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WO2018090805A1 - Skin-protection composition containing dendrobium-based ingredients - Google Patents

Skin-protection composition containing dendrobium-based ingredients Download PDF

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Publication number
WO2018090805A1
WO2018090805A1 PCT/CN2017/107798 CN2017107798W WO2018090805A1 WO 2018090805 A1 WO2018090805 A1 WO 2018090805A1 CN 2017107798 W CN2017107798 W CN 2017107798W WO 2018090805 A1 WO2018090805 A1 WO 2018090805A1
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Prior art keywords
skin
compound
resveratrol
independently selected
dihydro
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PCT/CN2017/107798
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French (fr)
Inventor
Hongjie Zhang
Siu Wai Tsang
Yu Zhu
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Hong Kong Baptist University HKBU
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Hong Kong Baptist University HKBU
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Priority claimed from US15/351,636 external-priority patent/US9738581B2/en
Application filed by Hong Kong Baptist University HKBU filed Critical Hong Kong Baptist University HKBU
Priority to MYPI2019002612A priority Critical patent/MY187342A/en
Priority to JP2019524017A priority patent/JP7051843B2/en
Priority to CN201780069402.9A priority patent/CN109952286B/en
Priority to TW106138517A priority patent/TWI732967B/en
Publication of WO2018090805A1 publication Critical patent/WO2018090805A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
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    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/67—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of saturated acids
    • C07C69/708—Ethers
    • C07C69/712—Ethers the hydroxy group of the ester being etherified with a hydroxy compound having the hydroxy group bound to a carbon atom of a six-membered aromatic ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
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    • C07C43/02—Ethers
    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/205—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring the aromatic ring being a non-condensed ring
    • C07C43/2055—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring the aromatic ring being a non-condensed ring containing more than one ether bond
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    • C07C43/20—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring
    • C07C43/215—Ethers having an ether-oxygen atom bound to a carbon atom of a six-membered aromatic ring having unsaturation outside the six-membered aromatic rings
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    • C07C69/22—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety
    • C07C69/30—Esters of acyclic saturated monocarboxylic acids having the carboxyl group bound to an acyclic carbon atom or to hydrogen having three or more carbon atoms in the acid moiety esterified with trihydroxylic compounds
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    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
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    • C07C69/54—Acrylic acid esters; Methacrylic acid esters
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    • C07C69/612—Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety
    • C07C69/614—Esters of carboxylic acids having a carboxyl group bound to an acyclic carbon atom and having a six-membered aromatic ring in the acid moiety of phenylacetic acid
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
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    • C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
    • C07C69/84—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring
    • C07C69/92—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring of monocyclic hydroxy carboxylic acids, the hydroxy groups and the carboxyl groups of which are bound to carbon atoms of a six-membered aromatic ring with etherified hydroxyl groups
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    • C07C2601/08—Systems containing only non-condensed rings with a five-membered ring the ring being saturated

Definitions

  • the present invention relates to a skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) isolated from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile for the management of melanogenesis, skin-darkening and skin-aging. More particularly, it relates to the usage of Dendrobium ingredients to reduce the formation of melanin in melanocytes. It also relates to the usage of Dendrobium ingredients to reduce the generation of reactive oxygen species and oxidative free radicals in melanocytes. The present invention further relates to the use of Dendrobium-derived extracts or ingredients in the formulation of skin-protection, skin-whitening and/or anti-skin aging products.
  • UV radiation ultraviolet
  • ROS reactive oxygen species
  • melanin-producing cells or melanocytes, located in the skin’s epidermis are prone to produce more melanin in order to minimize the indirect damages of body cells
  • the elevation of melanin production is actually a defense mechanism against UV radiation; however, the accumulation of melanin may result in deleterious biological effects, including abnormal pigmentation, skin darkening as well as aesthetic problems, e.g. freckles or chloasmata [Mishima Y, Imokawa G. Selective aberration and pigment loss in melanosomes of malignant melanoma cells in vitro by glycosylation inhibitors: premelanosomes as glycoprotein. Journal of Investigative Dermatology 1983; 81: 106-114] .
  • the formation of ROS and free radicals plays a pivotal mechanism leading to skin-darkening and skin-aging.
  • melanin refers to a group of endogenous pigments that give multitude of skin colors, i.e. from the basic skin tone to freckles, birth marks, age spots, also in eyes and hair.
  • melanins are produced by melanocytes and classified into three major types: eumelanin, pheomelanin and neuromelanin. They differ in chemical structures and physical properties, and thus they are elicited upon different biological responses against external stimuli.
  • Eumelanin and pheomelanin are the two types of melanin that can be found in the skin. Eumelanin provides primarily dark brown to black colors whereas pheomelanin produces reddish colors [Maresca V, Flori E, Picardo M.
  • tyrosinase is the major rate-limiting enzyme for regulating melanin production in epidermal melanocytes whereas tyrosinase-related proteins (TRPs) are melanogenic enzymes that control the proportion of carboxylated subunits in melanin biopolymers.
  • TRPs tyrosinase-related proteins
  • ⁇ -MSH alpha-melanocyte-stimulating hormone
  • the present invention provides a composition that efficiently attenuates the biosynthesis of melanin as well as oxidative substances. By any means of mechanism, the present invention is able to restrain melanin accumulation and/or oxidative damage in the skin, hence skin protection can be achieved.
  • the composition of present invention comprises stilbenoids and/or extracts obtained from plants of the genus Dendrobium (Orchidaceae family, commonly called “Shi Hu” ) , particularly from the species D. officinale and D. nobile.
  • the said stilbenoids, explicitly trans-resveratrol and dihydro-resveratrol, and extracts can be formulated into a cosmetic blend of skin-protection, skin-whitening and/or anti-skin aging products for human skin as they are found to attenuate the generation of ROS and oxidative free radicals, and/or reduce cellular melanin content in melanocytes.
  • the reduction of melanin formation may be associated with an inhibition of the activity of pigment-producing enzymes, namely tyrosinase, TRP-1 and TRP-2.
  • the objective of this invention relates to a skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) isolated from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile.
  • This invention also relates to the use of Dendrobium-derived extracts or ingredients in reducing melanin formation in melanocytes for the management of melanogenesis and skin-darkening.
  • this invention also relates to the use of Dendrobium-derived extracts or ingredients in the formulation of skin-protection, skin-whitening and/or anti-skin aging products.
  • Said compound has a formula of
  • R 2 and R 4 are each independently selected from -OR 11 and -OC (O) R 11 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 11 and -OC (O) R 11 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 13 , -C (O) R 14 , -C (O) N (R 13 ) R 14 , -C (O) OR 13 , -OC (O) R 14 , -S (O) 2 R 13 , -S (O) 2 N (R 13 ) R 14 , -N (R 13 ) R 14 ;
  • R 13 and R 14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need thereof comprising administering a composition comprising one or more compounds of formulae (i) to (viii) or dihydro-resveratrol [i.e., formula (2) ] to a subject in need thereof.
  • a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need thereof wherein the composition is administered to a subject in need thereof with a dosage of at least 1.622 mg/kg/day wherein said composition consists of a compound with a formula of
  • a third embodiment of the second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need wherein the said subject is human.
  • a fourth embodiment of the second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need wherein the composition is administered orally to said subject in need thereof.
  • said composition is administered to said subject orally.
  • a compound for use in treating pancreatogenic diabetes or Type 3c diabetes milieus in a subject in need thereof with a therapeutically effective amount of a compound of the following formula:
  • R 2 and R 4 are each independently selected from -OR 11 and -OC (O) R 11 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 11 and -OC (O) R 11 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 13 , -C (O) R 14 , -C (O) N (R 13 ) R 14 , -C (O) OR 13 , -OC (O) R 14 , -S (O) 2 R 13 , -S (O) 2 N (R 13 ) R 14 , -N (R 13 ) R 14 ;
  • R 13 and R 14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • a composition comprising compounds of the following formula:
  • R 2 and R 4 are each independently selected from -OR 11 and -OC (O) R 11 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 11 and -OC (O) R 11 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 13 , -C (O) R 14 , -C (O) N (R 13 ) R 14 , -C (O) OR 13 , -OC (O) R 14 , -S (O) 2 R 13 , -S (O) 2 N (R 13 ) R 14 , -N (R 13 ) R 14 ;
  • R 13 and R 14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • a method for treating pancreatogenic diabetes or Type 3c diabetes milieus
  • the composition is administered to a subject in need thereof with a dosage of at least 1.622 mg/kg/day wherein said composition consists of a compound with a formula of
  • a method for treating pancreatogenic diabetes or Type 3c diabetes milieus in a subject in need thereof wherein said subject is human.
  • a method for treating pancreatogenic diabetes or Type 3c diabetes milieus
  • the composition is administered orally to said subject in need thereof.
  • said compound is used in preparation of a composition for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in said subject, and said subject is human.
  • a composition for use in treating pancreatogenic diabetes comprising a therapeutically effective amount of a compound of one of the following formulae:
  • compositions for treating pancreatogenic diabetes or Type 3c diabetes milieus
  • the composition is administered to said subject with a dosage of at least 1.622 mg/kg/day
  • said composition consists of a compound with a formula of
  • a composition for treating pancreatogenic diabetes or Type 3c diabetes milieus
  • said subject is human.
  • R 2 , R 4 , and R 8 are each independently selected from -OR 11 , -OCH 2 R 12 , -OC (O) R 11 , -OCH 2 C (O) OR 12 and -OC (O) CH 2 R 12 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 12 and -OC (O) R 12 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently selected from – (CH 2 ) -hydrocarbyl, C 2-10 alkyl, alkenyl, cycloalkyl, aryl and heterocyclyl, each of these groups is optionally substituted with 1, 2, 3, 4 or 5 R 13 ;
  • R 12 is independently selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 13 ;
  • R 13 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 14 , -C (O) R 15 , -C (O) N (R 14 ) R 15 , -C (O) OR 14 , -OC (O) R 15 , -S (O) 2 R 14 , -S (O) 2 N (R 14 ) R 15 , -N (R 14 ) R 15 ;
  • R 14 and R 15 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • a compound of formula (4) wherein said compound is an optically pure stereoisomer, enantiomer, racemate or diastereomer thereof.
  • a pharmaceutical composition comprising an effective amount of the compound of formula (4) , the derivatives thereof, and a pharmaceutically acceptable carrier thereof.
  • a pharmaceutical composition comprising an effective amount of the skin whitening and skin protection compound of formula (4) , the derivatives thereof, and a pharmaceutically acceptable carrier thereof.
  • a compound of formula (4) wherein the compound is selected from DR1, DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10 or DR11 with the following formulae respectively:
  • a seventh embodiment of the fifth aspect of the present invention there is provided the use of the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) in the preparation of the composition for treatment, prevention or delay of progression of a skin darkening, wherein said subject is a human.
  • an eighth embodiment of the fifth aspect of the present invention there is provided a method of treating and preventing development and progression of a skin darkening comprising administering the compound of formula (4) and/or the derivatives thereof to a subject in need thereof.
  • a ninth embodiment of the fifth aspect of the present invention there is provided a method of treating and preventing development and progression of a skin darkening comprising administering the compound of formula (4) and/or the derivatives thereof to a subject in need thereof wherein said subject is a human.
  • a tenth embodiment of the fifth aspect of the present invention there is provided the use of the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) in the preparation of the composition for treatment, prevention or delay of progression of a skin darkening in a subject in need thereof, wherein the composition is applied topically.
  • the composition comprising the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) , wherein the composition is in the form of paste, solid, powder, particle, emulsion, a day cream, a night cream, a face lotion, a body lotion, a body butter, a skin peel, a mask, a shower gel, a sun cream, a sun lotion, an after sun cream, an after sun lotion, a lip balm, a lip gloss, or alike.
  • a skin whitening and skin protection against UV exposure, skin damage and aging compound of formula (5) in a first embodiment of a sixth aspect of the present invention there is provided a skin whitening and skin protection against UV exposure, skin damage and aging compound of formula (5) :
  • R 2 , R 4 , and R 8 are each independently selected from -OR 11 , -OCH 2 R 11 , -OC (O) R 11 , -OCH 2 C (O) OR 11 and -OC (O) CH 2 R 11 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 11 and -OC (O) R 11 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 13 , -C (O) R 14 , -C (O) N (R 13 ) R 14 , -C (O) OR 13 , -OC (O) R 14 , -S (O) 2 R 13 , -S (O) 2 N (R 13 ) R 14 , -N (R 13 ) R 14 ;
  • R 13 and R 14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • a skin whitening and skin protection against UV exposure, skin damage and aging compound with a formula of:
  • a seventh aspect of the present invention there is provided a method of synthesizing the compound of formulae DR1 to DR11 from dihydro-resveratrol according to the following synthesis schemes, respectively:
  • R is -OR 11 , -OCH 2 R 11 or -OCH 2 C (O) OR 11 ;
  • R 11 is hydrocarbyl
  • R is -R 11 or -CH 2 R 11 ;
  • R 11 is hydrocarbyl, which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is halogen or -OR 13 ;
  • R 13 is hydrogen or hydrocarbyl.
  • dihydro-resveratrol and RBr are added in dimethylformamide (DMF) and K 2 CO 3 to form a mixture.
  • the resulting mixture is stirred at room temperature until the starting material disappear on thin-layer chromatography (TLC) .
  • TLC thin-layer chromatography
  • the mixture is then diluted with H 2 O and washed with dichloromethane (DCM) three times to extract the organic phase.
  • dihydro-resveratrol and RCOCl are added into DCM and Et 3 N at 0 °C to form a mixture.
  • the resulting mixture is warmed to room temperature and stirred until the starting material disappear on TLC.
  • the mixture is then diluted with H 2 O and washed with DCM three times to extract the organic phase.
  • a method of treating acute inflammatory condition of the pancreas and associated systemic complications by administering to a subject in needs thereof a composition comprising an effective amount of a stilbenoid derivative which comprises a compound of formula (1) , wherein R 1 , R 2 and R 3 are each independently selected from an alkyl group.
  • a stilbenoid derivative which comprises a compound of formula (1) , wherein R 1 , R 2 and R 3 are each independently selected from an alkyl group.
  • alkyl alone or in combination with other groups, includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the term is further exemplified by such groups as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl and tert-butyl) , pentyl, hexyl and the like, and the derivatives or chemical variants thereof; or a mixture of said compound, the derivative and/or chemical variants thereof.
  • the stilbenoid derivative is trans-3, 5, 4'-trihydroxybibenzyl, or dihydro-resveratrol, which is a compound of formula (2) : and the derivatives or chemical variants thereof; or a mixture of said compound, the derivative and/or chemical variants thereof.
