34, about 2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23, about 2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56 or about
3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78. In various embodiments, the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23.
In various embodiments, composition comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90, about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12, about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34, about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23, about 1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about 2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56, about
1.39:0.21 :0.39:0.15:0.03:0.05:22.55:0.44:4.68 to about
3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78, about
1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90 to about
2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12, about
1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90 to about
2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34, about
1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90 to about
2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23, about
1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90 to about
2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56, about
1.74:0.26:0.49:0.19:0.04:0.06:18.79:0.37:3.90 to about
3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78, about
2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12 to about
2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34, about
2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12 to about
2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23, about
2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12 to about
2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56, about
2.09:0.31 :0.59:0.22:0.05:0.07:15.04:0.30:3.12 to about
3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78, about
2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34 to about
2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23, about
2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34 to about
2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56, about
2.43:0.37:0.69:0.26:0.05:0.08:11.28:0.22:2.34 to about
3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78, about
2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23 to about
2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56, about
2.48:0.37:0.70:0.27:0.06:0.08:10.74:0.21 :2.23 to about
3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78 or about
2.78:0.42:0.78:0.30:0.06:0.09:7.52:0.15:1.56 to about 3.13:0.47:0.88:0.34:0.07:0.10:3.76:0.07:0.78.
In various embodiments, the composition of any one of the above aspects comprises a 6g:8g:10:6s:C:B:D weight ratio of about 2.48:0.37 :0.70:0.27:10.74:0.21 :2.23, about 8:0.93:3.31:0.49:10.74:0.21:2.23 or about 7.1:0.74:2.69:0.35:10.74:0.21:2.23.ln various embodiments, the composition of any one of the above aspects comprises 6s and C, wherein the 6s:C weight ratio is about 1 :151 to about 1:11. In various embodiments, the 6s:C weight ratio is about 1:151 , about 1:101 , about 1:67, about 1:43, about 1:40, about 1:25 or about 1:11. In various embodiments, the 6s:C weight ratio is about 1:40.
In various embodiments, the composition of any one of the above aspects comprises 6s and C, wherein the 6s:C weight ratio is about 1:151 to about 1:101, about 1 :151 to about 1 :67, about 1:151 to about 1:43, about 1:151 to about 1:40, about 1 :151 to about 1:25, about 1:151 to about 1:11, about 1:101 to about 1 :67, about 1:101 to about 1 :43, about 1:101 to about 1 :40, about 1:101 to about 1 :25, about 1:101 to about 1 :11, about 1 :67 to about 1 :43, about 1 :67 to about 1 :40, about 1 :67 to about 1 :25, about 1 :67 to about 1 :11, about 1 :43 to about 1 :40, about 1 :43 to about 1 :25, about 1 :43 to about 1 :11, about 1 :40 to about 1 :25, about 1 :40 to about 1:11 or about 1 :25 to about 1:11.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 5:1:723 to about 5:1:54. In various embodiments, the 6s: 10s:C weight ratio is about 5: 1 :723, about 5: 1 :482, about 5: 1 :321 , about 5:1:207, about 5:1:193, about 5:1 :120 or about 5:1:54. In various embodiments, the 6s:10s:C weight ratio is about 5:1 :193.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s and C, wherein the 6s:10s:C weight ratio is about 5:1:723 to about 5:1:482, about 5:1:723 to about 5:1:321, about 5:1:723 to about 5:1:207, about 5:1 :723 to about 5:1 :193, about 5:1 :723 to about 5:1:120, about 5:1:723 to about 5:1:54, about 5:1 :482 to about 5:1 :321, about 5: 1 :482 to about 5: 1 :207, about 5: 1 :482 to about 5:1 :193, about 5: 1 :482 to about 5:1 :120, about 5:1 :482 to about 5:1:54, about 5:1 :321 to about 5:1:207, about 5:1 :321 to about 5:1:193, about 5:1:321 to about 5:1:120, about 5:1 :321 to about 5:1 :54, about 5:1:207 to about 5:1:193, about 5:1 :207 to about 5:1:120, about 5:1 :207 to about 5:1:54, about 5:1 :193 to about 5:1:120, about 5:1:193 to about 5:1:54 or about 5:1:120 to about 5:1:54.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 5:1:1:723 to about 5:1:1:54. In various embodiments, the 6s:8s: 10s:C weight ratio is about 5:1 :1 :723, about 5:1 :1 :482, about 5:1:1:321 , about 5:1 :1:207, about 5:1:1:193, about 5:1:1:120 or about 5:1 :1:54. In various embodiments, the 6s:8s:10s:C weight ratio is about 5:1 :1:193. In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s and C, wherein the 6s:8s:10s:C weight ratio is about 5:1:1:723 to about 5:1:1:482, about 5:1:1:723 to about 5:1:1:321, about 5:1:1:723 to about 5:1:1:207, about 5:1:1:723 to about 5:1:1:193, about 5:1:1:723 to about 5:1:1:120, about 5:1:1:723 to about 5:1:1:54, about 5:1:1:482 to about 5:1:1:321, about 5:1:1:482 to about 5:1:1:207, about 5:1:1:482 to about 5:1:1:193, about 5:1:1:482 to about 5:1:1:120, about 5:1:1:482 to about 5:1:1:54, about 5:1:1:321 to about 5:1:1:207, about 5:1:1:321 to about 5:1:1:193, about 5:1:1:321 to about 5:1:1:120, about 5:1:1:321 to about 5:1:1:54, about 5:1:1:207 to about 5:1:1:193, about 5:1:1:207 to about 5:1:1:120, about 5:1:1:207 to about 5:1:1:54, about 5:1:1:193 to about 5:1:1:120, about 5:1:1:193 to about 5:1:1:54 or about 5:1:1:120 to about 5:1:1:54.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 5:1:723:14:150 to about 5:1:54:1:11. In various embodiments, the 6s:10s:C:B:D weight ratio is about 5:1:723:14:150, about 5:1:482:9:100, about 5:1:321:6:67, about 5:1:207:4:43, about 5:1:193:4:40, about 5:1:120:2:25 or about 5:1:54:1:11. In various embodiments, the 6s:10s:C:B:D weight ratio is about 5:1:193:4:40.
In various embodiments, the composition of any one of the above aspects comprises 6s, 10s, C, B and D, wherein the 6s:10s:C:B:D weight ratio is about 5:1:723:14:150 to about 5:1:482:9:100, about 5:1:723:14:150 to about 5:1:321:6:67, about 5:1:723:14:150 to about 5:1:207:4:43, about 5:1:723:14:150 to about 5:1:193:4:40, about 5:1:723:14:150 to about 5: 1 : 120:2:25, about 5: 1 :723: 14: 150 to about 5: 1 :54: 1 : 11 , about 5: 1 :482:9: 100 to about 5:1:321:6:67, about 5:1:482:9:100 to about 5:1:207:4:43, about 5:1:482:9:100 to about 5:1:193:4:40, about 5:1:482:9:100 to about 5:1:120:2:25, about 5:1:482:9:100 to about 5: 1 :54: 1 : 11 , about 5: 1 :321 :6:67 to about 5: 1 :207:4:43, about 5: 1 :321 :6:67 to about 5:1:193:4:40, about 5: 1 :321 :6:67 to about 5:1:120:2:25, about 5: 1 :321 :6:67 to about 5: 1 :54: 1 : 11 , about 5: 1 :207:4:43 to about 5:1:193:4:40, about 5: 1 :207:4:43 to about 5: 1 : 120:2:25, about 5: 1 :207:4:43 to about 5: 1 :54: 1 : 11 , about 5:1:193:4:40 to about 5:1:120:2:25, about 5:1:193:4:40 to about 5:1:54:1:11 or about 5:1:120:2:25 to about 5:1:54:1:11.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 5:1:1:723:14:150 to about 5:1:1:54:1:11. In various embodiments, the 6s:8s:10s:C:B:D weight ratio is about 5:1:1 :723: 14: 150, about 5:1:1 :482:9: 100, about 5: 1 : 1 :321 :6:67, about 5:1:1 :207:4:43, about 5:1 :1:193:4:40, about 5:1:1 :120:2:25 or about 5:1:1:54:1:11. In various embodiments, the 6s:8s:10s:C:B:D weight ratio is about 5:1:1:193:4:40.
In various embodiments, the composition of any one of the above aspects comprises 6s, 8s, 10s, C, B and D, wherein the 6s:8s:10s:C:B:D weight ratio is about 5:1:1:723:14:150 to about 5:1:1:482:9:100, about 5:1 :1:723:14:150 to about 5:1 :1:321:6:67, about 5:1 :1:723:14:150 to about 5:1:1:207:4:43, about 5:1 :1:723:14:150 to about 5:1:1:193:4:40, about 5:1:1:723:14:150 to about 5:1:1 :120:2:25, about 5:1:1:723:14:150 to about
5: 1 : 1 :54: 1 : 11 , about 5:1:1 :482:9: 100 to about 5:1:1 :321 :6:67, about 5:1:1 :482:9: 100 to about 5:1 :1 :207:4:43, about 5:1:1 :482:9: 100 to about 5: 1 : 1 : 193:4:40, about 5:1:1 :482:9: 100 to about 5: 1 : 1 : 120:2:25, about 5:1:1 :482:9: 100 to about 5: 1 : 1 :54: 1 : 11 , about 5: 1 : 1 :321 :6:67 to about 5:1:1 :207:4:43, about 5: 1 : 1 :321 :6:67 to about 5: 1 : 1 : 193:4:40, about 5: 1 : 1 :321 :6:67 to about 5: 1 : 1 : 120:2:25, about 5: 1 : 1 :321 :6:67 to about 5: 1 : 1 :54: 1 : 11 , about 5:1:1 :207:4:43 to about 5: 1 : 1 : 193:4:40, about 5:1:1 :207:4:43 to about 5: 1 : 1 : 120:2:25, about 5:1:1 :207:4:43 to about 5: 1 : 1 :54: 1 : 11 , about 5: 1 : 1 : 193:4:40 to about 5:1 :1 : 120:2:25, about 5: 1 : 1 : 193:4:40 to about 5: 1 : 1 :54: 1 : 11 or about 5:1 :1 : 120:2:25 to about 5: 1 : 1 :54: 1 : 11.
In various embodiments, the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1 :16 to about 1:1. In various embodiments, the 6g:C weight ratio is about 1:16, about 1 :11, about 1:7, about 1 :5, about 1 :4, about 1:3 or about 1:1. In various embodiments, the 6g:C weight ratio is about 1 :4.
In various embodiments, the composition of any one of the above aspects comprises 6g and C, wherein the 6g:C weight ratio is about 1 :16 to about 1:11 , about 1:16 to about 1:7, about 1:16 to about 1:5, about 1:16 to about 1:4, about 1:16 to about 1:3, about 1:16 to about 1:1, about 1:11 to about 1:7, about 1:11 to about 1:5, about 1:11 to about 1:4, about 1:11 to about 1:3, about 1 :11 to about 1:1, about 1 :7 to about 1:5, about 1:7 to about 1:4, about 1 :7 to about 1 :3, about 1 :7 to about 1:1, about 1 :5 to about 1 :4, about 1 :5 to about 1 :3, about 1 :5 to about 1:1, about 1 :4 to about 1 :3, about 1 :4 to about 1 : 1 or about 1 :3 to about 1:1.
In various embodiments, the composition comprises 6g, 6s and C, wherein the 6g:6s:C weight ratio is about 9:1:151 to about 9:1:11. In various embodiments, the 6g:6s:C weight ratio is about 9:1:151, about 9:1 :101, about 9:1:67, about 9:1 :43, about 9:1:40, about 9:1:25 or about 9:1 :11. In various embodiments, the 6g:6s:C weight ratio is about 9:1:40.
In various embodiments, the composition of any one of the above aspects comprises 6g, 6s and C, wherein the 6g:6s:C weight ratio is about 9:1:151 to about 9:1:101, about 9:1:151 to about 9:1:67, about 9:1:151 to about 9:1:43, about 9:1:151 to about 9:1:40, about 9:1:151 to about 9:1:25, about 9:1:151 to about 9:1:11, about 9:1:101 to about 9:1:67, about 9:1:101 to about 9:1:43, about 9:1:101 to about 9:1:40, about 9:1:101 to about 9:1:25, about 9:1:101 to about 9: 1 : 11 , about 9: 1 :67 to about 9: 1 :43, about 9: 1 :67 to about 9: 1 :40, about 9: 1 :67 to about 9: 1 :25, about 9: 1 :67 to about 9:1:11, about 9: 1 :43 to about 9: 1 :40, about 9: 1 :43 to about 9: 1 :25, about 9: 1 :43 to about 9:1:11, about 9: 1 :40 to about 9: 1 :25, about 9: 1 :40 to about 9: 1 : 11 or about 9: 1 :25 to about 9: 1 : 11.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10 and C, wherein the 6g:10:C weight ratio is about 4:1:57 to about 4:1:4. In various embodiments, the 6g: 10:C weight ratio is about 4: 1 :57, about 4: 1 :38, about 4: 1 :26, about 4:1:16, about 4:1:15, about 4:1:10 or about 4:1:4. In various embodiments, the 6g:10:C weight ratio is about 4:1:15.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10 and C, wherein the 6g:10:C weight ratio is about 4:1:57 to about 4:1:38, about 4:1:57 to about 4:1:26, about 4:1:57 to about 4:1:16, about 4:1:57 to about 4:1:15, about 4:1:57 to about 4: 1 : 10, about 4: 1 :57 to about 4: 1 :4, about 4: 1 :38 to about 4: 1 :26, about 4: 1 :38 to about 4:1:16, about 4:1:38 to about 4:1:15, about 4:1:38 to about 4:1:10, about 4:1:38 to about 4:1:4, about 4: 1 :26 to about 4:1:16, about 4: 1 :26 to about 4:1:15, about 4: 1 :26 to about 4:1:10, about 4: 1 :26 to about 4:1:4, about 4: 1 : 16 to about 4:1:15, about 4: 1 : 16 to about 4:1:10, about 4:1:16 to about 4:1:4, about 4:1:15 to about 4:1:10, about 4:1:15 to about 4: 1 :4 or about 4: 1 : 10 to about 4:1:4.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 7:1:2:108 to about 7:1:2:8. In various embodiments, the 6g:8g:10:C weight ratio is about 7:1:2:108, about 7:1:2:72, about 7:1:2:48, about 7:1:2:31, about 7:1:2:29, about 7:1:2:18 or about 7: 1:2:8. In various embodiments, the 6g:8g:10:C weight ratio is about 7:1:2:29.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10 and C, wherein the 6g:8g:10:C weight ratio is about 7:1:2:108 to about 7:1:2:72, about 7:1:2:108 to about 7:1:2:48, about 7:1:2:108 to about 7:1:2:31, about 7:1:2:108 to about 7:1:2:29, about 7:1:2:108 to about 7:1:2:18, about 7:1:2:108 to about 7: 1 :2:8, about 7:1:2:72 to about 7:1 :2:48, about 7:1 :2:72 to about 7: 1 :2:31 , about 7: 1 :2:72 to about 7:1 :2:29, about 7:1:2:72 to about 7:1:2:18, about 7:1:2:72 to about 7: 1 :2:8, about 7:1:2:48 to about 7:1:2:31, about 7:1:2:48 to about 7:1:2:29, about 7:1:2:48 to about 7:1:2:18, about 7:1:2:48 to about 7: 1 :2:8, about 7:1 :2:31 to about 7:1 :2:29, about 7:1 :2:31 to about 7:1 :2:18, about 7:1 :2:31 to about 7:1 :2:8, about 7:1 :2:29 to about 7: 1 :2: 18, about 7:1 :2:29 to about 7: 1 :2:8 or about 7:1 :2:18 to about 7:1 :2:8.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 45:13:723:14:150 to about 45:13:54:1 :11. In various embodiments, the 6g:10:C:B:D weight ratio is about 45:13:723:14:150, about 45:13:482:9:100, about 45:13:321 :6:67, about 45:13:207:4:43, about 45:13:193:4:40, about 45:13:120:2:25 or about 45:13:54:1 :11. In various embodiments, the 6g:10:C:B:D weight ratio is about 45:13:193:4:40.
In various embodiments, the composition of any one of the above aspects comprises 6g, 10, C, B and D, wherein the 6g:10:C:B:D weight ratio is about 45:13:723:14:150 to about 45:13:482:9:100, about 45:13:723:14:150 to about 45:13:321 :6:67, about 45:13:723:14:150 to about 45:13:207:4:43, about 45:13:723:14:150 to about 45:13:193:4:40, about 45: 13:723: 14: 150 to about 45: 13: 120:2:25, about 45: 13:723: 14: 150 to about 45: 13:54: 1 : 11 , about 45:13:482:9:100 to about 45:13:321 :6:67, about 45:13:482:9:100 to about 45:13:207:4:43, about 45:13:482:9:100 to about 45:13:193:4:40, about 45:13:482:9:100 to about 45:13:120:2:25, about 45:13:482:9:100 to about 45:13:54:1 :11 , about 45:13:321 :6:67 to about 45:13:207:4:43, about 45:13:321 :6:67 to about 45:13:193:4:40, about
45: 13:321 :6:67 to about 45: 13: 120:2:25, about 45: 13:321 :6:67 to about 45: 13:54: 1 : 11 , about 45:13:207:4:43 to about 45:13:193:4:40, about 45:13:207:4:43 to about 45:13:120:2:25, about 45:13:207:4:43 to about 45:13:54:1 :11 , about 45:13:193:4:40 to about 45:13:120:2:25, about 45:13:193:4:40 to about 45:13:54:1 :11 or about 45:13:120:2:25 to about 45:13:54:1 :11.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, C, B and D, wherein the 6g:8g:10:C:B:D weight ratio is about 45:7:13:723:14:150 to about 45:7:13:54:1 :11. In various embodiments, the 6g:8g:10:C:B:D weight ratio is about 45:7:13:723:14:150, about 45:7:13:482:9:100, about 45:7:13:321 :6:67, about 45:7: 13:207:4:43, about 45:7: 13: 193:4:40, about 45:7: 13: 120:2:25 or about 45:7: 13:54: 1 : 11 . In various embodiments, the 6g:8g:10:C:B:D weight ratio is about 45:7:13:193:4:40.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, C, B and D, wherein the 6g:8g:10:C:B:D weight ratio is about 45:7:13:723:14:150 to about 45:7:13:482:9:100, about 45:7:13:723:14:150 to about 45:7:13:321 :6:67, about 45:7:13:723:14:150 to about 45:7:13:207:4:43, about 45:7:13:723:14:150 to about 45:7:13:193:4:40, about 45:7:13:723:14:150 to about 45:7:13:120:2:25, about 45:7: 13:723: 14: 150 to about 45:7: 13:54: 1 : 11 , about 45:7: 13:482:9: 100 to about 45:7: 13:321 :6:67, about 45:7: 13:482:9: 100 to about 45:7: 13:207:4:43, about 45:7:13:482:9:100 to about 45:7:13:193:4:40, about 45:7:13:482:9:100 to about 45:7: 13: 120:2:25, about 45:7: 13:482:9: 100 to about 45:7: 13:54: 1 : 11 , about 45:7: 13:321 :6:67 to about 45:7:13:207:4:43, about 45:7:13:321 :6:67 to about 45:7:13:193:4:40, about 45:7:13:321 :6:67 to about 45:7:13:120:2:25, about 45:7:13:321 :6:67 to about
45:7: 13:54: 1 : 11 , about 45:7: 13:207:4:43 to about 45:7: 13: 193:4:40, about 45:7: 13:207:4:43 to about 45:7: 13: 120:2:25, about 45:7: 13:207:4:43 to about 45:7: 13:54: 1 : 11 , about 45:7:13:193:4:40 to about 45:7:13:120:2:25, about 45:7:13:193:4:40 to about 45:7: 13:54: 1 : 11 or about 45:7: 13: 120:2:25 to about 45:7: 13:54: 1 : 11 .
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :723:14:150 to about 45:7:13:5:1 :1 :54:1 :11. In various embodiments, the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :723:14:150, about 45:7:13:5:1 :1 :482:9:100, about 45:7:13:5:1 :1 :321 :6:67, about 45:7:13:5:1 :1 :207:4:43, about 45:7:13:5:1 :1 :193:4:40, about 45:7:13:5:1 :1 :120:2:25 or about 45:7:13:5:1 :1 :54:1 :11. In various embodiments, the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :193:4:40.
In various embodiments, the composition of any one of the above aspects comprises 6g, 8g, 10, 6s, 8s, 10s, C, B and D, wherein the 6g:8g:10:6s:8s:10s:C:B:D weight ratio is about 45:7:13:5:1 :1 :723:14:150 to about 45:7:13:5:1 :1 :482:9:100, about 45:7:13:5:1 :1 :723:14:150 to about 45:7: 13:5: 1 : 1 :321 :6:67, about 45:7: 13:5: 1 : 1 :723: 14: 150 to about 45:7:13:5:1 :1 :207:4:43, about 45:7:13:5:1 :1 :723:14:150 to about 45:7:13:5:1 :1 :193:4:40, about 45:7: 13:5: 1 : 1 :723: 14: 150 to about 45:7: 13:5: 1 : 1 : 120:2:25, about 45:7: 13:5: 1 : 1 :723: 14: 150 to about 45:7: 13:5: 1 : 1 :54: 1 : 11 , about 45:7: 13:5: 1 : 1 :482:9: 100 to about 45:7: 13:5: 1 : 1 :321 :6:67, about 45:7: 13:5: 1 : 1 :482:9: 100 to about 45:7:13:5:1 :1 :207:4:43, about 45:7:13:5:1 :1 :482:9:100 to about 45:7:13:5:1 :1 :193:4:40, about 45:7: 13:5: 1 : 1 :482:9: 100 to about 45:7: 13:5: 1 : 1 : 120:2:25, about 45:7: 13:5: 1 : 1 :482:9: 100 to about 45:7: 13:5: 1 : 1 :54: 1 : 11 , about 45:7: 13:5: 1 : 1 :321 :6:67 to about 45:7: 13:5: 1 : 1 :207:4:43, about 45:7: 13:5: 1 : 1 :321 :6:67 to about 45:7: 13:5: 1 : 1 : 193:4:40, about 45:7:13:5:1 :1 :321 :6:67 to about 45:7:13:5:1 :1 :120:2:25, about 45:7:13:5:1 :1 :321 :6:67 to about 45:7:13:5:1 :1 :54:1 :11 , about 45:7:13:5:1 :1 :207:4:43 to about
45:7: 13:5: 1 : 1 : 193:4:40, about 45:7: 13:5: 1 : 1 :207:4:43 to about 45:7: 13:5: 1 : 1 : 120:2:25, about 45:7: 13:5: 1 : 1 :207:4:43 to about 45:7: 13:5: 1 : 1 :54: 1 : 11 , about 45:7: 13:5: 1 : 1 : 193:4:40 to about 45:7: 13:5: 1 : 1 : 120:2:25, about 45:7: 13:5: 1 : 1 : 193:4:40 to about 45:7: 13:5: 1 : 1 :54: 1 : 11 or about 45:7:13:5:1:1 :120:2:25 to about 45:7:13:5:1:1:54:1:11.
In various embodiments, the composition of any one of the above aspects comprises, consists essentially of, or consists of ginger and turmeric.
In various embodiments, the composition of any one of the above aspects comprises ginger from the rhizome of Zingiber officinale Roscoe. In various embodiments the ginger is fresh or dried.
In various embodiments, the composition of any one of the above aspects comprises a ginger extract derived from the rhizome of Zingiber officinale Roscoe. In various embodiments, the ginger extract is derived from dried or fresh ginger.
In various embodiments, the ginger extract is an extract of dried ginger and has a drug extract ratio of about 7:1 (7 parts dry ginger extracted to yield 1 part final extract), about 16:1 or about 12:1.
In various embodiments, the ginger extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting ginger with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication, liquid carbon dioxide and/or other organic solvents. In various embodiments, the ginger extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting ginger with 90 % ethanol, 100% ethanol or DCM. In various embodiments, the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1.
In various embodiments, the composition of any one of the above aspects comprises an artificial ginger composition comprising isolated, pure and/or synthetic 6g, 8g, 10g, 6s, 8s and/or 10s. In various embodiments, the 6g, 8g, 10g, 6s, 8s and/or 10s are synthetic, the 6g, 8g, 10g, 6s, 8s and/or 10s are isolated or purified from ginger, or the 6g, 8g, 10g, 6s, 8s and/or 10s are a combination of purified, isolated and/or synthetic.
In various embodiments, the composition of any one of the above aspects comprises turmeric from the rhizomes Curcuma longa L. In various embodiments, the turmeric fresh or dried. In various embodiments, the composition of any one of the above aspects comprises a turmeric extract derived from the rhizome of Curcuma longa L. In various embodiments, the turmeric extract is derived from fresh or dried turmeric. In various embodiments, the turmeric extract is an extract of dried turmeric and has a drug extract ratio (DER) of about 25:1 (25 parts dry turmeric extracted to yield 1 part final extract).
In various embodiments, the turmeric extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting turmeric with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol or warm water by sonication, liquid carbon dioxide, other organic solvents and/or liquid carbon dioxide. In various embodiments, the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1
In various embodiments, the composition comprises a turmeric extract with a drug extract ratio of 25:1 and the ginger extract has a drug extract ratio of about 7:1, about 16:1 or about 12:1.
In various embodiments, the composition of any one of the above aspects comprises pure, isolated and/or synthetic curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B). In various embodiments, the C, D and/or B are synthetic, the C, D and/or B are isolated and/or purified from turmeric, or the C, D and/or B are a combination of purified, isolated and/or synthetic. In various embodiments, the composition comprises C, D and B; C and D; C and B; or C.
In various embodiments, the composition of any one of the above aspects comprises ginger and turmeric, wherein the turmeric is a turmeric extract and the ginger is a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract has a drug to extract ratio (DER) of about 25:1 and wherein the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1.
In various embodiments, the composition of any one of the above aspects comprises ginger and turmeric, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1, and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1. In another aspect, the present invention provides a pharmaceutical composition comprising the composition of any one of the above aspects. In various embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition and/or pharmaceutical composition of any one of the above aspects.
In another aspect, the present invention provides a method of treating a disease or condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition and/or pharmaceutical composition of any one of the above aspects.
In another aspect, the present invention provides a method of conferring one or more health benefits to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition and/or pharmaceutical composition of any one of the above aspects.
In another aspect, the present invention provides a use of a composition and/or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for reducing inflammation.
In another aspect, the present invention provides a use of a composition and/or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for treating a disease or condition associated with inflammation.
In another aspect, the present invention provides a use of a composition and/or pharmaceutical composition of any one of the above aspects in the manufacture of a medicament for conferring one or more health benefits.
Any of the embodiments described herein may relate to any aspect of the invention.
Any of the features or embodiments or aspects described herein may be combined with one or more of the other features or embodiments or aspects as described herein.
The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. To those skilled in the art to which the invention relates, many changes in construction and widely differing embodiments and applications of the invention will suggest themselves without departing from the scope of the invention as defined in the appended claims. The disclosures and the descriptions herein are purely illustrative and are not intended to be in any sense limiting.
Further aspects of the invention, which should be considered in all its novel aspects, will become apparent to those skilled in the art upon reading of the following description which provides at least one example of a practical application of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:
Figure 1 shows an isobologram of G-T combinations (1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 5:2, 8:2, 9:1 , w/w) at IC50 on NO (A), IL-6 (B) and TNF (C) assays (n≥3).
Figure 2 shows Cl-Fa curves of G-T 5:2 on NO (A), IL-6 (B) and TNF (C) assays (n≥ 3) in RAW264.7 cells.
Figure 3 shows G, T and G-T 5:2 inhibited LPS activated iNOS expression in the RAW264.7 cells by immunofluorescent staining of iNOS (n=3). (A) Images were taken by using a confocal microscope with 40x magnification. Blue: DAPI in the nucleus, green: iNOS in the RAW264.7 cells. Scale bar = 20pm. (B) Quantification of iNOS positive and iNOS negative counting from the immunofluorescent staining. #p<0.05 of iNOS positive compared with blank. *p<0.05 of iNOS positive compared with LPS. ***p<0.001 of iNOS positive compared with LPS.
Figure 4 shows isobolograms (A) of G-T combinations (1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 5:2, 8:2, 9:1 , w/w) at IC50 and Cl-Fa curve (B) of G-T 5:2 on TNF (A) assay in THP-1 cells (n≥3).
Figure 5 shows the activation of Nrf2/H0-1 pathway by G-T 5:2. (A) Nrf2 activation of G, T, G-T 5:2 and tBHQ at selected concentrations (n 3). The maximum activation of Nrf2 by G-T 5:2 was significantly higher than that of G (p<0.05) and higher than T at 25 pg/mL. ***p<0.001 compared between G and T at 25 pg/ml; ###p<0.001 compared between G-T 5:2 and G at 25 pg/mL. (B) The Cl- Fa curve of G-T 5:2 on Nrf2 activation showed that there was strong synergy at all concentration levels of G-T 5:2 in activating Nrf2. (C) MCF 7 AREc32 cells were pre-incubated with DMEM medium (blank), 10 pg/mL of G, T and G-T 5:2 for 24h, and the total protein was collected for western blot analysis using antibodies to HO-1 and B-actin (control). The representative protein bands showed that G-T 5:2, G and T activated the expression of HO-1 (n=3). (D) Quantitative analysis of the protein image bands showed that G-T 5:2 significantly increased HO-1 protein expression relative to B-actin (p<0.05), which was higher than that of G and T. Data was presented as mean ± SEM (n=3). #p<0.05 vs. blank.
Figure 6 shows the effect of 10 pg/mL of G, T, GT (Figures 6A and 6C), and 5 pg/mL of S, C or SC (Figures 6B and 6D) on the TLR4-TRAF6-MAPK pathway in LPS- stimulated RAW264.7 cells. Cells were pre-treated with G, T, GT, S, C, SC 1 h prior to LPS (100 ng/mL) for various optimal time points. Protein expression levels of phosphorylated p38 (p-p38), JNK (p-JNK), CJUN (p-CJUN), and TLR4 and TRAF-6 were analysed by western blot using whole protein extract. All results (n = 3) were expressed as the mean ± SEM, # p<0.05 vs. blank control (B); *p < 0.05, **p <0.01 , ****p<0.0001 vs. LPS-stimulated, untreated cells (L).
Figure 7 shows representative protein bands of phosphorylation of p38, JNK and CJUN protein expression relative to their total protein expressions treated by media (Blank), LPS, G, T and G-T 5:2 (GT) in LPS-induced RAW264.7 cells (n=3). B- actin was used as internal control.
Figure 8 shows expression of p-p38 (A), pJNK (B) and pCJUN (C) quantified by Western blotting after 30 min LPS exposure and normalized to their corresponding total protein (n=3). # p<0.05 LPS vs. blank. ## p<0.01 LPS vs. blank. * p<0.05 compared with LPS, *** p<0.001 compared with LPS.
Figure 9 shows G, T, and G-T 5:2 inhibited LPS activated NF-KB p65 translocation in the RAW264.7 cells by immunofluorescence staining (n=3). (A). Images were taken by using a confocal microscope with 60x magnification. Blue: DAPI in the nucleus, red: NF-KB p65 in the RAW264.7 cells. Scale bar = 10pm. (B). Quantification of % of nuclei positive p65 of staining in unstimulated, LPS stimulated macrophages with and without various treatments. Bar represents 10 pm. Data points represent mean ± standard deviation from analysis of 9 separate images. #### p<0.0001 LPS vs. blank, **p<0.01 compared with LPS, ****p<0.0001 compared with LPS. Figure 10 shows dose-response curves of T/artificial turmeric (AT: mixed curcuminoids equivalent to their content in T) on NO assay (A) and cell viability (B), G-T/G- AT 5:2 on NO assay (C) and G-AT 5:2 (D) on NO production and cell viability. Their synergistic interaction was determined by Cl-Fa curves (E), n=3.
Figure 11 shows S, C and SC inhibited LPS activated iNOS expression in the RAW264.7 cells by immunofluorescent staining. (A) Images were taken by using a confocal microscope with 40x magnification. Blue: DAPI in the nucleus, green: iNOS in the RAW264.7 cells. Scale bar = 20pm. (B) Quantification of iNOS positive and negative counting. #p<0.05 LPS vs. blank. ****p<0.0001 of iNOS positive compared with LPS.
Figure 12 shows isobolograms at IC50, Cl-Fa curves and dose-response curves of SC on murine (A, B and C) and human TNF (D, E and F) assays, n=3.
Figure 13 shows isobolograms at IC50, Cl-Fa curves and dose-response curves of SC, S and C on murine (A, B and C) and human IL-6 (D, E and F) assays, n=3.
Figure 14 shows human TH P-1 cells (differentiated by 50 nM PM A) were treated media,
S (2.5 pg/mL), C (2.5 pg/mL) and SC (2.5 pg/mL), and stimulated with LPS (1 pg/mL) and IFN-y (50 ng/mL) for 24 hours (n=1). (A) Typical cytokine images in cells stimulated with LPS and IFN-y only, pre-treated with S, C and SC before the stimulation. (B) Cytokines that has been detected LPS and IFN-y. (C) The average expression levels of spot density for each cytokine, n=2.
Figure 15 shows isobolograms at IC50 and Cl-Fa curves of SC on Nrf2 activation (n=3).
Figure 16 shows S, C, and SC inhibited LPS activated NF-KB p65 translocation in the RAW264.7 cells by immunofluorescence staining (n=3). (A) Images were taken by using a confocal microscope with 60x magnification. Blue: DAPI in the nucleus, red: NF-KB p65 in the RAW264.7 cells. Scale bar = 10pm. (B) Data points represent mean ± standard deviation from analysis of 3 separate images. *** p<0.001 compared with LPS. # p<0.01 LPS vs. blank.
Figure 17 shows G, T and G-T 5:2 inhibited LPS activated NO expression (A), and thus reduced the endothelial barrier leakage (B) and restored the cell viability of neuron cells (C) in the tri-culture system, n=3. #p<0.05 vs. Blank.
Figure imgf000027_0001
p<0.0001 vs. Blank. *p<0.05 vs. LPS. ** p<0.01 vs. LPS. **** p<0.0001 vs. LPS. Figure 18 shows a simplified diagram of G-T 5:2 actions on the neuroinflammation triculture system.
Figure 19 shows effect of 10 pg/mL of G, T, GT, and 5 pg/mL of S, C or SC combinations on the mmu-microRNA(miR)-155-5p expression in LPS and IFN-y-stimulated RAW264.7ells. Cells were cultured in T25 cell flasks and were pre-treated with G, T, GT, S, C, SC 1 h prior to LPS (1 pg/mL) for 24h. The samples were then subjected to real-time PCR after RNA collection and cDNA synthesis. The mmu-miR-155-5p fold changes compared to untreated cells (B) were expressed as the mean ± SEM, # p<0.05 vs. blank control (B); *p < 0.05, **p <0.01 , ***p<0.001 vs. LPS-stimulated cells.
Figure 20 shows dose-response curves of NO production (% relative to control) in concentrations (0.3125 -10 pg /ml) of ginger solvent extracts in RAW264.7 cells tested by Griess assay.
Figure 21 shows individual and dual response curves of G with T in inhibiting NO. A. Dose-response curves of A. G(DCM), T and G(DCM)-T. B. G(ETOH), T and G(ETOH)-T.
Figure 22 shows the inhibitory effects of G(DCM)-T and G(ETOH)-T (5:2) on NO production (%) versus concentration (pg/ml) as shown in A and D, respectively. C and F, Isobologram curves for G(DCM)-T and G(ETOH)-T (5:2) on NO. B and E, Cl values verses NO inhibition effect (Fa) by ‘Calcusync’ software. (Dotted line is the reference line, where Cl value equals 1 ; solid line represents Cl values at different Fa)
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “comprising” as used in this specification means “consisting at least in part of”. When interpreting statements in this specification which include that term, the features, prefaced by that term in each statement, all need to be present, but other features can also be present. Related terms such as “comprise” and “comprised” are to be interpreted in the same manner.
It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1 , 1.1 , 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.
It will be appreciated that, unless the context clearly indicates otherwise, the term “weight ratio”, or similar, refers to w/w, such that a weight ratio of 2:1 could equivalently be expressed as 2:1 (w/w).
A “subject” may be human or a non-human animal. A "subject" to which administration is contemplated includes, but is not limited to, humans (i.e. , a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and/or other non-human animals, for example, mammals, including primates, commercially relevant mammals (such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female at any stage of development. The animal may be a transgenic animal or genetically engineered animal. In certain embodiments, the subject is non-human animal.
A "patient" as used herein will typically refer to a human subject in need of treatment of a disease.
As used herein, the terms "condition", "disease," and "disorder" are used interchangeably.
As used herein, the terms "treatment," "treat," and "treating" or variations thereof, is to be considered in its broadest context. It may refer to reducing, ameliorating, reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or condition described herein. As used herein, a "treatment" or "treating" of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or the delay, prevention, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. A composition useful in treatment as contemplated herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, provide improvement to a patient or subject's quality of life, or delay, prevent, or inhibit the onset of a disease, disorder, or condition. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
The term "preparation" is intended to include the formulation of the one or more active ingredients with encapsulating materials as a carrier which may provide a capsule in which the active component (with or without other carriers) is surrounded by a carrier, which is thus in association with it. Similarly, sachets are included. Tablets, powders, sachets, and capsules can be used as solid dosage forms suitable for oral administration. If desired for reasons of convenience or patient acceptance, pharmaceutical tablets may be provided in chewable form, using techniques well known in the art.
As used herein, the terms "administer," "administering," and "administration," refer to any method which, in sound medical practice, delivers the composition to a subject in such a manner as to provide a therapeutic effect, and may include implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a composition as herein described in or on a subject.
The term “therapeutically effective” with reference to an amount or dosage of a composition or medicament refers to an amount of a composition that is sufficient to effectively prevent, ameliorate or eliminate inflammation in a subject. The term should not be seen as limiting. It may refer to an amount of a dosage of a composition or medicament that optimises the antiinflammatory effects on a subject depending on desired application.
As used herein, the phrases an "effective amount" or a "therapeutically effective amount" of a composition, or of an active agent or ingredient or pharmaceutically active agent or ingredient (which are synonymous herein), refer to an amount of the composition or pharmaceutically active agent sufficient to have a therapeutic effect upon administration. A therapeutically effective amount of the composition or pharmaceutically active agent may, will, or is expected to elicit a response, such as an anti-inflammatory response, and/or provide a health benefit or therapeutic effect, and/or cause a relief of symptoms, and/or effect a treatment. Effective amounts of the composition or pharmaceutically active agent will vary with the particular condition or conditions being treated or the health benefit to be achieved, the severity of the condition, the duration of the treatment, the specific components of the composition being used, and like factors.
The term "enhancing" the biological activity, function, health, or condition of a cell or an organism refers to the process of augmenting, fortifying, strengthening, or improving.
As used herein the term “and/or” means “and” or “or”, or both.
As used herein “(s)” following a noun means the plural and/or singular forms of the noun.
An “artificial extract” in the context of the invention refers to a composition which comprises one or more of a purified, isolated or synthetic bioactive compound of a natural plant (e.g. ginger or turmeric).
As used herein, “or equivalent” in the phrase “dry weight or equivalent”, or variations thereof, refers to the quantity of extract extracted from a given quantity of dry weight raw material (dry herb). For example, an extract extracted from an equivalent quantity of dry weight raw material and/or an extract comprising the same or similar amount and/or ratio of bioactive compounds as the given quantity of dry weight raw material. It also refers to an equivalent ‘artificial extract’ comprising the same or a similar quantity and/or ratio of bioactive compounds as the given quantity of dry weight raw material (dry herb). In one example, the bioactive compounds are 6-gingerol, 8-gingerol, 10-gingerol, 6shogaol, 8-shogaol, 10- shogaol, curcumin, desmethoxycurcumin and/or bisdemethoxycurcumin.
As used herein, the phrase “drug extract ratio” or “DER” refers a ratio of dry raw material to final extract quantity. Where the final extract is a dry extract, the DER refers to a w/w ratio. Where the final extract is a liquid extract, the DER will generally be understood to refer to a w/v ratio.
The present invention relates to anti-inflammatory compositions comprising turmeric and ginger, and the use of such compositions to provide one or more health benefits and/or one or more anti-inflammatory responses in an animal subject.
Accordingly, in one aspect described herein is a composition comprising turmeric and ginger, wherein the composition elicits a synergistic anti-inflammatory effect. In one particular aspect, described herein is a composition comprising at least one turmeric extract and at least one ginger extract, wherein the composition elicits a synergistic antiinflammatory effect.