  • the invention includes all such variation and modifications.
  • the invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
  • Figure 1 shows the gain of water content in rats due to effect of pancreatic edema as a result of cerulein-induced acute pancreatitis.
  • the obtained weights are expressed as a ratio percentage of pancreatic weight to body mass.
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Figures 2A to 2F show the architecture and morphological alteration in pancreatic tissues of control group (Figure 2A) , cerulein group (Figure 2B) and dihydro-resveratrol treatment groups (D-Res) ( Figures 2C to 2F) by means of hematoxylin and eosin (H&E) staining. Images are shown with a scale bar of 50 ⁇ m.
  • Figures 3A to 3E show the architecture and morphological alteration in pulmonary tissues of control group (Figure 3A) , cerulein group (Figure 3B) and dihydro-resveratrol treatment groups (D-Res) ( Figures 3C to 3E) by means of H&E staining. Images are shown with a magnification of 200 ⁇ .
  • Figure 4A shows the measurement of MPO activity which represents neutrophil sequestration in pancreatic tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of colorimetric spectrophotometry.
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Figure 4B shows the measurement of MPO activity which represents neutrophil sequestration in pulmonary tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of colorimetric spectrophotometry.
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Figure 5A shows the measurement of TNF- ⁇ level in pancreatic tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of enzyme-linked immunosorbent assay (ELISA) .
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Figure 5B shows the measurement of TNF- ⁇ level in pulmonary tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of ELISA.
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Figure 6A shows the measurement of glutathione levels in pancreatic tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of colorimetric spectrophotometry.
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Figure 6B shows the ameliorative effect of dihydro-resveratrol (D-res) and trans-resveratrol (Res) on reducing water content as a result of pancreatic edema in rats with cerulein-induced acute pancreatitis.
  • the obtained weights are expressed as a ratio percentage of pancreatic weight to body mass.
  • a p-value of less than 0.05 is considered as statistically significant. *p ⁇ 0.05 when comparing with saline-treated control group (Con) whereas #p ⁇ 0.05 comparing with cerulein-treated control group.
  • Figure 7 shows the measurement of metabolic rates by means of MTT cell proliferation in pancreatic acinar cells treated with dihydro-resveratrol and trans-resveratrol. *p ⁇ 0.05 when comparing with cells treated without dihydro-resveratrol or trans-resveratrol.
  • Figures 8A to 8H shows eight derivatives (i.e. Compound i to Compound viii) from dihydro-resveratrol (i.e. Compound 2) .
  • Figure 8I shows the chemical structure of dihydro-resveratrol (i.e. Compound 2) .
  • Figure 9 shows Western blotting of LTC-14 PSCs pre-incubated with TGF- ⁇ (5 ng/mL) , and treated with trans-resveratrol (Resv) or dihydro-resveratrol or stilbene compounds i to viii at 20 ⁇ g/mL for 24 hours. Control was not treated with Resv or any stilbenoids.
  • Figure 10 shows the probing of ⁇ -SMA and FN1 in Western blotting of LTC-14 cells pre-incubated with TGF- ⁇ (5 ng/mL) , and treated with trans-resveratrol or dihydro-resveratrol at the indicated concentrations.
  • Figure 11 shows the green fluorescent signal (identified by arrow marks) of fibrotic filaments ⁇ -SMA in LTC-14 cells implying the degree of PSC activation.
  • Figure 12 shows the fluorescent signal of fibronectin (FN1) deposition in pancreatic tissue sections for an estimation of the degree of fibrosis with and without treatment of dihydro-resveratrol (D-Res, 20 mg/kg/day) or trans-resveratrol (Res, 20 mg/kg/day) in cerulein (Cer) -induced chronic pancreatitis mice.
  • D-Res dihydro-resveratrol
  • Res trans-resveratrol
  • Figure 13 shows the glucose response of the normal mice (Control) and chronic pancreatitis mice (Cer) with or without treatment of dihydro-resveratrol (D-Res, 20 mg/kg/day) in the intraperitoneal glucose tolerance test. At all time-points, a significant difference (p ⁇ 0.05) is achieved between the Cer group and the Cer+D-Res group.
  • FIG 14 shows the fluorescent signal of insulin in pancreatic tissue sections for an evaluation of pancreatic insulin-secreting cell (i.e. beta-cell) area. The comparison is made among the control mice, chronic pancreatitis mice and the chronic pancreatitis mice with treatment of dihydro-resveratrol (D-Res) at 20 mg/kg/day.
  • D-Res dihydro-resveratrol
  • D-Res dihydro-resveratrol
  • Res trans-resveratrol
  • Figure 16 shows the suppressive effect of the Dendrobium stilbenoids (25 ⁇ M) , which are dihydro-resveratrol (D-Res) , trans-resveratrol (Res) and compounds DR1 to DR11, on cellular melanin content in A375 melanocytes.
  • D-Res dihydro-resveratrol
  • Res trans-resveratrol
  • Figure 17 shows the suppressive effect of D. nobile extract (JCSH, 50 ⁇ g/mL) , D. officinale extract (TPSH, 50 ⁇ g/mL) , trans-resveratrol (Res, 25 ⁇ M) and dihydro-resveratrol (D-Res, 25 ⁇ M) on cellular melanin content in B16 melanocytes.
  • Ascorbic acid (AC) is served as a positive control.
  • Figure 19 shows the inhibitory effect of the Dendrobium stilbenoids (25 ⁇ M) , which are dihydro-resveratrol (D-Res) , trans-resveratrol (Res) and compounds DR1 to DR11, on cellular tyrosinase activity in A375 melanocytes.
  • D-Res dihydro-resveratrol
  • Res trans-resveratrol
  • Figure 20 shows the inhibitory effect of D. nobile extract (JCSH, 50 ⁇ g/mL) , D. officinale extract (TPSH, 50 ⁇ g/mL) , trans-resveratrol (Res, 25 ⁇ M) and dihydro-resveratrol (D-Res, 25 ⁇ M) on cellular tyrosinase activity in B16 melanocytes.
  • Ascorbic acid (AC) is served as a positive control.
  • Figure 21 shows suppressive effect of D. nobile extract (JCSH, L: 10; M: 25; H: 50 ⁇ g/mL) and D. officinale extract (TPSH, L: 10; M: 25; H: 50 ⁇ g/mL) , dihydro-resveratrol (D-Res, L: 25; H: 50 ⁇ M) , trans-resveratrol (Res, L: 25; H: 50 ⁇ M) and ascorbic acid (AC, H: 50 ⁇ g/mL) on protein levels of TRP-1, TRP-2, phospho-AKT and phospho-p38 in B16 melanocytes.
  • GAPDH is served as a loading reference.
  • Figure 23 shows the whitening effect of stilbenoid solution containing 2%trans-resveratrol (Res) or 2%dihydro-resveratrol (D-Res) on the arm of an individual human subject on day 7 and day 14.
  • hydrocarbyl as used herein includes reference to a moiety consisting exclusively of hydrogen and carbon atoms; such a moiety may comprise an aliphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.
  • hydrocarbyl groups include C 1-6 alkyl (e.g. C 1 , C 2 , C 3 or C 4 alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl) ; C 1-6 alkyl substituted by aryl (e.g.
  • benzyl or by cycloalkyl (e.g. cyclopropylmethyl) ; cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) ; aryl (e.g. phenyl, naphthyl or fluorenyl) and the like.
  • cycloalkyl e.g. cyclopropylmethyl
  • cycloalkyl e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl
  • aryl e.g. phenyl, naphthyl or fluorenyl
  • alkyl as used herein includes reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.
  • alkyl groups include “C 1-6 alkyl” and “C 2-10 alkyl” .
  • C 1-6 alkyl as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms.
  • C 2-10 alkyl as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
  • This term includes reference to groups such as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl or tert-butyl) , pentyl, hexyl and the like.
  • the alkyl moiety may have 1, 2, 3, 4, 5 or 6 carbon atoms.
  • alkenyl and C 2-6 alkenyl as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one double bond, of either E or Z stereochemistry where applicable. This term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like.
  • alkynyl and C 2-6 alkynyl as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one triple bond. This term includes reference to groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl and 3-hexynyl and the like.
  • alkoxy and C 1-6 alkoxy as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.
  • cycloalkyl as used herein includes reference to an alicyclic moiety having 3, 4, 5, 6, 7 or 8 carbon atoms.
  • the group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, bicyclo [2.2.2] octyl and the like.
  • aryl as used herein includes reference to an aromatic ring system comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms.
  • Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
  • Cyclic group means a ring or ring system, which may be unsaturated or partially unsaturated but is usually saturated, typically containing 5 to 13 ring-forming atoms, for example a 5-or 6-membered ring.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups. Cyclic group includes carbocyclyl and heterocyclyl moeities.
  • carbocyclyl as used herein includes reference to a saturated (e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon ring atoms.
  • carbocyclyl includes a 3-to 10-membered ring or ring system and, in particular, 5-or 6-membered rings, which may be saturated or unsaturated.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • a carbocyclic moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo [2.2.2] octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
  • heterocyclyl as used herein includes reference to a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulphur.
  • heterocyclyl includes a 3-to 10-membered ring or ring system and more particularly a 5-or 6-membered ring, which may be saturated or unsaturated.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • a heterocyclic moiety is, for example, selected from oxiranyl, azirinyl, 1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morph
  • heterocycloalkyl as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1 , 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur.
  • the group may be a polycyclic ring system but more often is monocyclic.
  • This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • heteroaryl as used herein includes reference to an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen and sulphur.
  • the group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but is more often monocyclic.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • This term includes reference to groups such as pyrimidinyl, furanyl, benzo [b] thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo [b] furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like.
  • halogen as used herein includes reference to F, Cl, Br or I.
  • halogen containing moiety includes reference to a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur which moiety includes at least one halogen.
  • the moiety may be hydrocarbyl for example C 1-6 alkyl or C 1-6 alkoxy, or carbocyclyl for example aryl.
  • substituted as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents.
  • optionally substituted as used herein means substituted or un-substituted. It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.
  • enantiomer as used herein means one of two stereoisomers that have mirror images of one another.
  • racemate as used herein means a mixture of equal amounts of enantiomers of a chiral molecule.
  • diastereomer as used herein means one of a class of stereoisomers that are not enantiomers, but that have different configurations at one or more of the equivalent chiral centers.
  • Example of diasteromers are epimers that differ in configuration of only one chiral center.
  • stereoisomer as used herein means one of a class of isomeric molecules that have the same molecular formula and sequence of bonded atoms, but different three-dimensional orientations of their atoms in space.
  • a prodrug is a medication that is administered as an inactive (or less than fully active) chemical derivative that is subsequently converted to an active pharmacological agent in the body, often through normal metabolic processes.
  • morphological alterations of organ architecture including interstitial edema, cellular damage, leukocyte infiltration and hemorrhage are characterized as the histological and/or pathological parameters.
  • aberrant MPO activity is often measured for assessing the severity of neutrophil-mediated inflammatory condition.
  • Both the local and systemic inflammatory responses can be further confirmed by the high levels of pro-inflammatory cytokines present in the homogenates of affected tissues.
  • glutathione depletion a defense mechanism, is one of the most common parameters for assessing the severity of tissue injury.
  • the subject to be treated by the method of this invention may be a human or an animal.
  • the present invention is applicable to various forms of acute pancreatitis, and particularly to the acute pancreatitis associated systemic complications including pulmonary injury.
  • Dihydro-resveratrol also known as trans-3, 5, 4'-trihydroxybibenzyl, is a derivative of polyphenol belonging to the family of stilbenoids, which are often obtained from plant extracts.
  • dihydro-resveratrol is a phytoalexin produced by various plant species including Orchidaceae and Cannabis sativa L. against abiotic and biotic challenges, particularly in the case of fungal infection as reported in Fritzemeier, K.H., Kindl, H. 1983. 9, 10-dihydrophenanthrenes as phytoalexins of Orchidaceae.
  • the present invention relates to the usage of a polyphenol derivative of the stilbenoid family with formula (1) :
  • R 1 , R 2 and R 3 are each independently selected from an alkyl group.
  • alkyl alone or in combination with other groups, includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the term is further exemplified by such groups as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl and tert-butyl) , pentyl, hexyl and the like, to ameliorate tissue injury of the pancreas and lungs.
  • the present invention further relates to the usage of a stilbene compound containing trans-3, 5, 4'-trihydroxybibenzyl, also known as dihydro-resveratrol, see Compound of formula (2) :
  • this particular stilbenoid derivative was obtained as white powders through dehydrogenation of trans-resveratrol.
  • the invention is concerned with a process for the manufacture of the above compound, pharmaceutical preparations which contain such compound, and the use of this compound for the production of pharmaceutical preparations.
  • mice with acute pancreatitis are shown to have lessened pancreatic water content due to an attenuation of pancreatic edema ( Figure 1) , lowered plasma level of ⁇ -amylase (Table 1) , more intact acinar morphology ( Figures 2C to 2F) and reduced thickening of alveolar wall and hemorrhage ( Figures 3C to 3E) .
  • Plasma ⁇ -amylase activities are expressed as U/ ⁇ l/minute. A p-value of less than 0.05 is considered as statistically significant and S. D. stands for standard derivation. *p ⁇ 0.05 when comparing with control group whereas #p ⁇ 0.05 comparing with cerulein group.
  • Cerulein-induced elevated levels of neutrophil sequestration which is quantified as the activity of MPO, are significantly suppressed in pancreatic and pulmonary tissues by the administration of dihydro-resveratrol ( Figures 4A and 4B) .
  • Cerulein-induced elevated levels of TNF- ⁇ in the pancreas and lungs are significantly suppressed by the administration of dihydro-resveratrol ( Figures 5A and 5B) .
  • Glutathione depletion is a distinctive sign of tissue injury.
  • the cerulein-induced declined levels of glutathione in the pancreas are significantly restored by the administration of dihydro-resveratrol (Figure 6A) .
  • dihydro-resveratrol completely and easily dissolves in 0.5% (weight/volume, w/v) methanol whereas trans-resveratrol, with vigorous shaking, dissolves in 2.5% (w/v) methanol (Table 2) .
  • the solubility of dihydro-resveratrol is at least 5 times higher than that of trans-resveratrol.
  • the ameliorative effect of dihydro-resveratrol was more promising than that of trans-resveratrol on reducing water content as a result of pancreatic edema in rats with cerulein-induced acute pancreatitis ( Figure 6B) .
  • Table 2 Solubility of Dihydro-resveratrol and Trans-resveratrol in methanol.