Ginger and turmeric comprise a variety of bioactive compounds, including gingerols shogaols and curcuminoids. As described herein, ginger and turmeric compounds 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s), 10-shogaol (10s); and one or more of curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B) , particularly 10s, 6s and C, and more particularly 6s and C, are considered by the inventors, without wishing to be bound by theory, to be at least part responsible for some of the synergistic anti-inflammatory activity associated with certain combinations of turmeric and ginger, or extracts thereof.
In various embodiments, the ginger in the composition described herein is fresh or dried ginger. In various embodiments, the ginger is a ginger extract, such as an extract derived from ginger or an artificial ginger extract. In various embodiments, the ginger extract is derived from dried or fresh ginger.
Ginger comprises bioactive compounds such as shogaols (6-, 8- and 10-shogaol) and gingerols (6-, 8- and 10-gingerol). In various embodiments, the ginger or ginger extract in the composition described herein comprises 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s) and/or 10-shogaol (10s).
In various embodiments, the ginger or ginger extract in the composition described herein comprises: at least two compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least three compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least four compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least five compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; or 6g, 8g, 10g, 6s, 8s and 10s.
Ginger and ginger extracts can be prepared using techniques well known in the art. In various embodiments, the ginger or ginger extract is derived from the whole or any part of the rhizome of Zingiber officinale Roscoe.
Zingiber officinale Roscoe in the composition described herein, or for the preparation of extracts in the composition described herein, may be gathered from the wild and/or cultivated.
In various embodiments, the ginger in the composition described herein is raw ginger or an extract derived from ginger. In various embodiments, the ginger extract is a ginger extract.
Suitable ginger, ginger extracts or raw materials suitable for making such extracts for use in the composition described herein are also commercially available. For example, from commercial suppliers of medicinal plants and spices, and/or extracts thereof. For example, ginger tincture, powder, tablets, liquid drops and capsules. Those skilled in the art would appreciate that other suppliers of suitable raw materials or extracts can be selected using the disclosure and examples presented herein.
Various methods to prepare extracts from Zingiber officinale Roscoe for use in the composition described herein are well known in the art, including solvent extraction methods. For example, solvent extraction comprising extracting ginger with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication, and/or extraction comprising other organic solvents. Another example includes supercritical fluid extraction using liquid carbon dioxide as the solvent. The extraction may also be a sequential extraction.
The ginger extract may be obtained by a solvent extraction method comprising extracting ginger with 90 % ethanol, 100% ethanol or DCM. In one example, the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1, extracted with dichloromethane (DCM) and has a DER of about 16:1 , or is extracted with 100% ethanol and has a DER of about 12:1.
Other examples of ginger extracts include (but are not limited to): ginger extracts with a drug to extract ratio (DER) of 1:2 (Liquid) obtained by extraction with90% aqueous ethanol, DER of 1:2 (Liquid) obtained by extraction with 60-70% aqueous ethanol or a DER of 30:1 (Liquid Cone.) obtained by extraction with 100% carbon dioxide. Accordingly, in various embodiments the ginger extract has a drug to extract ratio (DER) of 1:2 (Liquid) obtained by extraction with 90% aqueous ethanol, a DER of 1:2 (Liquid) obtained by extraction with 60- 70% aqueous ethanol or a DER of 30:1 (Liquid Cone.) obtained by extraction with 100% carbon dioxide
In various embodiments, the ginger extract is an artificial ginger extract. In various embodiments, the artificial ginger extract is a composition comprising one or more bioactive compounds of ginger. In various embodiments, the artificial ginger extract is a composition comprising one or more purified, isolated and/or synthetic bioactive compounds 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol and 10-shogaol.
In various embodiments, the artificial ginger extract comprises isolated, pure and/or synthetic 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6-shogaol (6s), 8-shogaol (8s) and/or 10- shogaol (10s). In various embodiments, the artificial ginger extract comprises: at least two compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least three compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least four compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; at least five compounds selected from 6g, 8g, 10g, 6s, 8s and 10s; or 6g, 8g, 10g, 6s, 8s and 10s.
In certain embodiments, the bioactive compounds 6-gingerol, 8-gingerol, 10-gingerol, 6- shogaol, 8-shogaol and/or 10-shogaol in the artificial ginger extract are isolated or purified from ginger, using techniques well known in the art.
In another embodiment, the bioactive compounds 6-gingerol, 8-gingerol, 10-gingerol, 6- shogaol, 8-shogaol and/or 10-shogaol are synthetic compounds manufactured using techniques well known in the art.
In various embodiments, the composition described herein comprises an artificial ginger extract with a combination of purified, isolated and/or synthetic 6g, 8g, 10g, 6s, 8s and/or 10s.
Curcuminoids are the major bioactive components in turmeric and include curcumin, desmethoxycurcumin and bisdemethoxycurcumin. Of these three, the most prevalent in turmeric is curcumin. In various embodiments, the turmeric or turmeric extract in the composition described herein comprises one or more of curcumin (C), desmethoxycurcumin (D) and bisdemethoxycurcumin (B). In various embodiments, the turmeric comprises C, D and B; C and D; C and B; or C.
In various embodiments, the turmeric in the composition described herein is from the rhizomes of Curcuma longa. In various embodiments, the turmeric is fresh or dried turmeric. In various embodiments, the turmeric a turmeric extract from the rhizomes of Curcuma longa. In various embodiments, the turmeric extract is an extract of dried turmeric. In various embodiments, the turmeric extract is a semi-refined turmeric extract from the rhizomes of Curcuma longa.
Turmeric and turmeric extracts in the compositions described herein can be prepared using techniques well known in the art.
Curcuma longa L. in the composition described herein, or for the preparation of turmeric extracts in the composition described herein, may be gathered from the wild and/or cultivated.
Suitable turmeric, turmeric extracts or raw materials suitable for making such extracts for use in the composition described herein are commercially available, for example from Integria Healthcare (Australia) Pty Ltd (Eight Mile Plains, QLD, Australia) or from other commercial suppliers of medicinal plants and spices, and/or extracts thereof. Those skilled in the art would appreciate that other suppliers of suitable raw materials or extracts can be selected using the disclosure and examples presented herein.
Various methods to prepare turmeric extracts from Curcuma longa L. for use in the composition described herein are well known in the art, including for example solvent extraction and sonication. In various embodiments, the turmeric extract is a semi-refined turmeric extract. In various embodiments, the turmeric extract is obtained by extraction with ethyl acetate (EA), 100% hexane, dichloromethane (DCM), alcohol, aqueous alcohol, ethanol, methanol, warm water by sonication and/or extraction comprising other organic solvents. Another example includes extracting turmeric with liquid carbon dioxide.
The turmeric extract may be an extract of dried turmeric with a DER of 25:1 (25 parts dry turmeric extracted to yield 1 part final extract). In one example, the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1. Other examples of suitable turmeric extracts include (but are not limited to) turmeric extracts with a drug extract ratio (DER) of 1:1 (Liquid) obtained by extraction with69% aqueous ethanol, a DER of 25:1 (Dry Cone.) obtained by extraction with99% ethyl acetate or a DER of 55:1 (Dry Cone.) obtained by extraction with93% aqueous ethanol. Accordingly, in various embodiments the turmeric in the composition described herein is an extract selected from a turmeric extract with a drug extract ratio (DER) of 1 :1 (Liquid) obtained by extraction with 69% aqueous ethanol, a DER of 25:1 (Dry Cone.) obtained by extraction with 99% ethyl acetate or a DER of 55:1 (Dry Cone.) obtained by extraction with 93% aqueous ethanol.
In various embodiments, the turmeric extract is an artificial turmeric extract. In various embodiments, the artificial turmeric extract is a composition comprising one or more of the bioactive compounds of turmeric. In various embodiments, the artificial turmeric extract is a composition comprising one or more of the bioactive compounds curcumin (C), desmethoxycurcumin (D) and bisdemethoxycurcumin (B). In various embodiments, the artificial turmeric composition comprises, consists essentially of or consists of C, D and B; C and D; C and B; or C.
In various embodiments, the C, D and/or B are synthetic. In various embodiments, the C, D and/or B are isolated and/or purified from turmeric. In various embodiments, the composition of the invention comprises an artificial turmeric extract with a combination of purified, isolated and/or synthetic C, D and/or B.
In certain embodiments, the bioactive compounds C, D and/or B in the artificial turmeric composition are isolated from turmeric by methods that are well known to a person skilled in the art.
In another embodiment, the bioactive compounds C, D and/or B are synthetic.
In various embodiments, the composition described herein comprises, consists essentially of, or consists of the bioactive ingredients: a) ginger, an extract derived from ginger and/or an artificial ginger extract; and b) turmeric, an extract derived from turmeric and/or an artificial turmeric extract.
In various embodiments, the composition described herein comprises, consists essentially of, or consists of the bioactive compounds: a) 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol and/or 10-shogaol; and b) curcumin, desmethoxycurcumin and/or bisdemethoxycurcumin.
It will be appreciated that in the context of the present invention a synergistic effect, such as a synergistic effect mediated by a composition described herein, is an effect that is of greater magnitude than the sum of the effects elicited by the individual active constituents alone. Similarly, it will be appreciated on reading this specification that synergism, such as that embodied in a synergistic response, can be assessed and determined in a number of different ways, including by methods described and exemplified herein, such as isobologram and combination index (Cl) models based on the Loewe Additivity principle well known in the art and described and exemplified herein.
It will also be appreciated that, in the context of the invention, a synergistic effect can be a lowered effective dosage compared to the effective dosage of the individual active constituents alone, a reduction in cytotoxicity compared to the cytotoxicity of the individual constitutions alone, or an enhanced anti-inflammatory activity compared to the antiinflammatory activity of the individual constitutions alone.
In various embodiments, the synergistic effect mediated by the composition described herein is an enhanced anti-inflammatory activity. In various embodiments, the composition described herein is an anti-inflammatory composition.
In various embodiments the anti-inflammatory effect is activation of the Nrf2/H0-1 antiinflammatory pathway, or inhibition of the MAPK/JNK, TLR4-TRAF6-MAPK and/or IKK/NFKB inflammatory pathways. In various embodiments the anti-inflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators. For example, pro- and/or secondary-inflammatory mediators. For example, in various embodiments the antiinflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more of IL-6, nitric oxide (NO), TNF, I L-1 a, I L-1 b, eotaxin, interferon-y (IFN-y), IL- 12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1, MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155. In various embodiments the antiinflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more of the above inflammatory mediators by one or more immune cells, including one or more mammalian immune cells. For example, pro- and/or secondary-inflammatory mediators. For example, in various embodiments the anti-inflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more of IL-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL- 13, G-CSF, GM-CSF, KC, MCP-1, MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL- 10, 1 L-17 and/or miRNA-155-5p by one or more immune cells, including one or more mammalian immune cells. In one example, the one or more immune cells are macrophages or monocytes.
In various embodiments the anti-inflammatory effect is a decrease in the expression and/or inhibition of elevated expression of one or more proinflammatory chemokines. In one example, the chemokines are associated with the acute inflammatory phase. In one example, the one or more chemokines are selected from CXCL5, IL-8 (CXCL8), CXCL10, CXCL11 , MIG, and/or MIP-3a.
Accordingly, in various embodiments the composition described herein elicits a synergistic activation of the Nrf2/H0-1 anti-inflammatory pathway, or inhibition of the MAPK/JNK, TLR4- TRAF6-MAPK and/or IKK/NFKB inflammatory pathways.
Accordingly, in various embodiments the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators. In various embodiments the composition described herein elicits a synergistic decrease in pro- and/or secondary-inflammatory mediators. In various embodiments the composition described herein elicits a synergistic decrease in any one or more of IL-6, NO, TNF, IL-1 a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1, MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p.
Accordingly, in various embodiments the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators by one or more immune cells, including one or more mammalian immune cells. In various embodiments the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of pro- and/or secondary-inflammatory mediators by one or more immune cells, including one or more mammalian immune cells. In various embodiments the composition described herein elicits a synergistic decrease in the expression and/or inhibition of elevated expression of IL-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p by one or more immune cells, including one or more mammalian immune cells. In one example, the one or more immune cells are macrophages or monocytes
In various embodiments, the synergistic effect, such as a synergistic effect mediated by a composition described herein, is an enhanced anti-neuroinflammatory activity.
In one aspect, the invention provides a method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition as described herein.
In another aspect, the invention provides a method of treating a disease or condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition as described herein.
In various embodiments, the inflammation, or the disease or condition associated with inflammation, is associated with an increase in expression of IL-6, NO, TNF, IL-1 a, I L-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p. In various embodiments, the inflammation or the disease or condition associated with inflammation, is associated with activation of MAPK/JNK, TLR4-TRAF6-MAPK and/or I KK/NFKB inflammatory pathways.
In various embodiments, the inflammation, or disease or condition associated with inflammation, is associated with an increase in the expression and/or elevated expression of one or more proinflammatory chemokines. In various embodiments, the chemokines are associated with the acute inflammatory phase. In one example, the one or more chemokines are selected from CXCL5, IL-8 (CXCL8), CXCL10, CXCL11 , MIG, and/or MIP-3a. In various embodiments, the inflammation, or the disease or condition associated with inflammation, is associated with a cytokine storm. In various embodiments, the cytokine storm is an LPS and IFN-y induced cytokine storm. In another particular embodiment, the cytokine storm is induced by a viral infection.
In various embodiments, the inflammation is chronic inflammation. In various embodiments, the chronic inflammation is neuroinflammation.
In various embodiments, the disease or condition associated with inflammation is associated with chronic inflammation. In various embodiments, the disease or condition associated with chronic inflammation is selected from the group consisting of: cardiovascular disease, cancer, diabetes, metabolic syndrome, autoimmune diseases, depression, declining cognitive function, Alzheimer’s and other dementias, and age-related macular degeneration. In various embodiments, the disease or condition associated with inflammation is associated with neuroinflammation.
In various embodiments, the disease or condition associated with inflammation is selected from the group consisting of: atherosclerosis, pain, depression, hyperlipidemia, atherogenesis, blood clots, hypertension, asthma, wounds, aging, hypoglycaemia, arthritis, cytokine storm, sepsis, ulcers.
In various embodiments, the inflammation is reduced by, or the disease or condition associated with inflammation treated by, a synergistic activation of the Nrf2/H0-1 antiinflammatory pathway, or inhibition of the MAPK/JNK, TLR4-TRAF6-MAPK and/or I KK/NFKB inflammatory pathways; a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators; a synergistic decrease in pro- and/or secondary-inflammatory mediator; a synergistic decrease in any one or more of interleukin (IL)-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p; a synergistic decrease in the expression and/or inhibition of elevated expression of one or more inflammatory mediators by one or more immune cells, including one or more mammalian immune cells; a synergistic decrease in the expression and/or inhibition of elevated expression of pro- and/or secondary-inflammatory mediators by one or more immune cells, including one or more mammalian immune cells; a synergistic decrease in the expression and/or inhibition of elevated expression of IL-6, NO, TNF, IL-1a, IL-1 b, eotaxin, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, IL-5, IL-9, IL-13, G-CSF, GM-CSF, KC, MCP-1 , MIP-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17 and/or miRNA-155-5p by one or more immune cells, including one or more mammalian immune cells; or anti-neuroinflammatory activity. In various embodiments, the one or more immune cells are macrophages or monocytes.
In one aspect, the invention provides a method of conferring one or more health benefits to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition as described herein.
In various embodiments, the one or more health benefits is selected from the group comprising: supporting healthy immune function, improving acute inflammation or pain, improving chronic inflammation or decreasing susceptibility to diseases linked to chronic inflammation.
In various embodiments, the one or more health benefits is selected from the group comprising: a decrease in inflammatory mediators, one or more anti-atherosclerotic effects, one or more immunological benefits, one or more analgesic effects, one or more antidementia effects, one or more anti-depressant effects, one or more cognitive benefits, one or more chemopreventive effects, one or more anti-cancer effects, one or more hypolipidemic effects, one or more anti-atherogenic effects, one or more anti-platelet and/or anti-thrombotic effects, one or more anti-diabetic effects, one or more anti-hypertensive effects, one or more anti-asthma effects, one or more wound-healing effects, one or more anti-aging effects, one or more hypoglycemic effects, one or more anti-arthritis effects, one or more anti-cytokine storm effects, one or more anti-sepsis effects and one or more anti-ulcer effects, wherein one or more of the above potential therapeutic effects are derived from the anti-inflammatory properties of the compositions described herein.
In certain embodiments, the subject is a mammal. The mammal may be of either sex and may be at any stage of development. In certain embodiments, the subject is a human. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In certain embodiments, the animal is a genetically engineered animal. In certain embodiments, the animal is a transgenic animal.
In various embodiments, the composition as described in any one or more of the embodiments described herein is a pharmaceutical composition. In various embodiments, the composition further comprises one or more pharmaceutically acceptable carriers.
It will be appreciated by those skilled in the art that the particular method of reducing inflammation, method of treating a disease or condition associated with inflammation, or method of conferring one or more health benefit as described herein, will employ a selected route of administration which will depend on a variety of factors, all of which are considered routinely when administering compositions to a subject. It will be further appreciated by one skilled in the art that the optimal course of administration of the composition described herein i.e. , the mode of administration, the dose and the daily number of doses of the composition of this invention given for a defined number of days, can be ascertained by those skilled in the art using conventional treatment tests.
The specific dose level selected for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the condition undergoing therapy.
Dosage levels of the active ingredients in the compositions may be varied so as to provide an amount of the active ingredient which is effective to achieve the desired therapeutic effect for a particular patient, composition, and mode of administration, without being toxic to the patient.
In an example, the composition is administered to deliver about 50 to 50,000 mg per day to an adult human subject. In another example, the composition is administered to deliver about 500 to 2000 mg per day of turmeric or about 500 to 2000 mg per day of curcumin to an adult human subject, and/or about 500 to 3000 mg per day of ginger to an adult human subject.
In various embodiments, the composition is formulated for topical application. In various embodiments, the topical composition is formulated as a cream, ointment, liniment, spray or plaster.
In various embodiments, the composition is formulated for oral administration. In various embodiments, the oral composition is a liquid, tablet, capsule, powder or wafer.
In various embodiments, the compositions described herein comprise one or more pharmaceutically acceptable carriers, excipients, or diluents, as required.
In various embodiments, the composition is a liquid composition. Liquid compositions include solutions, suspensions, and emulsions. In one example, the liquid composition comprises a solvent, for example an organic solvent or aqueous organic solvent. In one example, the liquid composition comprises a solvent, for example ethanol, methanol or ethyl acetate.
Solutions can be prepared by combining the active ingredient(s) with a suitable solvent, such as an organic solvent or aqueous organic solvent and optionally adding suitable colorants, flavors, stabilizers and thickening agents as desired.
In another embodiment, the composition is a solid preparation or dosage form, such as a powder, tablet, dispersible granule, capsule, sachet, or suppository.
A carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders or tablet disintegrating agents. It can also be an encapsulating material. In powders, the carrier will typically be a finely divided solid which is in admixture with the active ingredients. In tablets, the actives are mixed with a carrier having the necessary binding properties in suitable proportions and compacted to the shape and size desired. Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, silicas including colloidal silicas, glycollates, low melting point wax, cocoa butter, and the like.
In various embodiments, the composition described herein is formulated to increase the oral bioavailability of the 6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, curcumin, desmethoxycurcumin or bisdemethoxycurcumin, or combinations thereof, present in the composition. Methods of increasing bioavailability are well known to a person skilled in the art (see for example Stohs SJ et al., Molecules. 2020;25(6):1397). In various embodiments, the compositions described herein comprise a carrier selected from lipids (such as liposomes, phytosomes), cyclodextrin, whey protein, soy lecithin or soluble fibre (such as galactomannans from Fenugreek, also known as Trigonella foenum-graecum), micro-particle technology and nano-particle technology. In various embodiments, the compositions described herein are formulated for administration by a delivery system selected from micelles, liposomes, phospholipid complexes, microemulsions, nanoemulsions, emulsions, solid lipid nanoparticles, nanostructured lipid carriers, biopolymer nanoparticles and microgels.