  • the cytotoxicity of dihydro-resveratrol in pancreatic acinar cells is determined to be approximately 500 ⁇ M whereas that of trans-resveratrol is roughly 250 ⁇ M ( Figure 7) .
  • the cytotoxicity of dihydro-resveratrol was 50%lower than that of trans-resveratrol.
  • dihydro-resveratrol Preparation and structural identification of dihydro-resveratrol.
  • the molecular formula of dihydro-resveratrol was established as C 14 H 14 O 3 , which was obtained as white powders through hydrogenation of trans-resveratrol.
  • a solution of trans-resveratrol (10 g, 43.8 mmol) in anhydrous EtOH (150 ml) was stirred at room temperature under 5 atm H 2 pressure in the presence of 10%Pd/C (0.2 g) . The reaction was quenched after 8 hours (h) , by filtering off the catalyst.
  • Sprague-Dawley rats aged 28 days weighing in the range of 70 to 90 g were randomly assigned into 6 groups of 6 to 8 individuals.
  • the rats were housed with an ambient temperature of 23 ⁇ 2°C, a relative humidity of 60 to 80%and a 12-h light/dark cycle. Prior to the experiment, the rats were starved overnight but allowed with free access to water.
  • Experimental acute pancreatitis was induced in the rats by six hourly i. p. injections of cerulein at the supramaximally stimulating dose (50 ⁇ g/kg) followed by a single dose of LPS at 7.5 mg/kg 1 h after the last cerulein injection, and this group of rats was designated as the cerulein group.
  • the control group received injections of 0.9%saline instead of cerulein in the same volume and at same time intervals.
  • the treatment groups given with cerulein and oral doses of dihydro-resveratrol (10, 20 or 50 mg/kg) were designated as Cerulein + D-res 10 or 20 or 50 mg/kg.
  • the therapeutic intervention was given 30 minutes after the first cerulein injection for three consecutive hours.
  • pancreata were immediately removed, weighed, trimmed from fat and fixed in 4 %paraformaldehyde-phosphate buffered saline overnight at 4 °C. Samples were then processed, embedded in paraffin wax, sectioned and subjected to H&E staining. Levels of TNF- ⁇ in pancreatic and pulmonary samples were determined using commercial ELISA kits. Tissue homogenates were subjected to biochemical assays for the evaluation of MPO activity and glutathione content.
  • acini Functional intact acini were dissociated from pancreatic tissue using collagenase digestion with mild shearing forces.
  • Acini were cultured in Dulbecco’s modification of Eagle’s medium (GIBCO) supplemented with 5%fetal bovine serum (GIBCO) , 1%penicillin-streptomycin (GIBCO) in a 5%CO 2 , 95%air humidified atmosphere at 37°C.
  • LTC-14 cells were seeded at a density of 1 ⁇ 10 4 /well in a 96-well plate, and incubated with different concentrations of dihydro-resveratrol or trans-resveratrol (dissolved in DMSO) for 24 hours.
  • MTT reagent was added to the cells at the end of the 24-hour treatment period. After a 3-hour reaction time, MTT products were dissolved in DMSO and absorbance at 570 nm was taken.
  • pancreas to body in the acute pancreatitis rats was drastically increased by roughly 60%when compared with the non-cerulein induced controls due to the occurrence of pancreatic edema.
  • the oral administration of dihydro-resveratrol at an adequate dosage of not less than 20 mg/kg notably reduced the pancreatic edema as reflected by the significant decrease in the weight ratio of pancreas to body.
  • the ameliorative effect of dihydro-resveratrol on reducing pancreatic edema was more promising than that of trans-resveratrol, the accredited antioxidant.
  • the comparable dosage is 3.24 mg/kg based on the standard dosage conversion according to Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J 22 (3) : 659–661.
  • the levels of pro-inflammatory cytokine TNF- ⁇ as well as MPO activities were significantly reduced in the pancreatic and pulmonary tissues by the oral administration of dihydro-resveratrol.
  • the level of glutathione in cerulein-induced pancreatic tissue was depleted drastically by more than 50%when compared to the non-cerulein-treated control.
  • the oral administration of dihydro-resveratrol significantly suppressed glutathione depletion in the cerulein-induced pancreata.
  • the solubility of dihydro-resveratrol in a methanol-based solvent was at least 5 times higher than that of trans-resveratrol.
  • the cytotoxicity of dihydro-resveratrol was shown to be approximately 500 ⁇ M whereas that of trans-resveratrol was roughly 250 ⁇ M.
  • the cytotoxicity of dihydro-resveratrol was 50%lower than that of trans-resveratrol.
  • a method of treating acute inflammatory condition of the pancreas and associated systemic complications by administering to a subject in needs thereof a composition comprising an effective amount of a stilbenoid derivative which comprises a compound of formula (1) , wherein R 1 , R 2 and R 3 are each independently selected from an alkyl group.
  • a stilbenoid derivative which comprises a compound of formula (1) , wherein R 1 , R 2 and R 3 are each independently selected from an alkyl group.
  • alkyl alone or in combination with other groups, includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms.
  • the term is further exemplified by such groups as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl and tert-butyl) , pentyl, hexyl and the like, and the derivatives or chemical variants thereof; or a mixture of said compound, the derivative and/or chemical variants thereof.
  • a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the stilbenoid derivative is trans-3, 5, 4'-trihydroxybibenzyl, or dihydro-resveratrol, which is a compound of formula (2) : and the derivatives or chemical variants thereof; or a mixture of said compound, the derivative and/or chemical variants thereof.
  • a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the subject is a human or an animal.
  • a fourth embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the composition is administered orally.
  • a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the acute inflammatory condition of the pancreas comprises all forms of acute pancreatitis and associated systemic complications comprise pulmonary injury.
  • a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein said composition is administered at no less than 20 mg/kg to said subject for no less than 3 times a day.
  • a seventh embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein said composition is administered at no less than 3.24 mg/kg to said subject for no less than 3 times a day.
  • a method for preparing a compound of molecular formula C 14 H 14 O 3 and of formula (2) which is a stilbenoid derivative having a chemical name of trans-3, 5, 4'-trihydroxybibenzyl by hydrogenating of trans-resveratrol.
  • TGF- ⁇ has been reported by some previous studies as a potent inducer of PSC activation in which a series of fibrotic mediators, including FN1, are being up-regulated.
  • LTC-14 cells which are immortalized PSCs from rat
  • the expression levels of fibrotic filament ⁇ -SMA and ECM protein FN1 are remarkably elevated by the exogenous addition of recombinant TGF- ⁇ (5 ng/mL) .
  • the inventors discover that the administration of dihydro-resveratrol significantly attenuate the expression levels of ⁇ -SMA and FN1 in rat PSCs upon the challenge of TGF- ⁇ .
  • dihydro-resveratrol exerts similar suppressive effect in PSCs.
  • the inhibitory effect of dihydro-resveratrol is more significant.
  • dihydro-resveratrol exerts the most potent anti-fibrotic effect in PSCs despite they possess modest structural differences.
  • the present invention provides a compound for suppressing a fibrotic mediator of stellate cells present in an internal organ of a subject in need with a formula of
  • R 2 and R 4 are each independently selected from -OR 11 and -OC (O) R 11 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 11 and -OC (O) R 11 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 13 , -C (O) R 14 , -C (O) N (R 13 ) R 14 , -C (O) OR 13 , -OC (O) R 14 , -S (O) 2 R 13 , -S (O) 2 N (R 13 ) R 14 , -N (R 13 ) R 14 ;
  • R 13 and R 14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • the present invention further provides nine embodiments of compounds for suppressing a fibrotic mediator of stellate cells present in an internal organ of a subject in need with formula of:
  • the internal organ can be, for example, pancreas, liver, kidney and lung of a subject.
  • the subject can be a human subject.
  • LTC-14 cells were cultured at 37°C under a humidified condition of 95%air and 5%CO 2 in IMDM supplemented with 10%fetal bovine serum (FBS) . Cells used in all the experiment were among passages 9 to 25. LTC-14 cells were seeded at a density of 1 ⁇ 10 5 /well in a 12-well plate, and incubated with recombinant TGF- ⁇ at 5 ng/mL with dihydro-resveratrol at 0, 1, 5, 10 and 20 ⁇ g/mL in IMDM supplemented with 0.2%FBS for 24 hours. Cells were then harvested for protein extraction and Western blotting analysis or immunofluorescent staining.
  • FBS fetal bovine serum
  • Total proteins of the LTC-14 cells are extracted using RIPA lysis buffers containing protease inhibitors. Cell lysates were loaded and separated by SDS-polyacrylaminde gel electrophoresis. After wet electroblotting, proteins were transferred onto PVDF membranes (Bio-rad) , blocked with 5 %non-fat milk, probed with antibodies and visualized by utilization of an ECL kit (GE Healthcare) .
  • LTC-14 cells were seeded at a density of 1 ⁇ 10 5 onto the poly-L-lysine-coated cover slips in a 24-well plate, incubated with TGF- ⁇ at 5 ng/mL with dihydro-resveratrol at 0, and 10 ⁇ g/mL in IMDM supplemented with 0.2%FBS for 24 hours. Cells were then fixed, blocked with 3%BSA, probed with antibodies and mounted with fluorescence mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI) . Images were captured using the Nikon microscope and analyzed by the SPOT advanced software.
  • DAPI 6-diamidino-2-phenylindole
  • mice C57/BL6 mice aged 28 days weighing in the range of 20 to 25 g were randomly assigned into 4 groups of 6 to 8 individuals. The mice were housed with an ambient temperature of 23 ⁇ 2°C, a relative humidity of 60 to 80%and a 12-h light/dark cycle. Prior to the glucose tolerance test, the mice were starved overnight but allowed with free access to water. Experimental chronic pancreatitis was induced in the mice by four hourly i. p. injections of cerulein at the supramaximally stimulating dose (50 ⁇ g/kg) a day, 3 days a week, in a total of 6 weeks. The control group received injections of 0.9%saline instead of cerulein in the same volume and at same time intervals.
  • the treatment groups given with cerulein and oral doses of dihydro-resveratrol (20 mg/kg/day) were designated as Cer+D-res.
  • a dosage of dihydro-resveratrol at 50 mg/kg/day had also been attempted in the treatment course, but no statistically significant difference from the dose at 20 mg/kg/day in fibrosis formation was achieved. Nevertheless, no adverse effect was obtained from this higher dosage in the in vivo trial. Thus, it concludes that an effective dosage of dihydro-resveratrol is at least 20 mg/kg/day.
  • the group given with trans-resveratrol was designated as Cer+Res.
  • the drug intervention of both compounds was given from the first day of week 4 till the end of experiment, i.e.
  • mice were subjected to the intraperitoneal glucose tolerance test (IPGTT) .
  • IPGTT intraperitoneal glucose tolerance test
  • Mice had been starved for 14 hours prior to the IPGTT, in which a 15%(w/v) glucose solution was injected to individual animals at 1.5 g glucose per kg body weight.
  • About 1 ⁇ L of blood will be obtained from the tail vein, and blood glucose levels were monitored 30 min before (i.e. fasting level) and 10, 20, 30 and 60 min after glucose injection using a glucometer (Medisign, Korea) .
  • pancreases were immediately removed, weighed, trimmed from fat and fixed in 4%paraformaldehyde-phosphate buffered saline overnight at 4°C. Samples were then processed, embedded in paraffin wax, sectioned and subjected to immunostaining.
  • human equivalent dosage (mg/kg) Animal dose (mg/kg) multiplied by animal Km/human Km, where mouse Km is 3 and human Km is 37 (Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers)
  • the effective human equivalent dosage of dihydro-resveratrol of the present invention is at least 1.622 mg/kg/day.
  • LTC-14 cells were pre-incubated with TGF- ⁇ (5 ng/mL) , and treated with trans-resveratrol (Resv) or dihydro-resveratrol (D-Res) or stilbene compounds i-viii at 20 ⁇ g/mL for 24 hours. Control was not treated with Resv or any stilbenoids. Total proteins were extracted and analyzed using Western blotting. This is shown in Figure 9.
  • LTC-14 cells are pancreatic stellate cells.
  • ⁇ -SMA is the hallmark component of fibrogenesis whereas ⁇ -actin serves as a loading control.
  • the expression level of ⁇ -SMA implies the degree of PSC activation.
  • TGF- ⁇ was added since it is regarded as a potent inducer of fibrotic events.
  • Suppressive effect on ⁇ -SMA expression level is tested among dihydro-resveratrol and compounds i to viii in relation to trans-resveratrol (Resv) . All of the testing compounds exert suppressive effect of ⁇ -SMA expression level.
  • LTC-14 cells were pre-incubated with TGF- ⁇ (5 ng/mL) , and treated with trans-resveratrol or dihydro-resveratrol at the indicated concentrations. Total proteins were extracted and analyzed using Western blotting. This is shown in Figure 10.
  • FN1 is a major extracellular matrix protein produced during fibrogenesis or upon the activation of pancreatic stellate cells. Its expression level implies the degree of fibrogenesis. Suppressive effect on levels of FN1 and ⁇ -SMA is tested between dihydro-resveratrol (i.e. compound 2) and trans-resveratrol.
  • LTC-14 cells were pre-incubated with TGF- ⁇ (5 ng/mL) , and treated with dihydro-resveratrol at 20 ⁇ g/mL for 24 hours prior to immunofluorescent staining.
  • LTC-14 cells are pancreatic stellate cells.
  • ⁇ -SMA is the hallmark component of fibrogenesis whereas ⁇ -actin serves as a loading control.
  • the green fluorescent signal (identified by arrow marks in Figure 11) of ⁇ -SMA implies the degree of PSC activation.
  • TGF- ⁇ was added since it is regarded as a potent inducer of fibrotic events. Suppressive effect of dihydro-resveratrol on ⁇ -SMA expression level is tested.
  • Pancreatic tissue sections are stained with antibody against FN1; thus, the immunostaining signals imply the degree of FN1 deposition in the parenchyma.
  • the treatment with dihydro-resveratrol at 20 mg/kg/day reduces such deposition in chronic pancreatitis in a manner more significant to trans-resveratrol (Cer+Res) . This is shown in Figure 12.
  • C57/BL6 mice aged 28 days weighing in the range of 20 to 25 g were randomly assigned into 4 groups of 6 to 8 individuals. The mice were housed with an ambient temperature of 23 ⁇ 2°C, a relative humidity of 60 to 80%and a 12-h light/dark cycle.
  • dihydro-resveratrol 20 mg/kg/day
  • the fasting blood glucose levels of the chronic pancreatitis mice (Cer) become markedly higher than those of the control group, indicating these chronic pancreatitis mice develop hyperglycemia, a discernible feature of diabetes.
  • their impaired glucose tolerance has been significantly rectified by the 3-week dihydro-resveratrol treatment (Cer+D-Res) .
  • the hyperglycemic condition of the chronic pancreatitis mice is improved. This is shown in Figure 13.
  • dihydro-resveratrol Cer+D-Res
  • the severity of pancreatitis and the shrinkage and destruction of islets, explicitly beta cells are notably ameliorated.
  • the pancreatic beta-cell area is reflected by the immunofluorescent insulin signals.
  • a reduced beta-cell area or mass implicates the deficiency in glucose tolerance, or the development of diabetes.
  • the restoration of beta-cell area by the dihydro-resveratol treatment indicates this stilbenoid is beneficial to the treatment of pancreatogenic diabetes (i.e. Type3c DM) .
  • human equivalent dosage (mg/kg) Animal dose (mg/kg) multiplied by animal Km/human Km, where mouse Km is 3 and human Km is 37 (Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers)
  • the effective human equivalent dosage of dihydro-resveratrol of the present invention is at least 1.622 mg/kg/day.
  • Dendrobium plants commonly called “Shi Hu”
  • TCM Chinese medicine
  • Dendrobium-derived extracts or ingredients contain substantial amounts of various stilbenoids, such as trans-resveratrol and dihydro-resveratrol, which are potential compounds for combating oxidative stress in the human body.
  • stilbenoids such as trans-resveratrol and dihydro-resveratrol
  • the present invention relates to the use of Dendrobium-derived stilbenoids, particularly trans-resveratrol, dihydro-resveratrol or dihydro-resveratrol derivatives in reducing melanin formation with a potential to inhibit the generation of oxidative radicals and ROS.
  • the present composition is applied to the subject in need thereof via topical administration.
  • the present composition is in the form of a day cream, a night cream, a face lotion, a body lotion, a body butter, a skin peel, a mask, a shower gel, a sun cream, a sun lotion, an after sun cream or an after sun lotion.
  • the present composition comprises one or more extract (s) derived from Dendrobium plants.
  • the present composition comprises one or more stilbenoids with the following formula:
  • R 2 , R 4 , and R 8 are each independently selected from -OR 11 , -OCH 2 R 12 , -OC (O) R 11 , -OCH 2 C (O) OR 12 and -OC (O) CH 2 R 12 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, OR 12 and -OC (O) R 12 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently selected from – (CH 2 ) -hydrocarbyl, C 2-10 alkyl, alkenyl , cycloalkyl, aryl and heterocyclyl, Each of these groups is optionally substituted with 1, 2, 3, 4 or 5 R 13 ;
  • R 12 is independently selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 13 ;
  • R 13 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 14 , -C (O) R 15 , -C (O) N (R 14 ) R 15 , -C (O) OR 14 , -OC (O) R 15 , -S (O) 2 R 14 , -S (O) 2 N (R 14 ) R 15 , -N (R 14 ) R 15 ;
  • R 14 and R 15 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • the present composition comprises stilbenoid (s) with the following formula: which is dihydro-resveratrol, and the derivatives or chemical variants thereof; or a mixture of said compound, the derivative and/or chemical variants thereof, or with a formula of:
  • composition for skin whitening and skin protection comprises stilbenoid (s) with the following formula:
  • R 2 , R 4 , and R 8 are each independently selected from -OR 11 , -OCH 2 R 11 , -OC (O) R 11 , -OCH 2 C (O) OR 11 and -OC (O) CH 2 R 11 ;
  • R 1 , R 3 , R 5 , R 6 , R 7 , R 9 and R 10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR 11 and -OC (O) R 11 ; or R 2 and R 3 , or R 7 and R 8 may be taken together with the carbon atoms to which they are attached to form a cyclic group;
  • R 11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R 12 ;
  • R 12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR 13 , -C (O) R 14 , -C (O) N (R 13 ) R 14 , -C (O) OR 13 , -OC (O) R 14 , -S (O) 2 R 13 , -S (O) 2 N (R 13 ) R 14 , -N (R 13 ) R 14 ;
  • R 13 and R 14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;
  • a spectrophotometric decolorization assay complemented with pre-formed radical monocation of 2, 2'-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) , also known as ABTS is used.
  • ABTS (Abcam, USA) is dissolved in water to a 7 mM concentration and ABTS radical cation is produced by reacting the ABTS stock solution with 2.45 mM ammonium persulfate (Sigma-Aldrich, USA) and allowing the mixture to stand in the dark at room temperature for 16 hours before use.
  • the ABTS radical solution is diluted with ethanol to an absorbance of 0.70 at 734 nm and equilibrated at 30°C.
  • the dilutions of testing samples (0.1 mL) or DMSO (0.1 mL) are incubated with ABTS radical solution (0.9 mL) for 15 min prior to the absorbance taking at 734 nm.
  • DMSO is served as a vehicle treatment
  • Trolox (Abcam, USA) , a renowned derivative vitamin E, ranging from 0.001 to 0.05 mg/mL is used as a positive drug reference.
  • the antioxidant capacity of testing samples is expressed as the amount equivalent to Trolox in milligrams (mg) according to the Trolox standard curve.
  • the cellular melanin content and tyrosinase activity are determined in cultured B16 and A375 melanocytes.
  • melanocytes are melanin-producing cells whereas melanin refers to groups of endogenous pigments that give multitude of skin colors.
  • B16 cells (Shanghai Institutes for Biological Science, Chinese Academy of Sciences, China) and A375 cells (American Type Culture Collection, USA) are routinely grown in DMEM medium (Gibco, USA) supplemented with 10%heat-activated fetal bovine serum (FBS, Gibco, USA) and 1%penicillin/streptomycin (Gibco, USA) in a humidified atmosphere of 95%air and 5%CO 2 at 37°C.
  • melanocytes are seeded in 12-well plates (8 ⁇ 10 4 cells/well) and stimulated with alpha-melanocyte-stimulating hormone ( ⁇ -MSH, 100 nM) for 24 hours.
  • ⁇ -MSH alpha-melanocyte-stimulating hormone
  • ⁇ -MSH alpha-melanocyte-stimulating hormone
  • cells are then treated with different Dendrobium extract samples or stilbenoid samples (5 ⁇ L) or DMSO (5 ⁇ L, Sigma-Aldrich, USA) for another 24 hours.
  • DMSO serves as a vehicle treatment.
  • cells are trypsinized for detaching from the culturing plates. After centrifugation, the melanin pellet of each sample is incubated with 100 ⁇ L of 1 N Sodium hydroxide solution (Sigma-Aldrich, USA) for 1.5 hours at 70°C. After cooling to room temperature, the solution is centrifuged at 15,000 ⁇ g for 10 min. The supernatant (100 ⁇ L) of each sample is transferred to 96-well plates for a reading of absorbance taken at 405 nm. Relative melanin content of each sample is normalized with its protein content and presented as the percentage change over the DMSO-treated cells.
  • 1 N Sodium hydroxide solution Sigma-Aldrich, USA
  • D-Res dihydro-resveratrol
  • Res trans-resveratrol
  • Dendrobium extract samples or other stilbenoid samples compounds DR1 to DR11
  • melanocytes are seeded in 12-well plates (8 ⁇ 10 4 cells/well) and stimulated with ⁇ -MSH (100 nM) for 24 hours. Post the ⁇ -MSH stimulation, cells are treated with different testing samples (5 ⁇ L) or DMSO (5 ⁇ L) for another 24 hours. At the end of experiment, the cells are washed with ice-cold phosphate buffer saline (PBS, pH 6.8) (Gibco, USA) twice and then lysed with 150 ⁇ L of PBS (pH 6.8) containing 0.1%Triton X-100 on ice. The cell lysates are centrifuged at 15,000 ⁇ g for 15 min at 4°C.
  • PBS ice-cold phosphate buffer saline
  • B16 or A375 cells are seeded in 12-well plates (8 ⁇ 10 4 cells/well) and stimulated with ⁇ -MSH (100 nM) for 24 hours.
  • ⁇ -MSH 100 nM
  • cells are trypsinized for detaching from the culturing plates and subjected to cellular ROS detection assay (Abcam) according to manufacturer’s instruction.
  • Abcam cellular ROS detection assay
  • cells are stained with 20 ⁇ M DCFDA for 30 min at 37°C.
  • ITA° ITA°
  • L* luminance
  • b* yellow-blue component
  • Two areas (2 cm ⁇ 2 cm each) are selected as treated areas whereas regions surrounding the treated areas on the arms are regarded as control areas.
  • a volume of 200 ⁇ L of the testing stilbenoids (2%by weight dissolved in ethanol) is applied to the designated area twice a day, day and night.
  • individuals are tested with the skin colorimeter for recording the data about using the stilbenoids for 7 days.
  • individuals are tested with the skin colorimeter for recording the data about using the stilbenoids for 14 days.
  • the ITA° readings are presented in Table 5. Overt whitening effect from dihydro-resveratrol or trans-resveratrol is obtained from most individuals.
  • a set of representative images taken from a human individual who uses the topical treatment is shown in Figure 23.
  • the R group in RBr can be OR 11 , -OCH 2 R 11 or -OCH 2 C (O) OR 11 , where R 11 is hydrocarbyl.
  • the R group in RCOCl can be -R 11 or -CH 2 R 11 , where R 11 is hydrocarbyl, which is optionally substituted with 1, 2, 3, 4 or 5 R 12 , and where R 12 is halogen or -OR 13 , and where R 13 is hydrogen or hydrocarbyl.
  • the present invention relates to a skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) obtained from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile for the management of melanogenesis, skin-darkening and skin-aging. More particularly, it relates to the usage of Dendrobium ingredients and stilbenoids to reduce the formation of melanin in melanocytes. It also relates to the usage of Dendrobium ingredients and stilbenoids to reduce the generation of reactive oxygen species and oxidative free radicals. This invention relates to the use Dendrobium-derived extracts or ingredients or stilbenoids in the formulation of skin-protection, skin-whitening and/or anti-skin aging products.

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Abstract

A skin-whitening and skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) obtained from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile for the management of melanogenesis, skin-darkening and skin-aging. More particularly, it relates to the usage of Dendrobium ingredients and stilbenoids to reduce the formation of melanin in melanocytes. It also relates to the usage of Dendrobium ingredients and stilbenoids to reduce the generation of reactive oxygen species and oxidative free radicals. The use of Dendrobium-derived extracts or ingredients or stilbenoids in the formulation of skin-protection, skin-whitening and/or anti-skin aging products. Compounds for use in preparation of a composition for treating, preventing, and/or delaying the progress of skin darkening or for use in skin-whitening and skin-protection which are synthesized from dihydro-resveratrol and the related synthesis method are also provided.

Description

Skin-Protection Composition Containing Dendrobium-Based Ingredients
Inventors
Hongjie ZHANG (US) , Siu Wai TSANG (HK) and Yu ZHU (China)
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of (1) US Non-provisional Patent Application Serial Number 15/351,636 filed on November 15, 2016； (2) US Non-provisional Patent Application Serial Number 15/352,903 filed on November 16, 2016； and (3) US Non-provisional Patent Application Serial Number 15/633,780 filed on June 27, 2017, where the US Non-provisional Patent Application Serial Number 15/351,636 is a continuation-in-part application of US Non-provisional Patent Application Serial Number 14/740,410 filed on June 16, 2015； the US Non-provisional Patent Application Serial Number 15/352,903 is a continuation-in-part application of US Non-provisional Patent Application Serial Number 14/740,410 filed on June 16, 2015； the US Non-provisional Patent Application Serial Number 15/633,780 is a divisional application of the US Non-provisional Patent Application Serial Number 15/351,636 filed on November 15, 2016, which the disclosures are hereby incorporated by reference in their entirety.
FIELD OF INVENTION
The present invention relates to a skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) isolated from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile for the management of melanogenesis, skin-darkening and skin-aging. More particularly, it relates to the usage of Dendrobium ingredients to reduce the formation of melanin in melanocytes. It also relates to the usage of Dendrobium ingredients to reduce the generation of reactive oxygen species and oxidative free radicals in melanocytes. The present invention further relates to the use of Dendrobium-derived extracts or ingredients in the formulation of skin-protection, skin-whitening and/or anti-skin aging products.
BACKGROUND OF INVENTION
In general, light skin can become darker upon exposure to sunlight, which is the primary source of ultraviolet (UV) radiation in our daily life. Upon UV exposure, reactive oxygen species (ROS) and highly volatile free radicals are provoked. Consequently, our skin gets darkened since melanin-producing cells, or melanocytes, located in the skin’s epidermis are prone to produce more melanin in order to minimize the indirect damages of body cells [Sklar LR, Almutawa F, Lim HW, Hamzavi I. Effects  of ultraviolet radiation, visible light, and infrared radiation on erythema and pigmentation: a review. Photochemical & Photobiological Sciences 2013； 12: 54-64] . In other words, the elevation of melanin production is actually a defense mechanism against UV radiation； however, the accumulation of melanin may result in deleterious biological effects, including abnormal pigmentation, skin darkening as well as aesthetic problems, e.g. freckles or chloasmata [Mishima Y, Imokawa G. Selective aberration and pigment loss in melanosomes of malignant melanoma cells in vitro by glycosylation inhibitors: premelanosomes as glycoprotein. Journal of Investigative Dermatology 1983； 81: 106-114] . In this regard, the formation of ROS and free radicals plays a pivotal mechanism leading to skin-darkening and skin-aging. Scavenging of ROS and free radicals can definitely protect skin cells from the oxidative damages. Currently, topical antioxidants, e.g. vitamin A, vitamin C, vitamin E, ascorbic acid (AC) , butylated hydroxyanisole (BHA) and/or their derivatives, have been widely used in over-the-counter skincare products. However, these products are not effective skin-protection agents possessing both depigmentation and antioxidant properties. To this end, their effectiveness on skin protection is highly limited and restrained. There is a conception in the general public that dark and dull skin makes people look old, depressing and disfigured； therefore, people, especially in oriental countries, are eager to maintain a fair complexion and a pleasing appearance of their integumentary system.