Solid dosage forms are in certain embodiments formulated in single or divided dosage forms (such as multiple tablets). Those skilled in the art will appreciate that multiple tablets may be required to deliver certain doses and achieve the required patient compliance. In various embodiments of the methods or uses described herein, the ginger and turmeric are administered as a single formulation or administered as a separate ginger formulation and a separate turmeric formulation. In various embodiments of the method or use described herein, the ginger and turmeric are administered simultaneously, sequentially, or separately in separate dosage forms. The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.
Wherein in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.
It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the scope of the invention.
The foregoing description of the invention includes preferred forms thereof. Modifications may be made thereto without departing from the scope of the invention
EXAMPLES
The examples describe the analysis and preparation of various extracts and compositions, and analyses of the anti-inflammatory effects of such extracts and compositions.
EXAMPLE 1.0
MATERIALS AND METHODS
Preparation of herbal samples and their chemical compounds
Crude material of ginger and semi-refined powder of turmeric (LOT NO. E17E041) was obtained from Integria Healthcare (Australia) Pty Ltd, Eight Mile Plains, OLD, Australia. Crude material of ginger (100 g) was ground and extracted in 90% ethanol (1 L) by sonication. The supernatant was decanted and filtered. The collected supernatant was then dried by rotary evaporation. This ginger extract had a drug extract ratio of 7:1 (7 parts dry ginger was extracted to yield 1 part final extract).
The semi-refined powder of turmeric was dissolved in ethyl acetate and mixed by sonication. The supernatant was filtered and collected. This turmeric extract had a drug extract ratio of 25:1 (25 parts dry turmeric was extracted to yield 1 part final extract).
The filtered extracts of ginger (G) and turmeric (T) were dried and stored at -20°C until further use. The chemical profile of ginger and turmeric was analysed by high-performance liquid chromatography with established methods ( Lee, S., Khoo, C., Halstead, C. W., Huynh, T., & Bensoussan, A., 2007. Liquid chromatographic determination of 6-, 8-, 10- gingerol, and 6-shogaol in ginger (Zingiber officinale) as the raw herb and dried aqueous extract. Journal of AOAC international. 90, 5, 1219-1226; and ; XXII, U., & XVII, N., 1990. The United States Pharmacopeia. The National Formulary, 1483-1488). The validation parameters of the established method are shown in supplementary material 1. For the preparation of the combined extracts of ginger and turmeric (G-T), the individual extracts at the same concentration (50 mg/mL in dimethyl sulfoxide (DMSO)) were mixed by volume in different ratios (1 :9, 2:8, 3:7...9:1 , and 5:2), transferred to bioassays and tested in parallel with the single G and T extract.
Chemical standards for ginger including 6-gingerol (6g), 8-gingerol (8g), 10-gingerol (10g), 6- shogaol (6s), 8-shogaol (8s), 10-shogaol (10s) and those for turmeric including curcumin (C), demethyxocurcumin (D) and bisdemethyxocurcumin (B) were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China; purity >98%) and were used as marker compounds. The identity and purity of standards were verified via the established HPLC-PDA method. Their quantified amount is shown in supplementary material 2. The standard stock solutions of these reference compounds were prepared in DMSO and stored at -20°C until further use.
Cell culture
Murine RAW264.7 macrophages cells were cultured at 37°C in Dulbecco's Modified Eagle Medium (DM EM, Lonza, Victoria, Australia) supplemented with 5% foetal bovine serum (FBS; Life Technologies, Victoria, Australia), and 1% penicillin-streptomycin (Life Technologies, Victoria, Australia) in a humidified atmosphere containing 5% CO2 and 95% air. Human monocytic THP-1 cells were maintained in culture medium (RPMI 1640, Lonza) containing 10% of FBS and 1 % penicillin-streptomycin. THP-1 cells monocytes were differentiated into macrophages by 24 h incubation with 100 nM phorbol 12-myristate 13- acetate (PMA, Sigma, Australia).
Nitric oxide assay
The nitric oxide (NO) production stimulated by LPS and murine interferon-gamma (IFN-y; Sigma, Australia) in RAW264.7 cells was measured by its stable metabolite nitrite based on the Griess reaction ( Zhou, X., Razmovski-Naumovski, V., Chang, D., Li, C., Kam, A., Low, M., et al., 2016. Synergistic effects of danshen (salvia miltiorrhiza radix et rhizoma) and sanqi (notoginseng radix et rhizoma) combination in inhibiting inflammation mediators in RAW264. 7 cells. BioMed Research International. 2016.). Lipopolysaccharides from Escherichia coli 0111 :B4 purified by trichloroacetic acid extraction (LPS, Sigma, batch number 070M4018, Australia) and murine interferon (IFN)-y (Lonza, Australia) were used to stimulate inflammatory mediators including NO, tumor necrosis factor (TNF), and IL-6. Briefly, cells (density at 1 x lO6/mL) were seeded on 96-well cell culture plate (Corning Costar, Sigma, Australia) and incubated for 48 hours followed by pre-treatments with various extracts. After the cells were incubated for 2 hours, 50 ng/mL of LPS and 50 ng/mL murine IFN-y was added to the cells and co-incubated for another 24 hours. After the LPS and IFN-y stimulation, 100 pL of cells supernatant was collected and mixed with Griess reagent (1 % sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride in Milli-Q water) for NO measurement. The rest of the cell supernatant was used for TNF and interleukin (I L)-6 ELISA assay. The plate with mixed supernatant and Griess reagent was monitored under 540 nm using a microplate reader (BMG LABTECH FLUOstar OPTIMA, Mount Eliza, Victoria, Australia).
TNF and IL-6 ELISA assay
The cell supernatants from RAW264.7 cells treated with various samples were collected 24 hours after stimulation by LPS and IFN-y. The stored supernatants from RAW264.7 cells were analysed for TNF and IL-6 synthesis using commercial ELISA kits (Lonza, Australia) according to the manufacturer’s instructions. The absorbance was measured at 410 nm ( Zhou, X., 2016. Synergistic behaviour of Salvia and Notoginseng species in vascular diseases.).
The THP-1 cells were primed with PMA (100 nM, Sigma, Australia) and were co-incubated with various samples for 2 hours before the stimulation with LPS (1 pg/mL) for another 24 hours to enable the measurement of TNF using commercial ELISA kits (Lonza, Australia). Cell viability assays
The cytotoxicity of tested samples was controlled by methyl thiazolyl tetrazolium (MTT) and Alamar Blue assay. After the media was withdrawn from cells, MTT (0.12 mg/mL, Thermo Fisher Scientific, Australia) was added to the cells and incubated for 4 hours. The supernatant was then discarded and replaced with 100 pL dimethyl sulfoxide (DMSO, Sigma, Australia) and the optical density was measured using a microplate reader (BMG l_ABTECH FLUOstar OPTIMA, Mount Eliza, Victoria, Australia) at 510 nm. The density of formazan formed in control cells (medium with 0.1% DMSO) was taken as 100% of cell viability ( Zhou, X., Razmovski-Naumovski, V., Chang, D., Li, C., Kam, A., Low, M., et al., 2016., BioMed research international. 2016.).
For the Alamar Blue assay, 100 pL of 0.01 mg/mL of resazurin (Sigma, Australia) was added to the cells after the media was withdrawn. The cells with resazurin were incubated at 37°C for 2 h, and the viability of the cells was measured for fluorescence intensity (Ex: 530 nm and Em: 590 nm) in a microplate reader ( Liu, J., Chen, X., Ward, T., Pegram, M., & Shen, K., 2016., Tumor Biology. 37, 7, 9825-9835. 10.1007/s13277-015-4650-1).
Multi-plex assays
A release of 23 inflammatory mediators from RAW264.7 cells in response to LPS and IFN-y was quantified by the multiplex assay. Briefly, the cells were seeded at 1 x 106 cells/mL in a 96 well plate for 48 h. The samples of interest were added in DMSO (final concentration of 0.1 %). The supernatant was collected and centrifuged for the assay. The multi-bioplex assay was conducted using the Bio-Plex 200 system with a commercial kit (Bio-plex Pro Mouse Cytokine Grp I Panel 23-Plex from BIO-RAD, catalogue number. #M60009RDPD) according to the instructions from the supplier ( Kim, Y. J., & Park, W., 2016. Anti-Inflammatory Effect of Quercetin on RAW264.7 Mouse Macrophages Induced with Polyinosinic-Polycytidylic Acid. Molecules. 21 , 4, 450.). The 23 pro-inflammatory cytokines included I L-1 a, I L-1 b, eotaxin, IL- 6, interferon-y (IFN-y), IL-12 (P40), IL-12 (P70), RANTES, TNF, IL-5, IL-9, IL-13, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM- CSF), monocyte chemoattractant protein-1 (MCP-1), macrophage Inflammatory Protein-1 (MIP)-1a, MIP-1 b, IL-2, IL-3, IL-4, IL-10, IL-17. The up-regulation after the stimulation of LPS and IFN-y was analysed by unpaired t-test using GraphPad Prism 5.
In addition, THP-1 cells were seeded in a 24 well plate overnight, and incubated with S (2.5 pg/mL), C (2.5 pg/mL) and SC (2.5 pg/mL) for 1h followed by the stimulation with LPS (1 pg/mL) and human recombinant IFN-y (50 ng/mL). After 24h, the cell supernatant was collected and the total protein amount was determined by the Pierce BCA Protein Assay (Thermo Fisher Scientific, Australia). The cell viability was controlled by the Alamar blue assay. The equal amount of total protein collected from each sample was applied to the human XL Cytokine Array Kit (R&D, Australia) based on the manufacturers’ protocol.
Determination of synergistic, additive or antagonistic interactions
Potential interactions between ginger and turmeric extracts/compounds were determined using CompuSyn software 2.0 (Biosoft, USA) based on the Chou-Talalay’s method. The concentration-response curves of the individual and combined extract/compounds pertaining to the bioassays were constructed and input to the program. A Cl-fraction affected (Cl-Fa) curve and isobologram figure were then generated. The Cl values demonstrated the interaction: synergism (Cl < 1), additive effect (Cl = 1), and antagonism (Cl > 1) (Zhou, X., Seto, S. W., Chang, D., Kiat, H., Razmovski-Naumovski, V., Chan, K., et al., 2016., Frontiers in Pharmacology. 7, 201.). The Cl-Fa curve suggested the correlation between the Cl values and the effective levels on a certain biological target (i.e., the suppressive effect on NO) (Zhou, X., Razmovski-Naumovski, V., Kam, A., Chang, D., Li, C., Bensoussan, A., et al., 2017., Medicines. 4, 4, 85, and Zhou, X., Razmovski-Naumovski, V., Kam, A., Chang, D., Li, C. G., Chan, K., et al., 2019., BMC complementary and alternative medicine. 19, 1 , 50.).
Assessment of iNOS expression and NF-KB nuclear translocation
Intracellular inducible nitric oxide synthase (iNOS) expression and nuclear factor kappa B (NF- KB) nuclear translocation were assessed using fluorescence microscopy. RAW264.7 cells were plated in the 8 well glass chamber at 20,000 cells/well, incubated overnight, and then co-incubated with samples of interest or vehicle (0.1 % DMSO) in DM EM serum-free medium for 1 hour prior to LPS (50 ng/mL) for 30 minutes. Cells were then washed with ice-cold phosphate-buffered saline (PBS) and fixed using 2% paraformaldehyde for 30 minutes at room temperature. Triton X 100 (0.1%) was used to permeabilize the cells for 20 minutes. Cells were then washed again with PBS (three times) and blocked using goat serum for 1 hour followed by overnight incubation at 4°C with mouse inducible NO synthesis (iNOS) primary antibody (1 :100, Cell Signaling Technology, USA) or rabbit anti-p65 NFKB antibody (1 :200, Cell Signalling Technology, USA). Cells were washed with PBS and incubated with goat antimouse IgG conjugated with Alex Fluor 488 (green dye, 1 :1000, Thermo Fisher Scientific, Australia) or anti-rabbit IgG conjugated with Alexa Fluor 594 (red dye, 1 :1000, Thermo Fisher Scientific, Australia) for 1 hour in a dark room at room temperature. After washing with PBS, the chambers were removed and the anti-fade mounting media with DAPI dye (blue color, Sigma, Australia) was added before imaging with a LSM510 confocal microscope. Images were quantified and analysed using Image J. Samples were run in duplicates and over 10 representative cells were chosen at random in each treatment well to assess iNOS or NFKB location. The results were processed as the corrected total cell fluorescence (CTCF) which was corrected against the background (no fluoresce).
Nrf-2 luciferase assay
MCF-7 AREc32 cells (transfected with Nrf2) were cultured and seeded at a density of 1.0 x 106 cells/mL in 96 well plates. After 48 hours’ incubation, the cells were activated with tert- Butylhydroquinone (tBHQ, Sigma, Australia) as a positive control, samples of interest, or medium only (negative control). Cells were then incubated with Alamar Blue (0.01 mg/mL resazurin) to examine the cell viability. After reading the plates under excitation at 530 nm and emission at 590 nm, the Alamar Blue solution was replaced with a triton lysis buffer (tris HCI: 1.705%, tris base: 0.508%, 5M NaCI: 1.5%, 1M MgCI2: 0.3%, Triton X 100 pure liquid: 0.75%) for 30 minutes. The cells were then transferred to a new white micro-tire plate and mixed with luciferin buffer (D-luciferin 30 mg/mL: 0.525%, DTT 1 M: 3%, coenzyme A 10 mM: 1.5%, ATP 100 mM: 0.45%) for 100 pL per well. The absorbance was measured within 30 minutes at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The activation of Nrf2 was calculated by the fold increase compared to a negative control (cells with medium only). Cell viability was presented as a percentage relative to the negative control (%).
Western Blot Analysis
RAW264.7 cells were grown for 24 h in sterile petri-dish (94 x 16 mm) until confluent. Cells were then treated with various samples for 1 h before being activated with LPS (1 pg/mL). After incubation for various time points, cell lysates were harvested and subjected to protein estimation by the Bradford protein assay kit (Thermo Fisher Scientific, Australia). Lysates were separated by SDS-PAGE electrophoresis and electro-transferred to a PVDF membrane. After being blocked in PBST (PBS with 1 % tween 20) containing 5% skim milk powder for 20 minutes at room temperature, the membranes were incubated with anti-phospho-p38, anti- p38, anti-phospho JNK, anti-JNK, anti-phospho-cJUN, anti-cJUN, anti-HO-1 , anti- -actin, anti- TLR4, and anti-TRAF-6 (1 :1000, all from Cell Signalling Technology, USA) overnight at 4°C. Following a membrane wash with PBST buffer, immunoreactive bands were detected by incubating with secondary antibody conjugated with anti-mouse/anti-rabbit horseradish peroxidase (Cell Signalling Technology, USA) for 1 h. Immunoreactive bands were visualized by an ECL kit (Thermo Fisher Scientific, Australia). Specific bands were analysed and the intensity was quantified using Image J software. Tri-cultured neuroinflammation model
The tri-cultured neuroinflammation model was established using a transwell polycarbonate membrane cell culture inserts with a 6-well plate (Corning®Costar®Transwell® Cell culture inserts, Sigma, Australia) according to our previous published protocol (Zheng, Y.F., & Zhou X., 2020. A novel tri-culture model for neuroinflammation. Journal of Neurochemistry. 156:249-261). Firstly, the insert membrane of the transwell was placed upside down and coated with 1% polylysine (Sigma, Australia). Then 800 pL of murine microglia N11 cells (2.0 x 106 cells/mL) was seeded on the top of the transwell with DMEM containing 10% FBS and kept upside down in the incubator to ensure N11 cells adhered to the top of the transwell by surface tension. In the meantime, 2000 pL of murine neuro2A (N2A) cells (1.0 x 106 cells/mL) were seeded in another 6-well plate with DMEM containing 10% FBS. After 6 hours, the transwell with N11 cells was turned back and murine brain endothelial MVEC(B3) (MVEC) cells (1.0 x 106 cells/mL) were seeded inside the insert membrane with 1000 pL DMEM containing 10% FBS. The transwell was then placed in a 6-well plate with 1500 pL DMEM containing 10% FBS in each well. After 24 h of incubation, the transwell was combined with the 6-well plate containing N2A cells to construct a tri-culture model and the medium was replaced with DMEM-F12 (Lonza, Australia) with 10% FBS.
After 24 h, the medium in the lower compartment was replaced with serum-free DMEM-F12, and in the upper compartment was replaced with either 1000 pL of serum-free DMEM-F12 (normal control), 1 ug/mL LPS or G-T 5:2 at various concentrations for another 24 h before bioassays. After this 24 h incubation, the supernatant in the lower compartment was collected and used for the measurement of NO expression. The transwell was then placed in a fresh 6- well plate, and the upper and lower compartments were filled with 0.2 mL of Evans blue [0.25% Evans blue, (Sigma, Australia)] and 0.5 mL PBS, respectively. After 24 h incubation, the solution in the lower compartment was tested for absorbance at 450 nm using a microplate reader for a barrier leakage test. The N2A cells in the tri-culture system were incubated with Alamar Blue for a cell viability test. qPCR analysis
Total RNA was obtained from the RAW264.7 cells and harvested by the mirVana RNA isolation kit (Thermo Fisher Scientific, Australia). The quantity and purity of total RNA was determined by nanodrop spectrophotometer. The reverse transcription (RT) of approximately 10 ng of total RNA was performed using the TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Australia) to synthesise cDNA. Then the qPCR was performed after the TaqMan™ MicroRNA Assay using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, Australia). The primer sequences for murine microRNA-155-5p (mmu-miR-155-5p) and internal controls using U6 were also purchased from Thermo Fisher Scientific (Australia). The calculation of the relative fold gene expression of samples with qPCR was followed the delta-delta Ct method (also known as the 2-AACt method).
Statistical analysis
All data were expressed as mean ± SEM/STD (n≥ 3) and analysed by GraphPad Prism. Values of p<0.05 were considered statistically significant.
RESULTS
1.1 G-T combinations synergistically inhibited LPS and IFN-y -induced inflammatory mediators in RAW264.7 cells
In the presence of LPS (50 ng/mL) and IFN-y (50 U/mL), a significant amount of nitrite, IL-6 and TNF production was detected (p<0.05). The individual and combined activities of G-T combinations (1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 , 5:2, w/w) were then tested on NO, TNF and IL-6 assays to evaluate their anti-inflammatory activities and possible synergistic effects.
1.1.1 G-T combinations synergistically inhibited LPS and IFN-y-induced NO and iNOS expression in RAW264.7 cells
Both G (1 .63 - 50 pg/mL) and T (1.63 - 25 pg/mL) showed dose-independent inhibitory effects on NO release with IC50 values of 11.78 and 6.51 pg/mL, respectively. As shown in Table 1A, G-T combinations (1.63 - 50 pg/mL) at all tested ratios showed dose-dependent inhibitory effects with IC50 values ranging from 4.72 to 13.08 pg/mL. Synergistic effects at IC50 value were observed for G-T combined at the ratios from 4:6 to 9:1 , with their Cl values from 0.53 to 0.83. It was observed that when the proportion of ginger was lower than 40% (G-T 1 :9, 2:8 and 3:7), the interaction at IC50 became antagonism. Isobologram (Figure 1A) revealed that G-T (4:6 to 9: 1 , including 5:2) required lower doses than that of individual components to reach the same NO inhibitory effects at IC50 level, highlighting a synergistic interaction. Among all the ratios that represented synergy, G-T 7:3 and 6:4 appeared to be the two optimal combinations with low IC50 values of 5.02 and 4.72 pg/mL and Cl values of 0.53 and 0.54, respectively. G-T 5:2 also showed a synergistic effect with a Cl value of 0.61 and IC50 value of 5.83 pg/mL. The ratio of 5:2 is within the range of 7:3 to 8:2. Figure 2A demonstrated a synergistic NO inhibitory activity of G-T 5:2 (Cl < 1 ) at the inhibition level ranged from 0-80%. In addition, in activated RAW264.7 macrophage stimulated with LPS (1 pg/mL), most cells expressed iNOS green fluorescence (Figure 3A, p<0.0001 vs. vehicle control). The inhibitory effect of G (10 pg/mL) was not significant at 24 h post-treatment (Figure 3B), although there was a tendency to decrease the number of iNOS positive cells in activated cells. However, significant inhibitory activities were seen in T and G-T 5:2 (10 pg/mL, p<0.5 and p<0.001 , respectively) with G-T 5:2 showing the most prominent activity.