In fact, melanin refers to a group of endogenous pigments that give multitude of skin colors, i.e. from the basic skin tone to freckles, birth marks, age spots, also in eyes and hair. In the human body, melanins are produced by melanocytes and classified into three major types: eumelanin, pheomelanin and neuromelanin. They differ in chemical structures and physical properties, and thus they are elicited upon different biological responses against external stimuli. Eumelanin and pheomelanin are the two types of melanin that can be found in the skin. Eumelanin provides primarily dark brown to black colors whereas pheomelanin produces reddish colors [Maresca V, Flori E, Picardo M. Skin phototype: a new perspective. Pigment Cell & Melanoma Research 2015； 28: 378-389] . In melanogenesis, tyrosinase is the major rate-limiting enzyme for regulating melanin production in epidermal melanocytes whereas tyrosinase-related proteins (TRPs) are melanogenic enzymes that control the proportion of carboxylated subunits in melanin biopolymers. When exposed to sunlight, melanin deposition is accelerated as more alpha-melanocyte-stimulating hormone (α-MSH) is released from melanocytes. In addition to the overt skin darkening, such acceleration is indeed a defense mechanism against the UV-induced generation of ROS and/or free radicals, which consequently causes most of the wrinkles, freckles, rashes and age spots on the face. Collectively, the harmful effects of constant or excessive sunlight or UV exposure  include skin aging, skin damage and even skin cancer. The present invention provides a composition that efficiently attenuates the biosynthesis of melanin as well as oxidative substances. By any means of mechanism, the present invention is able to restrain melanin accumulation and/or oxidative damage in the skin, hence skin protection can be achieved.
The composition of present invention comprises stilbenoids and/or extracts obtained from plants of the genus Dendrobium (Orchidaceae family, commonly called “Shi Hu” ) , particularly from the species D. officinale and D. nobile. The said stilbenoids, explicitly trans-resveratrol and dihydro-resveratrol, and extracts can be formulated into a cosmetic blend of skin-protection, skin-whitening and/or anti-skin aging products for human skin as they are found to attenuate the generation of ROS and oxidative free radicals, and/or reduce cellular melanin content in melanocytes. The reduction of melanin formation may be associated with an inhibition of the activity of pigment-producing enzymes, namely tyrosinase, TRP-1 and TRP-2.
SUMMARY OF INVENTION
Accordingly, the objective of this invention relates to a skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) isolated from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile. This invention also relates to the use of Dendrobium-derived extracts or ingredients in reducing melanin formation in melanocytes for the management of melanogenesis and skin-darkening. Particularly, this invention also relates to the use of Dendrobium-derived extracts or ingredients in the formulation of skin-protection, skin-whitening and/or anti-skin aging products.
In a first embodiment of the first aspect of the present invention, there is provided a compound for use in suppressing a fibrotic mediator of stellate cells present in an internal organ of a subject in need thereof, or a use of the compound in preparation of a composition for suppressing a fibrotic mediator of stellate cells present in an internal organ of said subject. Said compound has a formula of
Figure PCTCN2017107798-appb-000001
wherein
R2 and R4 are each independently selected from -OR11 and -OC (O) R11；
R1, R3, R5, R6, R7, R8, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14；
R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof；
or a mixture of said compound, the derivative and/or chemical variants thereof.
In a second embodiment of the first aspect of the present invention there is provided a compound for use in suppressing a fibrotic mediator of stellate cells present in an internal organ of a subject in need thereof or the use of the compound in preparation of a composition for suppressing a fibrotic mediator of stellate cells present in an internal organ of said subject, wherein said compound has a formula of
Figure PCTCN2017107798-appb-000002
Figure PCTCN2017107798-appb-000003
In a first embodiment of a second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need thereof comprising administering a composition comprising one or more compounds of formulae (i) to (viii) or dihydro-resveratrol [i.e., formula (2) ] to a subject in need thereof.
In a second embodiment of the second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need thereof wherein the composition is administered to a subject in need thereof with a dosage of at least 1.622 mg/kg/day wherein said composition consists of a compound with a formula of
Figure PCTCN2017107798-appb-000004
In a third embodiment of the second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need wherein the said subject is human.
In a fourth embodiment of the second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need wherein the composition is administered orally to said subject in need thereof.
In a fifth embodiment of the second aspect of the present invention there is provided a method for suppressing fibrotic mediator (s) in stellate cells present in an internal organ of a subject in need thereof wherein the internal organ comprises pancreas, liver, kidney and lung.
In a sixth embodiment of the second aspect of the present invention, said composition is administered to said subject orally.
In a first embodiment of a third aspect of the present invention there is provided a compound for use in treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof with a therapeutically effective amount of a compound of the following formula:
Figure PCTCN2017107798-appb-000005
wherein
R2 and R4 are each independently selected from -OR11 and -OC (O) R11；
R1, R3, R5, R6, R7, R8, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14；
R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof；
or a mixture of said compound, the derivative and/or chemical variants thereof.
In a second embodiment of the third aspect of the present invention there is provided a method for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof comprising using a composition comprising compounds of the following formula:
Figure PCTCN2017107798-appb-000006
wherein
R2 and R4 are each independently selected from -OR11 and -OC (O) R11；
R1, R3, R5, R6, R7, R8, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14；
R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof；
or a mixture of said compound, the derivative and/or chemical variants thereof.
In a third embodiment of the third aspect of the present invention there is provided a method for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof wherein the composition is administered to a subject in need thereof with a dosage of at least 1.622 mg/kg/day wherein said composition consists of a compound with a formula of
Figure PCTCN2017107798-appb-000007
In a fourth embodiment of the third aspect of the present invention there is provided a method for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof wherein said subject is human.
In a fifth embodiment of the third aspect of the present invention there is provided a method for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof wherein the composition is administered orally to said subject in need thereof.
In a sixth embodiment of the third aspect, said compound is used in preparation of a composition for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in said subject, and said subject is human.
In a first embodiment of a fourth aspect of the present invention there is provided a composition for use in treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof comprising a therapeutically effective amount of a compound of one of the following formulae:
Figure PCTCN2017107798-appb-000008
Figure PCTCN2017107798-appb-000009
In a second embodiment of the fourth aspect of the present invention there is provided a composition for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof wherein the composition is administered to said subject with a dosage of at least 1.622 mg/kg/day wherein said composition consists of a compound with a formula of
Figure PCTCN2017107798-appb-000010
In a third embodiment of the fourth aspect of the present invention there is provided a composition for treating pancreatogenic diabetes (or Type 3c diabetes milieus) in a subject in need thereof wherein said subject is human.
In a first embodiment of a fifth aspect of the present invention there is provided a compound of formula (4) :
Figure PCTCN2017107798-appb-000011
wherein
R2, R4, and R8 are each independently selected from -OR11, -OCH2R12, -OC (O) R11, -OCH2C (O) OR12 and -OC (O) CH2R12；
R1, R3, R5, R6, R7, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR12 and -OC (O) R12； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently selected from – (CH2) -hydrocarbyl, C2-10 alkyl, alkenyl, cycloalkyl, aryl and heterocyclyl, each of these groups is optionally substituted with 1, 2, 3, 4 or 5 R13；
R12 is independently selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R13；
R13 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR14, -C (O) R15, -C (O) N (R14) R15, -C (O) OR14, -OC (O) R15, -S (O) 2R14, -S (O) 2N (R14) R15, -N (R14) R15；
R14 and R15 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or a pharmaceutically acceptable salt or prodrug thereof.
In a second embodiment of the fifth aspect of the present invention there is provided a compound of formula (4) wherein said compound is an optically pure stereoisomer, enantiomer, racemate or diastereomer thereof.
In a third embodiment of the fifth aspect of the present invention there is provided a pharmaceutical composition comprising an effective amount of the compound of formula (4) , the derivatives thereof, and a pharmaceutically acceptable carrier thereof.
In a fourth embodiment of the fifth aspect of the present invention there is  provided a pharmaceutical composition comprising an effective amount of the skin whitening and skin protection compound of formula (4) , the derivatives thereof, and a pharmaceutically acceptable carrier thereof.
In a fifth embodiment of the fifth aspect of the present invention there is provided a compound of formula (4) , wherein the compound is selected from DR1, DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10 or DR11 with the following formulae respectively:
Figure PCTCN2017107798-appb-000012
Figure PCTCN2017107798-appb-000013
Figure PCTCN2017107798-appb-000014
Figure PCTCN2017107798-appb-000015
or a pharmaceutically acceptable salt or prodrug thereof.
In a sixth embodiment of the fifth aspect of the present invention there is provided a use of the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) in preparation of a composition for treatment, prevention or delay of progression of a skin darkening in a subject in needs thereof.
In a seventh embodiment of the fifth aspect of the present invention there is provided the use of the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) in the preparation of the composition for treatment, prevention or delay of progression of a skin darkening,  wherein said subject is a human.
In an eighth embodiment of the fifth aspect of the present invention there is provided a method of treating and preventing development and progression of a skin darkening comprising administering the compound of formula (4) and/or the derivatives thereof to a subject in need thereof.
In a ninth embodiment of the fifth aspect of the present invention there is provided a method of treating and preventing development and progression of a skin darkening comprising administering the compound of formula (4) and/or the derivatives thereof to a subject in need thereof wherein said subject is a human.
In a tenth embodiment of the fifth aspect of the present invention there is provided the use of the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) in the preparation of the composition for treatment, prevention or delay of progression of a skin darkening in a subject in need thereof, wherein the composition is applied topically.
In an eleventh embodiment of the fifth aspect of the present invention there is provided the composition comprising the compound of formula (4) and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) , wherein the composition is in the form of paste, solid, powder, particle, emulsion, a day cream, a night cream, a face lotion, a body lotion, a body butter, a skin peel, a mask, a shower gel, a sun cream, a sun lotion, an after sun cream, an after sun lotion, a lip balm, a lip gloss, or alike.
In a first embodiment of a sixth aspect of the present invention there is provided a skin whitening and skin protection against UV exposure, skin damage and aging compound of formula (5) :
Figure PCTCN2017107798-appb-000016
wherein
R2, R4, and R8 are each independently selected from -OR11, -OCH2R11, -OC (O) R11, -OCH2C (O) OR11 and -OC (O) CH2R11；
R1, R3, R5, R6, R7, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14；
R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer, optically pure stereoisomer, racemate or diastereomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof
In a second embodiment of the sixth aspect of the present invention there is provided a skin whitening and skin protection against UV exposure, skin damage and aging compound with a formula of:
Figure PCTCN2017107798-appb-000017
Figure PCTCN2017107798-appb-000018
Figure PCTCN2017107798-appb-000019
Figure PCTCN2017107798-appb-000020
Figure PCTCN2017107798-appb-000021
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof.
In a seventh aspect of the present invention, there is provided a method of synthesizing the compound of formulae DR1 to DR11 from dihydro-resveratrol according to the following synthesis schemes, respectively:
Scheme 1 for DR1 to DR3:
Figure PCTCN2017107798-appb-000022
wherein
R is -OR11, -OCH2R11 or -OCH2C (O) OR11；
R11 is hydrocarbyl；
and
Scheme 2 for DR4 to DR11:
Figure PCTCN2017107798-appb-000023
wherein
R is -R11 or -CH2R11；
R11 is hydrocarbyl, which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is halogen or -OR13； and
R13 is hydrogen or hydrocarbyl.
In an embodiment, under Scheme 1, dihydro-resveratrol and RBr are added in dimethylformamide (DMF) and K2CO3 to form a mixture. The resulting mixture is stirred at room temperature until the starting material disappear on thin-layer chromatography (TLC) . The mixture is then diluted with H2O and washed with dichloromethane (DCM) three times to extract the organic phase. The combined organic layer is washed with saturated sodium chloride (NaCl) twice, dried over anhydrous Na2SO4, concentrated in vacuum and purified by preparative (prep-) TLC (PE/EA ＝ 5/1 or 3/1) to afford the desired compound (s) .
In other embodiment, under Scheme 2, dihydro-resveratrol and RCOCl are added into DCM and Et3N at 0 ℃ to form a mixture. The resulting mixture is warmed to room temperature and stirred until the starting material disappear on TLC. The mixture is then diluted with H2O and washed with DCM three times to extract the organic phase. The combined organic layer is washed with saturated NaCl twice, dried over anhydrous Na2SO4, concentrated in vacuum and purified by prep-TLC (PE/EA ＝5/1 or 3/1) to afford the desired compound (s) .
In other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications by administering to a subject in needs thereof a composition comprising an effective amount of a stilbenoid derivative which comprises a compound of formula (1) ,
Figure PCTCN2017107798-appb-000024
 wherein R1, R2 and R3 are each independently selected from an alkyl group. The term “alkyl” , alone or in combination with other groups, includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. The term is further exemplified by such groups as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl and tert-butyl) , pentyl, hexyl and the like, and the derivatives or chemical variants thereof； or a mixture of said compound, the derivative and/or chemical variants thereof.
In one embodiment of that other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the stilbenoid derivative is trans-3, 5, 4'-trihydroxybibenzyl, or dihydro-resveratrol, which is a compound of formula (2) : 
Figure PCTCN2017107798-appb-000025
 and the derivatives or chemical variants thereof； or a mixture of said compound, the derivative and/or chemical variants thereof.
In other embodiment of that other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the subject is a human or an animal.
In another embodiment of that other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the composition is administered orally.
In yet another embodiment of that other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the acute inflammatory condition of the  pancreas comprises all forms of acute pancreatitis and the associated systemic complications comprise pulmonary injury.
In thus another embodiment of that other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein said composition is administered at no less than 20 mg/kg to said subject for no less than 3 times a day.
In a further embodiment of that other aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein said composition is administered at no less than 3.24 mg/kg to said subject for no less than 3 times a day.
In another aspect of the present invention there is provided a method for preparing a compound of molecular formula C14H14O3 and of formula (2) , 
Figure PCTCN2017107798-appb-000026
 which is a stilbenoid derivative having a chemical name of trans-3, 5, 4'-trihydroxybibenzyl by hydrogenating of trans-resveratrol.
In an embodiment of that another aspect of the present invention there is provided a method of preparing the compound of molecular formula C14H14O3 and of formula (2) wherein the hydrogenating of trans-resveratrol comprises steps of；
stirring a solution of trans-resveratrol in anhydrous ethanol (EtOH) at room temperature under 5 atm H2 pressure in the presence of 10％Pd/C for 8 hours；
filtering off the catalyst from the stirred solution；
evaporating the filtrate in vacuum to produce a residue；
eluting the residue using silica gel chromatographic separation with petroleum ether and ethyl acetate (1: 1) to produce dihydro-resveratrol.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.
The invention includes all such variation and modifications. The invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" , will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises” , “comprised” , “comprising” and the like can have the meaning attributed to it in the corresponding Patent law； e.g., they can mean “includes” , “included” , “including” , and the like； and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
Furthermore, throughout the specification and claims, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” , will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
Other aspects and advantages of the invention will be apparent to those skilled in the art from a review of the ensuing description.