1.1.2 G-T combinations synergistically inhibited LPS and IFN-y-induced IL-6 expression in RAW264.7 cells
G and T (3.13-25 pg/mL) inhibited IL-6 in a dose-dependent manner with IC50 values of 32.91 and 16.10 pg/mL, respectively. As shown in Table 1 B, all the tested combinational extracts of G and T dose-dependently inhibited IL-6 expression with IC50 values ranging from 9.07 to 29.87 pg/mL. It was noticed that G-T (3:7, 4:6, 5:5, 6:4 and 5:2, w/w) with certain ratios exhibited stronger IL-6 inhibitory effects than their individual counterparts, evidenced by lower IC50 values (9.07 -13.98 pg/mL) than that of G (32.91 pg/mL) or T (16.10 pg/mL) alone. Among the combinational extracts, G-T (3:7 to 9:1) showed synergy with Cl values from 0.43 to 0.70. In particular, G-T 5:2 ratio exhibited strong synergy (Cl= 0.23) with the lowest IC50 value of 9.07 pg/mL. Similar to the results in the NO assay, when the proportion of ginger was lower than 30%, the interaction became antagonism as shown by isobologram (Figure 1 B). Figure 2B demonstrates synergy of G-T 5:2 in inhibiting IL-6 expressions (Cl<1) when Fraction affected (Fa) was in the range of 0.25 - 0.97 (25%-97% inhibitory level).
1.1.3 G-T combinations synergistically inhibited LPS and IFN-v-induced TNF expression in RAW264.7 cells
For the TNF assay, G did not show any significant TNF inhibitory activity within the working concentrations, and the IC50 was estimated to be 163.4 pg/mL. However, T showed a significant dose-dependent inhibitory effect with IC50 of 14.63 pg/mL. As shown in Table 1C, among all the tested G-T combinations, only G-T 5:5 showed a lower IC50 value than T at 12.04 pg/mL. However, isobologram suggested that most G-T combinations showed synergistic interaction in inhibiting TNF expression excluding G-T 1 :9, 2:8 and 3:7 at IC50 level (Figure 1C). Although the IC50 value of G-T 5:2 was higher than that of T (21.07 pg/mL vs. 14.63 pg/mL), G-T 5:2 showed synergy at all tested concentrations with Cl values of 0.39, 0.28 and 0.20 at 25, 50 and 75% inhibitory levels (Figure 2C). 1.1.4 G-T combinations showed lower cytotoxicity and higher therapeutic index against inflammation in RAW264.7 cells
G and T showed cytotoxicity on RAW264.7 cells when the concentration was higher than 50 and 25 pg/mL with LC50 values of 104.3 and 83.90 pg/mL, respectively. Compared to G and T, G-T combinations (6:4, 8:2, 9:1 and 5:2) showed lower cytotoxicity with higher LC50 values ranging from 104.8 to 115.8 pg/mL (Table 1A, 1 B and/or 1C). In particular, G-T 5:2 also showed a lower cytotoxicity level than G and T with LC50 estimated to be 115.8 pg/mL.
G-T combinations that showed the highest therapeutic index in NO, IL-6 and TNF were 6:4, 5:2 and 5:5, respectively. In particular, the therapeutic index of G-T 5:2 throughout the three assays consistently showed relatively high values of therapeutic index (19.86, 12.76 and 5.33, respectively). These results suggested the superiority of combined G and T not only on enhanced anti-inflammatory activity, but also on reduced cytotoxicity on RAW 264.7 cells.
Table 1A. Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS and IFN-y-induced NO in RAW264.7 cells (n>3).
Extracts Cell NO assay viability
LC50 IC50 Cl Synergistic Therapeutic
(jjg/mL) (pg/mL) value dosage range index at IC50 (pg/mL)
G 104.3 11.78 - - 8.85
T 83.90 6.51 - - 12.88
G-T 1:9 72.29 13.08 1.91 67.17-117.19 5.52
G-T 2:8 76.93 10.08 1.41 20.20 - 64.96 7.63
G-T 3:7 82.15 7.93 1.05 8.88 - 41.14 10.36
G-T 4:6 81.00 6.52 0.83 5.10 - 38.58 12.42
G-T 5:5 102.7 5.72 0.69 5.05 - 14.25 17.95
G-T 6:4 115.8 4.72 0.54 3.22 - 13.16 24.53
G-T 7:3 93.29 5.02 0.53 3.46 - 13.67 18.58
G-T 5:2 115.8 5.83 0.61 2.84 - 26.75 19.86
G-T 8:2 104.8 5.92 0.59 4.09 - 16.04 17.70
G-T 9:1 107.4 6.11 0.57 3.88 - 15.95 17.58 Table 1B. Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS and IFN-y-induced IL-6 in RAW264.7 cells (n>3).
Extracts Cell IL-6 assay viability
LC50 IC50 Cl Synergistic Therapeuti
(μ g/mL) (pg/mL) value dosage range c index at IC50 (pg/mL)
G 104.3 32.91 - - 3.17
T 83.90 16.10 - - 5.21
G-T 1:9 72.29 25.46 1.52 30.84 - 63.42 2.84
G-T 2:8 76.93 18.33 1.01 20.53 - 96.43 4.20
G-T 3:7 82.15 13.98 0.70 12.71 - 27.85 5.88
G-T 4:6 81.00 11.52 0.52 6.29 - 58.24 7.03
G-T 5:5 102.7 14.47 0.59 7.22 - 93.33 7.10
G-T 6:4 115.8 12.40 0.45 5.82 - 61.82 9.34
G-T 7:3 93.29 16.94 0.53 10.67 - 84.61 5.51
G-T 5:2 115.8 9.07 0.23 4.08 - 113.30 12.76
G-T 8:2 104.8 16.53 0.43 4.51 - 150.02 6.34
G-T 9:1 107.4 29.87 0.64 3.26 - 582.10 3.60
Table 1C. Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS and IFN-y-induced TNF assays in RAW264.7 cells (n>3).
Cell TNF assay
Extracts viability
LC50 IC50 Cl value Synergistic Therapeut
( g/mL) (pg/mL) at IC50 dosage range ic index
(pg/mL)
G 104.3 163.4 - - 0.64
T 83.90 14.63 - - 5.73
G-T 1:9 72.29 22.20 1.86 47.75 - 94.25 3.26
G-T 2:8 76.93 19.02 1.25 25.25 - 52.54 4.04 G-T 3:7 82.15 20.51 1.13 24.12 - 38.27 4.01
G-T 4:6 81.00 18.30 0.73 16.37 - 28.27 4.43
G-T 5:5 102.7 12.04 0.31 4.13 - 27.48 8.53
G-T 6:4 115.8 22.81 0.57 2.48 - 223.09 5.08
G-T 7:3 93.29 24.79 0.24 5.32 - 28.57 3.76
G-T 5:2 115.8 20.07 0.28 5.32 - 28.57 5.77
G-T 8:2 104.8 29.92 0.49 1.75 - 824.51 3.50
G-T 9:1 107.4 31.91 0.41 6.34-340.30 3.37
1.2 G-T combinations synergistically inhibited LPS-induced pro-inflammatory cytokines in THP-1 cells
The inhibitory effect of G-T combinations on TNF was investigated in human monocytic THP- 1 cells. As shown in Table 2, G (1 .63 - 25 pg/mL) did not show any significant TNF inhibitory activity, whereas T (1.63 - 25 pg/mL) dose-dependently inhibited TNF in response to LPS with IC50 of 8.54 pg/mL. G-T combinations showed a TNF inhibitory effect in a dose-dependent manner with IC50 ranging from 4.79 to 57.13 pg/mL. The potency for G-T combinations 1 :9, 2:8, 3:7, 4:6, 5:5, 6:4 were stronger (with lower IC50 values) than the single contributor of G and T. Isobologram showed that all G-T combinations showed synergistic interaction in inhibiting TNF expression at IC50 level (Figure 4A). G-T combinations (3:7, 4:6, 5:5, 6:4, 7:3, 5:2, 8:2, 9:1) showed lower cytotoxicity than T with LC50 ranging from 52.03 - 118.90 pg/mL. The therapeutic index values for most G-T combinations (6:4 to 1 :9) were higher than G (no effect) and T (5.53). Particularly, G-T 6:4 was calculated to have the highest therapeutic index of 10.70, followed by G-T 5:2 at 8.85. G-T 5:2 (1.63 - 25 pg/mL) showed a comparable effect as T with a slightly lower IC50 value of 8.32 pg/mL. However, the cytotoxicity of G-T 5:2 was much lower than that of T (73.61 pg/mL vs. 47.24 pg/mL) which led to a superior therapeutic index (8.85 vs. 5.53). As analysed by Cl model, G-T 5:2 showed a strong synergy within the tested concentration (Figure 4B), with Cl values of 0.25, 0.32 and 0.41 at 25%, 50% and 75% inhibitory level, respectively.
Table 2. Cell viability, IC50, Cl values, synergistic dosages and therapeutic indexes of G, T and G-T combinations with various ratios on LPS-induced TNF assays in THP-1 cells (n>3).
Extracts Cell TNF assay viability LC50 IC50 Cl value Synergistic dosage Therapeutic
(pg/mL) (pg/mL) at IC50 range (pg/mL) index
G 123.8 . . .
T 47.24 8.54 - - 5.53
G-T 1:9 40.94 7.40 0.87 6.59 - 37.87 5.53
G-T 2:8 41.16 5.99 0.56 0.98 - 52.71 6.87
G-T 3:7 45.30 5.72 0.11 1.32 - 30.90 7.92
G-T 4:6 52.03 8.43 0.59 0.87 - 81.94 6.17
G-T 5:5 53.16 8.37 0.49 0.77 - 134.16 6.35
G-T 6:4 51.26 4.79 0.23 0.54 - 54.21 10.70
G-T 7:3 52.61 13.24 0.39 0.58 - 96.56 3.97
G-T 5:2 73.61 8.32 0.32 0.68 - 185.96 8.85
G-T 8:2 56.35 15.58 0.37 5.18 - 40.16 3.62
G-T 9:1 118.90 57.13 0.67 5.84 - 77.43 2.08
1.3 G-T 5:2 synergistically inhibited LPS and IFN-y-induced 17 pro-inflammatory cytokines on multi-plex assay in RAW264.7 cells
To determine the potency and synergy of G-T 5:2 in a broader range, the inhibitory effect of G, T and G-T 5:2 were tested in a panel of 23 inflammatory mediators on RAW264.7 cells.
The activation with LPS and IFN-y for 24h led to a significant up-regulation of the pro- inflammatory mediators, secondary inflammatory mediators, and anti-inflammatory mediators at different levels (Table 3). The activation also enhanced the production of anti-inflammatory cytokines such as IL-10 and IL-13. The upregulation was not detected for MIP-1b. Therefore, data could not be analysed for this mediator.
Table 3. Cytokine & chemokine responses to the activation of LPS and IFN-y in RAW264.7 cells. Data are shown as mean concentration (pg/mL) ± STD from duplicates and fold change.
Inflammatory mediators Concentration in RAW264.7 macrophages (pg/mL)
Non-activated Activated Upregulation (fold)
Pro- IL-1a (53) 20.2511.72 825.30194.31 9.17** inflammatory IL-1b (19) 29.23123.03 263.00131.48 9.00** mediators Eotaxin (74) 16.5811.19 44.5911.87 2.69**
IL-6 (38) *1512.25±131.88 *254161321.03 16.81** IFN-y (34) 11.9010.42 29.76±0.61 2.50***
IL-12 (p40) 17.9610.60 527.99±40.16 29.41**
(76)
IL-12 (P70) 45.2812.95 129.8311.12 2.87*
(78)
RANTES 5141.161425.11 16178.34±1007.06 3.14*
TNF *1598.751240.06 24104.75±127.63 15.08***
Secondary IL -5 (52) 3.91±0.23 10.4110.07 2.66* inflammatory IL-9 (33) 7.65±0.53 18.36±0.06 2.4* mediators IL-13 (37) 496.02±14.91 1044.12±5.27 2.10*
G-CSF (54) 16228.641452.66 97776.79±2777.44 6.02**
GM-CSF (73) 34.8811.07 2231.431202.15 63.97***
KC (57) 5.2710.35 29.79±0.91 5.65**
MCP-1 1753.591139.79 45061.93±61.53 25.70***
MIP-1a *18800.75178.13 23442.75192.98 1.25*
MIP-1b *23467.5153.03 23496.5116.26 1.00
Anti- IL-2 (36) 5.9910.40 69.3815.02 11.59** inflammatory IL-3 (18) 1.6710.05 6.0610.08 3.64** mediators IL-4 (39) 4.6510.00 7.5710.24 1.63*
IL-10 (56) 19.6311.30 82.1217.45 4.18*
IL-17 (72) 3.0110.06 7.3710.52 2.45*
*p<0.05, **p<0.01, ***p<0.001.
Cells were pre-treated with G, T, and G-T 5:2 (1.625 - 25 pg/mL) for 2 hours prior to the stimulation of LPS and IFN-y in RAW264.7 cells, and the release of 23 mediators were measured. The data of pro-inflammatory and secondary inflammatory mediators is shown in Table 4. G did not demonstrate significant inhibitory effects on most cytokines, with prominent inhibitory effects on GM-CSF (73), KC (57) and MCP-1 only with IC50 values ranging from 5.97 to 16.69 pg/mL. In contrast, T showed potent activities on most of the cytokines (excluding MIP-1b) with the IC50 values ranged from 2.15 to 7.26 pg/mL. G-T 5:2 showed dose-dependent inhibitory activities on most of the tested mediators (excluding G-CSF) with IC50 ranging from 3.84 to 13.62 pg/mL. In addition, the doses required to reach 50% of G and T in the combined extract to reduce cytokines were generally lower than that of individual extracts.
Based on Cl model, synergy was detected for most of the tested cytokines with Cl values ranging from 0.46 to 0.99. Only IL-12 (p40), RANTES and G-CSF were found to have additive or antagonistic effects with Cl at 1.55, 1.02 and 15.43, respectively. Table 4. Effect of G, T and G-T 5:2 on cytokine and chemokine levels in RAW264.7 cells. Data shown are mean IC50 from duplicates and Cl values at IC50.
Inflammatory mediators Dose required to reach Dose required to reach Cl at 50% (individual extract) 50% (combined extract) IC50
G T G T
Pro- IL-1a (53) 36.68 2.37 3.51 1.40 0.69 inflammatory IL-1b (19) 22.34 3.26 3.6 1.44 0.60 mediators Eotaxin (74) 58.03 4.25 4.57 1.83 0.51
IL-6 (38) 858.35 7.26 8.21 3.28 0.46
IFN-y (34) 107.95 2.89 4.49 1.80 0.66
IL-12 (P70) 3.81 1.52 0.41
3.68
(78) TNF 449.64 5.28 9.91 3.97 0.77
IL-12 (p40) 9.28 3.71 1.55
2.40
(76)
RANTES 25331.4 3.17 8.11 3.25 1.02
Secondary IL -5 (52) - 3.00 5.69 2.27 0.76 inflammatory IL-9 (33) 3806.18 3.58 5.35 2.14 0.60 mediators GM-CSF 2.97 1.19 0.63
16.69 2.61
(73) KC (57) 9.77 2.15 3.11 1.24 0.90
MCP-1 5.97 2.21 2.74 1.10 0.95
MIP-1a 111.41 4.33 9.73 3.89 0.99
G-CSF (54) 214.57 4.84 176.74 70.69 15.43
MIP-1b NA NA NA NA NA
NA = not available.
1.4 Synergistic anti-inflammatory activity of G-T 5:2 by regulating key proteins involved in triggering inflammation
1.4.1 G-T 5:2 activated the Nrf-2/H0-1 pathway
The Nrf2/H0-1 mediated signalling pathway is the main cellular defence mechanism against oxidative stress and inflammation. The effect of G-T 5:2 on Nrf2 pathway was investigated using the Nrf2 luciferase assay in MCF AREc32 cells. To validate the Nrf2 luciferase assay, a prototypical and potent Nrf2 activator, tert-Butylhydroquinone (tBHQ), was used as a positive control. The results show that tBHQ (0.13 - 4.16 pg/mL) increased the luminescence signal of Nrf2 in a concentration-dependent manner. At 25 pM the luciferase activity was increased 11.45±1.31 fold.
Extracts of G, T and G-T 5:2 were tested in the Nrf2 luciferase assay and the comparison of their induction on Nrf2 at 25 and 12.5 pg/mL are shown in Figure 5A. At 25 pg/mL, G-T 5:2 produced a higher maximum-fold induction of Nrf2 (11.57 ± 3.30) compared to that of G or T (2.04 ± 0.22 and 10.11 ± 1.60, respectively). Turkey non-parametric analysis showed that the maximum-fold activation of G-T 5:2 was significantly higher than that of G (p<0.001), but comparable to that of T and tBHQ (at 4.16 pg/mL). Strong synergistic interaction was suggested from the changes of Cl and isobologram models (Figure 5B). Cl values were consistently lower than 1 at all tested concentrations with Cl values at 0.13, 0.06 and 0.03 at Fa 0.25, 0.5 and 0.75, respectively.
As shown in Figure 5C, cells with medium only (Blank) did not show significant expression of HO-1, however, the co-incubation of G-T 5:2, G and T triggered expression of HO-1. Figure 5D showed the quantitative analysis of the HO-1 upregulation by G, T and G-T 5:2. The activated expression of HO-1 by G-T 5:2 was 1.92±0.37 times that of blank control (p<0.05). G and T also showed an increasing trend of HO-1 expressions, however, did not reach statistical significance, with 1.46±0.32 and 1.03±0.19 fold, respectively. Taken together with the synergistically increased expression of Nrf2 by G-T 5:2, the synergistic anti-inflammatory activity of G-T 5:2 maybe at least partially associated with the activation of the Nrf2-H0-1 pathway.
1.4.2 Investigation of G-T 5:2 on LPS-induced TLR4 and TRAF6 protein expressions
The Toll-like receptor-4 (TLR-4), a key pattern recognition receptor, recognizes and binds LPS which contacts down-stream tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6), then activates mitogen-activated protein kinases (MAPKs) and nuclear transcription factor-kappa B (NF-KB) signalling pathways. The transcription of NF-KB is the central mediator of the innate immune response leading to inflammation that induces the expressions of many pro-inflammatory mediators and cytokines including iNOS, IL-6 and TNF. Thus, the effects of G-T 5:2 on TLR4 and TRAF6 in the MyD88 pathway were investigated, to see if there is any crosstalk between G and T in the upstream molecular targets.
Three experiments were conducted to examine the modulatory effects of G, T and G-T 5:2 on the protein levels of TLR4 and TRAF6. As shown in Figure 6 (A and C), the stimulation of RAW264.7 cells with LPS significantly increased the expression of TLR4 and TRAF-6 compared to the unstimulated cells (p<0.05). G and T were both shown to individually inhibit the LPS-induced upregulation of TLR4 and TRAF-6 (p<0.05). G-T 5:2 also showed substantial downregulatory effects on TLR4 and TRAF-6 (p<0.05).
1.4.3 G-T 5:2 inhibited LPS-induced MAPK/JNK/CJUN pathway and NF-KB transcription
Figure 7 shows the three trials and data analysis of the individual and combined effects of G- T in regulating the key protein kinase and expressions in the MAPK/JNK/CJUN pathways.
RAW264.7 co-incubated with LPS (1 pg/mL) for 40 minutes induced the phosphorylation of p38, JNK (p<0.05) and c-JUN (p<0.01). Total protein of p38, JNK, CJUN and beta-actin was used as loading controls which showed that the protein loading for all samples was comparable. The inhibition of kinase was expressed as the ratio between phosphorylated protein production to its total production (n=3).
After three trials, a trend of inhibition of p-p38 was observed with G, T and G-T 5:2, with G-T 5:2 showing the highest inhibition level (Figure 8A). The inhibition of pJNK was more prominent, with G-T 5:2 showing a significant inhibitory level (p<0.05, Figure 8B). Furthermore, G-T 5:2 also inhibited pCJUN, which was more potent than G or T (p<0.001, Figure 8C).
DAPI blue was used to stain the cell nucleus and fluor 594 red fluorescence represents p65 expression within the cell. In the absence of LPS, p65 was observed almost exclusively in the cytoplasm outside the nucleus (Figure 9A, blank). With stimulation by LPS (100 ng/mL) for 30 min, a dramatic increase of p65 was seen in the nuclei as indicated by the overlap of the p65- fluor 594 red fluorescence with the DAPI blue staining in the nucleus.
Significant reductions of p65 translocation were seen in the cells treated with G, T and G-T (n=3). As shown in Figure 9B, G and G-T (5:2) at 10 pg/mL significantly reduced the p65 positive expression in the nuclei (p<0.01 and p<0.0001 vs. vehicle + LPS, respectively). Noticeably, the highest inhibitory level was seen in G-T.
1.5 Key compounds contributing to the synergistic activity of G-T 5:2 against inflammation in RAW264.7 and THP-1 cells
1.5.1 Interactions among gingerols, shogaols and curcumin in inhibiting LPS-induced NO expression in RAW264.7 cells
To investigate the key compound(s) that play an important role in the potent and synergistic activities of G-T, individual and different forms of compound(s) combinations were investigated using the same bioassays as the G-T combinations. Individual compounds of 6g, 8g and 10g did not show any significant activity in inhibiting LPS-induced NO and IL-6 (p>0.05), whereas 6s, 8s and 10s inhibited NO and IL-6 dose-dependently with IC50 values ranging from 1.96 to 11.73 pg/mL. Curcumin (C), desmethoxycurcumin (D) and bisdemethoxycurcumin (B) also dose-dependently inhibited NO and IL-6 with IC50 values that ranged from 3.69 to 8.30 pg/mL. The summary of IC50 values of individual compounds is shown in Supplementary material 3.
For the combinations of compounds, the results suggested that pair-wise combinations of single compounds in G and T either combined by their content in G-T 5:2 or by IC50 values on NO and IL-6 assays generally exhibited antagonistic effects (Supplementary material 4 and 5). Moreover, pair-wise combinations of G with curcuminoids (G-C, G-D, G-B), T with gingerols (T-6g, T-8g, T-10g) or shogaols (T-6s, T-8s, T-10s) in 5:2 also displayed antagonistic effects (Supplementary material 6). An artificial T (AT, constructed by C, B and D based on their content in T) was tested in the NO assay together with G at 5:2, and the dose-response curves and IC50 values were comparable to that of original T and G-T 5:2 (Figure 10). Since C is the dominant compound that accounted for approximately 60% of the total weight of T, the compounds combination was limited to 2 gingerols -C, and 2 shogaols - C for the synergistic investigation.
Combinations of 6s8s-C, 6s10s-C and 8s10s-C (0.1 -25 pg/mL) based on their content in G- T 5:2 were tested on NO assay and the results were compared with combinations of 6g8g-C, 6g10g-C, and 8g10g-C 5:2 (0.4 -15 pg/mL). The results suggested that the IC50 values of 2 shogaols-C 5:2 combinations (ranging from 3.47 to 6.18 pg/mL) were generally lower than that of 2 gingerols-C 5:2 combinations (higher than 15.17 pg/mL). Moreover, the dose required for C (3.33 -6.01 pg/mL) in the 2 shogaols-C 5:2 combination was lower than using C alone (7.56 pg/mL) to reach the same effect level, indicating positive interactions between 2 shogaols and curcumin (supplementary material 7).
In addition, the inventors further demonstrated the effect of SC in inhibiting iNOS on RAW264.7 cells using fluorescent staining. As shown in Figure 11, S, C and SC at 2.5 pg/mL all significantly inhibited iNOS expression with p<0.0001. The most prominent activity was seen in SC with almost no iNOS positive cells observed.
None of the combinations within the tested concentration ranges exhibited any cytotoxicity detected by MTT assay. 1.5.2 6s10sC (SC) synergistically inhibited LPS-induced TNF in RAW264.7 and THP-1 cells
The combination of 6s10sC 5:2 (SC) was selected and tested in LPS induced-TNF assay in RAW264.7 and THP-1 cells. The results of SC were compared with that of individual components and G-T extracts to see whether it represented a higher level of bioactivity. According to the calculations in supplementary material 2, the concentrations of 6s, 10s and C in G-T 5:2 at 50 mg/mL were 0.27, 0.08 and 10.74 pg/mL, respectively. Thus, the ratio in SC was 0.27:0.08:10.74, and the concentrations in 10 pg/mL were 0.24, 0.07 and 9.68 pg/mL, respectively. The SC mixture at 10 pg/mL was then series diluted with DMSO before being subjected to the cellular assays.
In RAW264.7 cells, SC showed the highest TNF inhibitory effect with IC50 value of 7.03 pg/mL compared to 6s10s (S) or C. S (0-0.45 pg/mL) failed to exhibit any significant activity whereas C (0-18.53 pg/mL) exhibited dose-dependent activity with IC50 of 8.69 pg/mL. Interestingly, a low amount of S (0.23 pg/mL) mixed with C (6.80 pg/mL) did not dilute the effect of C, but reached the same level of C when it was used at 8.69 pg/mL. Both isobologram and Cl showed strong synergy of SC, with Cl value consistently lower than 1 (Figure 12A and B). In addition, the IC50 value of SC was much lower than that of the extracts G, T and G-T 5:2 (14.63-98.05 pg/mL). Thus, SC showed the most potent TNF inhibition and the effect was consistently higher compared to single compounds and extracts (Figure 12C). These results indicated that these three compounds together play key roles in contributing to the activities of the extract. The MTT assay revealed that the inhibitory activity of SC (0.34 - 10.93 pg/mL) was not generated from cytotoxicity. Although the LC50 value of SC (28.96 pg/mL) was lower than that of C (34.79 pg/mL), the therapeutic index of SC (4.12) was slightly higher than C (4.00). The IC50, LC50 and therapeutic index values of SC reducing TNF in RAW264.7 cells are shown in Table 5A.
The anti-inflammatory activities of SC were further tested on LPS-induced TNF in THP-1 cells (Table 5B). Our results revealed that both SC and C exhibited strong inhibitory effects with comparable IC50 values at 4.71 and 5.11 pg/mL, respectively. Similarly, the additional S (0.15 pg/mL) with C (4.56 pg/mL) reached the same level as using C alone (5.11 pg/mL). Synergy on human TNF was also demonstrated as shown in Figure 12D and E, although not as strongly as the effect on murine TNF. Isobologram showed synergy of SC at IC50 level and Cl-Fa curve revealed that synergy occurred when the inhibition level was lower than 50%, with Cl values of 1.59, 0.97, and 0.60 at 25%, 50% and 75% inhibitory levels, respectively (Figure 12D and E). When comparing individual and combination compounds to G-T extracts, the IC50 value of SC (equivalent to 21.55 pg/mL of G-T 5:2) was markedly lower than that of G, T and G-T 5:2 (no inhibition, 8.54 and 8.32 pg/mL, respectively), as shown in Figure 12F. An Alarmar blue assay revealed that SC did not exhibit any cytotoxicity on TH P-1 cells within the tested concentration and its highest safety dosage was observed at 22 pg/mL. C was found to exhibit higher toxicity on THP1-1 cells, with LC50 value at 8.55 pg/mL and highest safety dosage at 9.36 pg/mL. Due to its high toxicity, the therapeutic index of C (1 .67) was lower than SC (4.84), despite it exerting a comparable TNF inhibitory effect as SC.
Table 5A. Results of SC in comparison to S, C, G-T 5:2, G and T on LPS stimulated TNF expressions and cell viability determined by MTT in RAW264.7 (n≥ 3).
Figure imgf000062_0001
Table 5B. Results of SC in comparison to S, C, G-T 5:2, G and T on LPS stimulated TNF expressions and cell viability determined by Alamar Blue in THP-1 cells (n^3).
Figure imgf000062_0002
1.5.3 SC synergistically inhibited LPS-induced IL-6 expression in RAW264.7 and THP-1 cells
In the presence of LPS for 24 hours, IL-6 production in RAW264.7 cells reached approximately 195.414 ng/mL, detected by a commercial IL-6 ELISA kit used according to the manufacturer’s instructions.
As shown in Figure 13C, C, S, C and SC all showed IL-6 inhibitory activities in a dosedependent manner without the impairment of cell viability on RAW264.7 cells within the tested concentrations. The IC50 values of SC (2.37 pg/mL) were lower than that of S (2.95 pg/mL) or C (5.15 pg/mL) (Table 6). Cl and isobologram analysis revealed that synergy was demonstrated at all tested concentrations, and the Cl value was 0.53 at 50% inhibitory level (Figure 13A and B).
To compare with the original extract, the maximum inhibition of compounds was observed at 10 pg/mL which was markedly lower than that of the extracts (maximum inhibitions of G, T and G-T 5:2 were observed at 50, 50 and 25 pg/mL). The IC50 value of SC (2.37 pg/mL) was also markedly lower than that of G-T 5:2 (9.07 pg/mL).
The anti-inflammatory activities of SC were further tested on LPS-induced IL-6 in THP-1 cells (Table 6). In the presence of LPS (1 pg/mL) for 24h, IL-6 production reached approximately 139.01 ±37.49 ng/mL. Samples on human IL-6 were tested three times with duplicates. As shown in Figure 13F, S, C and SC all showed IL-6 inhibitory activities in a dose-dependent manner within the non-cytotoxic concentrations on THP-1 cells. The IC50 values of S, C and SC were determined at 2.88, 2.13 and 1.45 pg/mL as shown in Table 6. Generally, the IL-6 levels in SC were consistently lower than that of S and C. Cl and isobologram analysis suggested that there was synergy at all tested concentrations, and the Cl value was 0.24 at 50% inhibitory level (Figure 13D-E). Together with the results of the murine IL-6 assay, SC has consistently showed synergistic and potent activity in inhibiting LPS induced IL-6 expressions on both RAW264.7 cells and THP-1 cells. Since the ratio of 6-shogaol, 10- shogaol and curcumin in SC was equivalent to that in G-T 5:2, the potent activity suggests that they were the key compounds contributing to the IL-6 inhibition of G-T 5:2.
Compared with the original extract, the IC50 values of compounds were all lower than that of the extracts (IC50 values of G, T and G-T 5:2 were 24.75, 5.16, 3.63 pg/mL).
Table 6. IC50 values of SC in comparison to S, C, G-T 5:2, G and T on LPS stimulated IL-6 expressions and Cl values in both RAW264.7 and THP-1 cells (n>3). Samples Murine IL-6 (n=3) Human IL-6 (n=3)
Cl IC50 values IC50 values Cl value vai ue
SC (6-shogaol, 10-shogaol, 2.37 0.53 1.45 0.24 and curcumin) as (0.06+0.02+2.30) (0.04+0.01+1.40) equivalent to G-T 5:2 (0.27:0.08:10.74)
S (6-shogaol and 10- 2.95 2.88 shogaol)
C (curcumin) 5.15 2.13
G (ginger) 32.91 0.16 24.75 0.43
T (turmeric) 16.10 5.16
G-T 5:2 9.07 3.63
1.6 Assessment of S, C and SC on human multiple cytokines
In most biological processes, multiple cytokines operate in a large network in the immune system, where the action of one cytokine is regulated by the presence or absence of other cytokines. After determining the synergistic effect of SC on single cytokines such as TNF and IL-6 in earlier examples, the anti-cytokine effect of SC was demonstrated in a broader range using human XL Cytokine Array Kit (R&D, Australia). In this cytokine array assay, differences of up to 102 cytokines between samples can be determined simultaneously. Briefly, THP-1 cells were seeded in a 24 well-plate overnight, and incubated with RPMI media, S (2.5 pg/mL), C (2.5 pg/mL) and SC (2.5 pg/mL) for 1 hour followed by stimulation with LPS (1 pg/mL) and human IFN-y (50 ng/mL). After 24h, the cell supernatant was collected and the total protein amount was determined by the Pierce BCA Protein Assay (ThermoFisher, Australia). Then equal amounts of total protein from each sample was applied to the cytokine array kit to conduct the assay based on the manufacturers’ protocol.
Typical cytokine images in cells stimulated with LPS and IFN-y only, pre-treated with S, C and SC before the stimulation are shown in Figure 14A. Cytokines detected with LPS and IFN-y stimulation are listed in Figure 14B. The average expression levels of spot density for each cytokine are shown in Figure 14C.
We detected 25 cytokines in LPS and IFN-y stimulated THP-1 cells on membrane arrays containing 101 different cytokine antibodies. As expected from the literature, most of the expressed cytokines induced by LPS and IFN- y were proinflammatory chemokines, namely CXCL5, IL-8 (CXCL8), CXCL10, CXCL11 , MIG, and MIP-3a. From the semi-quantification of these cytokine expressions, S showed a weak inhibition of IL- 6, but a strong inhibition of CXCL11 (see Figure 14A and 14C). C inhibited most of the LPS- induced cytokines, except for CXCL11 . SC showed the greatest inhibitory effects against all these cytokines.
CXCL family contains chemokines for the recruitment of the first line of innate immune effector cells to sites of infection and inflammation which contribute to elimination of pathogens, but it may also contribute significantly to disease-associated processes, including tissue injury. For instance, IL-8 is a classical inflammatory cytokine (chemokine) that stimulates neutrophil chemotaxis during bacterial exposure to help eliminate a pathogen. These actions have a relatively rapid onset in the initial stage of inflammation. CXCL10 and CXCL11 are mainly induced by IFN-y and their actions have been associated with the migration of Th1-polarized effector T cells to inflamed sites during the adaptive immune response.
Our results showed that SC demonstrated greater inhibitory effects in inhibiting a range of chemokines under the acute inflammatory phase, and the effect was greater than S or C alone. The inhibitory effects covered chemokines and main cytokines (IL-6 and TNF). Previous studies have demonstrated the potential use of C against cytokine storm. So far, there has not been any study on the possible inhibitory effect of shogaol or ginger in inhibiting CXCL11 . Our results suggested that SC is a better candidate for a broad anti-inflammatory and anticytokines action and has great potential against LPS and IFN-y induced cytokine storm.
1.7 Synergistic anti-inflammatory activity of SC by regulating key proteins involved in triggering inflammation
1.7.1 SC activated Nrf-2 expression
Compounds of SC, S and C were tested with the Nrf2 luciferase assay for the evaluation of Nrf2 activation. tBHQ was used as positive control and Nrf2 activation by treatments was compared to that of a negative control (cell with medium only). The results show that SC, S and C activated Nrf2 in a dose-dependent manner. The maximum-induction of Nrf2 induced by SC at 16.40 pg/mL was 13.27 ± 1.54 times which was markedly higher than that of the maximum induction of Nrf2 by S and C at 6.17 ± 1.00 and 6.65 ± 0.36 times, respectively (p<0.05). The maximum induction of Nrf2 by SC was comparable to that of tBHQ. At the highest induction level, SC consisted of S (0.53 pg/mL) and C (15.87 pg/mL), of which the individual component of S showed no significant effect whereas C was likely to have cytotoxicity. Moreover, isbologram and Cl-Fa curves revealed that there was a synergistic interaction in inducing Nrf2 by SC at all tested concentration levels (Figure 15). Cl values were consistently lower than 1 at all Fa values, with Cl values at 0.55, 0.57 and 0.60 at Fa 0.25, 0.5 and 0.75, respectively. This result highlights the possibility that the synergistic interaction of SC in reducing LPS-induced inflammation is attributed to its synergistic Nrf2 activation which strengthened the cellular defence against inflammation.
1.7.2 SC inhibited LPS-induced NF-KB (p65) nuclear translocation in RAW264.7 cells
DAPI blue was used to stain the cell nucleus and fluor 594 red fluorescence represents p65 expression within the cell. In the absence of LPS, p65 was observed almost exclusively in the cytoplasm outside the nucleus (Figure 16A, blank). With stimulation by LPS (100 ng/mL) for 30 min, a dramatic increase of p65 was seen in the nuclei as indicated by the overlap of the p65-fluor 594 red fluorescence with the DAPI blue staining in the nucleus.
The inhibitory effect of p65 translocation was compared among S, C and SC at 5 pg/mL. As shown in Figure 16A and B, S did not show significant inhibition in p65 translocation, whereas C showed an inhibitory effect of p65 translocation to some extent. SC showed the most potent inhibitory activity in p65 translocation with a prominent amount of p65 expressed outside the nucleus. The statistical analysis showed that the nuclear p65 localization was significantly reduced by SC (p<0.01 vs. vehicle + LPS), and the reduction was greater than that of S and C at 5 pg/mL (p <0.5).
1.7.3 SC on LPS-mediated MAPK pathway
In earlier examples, it was demonstrated that G-T (5:2) significantly inhibited the phosphorylation of p38, JNK and CJUN in the LPS-stimulated MyD88 dependent signalling pathway. The effects of the key contributing compounds, SC, on the phosphorylation of p38MAPK, JNK and cJUN were demonstrated.
As shown in Figure 6, the stimulation from LPS significantly increased the expression of p- JNK/JNK and p-CJUN/CJUN in RAW264.7 cells compared to untreated cells (p<0.05). However, LPS did not significantly upregulate the expression of p-p38/p38 (p>0.05). Individual S or C inhibited the key proteins in this path generally, and pJNK/JNK in particular (p<0.0001). C also showed significant inhibition in p-cJUN/cJUN (p<0.05). SC inhibited the expression of all key proteins except p-p38/p38, with the most significant inhibitory activity seen against p- JNK/JNK (p<0.0001). 1.8 Assessment of G-T 5:2 on the neuroinflammation tri-culture system
As shown in Figure 17, NO expression in N11 cells was increased significantly after 24 h LPS exposure (p<0.0001). G-T 5:2 (10-20 pg/mL) treatment markedly attenuated the increase of NO (p<0.001) in the tri-culture system. In addition, the integrity of MVEC cells in the tri-culture system was damaged after LPS exposure for 24h at 1 pg/mL as evidenced by significant increased leakage (p<0.05), but it was restored by G-T 5:2 treatment (10-20 pg/mL) significantly (p<0.001). Furthermore, G-T 5:2 (10-20 pg/mL) protected neuron survival by restoring the cell viability impaired by cytokine release in the tri-culture system (p<0.05). Hence, the preliminary data of G-T 5:2 suggested that G-T 5:2 protects the neuron survival and endothelial barrier via reduced NO expression from the N11 microglia cells in the established neuroinflammatory tri-culture system. The proposed diagram explaining how G-T 5:2 protected neuron survival through inhibiting LPS-induced proinflammation and reducing the barrier leakage in the tri-culture system is shown in Figure 18.
1.9 The modulatory effect of G-T and SC on murine microRNA 155-5p expression
Small endogenous RNA molecules known as microRNAs (miRs or miRNAs) have been shown to play an important role in regulating the inflammatory response by specifically binding to the 3’IITR of target miRNAs. Among them, miR-155 has been implicated in the control of the inflammatory response, which creates a negative feedback loop in LPS-induced signalling pathways. Here the combined effect of G-T and SC on the production miR-155 by using miR- 155 qPCR, and any crosstalk at the miRNA level that contributes to the consequent synergistic anti-cytokine effects was investigated.
As shown in Figure 19, three individual experiments were conducted to examine the modulatory effects of GT and SC on mmu-miR-155-5p. The averaged AACt value of LPS (1 pg/mL) was 6.78 ± 0.53, which resulted in up to 123.62±35.67 fold increase in mmu-miR-155- 5p expression. All the individual and combined treatments substantially attenuated the increased expression of mmu-miR-155-5p, and the significance was shown in T, GT (G-T 5:2), C and SC. In particular, GT and SC showed greater inhibitory effects than that of G or C, respectively. SC almost restored the level of mmu-miR-155-5p to a comparable level to that of untreated cells. The synergistic effect via inhibiting TLR4-TRAF6-MAPK signalling was likely to be attributed to the down-regulated miroRNA-155-5p expression analysed by realtime PCR.
MiR-155 is rapidly activated by NFKB which then induces the IKK signalosome complex and amplifies the signal of the downstream inflammatory response and the excessive release of the proinflammatory cytokines (Mahesh G and Biswas R., Journal of Interferon and Cytokine research. 2019. 39 (6): 321-330). Thus, the synergistic decrease in microRNA 155-5p of SC composition was likely to be attributed to the supressed NFKB pathway and led to further supressed downstream inflammatory response.