BRIEF DESCRIPTION OF DRAWINGS
The above and other objects and features of the present invention will become apparent from the following description of the present invention, when taken in conjunction with the accompanying drawings, in which:
Figure 1 shows the gain of water content in rats due to effect of pancreatic edema as a result of cerulein-induced acute pancreatitis. The obtained weights are  expressed as a ratio percentage of pancreatic weight to body mass. A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with cerulein group.
Figures 2A to 2F show the architecture and morphological alteration in pancreatic tissues of control group (Figure 2A) , cerulein group (Figure 2B) and dihydro-resveratrol treatment groups (D-Res) (Figures 2C to 2F) by means of hematoxylin and eosin (H&E) staining. Images are shown with a scale bar of 50 μm.
Figures 3A to 3E show the architecture and morphological alteration in pulmonary tissues of control group (Figure 3A) , cerulein group (Figure 3B) and dihydro-resveratrol treatment groups (D-Res) (Figures 3C to 3E) by means of H&E staining. Images are shown with a magnification of 200×.
Figure 4A shows the measurement of MPO activity which represents neutrophil sequestration in pancreatic tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of colorimetric spectrophotometry. A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with cerulein group.
Figure 4B shows the measurement of MPO activity which represents neutrophil sequestration in pulmonary tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of colorimetric spectrophotometry. A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with cerulein group.
Figure 5A shows the measurement of TNF-α level in pancreatic tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of enzyme-linked immunosorbent assay (ELISA) . A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with cerulein group.
Figure 5B shows the measurement of TNF-α level in pulmonary tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of ELISA. A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with  cerulein group.
Figure 6A shows the measurement of glutathione levels in pancreatic tissues of control group, cerulein group and dihydro-resveratrol treatment groups (D-Res) by means of colorimetric spectrophotometry. A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with cerulein group.
Figure 6B shows the ameliorative effect of dihydro-resveratrol (D-res) and trans-resveratrol (Res) on reducing water content as a result of pancreatic edema in rats with cerulein-induced acute pancreatitis. The obtained weights are expressed as a ratio percentage of pancreatic weight to body mass. A p-value of less than 0.05 is considered as statistically significant. *p< 0.05 when comparing with saline-treated control group (Con) whereas #p< 0.05 comparing with cerulein-treated control group.
Figure 7 shows the measurement of metabolic rates by means of MTT cell proliferation in pancreatic acinar cells treated with dihydro-resveratrol and trans-resveratrol. *p< 0.05 when comparing with cells treated without dihydro-resveratrol or trans-resveratrol.
Figures 8A to 8H shows eight derivatives (i.e. Compound i to Compound viii) from dihydro-resveratrol (i.e. Compound 2) .
Figure 8I shows the chemical structure of dihydro-resveratrol (i.e. Compound 2) .
Figure 9 shows Western blotting of LTC-14 PSCs pre-incubated with TGF-β (5 ng/mL) , and treated with trans-resveratrol (Resv) or dihydro-resveratrol or stilbene compounds i to viii at 20 μg/mL for 24 hours. Control was not treated with Resv or any stilbenoids.
Figure 10 shows the probing of α-SMA and FN1 in Western blotting of LTC-14 cells pre-incubated with TGF-β (5 ng/mL) , and treated with trans-resveratrol or dihydro-resveratrol at the indicated concentrations.
Figure 11 shows the green fluorescent signal (identified by arrow marks) of fibrotic filaments α-SMA in LTC-14 cells implying the degree of PSC activation.
Figure 12 shows the fluorescent signal of fibronectin (FN1) deposition in pancreatic tissue sections for an estimation of the degree of fibrosis with and without treatment of dihydro-resveratrol (D-Res, 20 mg/kg/day) or trans-resveratrol (Res, 20 mg/kg/day) in cerulein (Cer) -induced chronic pancreatitis mice.
Figure 13 shows the glucose response of the normal mice (Control) and chronic pancreatitis mice (Cer) with or without treatment of dihydro-resveratrol (D-Res, 20 mg/kg/day) in the intraperitoneal glucose tolerance test. At all time-points, a significant difference (p<0.05) is achieved between the Cer group and the Cer+D-Res group.
Figure 14 shows the fluorescent signal of insulin in pancreatic tissue sections for an evaluation of pancreatic insulin-secreting cell (i.e. beta-cell) area. The comparison is made among the control mice, chronic pancreatitis mice and the chronic pancreatitis mice with treatment of dihydro-resveratrol (D-Res) at 20 mg/kg/day.
Figure 15 shows the suppressive effect of the Dendrobium stilbenoids (25 μM) , which are dihydro-resveratrol (D-Res) , trans-resveratrol (Res) and compounds DR1 to DR11, on cellular melanin content in B16 melanocytes. Ascorbic acid (AC) and BHA are served as positive controls. Data are expressed as mean ± SEM (n＝3) . *P<0.05 and **p<0.01 vs DMSO-treated cells.
Figure 16 shows the suppressive effect of the Dendrobium stilbenoids (25 μM) , which are dihydro-resveratrol (D-Res) , trans-resveratrol (Res) and compounds DR1 to DR11, on cellular melanin content in A375 melanocytes. Ascorbic acid (AC) and BHA are served as positive controls. Data are expressed as mean ± SEM (n＝3) . *P<0.05 and **p<0.01 vs DMSO-treated cells.
Figure 17 shows the suppressive effect of D. nobile extract (JCSH, 50 μg/mL) , D. officinale extract (TPSH, 50 μg/mL) , trans-resveratrol (Res, 25 μM) and dihydro-resveratrol (D-Res, 25 μM) on cellular melanin content in B16 melanocytes. Ascorbic acid (AC) is served as a positive control. Data are expressed as mean ± SEM (n＝3) . *P<0.05 vs DMSO-treated cells.
Figure 18 shows the inhibitory effect of the Dendrobium stilbenoids (25 μM) , which are dihydro-resveratrol (D-Res) , trans-resveratrol (Res) and compounds DR1 to DR11, on cellular tyrosinase activity in B16 melanocytes. Ascorbic acid (AC) and BHA  are served as positive controls. Data are expressed as mean ± SEM (n＝3) . *P<0.05 and **p<0.01 vs DMSO-treated cells.
Figure 19 shows the inhibitory effect of the Dendrobium stilbenoids (25 μM) , which are dihydro-resveratrol (D-Res) , trans-resveratrol (Res) and compounds DR1 to DR11, on cellular tyrosinase activity in A375 melanocytes. Ascorbic acid (AC) and BHA are served as positive controls. Data are expressed as mean ± SEM (n＝3) . *P<0.05 vs DMSO-treated cells.
Figure 20 shows the inhibitory effect of D. nobile extract (JCSH, 50 μg/mL) , D. officinale extract (TPSH, 50 μg/mL) , trans-resveratrol (Res, 25 μM) and dihydro-resveratrol (D-Res, 25 μM) on cellular tyrosinase activity in B16 melanocytes. Ascorbic acid (AC) is served as a positive control. Data are expressed as mean ± SEM (n＝3) . *P<0.05 and **p<0.01 vs DMSO-treated cells.
Figure 21 shows suppressive effect of D. nobile extract (JCSH, L: 10； M: 25； H: 50 μg/mL) and D. officinale extract (TPSH, L: 10； M: 25； H: 50 μg/mL) , dihydro-resveratrol (D-Res, L: 25； H: 50 μM) , trans-resveratrol (Res, L: 25； H: 50 μM) and ascorbic acid (AC, H: 50 μg/mL) on protein levels of TRP-1, TRP-2, phospho-AKT and phospho-p38 in B16 melanocytes. GAPDH is served as a loading reference.
Figure 22 shows suppressive effect of D. nobile extract (JCSH, high: 50 μg/mL； low: 25 μg/mL) , D. officinale extract (TPSH, high: 50 μg/mL； low: 25 μg/mL) , trans-resveratrol (Res, high: 50 μM； low: 25 μM) , dihydro-resveratrol (D-R, high: 50 μM； low: 25 μM) and ascorbic acid (AC, 50 μM) on ROS generation in B16 melanocytes. Fluorescent signals are measured at Ex488nm. Data are expressed as mean ± SEM (n＝3) . *p<0.05 vs the sole TBHP treatment (i.e. no Dendrobium extract or compound) .
Figure 23 shows the whitening effect of stilbenoid solution containing 2％trans-resveratrol (Res) or 2％dihydro-resveratrol (D-Res) on the arm of an individual human subject on day 7 and day 14.
DETAILED DESCRIPTION OF INVENTION
The present invention is not to be limited in scope by any of the specific embodiments described herein. The following embodiments are presented for exemplification only.
Definitions
Hydrocarbyl
The term "hydrocarbyl" as used herein includes reference to a moiety consisting exclusively of hydrogen and carbon atoms； such a moiety may comprise an aliphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of hydrocarbyl groups include C1-6 alkyl (e.g. C1, C2, C3 or C4 alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl) ； C1-6 alkyl substituted by aryl (e.g. benzyl) or by cycloalkyl (e.g. cyclopropylmethyl) ； cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) ； aryl (e.g. phenyl, naphthyl or fluorenyl) and the like.
Alkyl
The term "alkyl" as used herein includes reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Examples of alkyl groups include "C1-6 alkyl" and "C2-10 alkyl" . The term "C1-6 alkyl" as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. The term "C2-10 alkyl" as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. This term includes reference to groups such as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl or tert-butyl) , pentyl, hexyl and the like. In particular, the alkyl moiety may have 1, 2, 3, 4, 5 or 6 carbon atoms.
Alkenyl
The terms "alkenyl" and "C2-6 alkenyl" as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one double bond, of either E or Z stereochemistry where applicable. This term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like.
Alkynyl
The terms "alkynyl" and "C2-6 alkynyl" as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one triple bond. This term includes reference to groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl and 3-hexynyl and the like.
Alkoxy
The terms "alkoxy" and "C1-6 alkoxy" as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.
Cycloalkyl
The term "cycloalkyl" as used herein includes reference to an alicyclic moiety having 3, 4, 5, 6, 7 or 8 carbon atoms. The group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, bicyclo [2.2.2] octyl and the like.
Aryl
The term "aryl" as used herein includes reference to an aromatic ring system comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms. Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
Cyclic group
"Cyclic group" means a ring or ring system, which may be unsaturated or partially unsaturated but is usually saturated, typically containing 5 to 13 ring-forming atoms, for example a 5-or 6-membered ring. The ring or ring system may be substituted with one or more hydrocarbyl groups. Cyclic group includes carbocyclyl and heterocyclyl moeities.
Carbocyclyl
The term "carbocyclyl" as used herein includes reference to a saturated (e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon ring atoms. In particular, carbocyclyl includes a 3-to 10-membered ring or ring system and, in particular, 5-or 6-membered rings, which may be saturated or unsaturated. The ring or ring system may be substituted with one or more hydrocarbyl groups. A carbocyclic moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo [2.2.2] octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
Heterocyclyl
The term "heterocyclyl" as used herein includes reference to a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulphur. In particular, heterocyclyl includes a 3-to 10-membered ring or ring system and more particularly a 5-or 6-membered ring, which may be saturated or unsaturated. The ring or ring system may be substituted with one or more hydrocarbyl groups.
A heterocyclic moiety is, for example, selected from oxiranyl, azirinyl, 1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, especially thiomorpholino, indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4/V-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazoiyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl and the like.
Heterocycloalkyl
The term "heterocycloalkyl" as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1 , 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur. The group may be a polycyclic ring system but more often is monocyclic. This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like. The ring or ring system may be substituted with one or more hydrocarbyl groups.
Heteroaryl
The term "heteroaryl" as used herein includes reference to an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen and sulphur. The group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but  is more often monocyclic. The ring or ring system may be substituted with one or more hydrocarbyl groups. This term includes reference to groups such as pyrimidinyl, furanyl, benzo [b] thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo [b] furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like.
Halogen
The term "halogen" as used herein includes reference to F, Cl, Br or I.
Halogen Containing Moiety
The expression "halogen containing moiety" as used herein includes reference to a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur which moiety includes at least one halogen. The moiety may be hydrocarbyl for example C1-6 alkyl or C1-6 alkoxy, or carbocyclyl for example aryl.
Substituted
The term "substituted" as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents. The term "optionally substituted" as used herein means substituted or un-substituted. It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.
Enantiomer
The term “enantiomer” as used herein means one of two stereoisomers that have mirror images of one another.
Racemate
The term “racemate” as used herein means a mixture of equal amounts of enantiomers of a chiral molecule.
Diastereomer
The term “diastereomer” as used herein means one of a class of stereoisomers that are not enantiomers, but that have different configurations at one or  more of the equivalent chiral centers. Example of diasteromers are epimers that differ in configuration of only one chiral center.
Stereoisomer
The term “stereoisomer” as used herein means one of a class of isomeric molecules that have the same molecular formula and sequence of bonded atoms, but different three-dimensional orientations of their atoms in space.
Prodrug
A prodrug is a medication that is administered as an inactive (or less than fully active) chemical derivative that is subsequently converted to an active pharmacological agent in the body, often through normal metabolic processes.
Independently
Where two or more moieties are described as being "each independently" selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.
Embodiments of the present invention are described below. Preferred features of each aspect of the present invention are as for each of the other aspects mutatis mutandis. Moreover, it will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments.
Among the several established animal models, repetitive intraperitoneal (i.p. ) injection of cholecystokinin secretagogue, cerulein, is the most widely used and a highly reproducible method for the production of an experimental acute pancreatitis. Followed by a single shot of lipopolysaccharide (LPS) , pulmonary injury characterized by neutrophil sequestration in the lung tissues and increased permeability of the alveolar membrane barrier is often observed as an acute pancreatitis associated complication. For the diagnosis of the onset of acute pancreatitis, bulky leakage of digestive enzymes, namely α-amylase, into the bloodstream is regarded as the principal pathological parameter. For evaluating the severity of acute pancreatitis and the associated pulmonary injury, morphological alterations of organ architecture including interstitial edema, cellular damage, leukocyte infiltration and hemorrhage are characterized as the histological and/or pathological parameters. Besides histological examination, aberrant MPO activity is often measured for assessing the severity of  neutrophil-mediated inflammatory condition. Both the local and systemic inflammatory responses can be further confirmed by the high levels of pro-inflammatory cytokines present in the homogenates of affected tissues. Moreover, glutathione depletion, a defense mechanism, is one of the most common parameters for assessing the severity of tissue injury.
The subject to be treated by the method of this invention may be a human or an animal. The present invention is applicable to various forms of acute pancreatitis, and particularly to the acute pancreatitis associated systemic complications including pulmonary injury.
Dihydro-resveratrol, also known as trans-3, 5, 4'-trihydroxybibenzyl, is a derivative of polyphenol belonging to the family of stilbenoids, which are often obtained from plant extracts. In fact, dihydro-resveratrol is a phytoalexin produced by various plant species including Orchidaceae and Cannabis sativa L. against abiotic and biotic challenges, particularly in the case of fungal infection as reported in Fritzemeier, K.H., Kindl, H. 1983. 9, 10-dihydrophenanthrenes as phytoalexins of Orchidaceae. Biosynthetic studies in vitro and in vivo proving the route from L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene and dihydrophenanthrenes. Eur J Biochem 133, 545-550.
The present invention relates to the usage of a polyphenol derivative of the stilbenoid family with formula (1) :
Figure PCTCN2017107798-appb-000027
wherein R1, R2 and R3 are each independently selected from an alkyl group. The term “alkyl” , alone or in combination with other groups, includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. The term is further exemplified by such groups as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl and tert-butyl) , pentyl, hexyl and the like, to ameliorate tissue injury of the pancreas and lungs.
The present invention further relates to the usage of a stilbene compound  containing trans-3, 5, 4'-trihydroxybibenzyl, also known as dihydro-resveratrol, see Compound of formula (2) :
Figure PCTCN2017107798-appb-000028
to ameliorate tissue injury of the pancreas and lungs. In the present invention, this particular stilbenoid derivative was obtained as white powders through dehydrogenation of trans-resveratrol.
Further, the invention is concerned with a process for the manufacture of the above compound, pharmaceutical preparations which contain such compound, and the use of this compound for the production of pharmaceutical preparations.
The oral administration of dihydro-resveratrol at an adequate dosage of not less than 20 mg/kg is shown to significantly ameliorate the severity of acute pancreatitis and associated pulmonary injury in cerulein-treated rats. In terms of pathological parameters, rats with acute pancreatitis are shown to have lessened pancreatic water content due to an attenuation of pancreatic edema (Figure 1) , lowered plasma level of α-amylase (Table 1) , more intact acinar morphology (Figures 2C to 2F) and reduced thickening of alveolar wall and hemorrhage (Figures 3C to 3E) .
Table 1: Plasma α-amylase activities are expressed as U/μl/minute. A p-value of less than 0.05 is considered as statistically significant and S. D. stands for standard derivation. *p< 0.05 when comparing with control group whereas #p< 0.05 comparing with cerulein group.
Figure PCTCN2017107798-appb-000029
Cerulein-induced elevated levels of neutrophil sequestration, which is quantified as the activity of MPO, are significantly suppressed in pancreatic and pulmonary tissues by the administration of dihydro-resveratrol (Figures 4A and 4B) . Cerulein-induced elevated levels of TNF-α in the pancreas and lungs are significantly  suppressed by the administration of dihydro-resveratrol (Figures 5A and 5B) .
Glutathione depletion is a distinctive sign of tissue injury. The cerulein-induced declined levels of glutathione in the pancreas are significantly restored by the administration of dihydro-resveratrol (Figure 6A) .
In the present invention, dihydro-resveratrol completely and easily dissolves in 0.5％ (weight/volume, w/v) methanol whereas trans-resveratrol, with vigorous shaking, dissolves in 2.5％ (w/v) methanol (Table 2) . Thus, the solubility of dihydro-resveratrol is at least 5 times higher than that of trans-resveratrol. The ameliorative effect of dihydro-resveratrol was more promising than that of trans-resveratrol on reducing water content as a result of pancreatic edema in rats with cerulein-induced acute pancreatitis (Figure 6B) .
Table 2: Solubility of Dihydro-resveratrol and Trans-resveratrol in methanol.
  Trans-resveratrol Dihydro-resveratrol
Methanol required (w/v) 2.5％ 0.5％
From the evaluation of mitochondrial metabolic rates by means of MTT assay, the cytotoxicity of dihydro-resveratrol in pancreatic acinar cells is determined to be approximately 500 μM whereas that of trans-resveratrol is roughly 250 μM (Figure 7) . Thus, the cytotoxicity of dihydro-resveratrol was 50％lower than that of trans-resveratrol.
Experiments
Preparation and structural identification of dihydro-resveratrol. The molecular formula of dihydro-resveratrol was established as C14H14O3, which was obtained as white powders through hydrogenation of trans-resveratrol. A solution of trans-resveratrol (10 g, 43.8 mmol) in anhydrous EtOH (150 ml) was stirred at room temperature under 5 atm H2 pressure in the presence of 10％Pd/C (0.2 g) . The reaction was quenched after 8 hours (h) , by filtering off the catalyst. The filtrate was evaporated in vacuum and the residue was subjected to silica gel chromatographic separation eluting with petroleum ether and ethyl acetate (1: 1) to afford dihydro-resveratrol as white amorphous powder (9.6 g, 95％yield) : HR-ESIMS ( [M+1] + m/z 231.1026, calcd 231. 1016 for C14H15O3) ； 1H NMR (methanol-d4, 400 MHz) δ 6.96 (2H, ABd, J ＝ 8.3 Hz) , 6.67 (2H, ABd, J ＝ 8.4 Hz) , 6.13 (2H, brd, J ＝ 2.2 Hz) , 6.09 (1H, brt, J ＝ 2.2 Hz) , 2.74 (2H, brdd, J ＝ 8.5, 5.6) , 2.67 (2H, brdd, J ＝ 8.3, 5.2) ； 13C NMR (methanol-d4, 100 MHz) δ 159.2 (2C, s) , 156.3 (1C, s) , 145.6 (1C, s) , 134.1 (1C, s) , 130.3 (2C, d) , 116.0  (2C, d) , 108.1 (2C, d) , 101.1 (1C, d) , 39.6 (2C, t) , 38.0 (2C, t) .
Evaluation of biological activities. Sprague-Dawley rats aged 28 days weighing in the range of 70 to 90 g were randomly assigned into 6 groups of 6 to 8 individuals. The rats were housed with an ambient temperature of 23 ± 2℃, a relative humidity of 60 to 80％and a 12-h light/dark cycle. Prior to the experiment, the rats were starved overnight but allowed with free access to water. Experimental acute pancreatitis was induced in the rats by six hourly i. p. injections of cerulein at the supramaximally stimulating dose (50 μg/kg) followed by a single dose of LPS at 7.5 mg/kg 1 h after the last cerulein injection, and this group of rats was designated as the cerulein group. The control group received injections of 0.9％saline instead of cerulein in the same volume and at same time intervals. The treatment groups given with cerulein and oral doses of dihydro-resveratrol (10, 20 or 50 mg/kg) were designated as Cerulein + D- res  10 or 20 or 50 mg/kg. The therapeutic intervention was given 30 minutes after the first cerulein injection for three consecutive hours. Upon scarification, pancreata were immediately removed, weighed, trimmed from fat and fixed in 4 ％paraformaldehyde-phosphate buffered saline overnight at 4 ℃. Samples were then processed, embedded in paraffin wax, sectioned and subjected to H&E staining. Levels of TNF-α in pancreatic and pulmonary samples were determined using commercial ELISA kits. Tissue homogenates were subjected to biochemical assays for the evaluation of MPO activity and glutathione content.
Functional intact acini were dissociated from pancreatic tissue using collagenase digestion with mild shearing forces. Acini were cultured in Dulbecco’s modification of Eagle’s medium (GIBCO) supplemented with 5％fetal bovine serum (GIBCO) , 1％penicillin-streptomycin (GIBCO) in a 5％CO2, 95％air humidified atmosphere at 37℃. LTC-14 cells were seeded at a density of 1 × 104/well in a 96-well plate, and incubated with different concentrations of dihydro-resveratrol or trans-resveratrol (dissolved in DMSO) for 24 hours. MTT reagent was added to the cells at the end of the 24-hour treatment period. After a 3-hour reaction time, MTT products were dissolved in DMSO and absorbance at 570 nm was taken.
Results
After the induction of cerulein, the weight ratio of pancreas to body in the acute pancreatitis rats was drastically increased by roughly 60％when compared with the non-cerulein induced controls due to the occurrence of pancreatic edema. The oral administration of dihydro-resveratrol at an adequate dosage of not less than 20 mg/kg notably reduced the pancreatic edema as reflected by the significant decrease in the  weight ratio of pancreas to body. The ameliorative effect of dihydro-resveratrol on reducing pancreatic edema was more promising than that of trans-resveratrol, the accredited antioxidant. Regarding the human dosage, the comparable dosage is 3.24 mg/kg based on the standard dosage conversion according to Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J 22 (3) : 659–661.
When oral administration of dihydro-resveratrol was given, the focal expansion of the interlobular septae, cytoplasmic shrinkage and leukocyte infiltration in pancreatitic parenchyma was remarkably reduced whereas the pulmonary wall thickening and hemorrhage in lung tissues were significantly improved in the rats with cerulein-induced acute pancreatitis.
For a relief of inflammatory conditions of the pancreas and lungs, the levels of pro-inflammatory cytokine TNF-α as well as MPO activities were significantly reduced in the pancreatic and pulmonary tissues by the oral administration of dihydro-resveratrol.
The level of glutathione in cerulein-induced pancreatic tissue was depleted drastically by more than 50％when compared to the non-cerulein-treated control. The oral administration of dihydro-resveratrol significantly suppressed glutathione depletion in the cerulein-induced pancreata.
The solubility of dihydro-resveratrol in a methanol-based solvent was at least 5 times higher than that of trans-resveratrol. By assessing the mitochondrial metabolic rates of acini, the cytotoxicity of dihydro-resveratrol was shown to be approximately 500 μM whereas that of trans-resveratrol was roughly 250 μM. Thus, the cytotoxicity of dihydro-resveratrol was 50％lower than that of trans-resveratrol.
In a first embodiment of a first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications by administering to a subject in needs thereof a composition comprising an effective amount of a stilbenoid derivative which comprises a compound of formula (1) ,
Figure PCTCN2017107798-appb-000030
 wherein R1, R2 and R3 are each independently selected from an alkyl group. The term “alkyl” , alone or in combination with other groups, includes reference to a straight chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. The term is further exemplified by such groups as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl and tert-butyl) , pentyl, hexyl and the like, and the derivatives or chemical variants thereof； or a mixture of said compound, the derivative and/or chemical variants thereof.
In a second embodiment of a first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the stilbenoid derivative is trans-3, 5, 4'-trihydroxybibenzyl, or dihydro-resveratrol, which is a compound of formula (2) : 
Figure PCTCN2017107798-appb-000031
 and the derivatives or chemical variants thereof； or a mixture of said compound, the derivative and/or chemical variants thereof.
In a third embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the subject is a human or an animal.
In a fourth embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the composition is administered orally.
In a fifth embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein the acute inflammatory condition of the  pancreas comprises all forms of acute pancreatitis and associated systemic complications comprise pulmonary injury.
In a sixth embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein said composition is administered at no less than 20 mg/kg to said subject for no less than 3 times a day.
In a seventh embodiment of the first aspect of the present invention there is provided a method of treating acute inflammatory condition of the pancreas and associated systemic complications wherein said composition is administered at no less than 3.24 mg/kg to said subject for no less than 3 times a day.
In a first embodiment of a second aspect of the present invention there is provided a method for preparing a compound of molecular formula C14H14O3 and of formula (2) , 
Figure PCTCN2017107798-appb-000032
 which is a stilbenoid derivative having a chemical name of trans-3, 5, 4'-trihydroxybibenzyl by hydrogenating of trans-resveratrol.
In a second embodiment of the second aspect of the present invention there is provided a method of preparing the compound of molecular formula C14H14O3 and of formula (2) wherein the hydrogenating of trans-resveratrol comprises steps of:
stirring a solution of trans-resveratrol in anhydrous EtOH at room temperature under 5 atm H2 pressure in the presence of 10％Pd/C for 8 hours；
filtering off the catalyst from the stirred solution；
evaporating the filtrate in vacuum to produce a residue；
eluting the residue using silica gel chromatographic separation with petroleum ether and ethyl acetate (1: 1) to produce dihydro-resveratrol.
PREFERRED EMBODIMENTS OF THE PRESENT INVENTION
TGF-β has been reported by some previous studies as a potent inducer of PSC activation in which a series of fibrotic mediators, including FN1, are being up-regulated. In cultured LTC-14 cells, which are immortalized PSCs from rat, the expression levels of fibrotic filament α-SMA and ECM protein FN1 are remarkably elevated by the exogenous addition of recombinant TGF-β (5 ng/mL) . In one further embodiment of the present invention, the inventors discover that the administration of dihydro-resveratrol significantly attenuate the expression levels of α-SMA and FN1 in rat PSCs upon the challenge of TGF-β. The derivatives of dihydro-resveratrol exert similar suppressive effect in PSCs. When compared to the renowned anti-oxidant trans-resveratrol, the inhibitory effect of dihydro-resveratrol is more significant. Among the testing stilbenoids, dihydro-resveratrol exerts the most potent anti-fibrotic effect in PSCs despite they possess modest structural differences.
The present invention provides a compound for suppressing a fibrotic mediator of stellate cells present in an internal organ of a subject in need with a formula of
Figure PCTCN2017107798-appb-000033
wherein
R2 and R4 are each independently selected from -OR11 and -OC (O) R11；
R1, R3, R5, R6, R7, R8, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14；
R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents  independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof；
or a mixture of said compound, the derivative and/or chemical variants thereof.
The present invention further provides nine embodiments of compounds for suppressing a fibrotic mediator of stellate cells present in an internal organ of a subject in need with formula of:
Figure PCTCN2017107798-appb-000034
or 4- [2- (3-Hydroxy-5-methoxyphenyl) ethyl] -2, 6-dimethoxy-phenol；
Figure PCTCN2017107798-appb-000035
or Tristin；
Figure PCTCN2017107798-appb-000036
or 3, 3', 5-Trihydroxydibenzyl；
Figure PCTCN2017107798-appb-000037
or 5- [2- (4-Hydroxy-3-methoxyphenyl) ethyl] -2, 3-dimethoxy-phenol；
Figure PCTCN2017107798-appb-000038
or 5- [2- (3-Hydroxyphenyl) ethyl] -2, 3-dimethoxy-phenol；
Figure PCTCN2017107798-appb-000039
or Crepidatin；
Figure PCTCN2017107798-appb-000040
or Moscatilin；
Figure PCTCN2017107798-appb-000041
or Aloifol I；
Figure PCTCN2017107798-appb-000042
or dihydro-resveratrol.
The internal organ can be, for example, pancreas, liver, kidney and lung of a subject. The subject can be a human subject.