EXAMPLE 2.0
MATERIALS AND METHODS
Solvent extraction of ginger
Ginger raw material (fresh ginger root) was ground to powder and then filtered using a laboratory sieve (500 microns, 200 mm diameter, ENDECOTTS, UK). The dried powder (50g) was weighed and then dissolved in 500 ml of 100% ethanol or DCM. The solution was first shaken manually to ensure the powder is evenly spread in the solvent, then subjected to sonication (Thermoline Scientific, Power Sonic420, Korea) at room temperature for 30 minutes on maximum power to maximise the solubility in the solvents. The solution was then filtered using a funnel with a filter paper (Whatman, circle filter paper 125mm Grade 4: 20-25 pm, Sigma-Aldrich, Germany), then the filtered solution was collected into a fresh 500ml round bottom flask. The flask was weighed before and after the evaporation to calculate the extraction yield. The filtered solution in the flask was then evaporated using a rotary evaporator (Buchi R-215 Rotavapor System including Buchi B-491 Heating Bath, BUCHI, Germany) under the vacuum (Buchi V-850 vacuum controller, BUCHI, Germany) at 40 °C. The extract was then subjected to freeze-dry using a freeze drier (Christ™ LDplus freeze-dryer alpha 1-4 Id plus, Germany) overnight. The dried extract was dried using nitrogen gas. Finally, the dried extract was collected and resuspended in methanol and stored in a -20 °C freezer for the subsequent analysis or stored into a -80 °C freezer for further use. The collected extract was then labelled as DCM (G(DCM)) or EtOH (G(ETOH)) extracts.
Extraction of turmeric
Turmeric semi-refined granule powder (extracted by ethyl acetate) was provided by Integria Healthcare, Australia, which contains curcumin, demethxycurcumin, and bisdemethoxycurcumin only. The turmeric granule (50g) was re-suspended in ethyl acetate (500 ml) and sonicated for 30 min. The solution was filtered and concentrated by the rotary evaporator following the same procedure as for ginger. The extract was stored under -20°C for subsequent analysis or -80°C until further use. This turmeric extract had a drug extract ratio of 25:1 (25 parts dry turmeric was extracted to yield 1 part final extract). High performance liquid chromatography
Preparation of ginger extracts/fractions and reference standards: Ginger extracts were dissolved in methanol (HPLC grade, Honeywell B&J ACS/HPLC, Korea) to a final concentration of 1 mg/ml and filtered using syringe filter 4.5 um (Sigma-Aldrich, Sydney, Australia) before subjecting to HPLC analysis. The reference standards of 6-gingerol (6g), 8- gingerol (8g), 10-gingerol (10), 6-shogaol (6s), 8-shogaol (8s), and 10-shogaol (10s) were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China; purity > 98%). The reference compounds' standard stock solution was prepared in methanol at 0.8 mg/ml and stored at - 80 °C until further use. A mixed standard solution containing all six compounds was prepared with each standard concentration at 0.167 mg/ml. It was serially diluted in a 1 :2 ratio to form a standard calibration curve. The mixed standards were prepared at concentrations that produced comparable peak sizes.
HPLC system and method: High-performance liquid chromatography-photodiode array detection (HPLC-PDA) was used to profile the phytochemical composition of the ginger extracts. The HPLC-PDA analysis was performed on the Shimadzu UFLC system (Shimadzu, Australia), comprising an LC-30AD pump, SIL-30ACHT autosampler, SPD-M20A PDA detector and DGU-20A5 inline solvent degasser. The system was controlled using Class-VP 7.4SP4 software. HPLC analysis of the extracts was performed using an Alltech Alltima (Alltech Australia) reverse phase C18 column (46 x 150 mm I.D., 5 pm). The HPLC-PDA analysis of ginger was a modification method from Jung et al. (2019). Solution A (Acetonitrile, dilute phosphoric acid (0.1 %), and methanol (55:44:1) was used as mobile phase in isocratic flow at rate 1 ml/min for 60 min followed by washing method involving solution B (100% Acetonitrile) in between each run as shown in Table 7. The column temperature was kept at 30 °C throughout the analysis. The samples were kept at 4 °C. The injection volume was 25 pl. The PDA (UV 200-500 nm) was recorded, and UV 280 nm was used to quantify the gingerols and shogaols peaks.
Table 7. Chromatographic gradient
TIME SOLUTION A SOLUTION B
Figure imgf000069_0001
0 i 100 0
2 i o 100
12 i 0 100
14 i 100 0
29 i 100 0 Solution A; Acetonitrile, dilute phosphoric acid (1 in 1000), and methanol (55:44:1), solution B; 100% Acetonitrile (Maged, 2017).
HPLC method validation: Eight-point calibration curves were constructed with linear ranges of 0.004 - 1 mg/ml for HPLC-PDA. Furthermore, eight replicates of the calibration standards were prepared, and each was analysed in triplicate. Regression equations, y = ax + b, were obtained from the calibration curve, where x and y were the reference standard's concentrations and the peak area, respectively. The R2 value determined the linearity of the calibration curve. The LOD and LOQ were calculated according to the equations, LOD = 3.33 A~ (standard deviation [SD] of y-intercept/mean of the slope) and LOQ= 10 A~ (SD of y- intercept/mean of the slope) (Chan et al., 2004). The amounts of each analyte were calculated using the corresponding Regression equations. The relative standard deviation (RSD) was used as a repeatability metric. The intra-day precision was assessed by analysing four concentrations of each marker compound three times within a day, and the inter-day reproducibility was assessed over three days (Chan et al., 2004).
Cell culture
The RAW264.7 cell line (provided from the NICM Health Research Institute) was preserved in Dulbecco's Modified Eagle Medium (DMEM) (Lonza, Australia) having 4.5 g/l D-glucose and supplemented with 2 mM I- GlutaMax, 100 U penstrep, and 5% FBS (foetal bovine serum) French origin (Bovogen Biologicals, Keilor East, Australia). Then it was incubated at 37°C, 5% CO2 in 95% air in a vented flask T75 cm2 (Sigma-Aldrich, Sydney, Australia). The cells were sub-cultured every 3-4 days to sustain a controllable number.
The viable and total cell number was analysed by the Vi-CELL XR cell viability analyser (Beckman Coulter, Australia). The cells with a passage under 30 were used for the bioassays.
An example of the counting parameters used for RAW264.7 cells is shown in Table 8.
Table 8. A typical example of RAW264.7 cell counting parameters (n=3)
CONDUCTED VIABILITY % VIABLE AVG. TOTAL TRIAL CELLS/ML DIAMETER CELLS/ML
Figure imgf000070_0001
[MICRONS)
*T#1 85.6 4x 106 . 9.79 4.68x 106
*T#2 95.3 4.88x 106 9.55 5.13x 106
*T#3 90.8 3.92x 106 9.36 4.32x 106
(per 100 images) Raw264.7 cells were diluted to (23.52 x 106 , 24 x 106 and 29.28 x 106 cells/ml), respectively, in complete DMEM. The cells (23.52x10A4/well, 100 pl, 24x10A4/well, 100 pl and 29.28x10A4/well 100 pl) were seeded in 96 well plates and incubated for 48 h at 37°C (5% CO2) to allow attachment of the cells and reach consistent confluence.
The ginger solvents were prepared by weighing (10 mg) of each extract, then were dissolved in 1 ml of DMSO in 1.5 ml centrifuge tube. After the solvent was added, the solutions were vortexed for > 10 min or until dissolved. In order to make the combination of ginger solvent’s with turmeric extract (G(DCM)-T and G(ETOH)-T), (50 ml) of each ginger extract solution were mixed with (20 ml) of turmeric extract (5:2) ratio, before preparing the drug 96 well plate for dilution. Drugs 96 well plate was prepared to dilute ginger solution prior adding into cells, the first row of the plate was filled with 198 ml of fresh DMEM serum-free mixed with 2 ml of each extract solutions that were prepared in DMSO to produce a final volume of 200 ml in the first row of wells. After filling the rest of the wells with 100 ml of fresh DMEM serum-free, the drugs solution was mixed in each well by multichannel pipette and diluted in (1 :2) ratio to produce six serial concentrations (10, 5, 2.5, 1.25, 0.625 and 0.3125 mg/ml), before adding to the cell plates. After seeding the cells for 48 h, the old media was discarded and replaced with another (170 pl) of fresh DMEM serum-free media, the prepared DMSO with ginger solvent’s and turmeric extract solutions were added (20 pl) into each well.
Cells were then pre-treated with various extracts for 2 hours and stimulated with lipopolysaccharides (E. Coli strain 0111 : B4, 3, 3, 5, 5- tetramethylbenzidine, Sigma, Australia). LPS was prepared by dissolving LPS powder into sterile Milli-Q water to make a final concentration of 1 mg/ml. After dissolving 100 pl of LPS stock solution (1 mg/ml) in 49.9 ml of sterile Milli-Q water, 1 ml of the diluted LPS (2 pg/ml), was then mixed with fresh DMEM serum- free media prior to the cell activation. LPS (10 pl) was added to a final volume of 200 pl in the cells to give a final concentration of 50 ng/ml in each well. The cells were then incubated for another 24 h for the following bioassays.
Nitric Oxide (NO) assay - Griess method
The Griess method is a quantitative colourimetric method for the measurement of nitrites. Sodium nitrite was used as a standard to quantify NO release in this model. Briefly, sulfanilic acid (sulphanilamide dissolved in 5% phosphoric acid) is quantitatively converted to a diazonium salt by the reaction with nitrite (stimulated from the cells) in acid solution (Miranda et al., 2001). Thus, the concentration of NO in the cell culture medium can be measured by the Griess reagent proportional to the quantity of nitrite (the end product of NO). In the bioassay, the cell supernatant (100 pl) was collected 16h after the activation with LPS and IFN- and transferred to a new 96 well plate. The Griess reagent was then added to the supernatant with 50 pl of 1% sulfanilamide in 2.5% phosphoric acid and 50 pl of 0.1% N-(1 naphthyl) ethylenediamine dihydrochloride. The Griess reagent with the presence of NO produces a violet colour immediately, and the colour density measured under 545 nm absorbance is proportional to the production of NO. The amount of nitrite was normalised to the control wells, leading to non-stimulated cells providing 0 % NO production and LPS stimulated cells that provide 100 % with NO production.
Cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The reduction of MTT colourimetric assay was used to measure the viability of RAW264.7 cells. MTT is a powdered yellow tetrazole. As the MTT solution is applied to the cells, it reaches the living cells and progresses into the mitochondria, reducing the NAD(P)H-dependent cellular oxidoreductase enzymes that are only present in viable cells (Liu et al., 1997). It then transforms into an insoluble dark purple formazan complex (Liu et a!., 1997). As a result, the purple colour density, which represents the amount of insoluble formazan produced in viable cells, can be used to indicate cell viability. The insoluble formazan is usually solubilized with organic solvents such as isopropanol and dimethyl sulfoxide (DMSO), and this colour measurement can be detected using a microplate reader at a wavelength of 510-590 nm. The cell viability of the measured sample is determined using the equation shown below:
Absorbance of treated cells
Cell viability % = — - - - - - - — — — - - x l00%
Absorbance of untreated cells (control)
In the MTT assay using RAW264.7 cells, MTT solution (50 pl of 0.12 mg/ml in complete medium) was added to the cells and incubated for 2 hours at 37 °C with 5% CO2. Then, the MTT solution was discarded, and 100 pl of DMSO was added to dissolve the formazan crystals. The plate was then shaken for approximately 3 minutes before the absorbance was measured at 545 nm using the FlUOstar omega microplate reader (BMG LABTECH, The Microplate Reader Company, Australia).
Determination of Synergistic, Additive, or Antagonistic Interactions
The determination of possible interaction of the combined extract was conducted using the combined index (Cl) and isobologram models based on Chou-Talalay’s method (Chou, 2006). In this present study, compusyn software 2.0 (Biosoft, USA) was used to analyse the raw data and generate single and dual dose-response curves. The program will construct the combination index-fraction affected (Cl-Fa) curve and isobologram figures for the synergy determination. The Cl-Fa curve illustrates the association between the Cl value and the effective level on a particular biological target. The Cl value of (Cl < 1) indicated synergism, (Cl =1) represent additive effect, and (Cl > 1) denoted antagonism.
For statistical analysis, all data were expressed as mean ± SD (n > 3). Non-parametric t-test and One-way ANOVA were used to analyse statistical differences, and p<0.05 was considered statistically significant.
RESULTS
2.1 Ginger extraction yield
The extraction yields of ginger solvents extracts and fractionation are shown in Table 9.
Table 9. Extracted yield of ginger extracts by five different solvents
Extract Sample weight (g Yield%
Dichioromethane extract 3.15 6.30
Ethanol extract 4.32 8.64
2.2 Quantification of major chemical constituents in ginger extracts
The content of the major compounds in ginger fractions is shown in Table 10.
Table 10A. Contents (mg/g, mean ± SD, n = 3) of the major compounds in ginger fractions analysed by HPLC-PDA.
Marker Content in the solvent extracts (mg/g) compounds
Figure imgf000073_0001
extract) extract)
Figure imgf000073_0002
Table 10B. Calculation of constituients based on contents (mg/g, mean ± SD, n = 3) of the six marker compounds in ginger fractions analysed by HPLC-PDA.
Figure imgf000074_0002
In the solvent extractions, a high total amount of the marker compounds was observed in the ethanol extract (G(ETOH)) at 356.2 mg/g and DCM extract (G(DCM)) at 304.4 mg/g. 8s and 10s were not detected in the extracts, likely due to the low sensitivity of the instrument.
2.3 Anti-inflammatory effects of ginger solvent extracts on LPS induced nitric oxide production in RAW264.7 cells
2.3.1 LPS-induced NO release from RAW264.7 cells.
Without the LPS stimulation, untreated RAW264.7 cells have a low amount of NO (4.5 ± 2.4 pg/ml), and with LPS stimulation, the NO release went up to (121.1 ± 10.2 pg/ml), which was significantly higher (p<0.05, n>3). An MTT assay was conducted to examine the extracts' safe concentration range.
Table 11. Cell viability (% relative to control) in concentrations (0.3125 -10 g /ml) of ginger solvent extracts in RAW264.7 cells tested by MTT assay.
Average cell viability (%) relative to untreated vehicle control
Concentrations G(DCM) G(ETOH)
Figure imgf000074_0001
10 96.31 100
5 93.35 97.77
2.5 97 .34 98.35
1.25 98.01 98.86 0.625 92.32 90.91
0.3125 89.14 92.02
2.3.2 Effects of ginger solvent extracts in inhibiting LPS-induced NO in RAW264.7 cells
The solvent extracts of ginger were tested in the LPS-induced NO assay on RAW264.7 cells, and their IC50 values are shown in Table 12 and Figure 20.
Table 12. Effects of ginger solvent’s extracts on NO production and their IC50 values in LPS-stimulated RAW264.7 macrophages. NO production was measured by the Griess reaction assay and expressed as averaged relative percentage ± STD to the vehicle control.
Average NO production (%) relative LPS stimulated RAW264.7 cells
Concentrations (pG/ML) G(DCM) G(ETOH)
10 17.9 ± 19.2 14.5 ± 10.4
5 7679 + 38.7 74.7 ± 7.5
2.5 99.6± 22.7 85.1 ± 8.2
1.25 102.5 ± 20.9 86.6 ±7.5
0.625 106.6 ± 18.2 90.4 ± 4.5
0.3125 102 ± 17.7 94.1 ± 6.8
IC50 values (pG/ML) 6.80 6.41
Figure 20. Dose-response curves of NO production (% relative to control) in concentrations (0.3125 -10 g /ml) of ginger solvent extracts in RAW264.7 cells tested by Griess assay.
2.4 Bioactivity and synergistic interaction between ginger extracts/fractions and turmeric extract
2.4.1 Combined activities of ginger extracts and turmeric extract in inhibiting LPS-induced NO in RAW264.7 cells.
The results have shown that the IC50 values G(DCM) and G(ETOH) were (6.8 and 6.41 pg/ml, respectively) as shown in table 13 and Figure 21.
Table 13. Individual and combined IC50 values of G and T in inhibiting LPS induced NO expression on RAW264.7 cells.
Samples IC50 (pG/ML) Cl at IC50 G(DCM) 6.80
G(ETOH) 6.41
G(DCM)-T 5.47 (3.91 + 1.56) 0.70
G(ETOH)-T 5.00 (3.57+1.43) 0.81
T 5.95
Figure 21 shows Individual and dual response curves of G with T in inhibiting NO. A. Doseresponse curves of A. G(DCM), T and G(DCM)-T. B. G(ETOH), T and G(ETOH)-T.
2.4.2 Cytotoxicity of ginger extracts/fractions and turmeric extract on RAW264.7 cells
The cell viability shown in table 14 proves that the inhibitory effects of NO for both solvent extractions were not due to cytotoxicity. MTT assay was conducted to examine the safe concentration range for all the extracts.
Table 14. Cell viability (% relative to control) verses concentrations (μg /ml) of combinations G(DCM) and G(ETOH) with T on RAW264.7 cells tested by MTT assay.
Average cell viability (%) relative to untreated vehicle control
Concentrations G(DCM)-T G(ETOH)-T
. feG/ML) .
10 97.16 104.85
5 95.97 101
........................ 2.5 99.01 99.15
0.625 93.56 92.54
0.3125 93.37 93.07
2.4.3 Synergistic effect of ginger solvent’s extract on anti-inflammation
Both G(ETOH)-T and G(DCM)-T exhibited synergistic interaction with Cl values at 0.81 and 0.70 at the Fa50 level.
Figure 22 shows the inhibitory effects of G(DCM)-T and G(ETOH)-T (5:2) on NO production (%) versus concentration (pg/ml) as shown in A and D, respectively. C and F, Isobologram curves for G(DCM)-T and G(ETOH)-T (5:2) on NO. B and E, Cl values verses NO inhibition effect (Fa) by ‘Calcusync’ software. (Dotted line is the reference line, where Cl value equals 1; solid line represents Cl values at different Fa)
Table 15. Cl-Fa levels for (G(DCM)-T and G(ETOH)-T) combinations at Fa 0.25, 0.5, 0.75 and 0.95. Combinations Cl at Fa 0.25 Cl at Fa 0.5 Cl at Fa 0.75 Cl at Fa 0.95
. G(DCM)-T . 0.497. 0.835. 1.442 3.826
''''G(ETOH)-T 0.871 0.813 0 791 0'831
Table 15 represent G-T combinations of synergistic effects at different concentrations level. The Cl values of G(ETOH)-T were below 1 at all Fa levels, which showed synergy when Fa was at 0.25, 0.5, 0.75 and 0.95 respectively, the Cl values of G(DCM)-T at Fa level 0.25 and 0.5 were below 1 , which shows synergy effect.
SUPPLEMENTARY MATERIALS
Supplementary material 1. Validation parameters for the HPLC-PDA method used to quantify ginger (G) and turmeric (T) extracts
Compounds Regression R2 LOD LOQ. Precision, Precision, equation (pg/mL) (pg/mL) RSD% RSD%
Intra-day inter-day
Figure imgf000077_0001
Zingerol Y=81367x +617.45 0.99 1.59 4.79 0.44 3.32
6-gingerol (6g) Y=60921x + 1312 0.99 0.99 2.97 0.14 0.18
8-gingerol (8g) Y= 51224x + 1547.7 0.99 0.92 2.76 0.19 0.28
10-gingerol (10g) Y = 47288x + 1217.4 0.99 1.59 4.78 0.17 0.49
6-shogaol (6s) Y = 79816x + 74.868 1.00 0.21 0.64 0.16 0.85
8-shogaol (8s) Y = 73090x + 73.626 1.00 0.28 0.85 0.12 0.38
10-shogaol (10s) Y = 74753x + 54.276 1.00 0.19 0.57 0.07 0.25
Curcumin (C) Y=67047x - 1803.5 0.99 8.88 26.67 0.10 1.27
Desmethoxycurcumin (D) Y=17556x + 32.161 1.00 0.23 0.68 0.27 0.88
Bisdesmethoxycurcumin Y= 43107x + 54.821 1.00 0.17 0.51 0.22 1.04
Figure imgf000077_0002
Supplementary material 2. Content of bioactive compounds in ginger and turmeric extracts quantified by HPLC-PDA (n>3) and their content in G-T 5:2 dried extract in the cell-based assays.
Herb Com Content in G/T (mg/g) Concentratio Concentration Concentratio al poun determined by HPLC- n (mg/mL) in (mg/mL) in G-T n (pg/mL) in samp ds PDA G/T at 50 5:2 at 50 mg/mL G-T 5:2 at 10 les mg/mL (compounds in pg/mL# ginger x 5/7; (diluted by compounds in 5000 times turmeric x 2/7) when
Figure imgf000077_0003
subjecting to cells)
G 6g 69.57 3.48 2.48 0.50
8g 10.43 0.52 0.37 0.07
10g 19.62 0.98 0.70 0.14
6s 7.48 0.37 0.27 0.05
8s 1.56 0.08 0.06 0.01
10s 2.30 0.12 0.08 0.02
T C 751.76 (1st trial: 740.63) 37.59 (37.03) 10.74 (10.58) 2.15 (2.12)
B 14.77 (1st trial: 19.22) 0.74 (0.96) 0.21 (0.27) 0.04 (0.05)
D 156.15 (1st trial: 475.6) 7.81 (23.78) 2.23 (6.79) 0.45 (1.36)
*These values are used as ratios for the compound’s combination by content (refer to supplementary material 5).
# 10 pg/mL refers to a working concentration of G-T 5:2 in the cell-based assays (western blot, p65 transcription, etc.)
Supplementary material 3. Individual activities of bioactive compounds (0-20 pg/mL) on NO and IL-6 assays (n=3) without causing cytotoxicity.
Bioactive IC50 values for NO assay (pg/mL) IC50 values for IL-6 assay (pg/mL) compounds
Figure imgf000078_0001
6s 2.90 11.73
8s 1.96 10.05
10s 3.69 5.42
C 5.87 6.38
D 8.30 6.45
B 16.14 5.09
*NA: Not available
Supplementary material 4. Interactions of paired bioactive compounds combined by their IC50 values (n=3). Based on the IC50 values reported in supplementary material 3, compounds were mixed from 4 times of their IC50 values (4X) with equal volume, and series diluted to 2X, X, 1/2X, 1/4X, 1/8X before subjecting to the NO and IL-6 assays. Since the IC50 values of 6g, 8g, 10g were not available, their maximum concentration (20 pg/mL) was used for the preparation of mixture.
Compounds NO assay IL-6 assay combination Ratio of the Concentrations IC50 (pg/mL) Cl at IC50 IC50 Cl at by IC5o(l:l) compounds (pg/mL) when IC50 in the subjecting to mixture the cells (1/8X
— 2X)
Figure imgf000079_0001
Figure imgf000079_0002
Supplementary material 5. IC50 and Cl values for compounds combination in inhibiting NO and IL-6 on RAW264.7 cells (n=3). The pair-wise combination was prepared by mixing compounds based on their ratios (content in G-T 5:2 at 50 mg/mL) to reach 10 pg/mL followed by two-fold serial dilutions before subjecting to cells.
Compou NO assay IL-6 assay nds Ratio of Compositio IC50 (pg/mL) Cl at IC50 (pg/mL) Cl at combina the n in the IC50 IC50 tion by compoun mixture in their ds by their 10 pg/mL* content content in when in G-T G-T 5:2 subjecting 5:2 to cells
6g - 6s 2.48:0.27 9.02 + 0.98 91.24 (82.28 + 8.96) 6.68 72.00 (64.93 + 7.07) 2546.97
6g - 8s 2.48:0.06 9.77 + 0.23 198.84 (194.14 + 4.70) 3.19 73.29 (71.56 + 1.73) 2806.94
6g - 10s 2.48:0.09 9.65 + 0.35 126.99 (122.54 + 4.45) 4.69 74.00 (71.41 + 2.59) 2805.09
8g - 6s 0.37:0.27 5.78 + 4.22 8.47 (4.90 + 3.57) 2.85 18.43 (10.65 + 2.34
14.49)
8g - 8s 0.37:0.06 8.60 + 1.40 84.05 (72.32 + 11.73) 10.69 6.17 (3.57 + 2.60) 0.27
8g - 10s 0.70:0.09 8.86 + 1.14 59.73 (52.93 + 6.80) 14.26 13.07 (11.58 + 1.49) 0.52 10g - 6s 0.70:0.27 7.22 + 2.78 22.06 (15.92 + 6.14) 5.81 27.90 (20.13 + 7.77) 3.63 10g - 8s 0.70:0.06 9.21 + 0.79 1174.99 (1082.23 + 92.76) 146.35 313.22 (288.49 + 26.45 24.73)
10g - 0.70:0.09 8.86 + 1.14 24.52 (21.73 + 2.79) 4.64 22.56 (19.99 + 2.57) 1.81
10s
6g - B 2.48:0.27 9.02 + 0.98 43.93 (39.62+4.31) 2.15 4655.57 (4198.48 + 164289
457.09)
6g - C 2.48:10.5 1.90 + 8.10 17.21 (3.27 + 13.94) 5.23 35.73 (6.78 + 28.95) 273.27 8
6g - D 2.48:6.79 2.68 + 7.32 9.44 (2.53 + 6.91) 3.69 18.32 (4.90 + 13.42) 198.04
8g - B 0.37:0.27 5.78 + 4.22 27.33 (15.80 + 11.53) 6.28 2904.95 (1679.42 + 409.32
1225.53)
8g - C 0.37:10.5 0.34 + 9.66 12.31 (0.42 + 11.89) 4.49 34.43 (1.16 + 33.27) 8.44 8
8g - D 0.37:6.79 0.52 + 9.48 4.89 (0.25 + 4.64) 2.48 22.11 (1.14 + 20.97) 9.50
10g - B 0.70:0.27 7.22 + 2.78 17.34 (12.51 + 4.83) 3.35 4.67E11 6.51E10
(3.37E11+1.30E11)
10g - C 0.70:10.5 0.62 + 9.38 10.02 (0.62 + 9.40) 3.58 38.54 (2.39 + 36.15) 9.32
8
10g - D 0.70:6.79 0.93 + 9.07 11.86 (1.11 + 10.75) 5.82 54.83 (5.12 + 49.71) 22.87 6s- B 0.27:0.27 5.00 + 5.00 2.70 (1.35 + 1.35) 1.67 30.14 (15.07 + 9.58
15.07)
6s - C 0.27:10.5 0.25 + 9.75 16.06 (0.40 + 15.66) 6.19 21.00 (0.52 + 20.48) 5.35 8
6s - D 0.27:6.79 0.38 + 9.62 9.06 (0.35 + 8.71) 4.91 37.59 (1.44 + 36.15) 16.83
8s- B 0.06:0.27 1.82 + 8.18 112.59 (20.47 + 92.12) 59.19 NA 3.10
8s - C 0.06:10.5 0.06 + 9.94 13.19 (0.07 + 13.12) 4.98 37.53 (0.21 + 37.32) 9.54
8
8s - D 0.06:6.79 0.09 + 9.91 6.33 (0.06 + 6.27) 3.38 22.63 (0.20 + 22.43) 10.24 10s - B 0.09:0.27 2.50 + 7.50 40.32 (10.08 + 30.24) 25.73 9.70 (2.43 + 7.28) 3.04
10s - C 0.09:10.5 0.08 + 9.92 10.02 (0.08 + 9.94) 3.82 20.73 (0.17 + 20.56) 5.25
8
10s - D 0.09:6.79 0.13 + 9.87 9.68 (0.13 + 9.55) 5.23 28.84 (0.38 + 28.46) 13.00
*10 pg/mL was the highest concentration when subjecting to the cells for the bioassays, and followed by five two-fold serial dilutions.
Supplementary material 6. IC50 and Cl values for extract - compounds combination (combined by their content in G-T 5:2) in inhibiting NO and IL-6 on RAW264.7 cells (n=3). G or T was prepared in 50 mg/mL, and the single compounds (B, C, D, 6g, 8g, 10g, 6s, 8s and 10s) were prepared in the concentration equivalent to their content in G or T at 50 mg/mL. Then they were mixed by taking 50 pL from G or its derived compounds (6g, 8g, 10g, 6s, 8s and 10s) with 20 pL from T or its derived compounds (C, B or D). The pair-wise mixture was then diluted by 1:1000 followed by two-fold serial dilutions before subjecting to cells.
Extract and Ratio of the Composition NO assay IL-6 assay compounds components in in the mixture combination the mixture at highest by their based on their working content in G- concentrations concentration T 5:2 (mg/mL) in G/T when at 50 mg/mL subjecting to cells (pg/mL)
IC50 (pg/mL) Cl IC50 (pg/mL) Cl at at IC50
IC50
G - B 35.71 :0.27 35.98 (35.71 11.47 (11.38 + 0.09) 1.62 143.29 (142.21 2.73
+ 0.27) + 1 .08)
G - C 35.71 :10.58 46.29 (35.71 9.57 (7.38 + 2.19) 1.07 30.14 (23.25 + 2.13
+ 10.58) 6.89
G - D 35.71 :6.79 42.50 (35.71 9.89 (8.31 +1.58) 1.58 53.32 (44.80 + 4.61
+ 6.79) 8.52)
T - 6g 14.29:2.48 16.77 (14.29 10.24 (8.73+ 1.51) 1.34 16.55 (14.10 + 96.70
+ 2.48) 2.45)
T - 8g 14.29:0.37 14.66 (14.29 11.91 (11.61 + 0.30) 1.79 22.77 (22.20 + 1.39
+ 0.37) 0.57)
T - 10g 14.29:0.70 14.99 (14.29 8.08 (7.70 + 0.38) 1.21 16.21 (15.45 + 1.02
+ 0.70) 0.76)
T - 6s 14.29:0.26 14.55 (14.29 9.49 (9.32 + 0.05) 1.56 17.21 (16.90 + 1.16
+ 0.26) 0.31)
T - 8s 14.29:0.06 14.35 (14.29 10.97 (10.92 + 0.05) 1.71 42.02 (41.84 + 2.68
+ 0.06) 0.18)
T - 10s 14.29:0.09 14.38 (14.29 13.17 (13.09 + 0.08) 2.10 NA NA
+ 0.09)
G 11.78 / 32.91 /
T 6.51 / 16.10 /
6g NA / NA /
8g NA / NA /
10g NA / NA /
6s 2.90 / 11.73 /
8s 1.96 / 10.05 /
10s 3.69 / 5.42 / B 5.87 I 6.38 /
C 8.30 I 6.45 I
D 16.14 I 5.09 /
Supplementary material 7. Inhibitory effects of mixed gingerols or shogaols with curcumin (content equivalent to G-T 5:2) on LPS stimulated NO expressions in RAW264.7 cells (n>3).
Mixtures Ratio of Compos IC50 IC50 IC50 Dose Dose Dose gingerols/shogao components in ition in (pg/mL value value of of equivalent
Is - curcumin the mixture the ) s of of C ginger curcu to G-T 5:2 mixture ginge (pg/mL ols/sho min extract in 10 rols/s ) gaols (pg/mL (pg/mL) pg/mL hoga (pg/mL ) in the when ols ) in the mixtur subjecti (pg/ mixtur e at ng to mL) e at IC50 cells IC50
Figure imgf000082_0001
6g8g C 5:2 2.48:0.37:10.7 1.82+0. NA NA 7.56 NA NA NA
4 27+7.90
6g10g C 5:2 2.48:0.70:10.7 1.78+0. 15.17 NA 7.56 3.47 11.71 54.49
4 50+7.72
8g10g C 5:2 0.37:0.70:10.7 0.31 +0. 16.96 NA 7.56 1.54 15.47 71.80
4 59+9.09
6s8s C 5:2 0.27:0.06:10.7 0.24+0. 6.18 NA 7.56 0.18 6.01 27.86
4 05+9.70
6s10s C 5:2 0.27:0.08:10.7 0.24+0. 3.47 NA 7.56 0.11 3.33 15.64
4 07+9.68
8s10s C 5:2 0.06:0.08:10.7 0.06+0. 4.02 NA 7.56 0.05 3.97 19.00
Figure imgf000082_0002
NA: Values not available

Claims

82 Claims
1. A composition comprising turmeric and ginger, wherein the gingerturmeric dry weight or equivalent ratio is about 7:225 to about 63:25.
2. The composition of claim 1 comprising a gingerturmeric dry weight or equivalent ratio of about 7:225 to about 63:25, about 7:100 to about 28:25, about 3:25 to about 7:10, about 14:75 to about 63:25, about 7:25 to about 28:25, about 14:75 to about 28:25, about 21 :50 to about 7:10, about 49:75 to about 28:25, about 49:75 to about 7:10, about 7:10 to about 63:25 or about 7:10 to about 28:25.
3. The composition of claim 1 comprising a gingerturmeric dry weight or equivalent ratio of about 7:225, about 7:100, about 3:25, about 14:75, about 7:25, about 21 :50, about 49:75, about 7:10, about 28:25 and/or about 63:25.
4. The composition of claim 1 comprising a gingerturmeric dry weight or equivalent ratio of about 7:10.
5. A composition comprising 6-shogaol (6s) and curcumin (C), wherein the 6s:C weight ratio is about 0.15:22.55 to about 0.34:3.76.
6. The composition of claim 1 or claim 5 wherein the 6s:C weight ratio is about 0.15:22.55 to about 0.19:18.79, about 0.15:22.55 to about 0.22:15.04, about 0.15:22.55 to about 0.26:11.28, about 0.15:22.55 to about 0.27:10.74, about 0.15:22.55 to about 0.30:7.52, about 0.15:22.55 to about 0.34:3.76, about 0.19:18.79 to about 0.22:15.04, about 0.19:18.79 to about 0.26:11.28, about 0.19:18.79 to about 0.27:10.74, about 0.19:18.79 to about 0.30:7.52, about 0.19:18.79 to about 0.34:3.76, about 0.22: 15.04 to about 0.26: 11 .28, about 0.22: 15.04 to about 0.27:10.74, about 0.22:15.04 to about 0.30:7.52, about 0.22:15.04 to about 0.34:3.76, about 0.26:11.28 to about 0.27:10.74, about 0.26:11.28 to about 0.30:7.52, about 0.26:11.28 to about 0.34:3.76, about 0.27:10.74 to about 0.30:7.52, about 0.27:10.74 to about 0.34:3.76 or about 0.30:7.52 to about 0.34:3.76.
7. The composition of claim 6 wherein the 6s:C weight ratio is about 0.27:10.74 to about 0.49:10.74. 83 The composition of claim 6 or claim 7 further comprising 10-shogaol (10s), wherein the 6s:10s:C weight ratio is about 0.15:0.05:22.55 to about 0.34:0.10:3.76. The composition of claim 8 wherein the 6s:10s:C weight ratio is about 0.15:0.05:22.55 to about 0.19:0.06:18.79, about 0.15:0.05:22.55 to about 0.22:0.07: 15.04, about 0.15:0.05:22.55 to about 0.26:0.08: 11 .28, about 0.15:0.05:22.55 to about 0.27:0.08:10.74, about 0.15:0.05:22.55 to about 0.30:0.09:7.52, about 0.15:0.05:22.55 to about 0.34:0.10:3.76, about 0.19:0.06:18.79 to about 0.22:0.07:15.04, about 0.19:0.06:18.79 to about 0.26:0.08:11.28, about 0.19:0.06:18.79 to about 0.27:0.08:10.74, about 0.19:0.06:18.79 to about 0.30:0.09:7.52, about 0.19:0.06:18.79 to about 0.34:0.10:3.76, about 0.22:0.07:15.04 to about 0.26:0.08:11.28, about 0.22:0.07:15.04 to about 0.27:0.08:10.74, about 0.22:0.07:15.04 to about 0.30:0.09:7.52, about 0.22:0.07:15.04 to about
0.34:0.10:3.76, about 0.26:0.08: 11 .28 to about 0.27:0.08: 10.74, about 0.26:0.08: 11.28 to about 0.30:0.09:7.52, about 0.26:0.08: 11.28 to about 0.34:0.10:3.76, about 0.27:0.08:10.74 to about 0.30:0.09:7.52, about 0.27:0.08:10.74 to about 0.34:0.10:3.76 or about 0.30:0.09:7.52 to about 0.34:0.10:3.76. The composition of any one of the preceding claims wherein the composition comprises, consists essentially of, or consists of ginger and turmeric. The composition of any one of the preceding claims wherein the composition comprises ginger and turmeric, and wherein the ginger is from the rhizome of Zingiber officinale Roscoe. The composition of any one of the preceding claims wherein the composition comprises ginger and turmeric, and wherein the ginger is fresh or dried. The composition of any one of the preceding claims wherein the composition comprises ginger and turmeric, and wherein the ginger is an extract derived the rhizome of Zingiber officinale Roscoe. The composition of claim 13 wherein the ginger extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting ginger with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, 84 aqueous alcohol, ethanol, methanol, warm water by sonication, liquid carbon dioxide and/or other organic solvents. The composition of claim 14 wherein the ginger extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting ginger with 90 % ethanol, 100% ethanol or dichloromethane (DCM). The composition of claim 15 wherein the ginger extract is obtained by extraction with 90% ethanol and has a drug extract ratio (DER) of about 7:1, is extracted with DCM and has a DER of about 16:1, or is extracted with 100% ethanol and has a DER of about 12:1. The composition of any one of claims 5 to 9 wherein the composition comprises isolated, pure and/or synthetic 6g, 8g, 10g, 6s, 8s and/or 10s. The composition of claim 5 to 9 or 17 wherein the 6g, 8g, 10g, 6s, 8s and/or 10s are synthetic, the 6g, 8g, 10g, 6s, 8s and/or 10s are isolated or purified from ginger, or the 6g, 8g, 10g, 6s, 8s and/or 10s are a combination of purified, isolated and/or synthetic. The composition of any one of claims 1 to 16 wherein the composition comprises ginger and turmeric, and wherein the turmeric is from the rhizomes Curcuma longa L. The composition of any one of claims 1 to 16 or 19 wherein the composition comprises ginger and turmeric, and wherein the turmeric is a turmeric extract. The composition of claim 20 wherein the turmeric extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting turmeric with 100% hexane, dichloromethane (DCM), ethyl acetate (EA), alcohol, aqueous alcohol, ethanol, methanol or warm water by sonication, liquid carbon dioxide, other organic solvents and/or liquid carbon dioxide. The composition of claim 21 wherein the turmeric extract is obtained by a solvent extraction method, wherein the solvent extraction method comprises extracting turmeric with ethyl acetate and has a DER of 25:1. 85 The composition of claim 20 wherein the turmeric extract has a drug extract ratio of 25:1 and the ginger extract has a drug extract ratio of about 7:1, about 16:1 or about 12:1. The method of claim any one of claims 19 to 23, wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1. The composition of any one of claims 5 to 9, 18 or 19, wherein the composition comprises pure, isolated and/or synthetic curcumin (C), desmethoxycurcumin (D) and/or bisdemethoxycurcumin (B). The composition of claim 25, wherein the C, D and/or B are synthetic, the C, D and/or B are isolated and/or purified from turmeric, or the C, D and/or B are a combination of purified, isolated and/or synthetic. The composition of claims 25 or 26 wherein the artificial turmeric composition comprises C, D and B; C and D; C and B; or C. The composition of any one of claims 5 to 16 or 19 to 24, wherein the composition comprises turmeric and ginger, and wherein the gingerturmeric dry weight or equivalent ratio is about 7:10. The composition of claims 4 or 28 wherein the turmeric is a turmeric extract and the ginger is a ginger extract, wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract has a drug to extract ratio (DER) of about 25:1 and wherein the ginger extract has a DER of about 7:1 , about 16:1 or about 12:1. The composition of claim 29 wherein the ginger extractturmeric extract ratio is about 5:2, and wherein the turmeric extract is obtained by extraction with ethyl acetate and has a DER of 25:1 , and wherein the ginger extract is obtained by extraction with 90% ethanol and has a DER of about 7:1 , extracted with dichloromethane (DCM) and has a DER of about 16:1 , or is extracted with 100% ethanol and has a DER of about 12:1. A pharmaceutical composition comprising the composition of any one of the preceding claims. 86 The composition of any one of claims 1 to 30 or the pharmaceutical composition of claim 31 , comprising one or more pharmaceutically acceptable carriers. A method of reducing inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of the claims 1 to 30 or pharmaceutical composition of claim 31 or 32. A method of treating a disease or condition associated with inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 1 to 30 or pharmaceutical composition of claim 31 or 32. A method of conferring one or more health benefits to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 1 to 30 or pharmaceutical composition of claim 31 or 32. Use of a composition of any one of claims 1 to 30 or pharmaceutical composition of claim 31 or 32 in the manufacture of a medicament for reducing inflammation. Use of a composition of any one of claims 1 to 30 or pharmaceutical composition of claim 31 or 32 in the manufacture of a medicament for treating a disease or condition associated with inflammation. Use of a composition of any one of claims 1 to 30 or pharmaceutical composition of claim 31 or 32 in the manufacture of a medicament for conferring one or more health benefits.
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