Experiments:
LTC-14 cells were cultured at 37℃ under a humidified condition of 95％air and 5％CO2 in IMDM supplemented with 10％fetal bovine serum (FBS) . Cells used in all the experiment were among passages 9 to 25. LTC-14 cells were seeded at a density of 1 × 105/well in a 12-well plate, and incubated with recombinant TGF-β at 5 ng/mL with dihydro-resveratrol at 0, 1, 5, 10 and 20 μg/mL in IMDM supplemented with 0.2％FBS for 24 hours. Cells were then harvested for protein extraction and Western blotting analysis or immunofluorescent staining.
Total proteins of the LTC-14 cells are extracted using RIPA lysis buffers containing protease inhibitors. Cell lysates were loaded and separated by SDS-polyacrylaminde gel electrophoresis. After wet electroblotting, proteins were transferred onto PVDF membranes (Bio-rad) , blocked with 5 ％non-fat milk, probed with antibodies and visualized by utilization of an ECL kit (GE Healthcare) .
For immunofluorescent staining of α-SMA, LTC-14 cells were seeded at a  density of 1 × 105 onto the poly-L-lysine-coated cover slips in a 24-well plate, incubated with TGF-β at 5 ng/mL with dihydro-resveratrol at 0, and 10 μg/mL in IMDM supplemented with 0.2％FBS for 24 hours. Cells were then fixed, blocked with 3％BSA, probed with antibodies and mounted with fluorescence mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI) . Images were captured using the Nikon microscope and analyzed by the SPOT advanced software.
Evaluation of biological activities. C57/BL6 mice aged 28 days weighing in the range of 20 to 25 g were randomly assigned into 4 groups of 6 to 8 individuals. The mice were housed with an ambient temperature of 23 ± 2℃, a relative humidity of 60 to 80％and a 12-h light/dark cycle. Prior to the glucose tolerance test, the mice were starved overnight but allowed with free access to water. Experimental chronic pancreatitis was induced in the mice by four hourly i. p. injections of cerulein at the supramaximally stimulating dose (50 μg/kg) a day, 3 days a week, in a total of 6 weeks. The control group received injections of 0.9％saline instead of cerulein in the same volume and at same time intervals. The treatment groups given with cerulein and oral doses of dihydro-resveratrol (20 mg/kg/day) were designated as Cer+D-res. A dosage of dihydro-resveratrol at 50 mg/kg/day had also been attempted in the treatment course, but no statistically significant difference from the dose at 20 mg/kg/day in fibrosis formation was achieved. Nevertheless, no adverse effect was obtained from this higher dosage in the in vivo trial. Thus, it concludes that an effective dosage of dihydro-resveratrol is at least 20 mg/kg/day. The group given with trans-resveratrol was designated as Cer+Res. The drug intervention of both compounds was given from the first day of week 4 till the end of experiment, i.e. in a total of 3 weeks. At the end of the 6-week experiment, mice were subjected to the intraperitoneal glucose tolerance test (IPGTT) . Mice had been starved for 14 hours prior to the IPGTT, in which a 15％(w/v) glucose solution was injected to individual animals at 1.5 g glucose per kg body weight. About 1 μL of blood will be obtained from the tail vein, and blood glucose levels were monitored 30 min before (i.e. fasting level) and 10, 20, 30 and 60 min after glucose injection using a glucometer (Medisign, Korea) . Upon scarification, pancreases were immediately removed, weighed, trimmed from fat and fixed in 4％paraformaldehyde-phosphate buffered saline overnight at 4℃. Samples were then processed, embedded in paraffin wax, sectioned and subjected to immunostaining.
According to the dose translation formula, human equivalent dosage (mg/kg) ＝ Animal dose (mg/kg) multiplied by animal Km/human Km, where mouse Km is 3 and human Km is 37 (Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) , the  effective human equivalent dosage of dihydro-resveratrol of the present invention is at least 1.622 mg/kg/day.
In yet another embodiment of the present invention, 8 other derivatives (compounds i to viii) of dihydro-resveratrol and dihydro-resveratrol (compound 2) are shown in Figures 8A –8I. In this embodiment, each compound is experimented accordingly as follows:
Experiment with the embodiments of compounds in Figures 8A –8I
LTC-14 cells were pre-incubated with TGF-β (5 ng/mL) , and treated with trans-resveratrol (Resv) or dihydro-resveratrol (D-Res) or stilbene compounds i-viii at 20 μg/mL for 24 hours. Control was not treated with Resv or any stilbenoids. Total proteins were extracted and analyzed using Western blotting. This is shown in Figure 9.
LTC-14 cells are pancreatic stellate cells. α-SMA is the hallmark component of fibrogenesis whereas β-actin serves as a loading control. Thus, the expression level of α-SMA implies the degree of PSC activation. TGF-β was added since it is regarded as a potent inducer of fibrotic events. Suppressive effect on α-SMA expression level is tested among dihydro-resveratrol and compounds i to viii in relation to trans-resveratrol (Resv) . All of the testing compounds exert suppressive effect of α-SMA expression level.
LTC-14 cells were pre-incubated with TGF-β (5 ng/mL) , and treated with trans-resveratrol or dihydro-resveratrol at the indicated concentrations. Total proteins were extracted and analyzed using Western blotting. This is shown in Figure 10.
FN1 is a major extracellular matrix protein produced during fibrogenesis or upon the activation of pancreatic stellate cells. Its expression level implies the degree of fibrogenesis. Suppressive effect on levels of FN1 and α-SMA is tested between dihydro-resveratrol (i.e. compound 2) and trans-resveratrol.
LTC-14 cells were pre-incubated with TGF-β (5 ng/mL) , and treated with dihydro-resveratrol at 20 μg/mL for 24 hours prior to immunofluorescent staining.
LTC-14 cells are pancreatic stellate cells. α-SMA is the hallmark component of fibrogenesis whereas β-actin serves as a loading control. Thus, the green fluorescent signal (identified by arrow marks in Figure 11) of α-SMA implies the degree of PSC activation. TGF-β was added since it is regarded as a potent inducer of fibrotic events.  Suppressive effect of dihydro-resveratrol on α-SMA expression level is tested.
Pancreatic tissue sections are stained with antibody against FN1； thus, the immunostaining signals imply the degree of FN1 deposition in the parenchyma. The treatment with dihydro-resveratrol at 20 mg/kg/day (Cer+D-Res) reduces such deposition in chronic pancreatitis in a manner more significant to trans-resveratrol (Cer+Res) . This is shown in Figure 12.
Evaluation of biological activities. C57/BL6 mice aged 28 days weighing in the range of 20 to 25 g were randomly assigned into 4 groups of 6 to 8 individuals. The mice were housed with an ambient temperature of 23 ± 2℃, a relative humidity of 60 to 80％and a 12-h light/dark cycle. When oral administration of dihydro-resveratrol (20 mg/kg/day) is given, the fasting blood glucose levels of the chronic pancreatitis mice (Cer) become markedly higher than those of the control group, indicating these chronic pancreatitis mice develop hyperglycemia, a discernible feature of diabetes. Importantly, their impaired glucose tolerance has been significantly rectified by the 3-week dihydro-resveratrol treatment (Cer+D-Res) . As a result, the hyperglycemic condition of the chronic pancreatitis mice is improved. This is shown in Figure 13.
When oral administration of dihydro-resveratrol (Cer+D-Res) is given at 20 mg/kg/day, the severity of pancreatitis and the shrinkage and destruction of islets, explicitly beta cells, are notably ameliorated. As shown in Figure 14, the pancreatic beta-cell area is reflected by the immunofluorescent insulin signals. A reduced beta-cell area or mass implicates the deficiency in glucose tolerance, or the development of diabetes. Thus, the restoration of beta-cell area by the dihydro-resveratol treatment indicates this stilbenoid is beneficial to the treatment of pancreatogenic diabetes (i.e. Type3c DM) .
According to the dose translation formula, human equivalent dosage (mg/kg) ＝ Animal dose (mg/kg) multiplied by animal Km/human Km, where mouse Km is 3 and human Km is 37 (Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) , the effective human equivalent dosage of dihydro-resveratrol of the present invention is at least 1.622 mg/kg/day.
OTHER PREFERRED EMBODIMENTS OF THE PRESENT INVENTION
Dendrobium plants, commonly called “Shi Hu” , are widely used in the traditional Chinese medicine (TCM) system as well as in folk medicines for treating  various kinds of diseases, such as chronic atrophic gastritis, diabetes and cardiovascular disease. Dendrobium-derived extracts or ingredients contain substantial amounts of various stilbenoids, such as trans-resveratrol and dihydro-resveratrol, which are potential compounds for combating oxidative stress in the human body. However, the uses of these compounds for skin-protection or skin whitening have not been disclosed.
As oriental cosmetics prefer plant-based composition, the present invention relates to the use of Dendrobium-derived stilbenoids, particularly trans-resveratrol, dihydro-resveratrol or dihydro-resveratrol derivatives in reducing melanin formation with a potential to inhibit the generation of oxidative radicals and ROS. The present composition is applied to the subject in need thereof via topical administration. The present composition is in the form of a day cream, a night cream, a face lotion, a body lotion, a body butter, a skin peel, a mask, a shower gel, a sun cream, a sun lotion, an after sun cream or an after sun lotion.
The present composition comprises one or more extract (s) derived from Dendrobium plants.
The present composition comprises one or more stilbenoids with the following formula:
Figure PCTCN2017107798-appb-000043
wherein
R2, R4, and R8 are each independently selected from -OR11 , -OCH2R12, -OC (O) R11, -OCH2C (O) OR12 and -OC (O) CH2R12；
R1, R3, R5, R6, R7, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, OR12 and -OC (O) R12； or R2 and R3, or R7 and R8 may be taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently selected from – (CH2) -hydrocarbyl, C2-10 alkyl, alkenyl , cycloalkyl, aryl and heterocyclyl, Each of these groups is optionally substituted with 1, 2, 3, 4 or 5 R13；
R12 is independently selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R13；
R13 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR14, -C (O) R15, -C (O) N (R14) R15, -C (O) OR14, -OC (O) R15, -S (O) 2R14, -S (O) 2N (R14) R15, -N (R14) R15；
R14 and R15 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof.
The present composition comprises stilbenoid (s) with the following formula: 
Figure PCTCN2017107798-appb-000044
 which is dihydro-resveratrol, and the derivatives or chemical variants thereof； or a mixture of said compound, the derivative and/or chemical variants thereof, or with a formula of:
Figure PCTCN2017107798-appb-000045
Figure PCTCN2017107798-appb-000046
Figure PCTCN2017107798-appb-000047
Figure PCTCN2017107798-appb-000048
Figure PCTCN2017107798-appb-000049
The present composition for skin whitening and skin protection comprises stilbenoid (s) with the following formula:
Figure PCTCN2017107798-appb-000050
wherein
R2, R4, and R8 are each independently selected from -OR11, -OCH2R11, -OC (O) R11, -OCH2C (O) OR11 and -OC (O) CH2R11；
R1, R3, R5, R6, R7, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11； or R2 and R3, or R7 and R8 may be  taken together with the carbon atoms to which they are attached to form a cyclic group；
R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12；
R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14；
R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy；
or an enantiomer thereof；
or a pharmaceutically acceptable salt or prodrug thereof.
To determine the antioxidant capacity, a spectrophotometric decolorization assay complemented with pre-formed radical monocation of 2, 2'-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) , also known as ABTS, is used. In this assay, ABTS (Abcam, USA) is dissolved in water to a 7 mM concentration and ABTS radical cation is produced by reacting the ABTS stock solution with 2.45 mM ammonium persulfate (Sigma-Aldrich, USA) and allowing the mixture to stand in the dark at room temperature for 16 hours before use. When testing the Dendrobium extract samples or stilbenoid samples, the ABTS radical solution is diluted with ethanol to an absorbance of 0.70 at 734 nm and equilibrated at 30℃. The dilutions of testing samples (0.1 mL) or DMSO (0.1 mL) are incubated with ABTS radical solution (0.9 mL) for 15 min prior to the absorbance taking at 734 nm. DMSO is served as a vehicle treatment whereas Trolox (Abcam, USA) , a renowned derivative vitamin E, ranging from 0.001 to 0.05 mg/mL is used as a positive drug reference. The antioxidant capacity of testing samples is expressed as the amount equivalent to Trolox in milligrams (mg) according to the Trolox standard curve.
Upon the incubation with dihydro-resveratrol (D-Res) , trans-resveratrol (Res) , Dendrobium extract samples or other stilbenoid samples (compounds DR1 to DR11) , the pre-formed ABTS radicals have been scavenged, and their antioxidant capacity is normalized to the Trolox positive standards as illustrated in Table 3.
Table 3. Antioxidant capacity of stilbenoids equivalent to amount of Trolox (mg) :
Figure PCTCN2017107798-appb-000051
Figure PCTCN2017107798-appb-000052
The cellular melanin content and tyrosinase activity are determined in cultured B16 and A375 melanocytes. In fact, melanocytes are melanin-producing cells whereas melanin refers to groups of endogenous pigments that give multitude of skin colors. B16 cells (Shanghai Institutes for Biological Science, Chinese Academy of Sciences, China) and A375 cells (American Type Culture Collection, USA) are routinely grown in DMEM medium (Gibco, USA) supplemented with 10％heat-activated fetal bovine serum (FBS, Gibco, USA) and 1％penicillin/streptomycin (Gibco, USA) in a humidified atmosphere of 95％air and 5％CO2 at 37℃.
To determine the cellular melanin content, melanocytes are seeded in 12-well plates (8×104 cells/well) and stimulated with alpha-melanocyte-stimulating hormone (α-MSH, 100 nM) for 24 hours. Such stimulation is aimed to accelerate the cellular formation of melanin in the melanocytes, so that the reducing ability of the testing compounds or extracts on melanin formation becomes more apparent. Followed by the α-MSH stimulation, cells are then treated with different Dendrobium extract samples or stilbenoid samples (5 μL) or DMSO (5 μL, Sigma-Aldrich, USA) for another 24 hours. DMSO serves as a vehicle treatment. At the end of experiment, cells are trypsinized for detaching from the culturing plates. After centrifugation, the melanin pellet of each sample is incubated with 100 μL of 1 N Sodium hydroxide solution (Sigma-Aldrich, USA) for 1.5 hours at 70℃. After cooling to room temperature, the solution is centrifuged at 15,000 × g for 10 min. The supernatant (100 μL) of each sample is transferred to 96-well plates for a reading of absorbance taken at 405 nm. Relative melanin content of each sample is normalized with its protein content and presented as the percentage change over the DMSO-treated cells.
Upon the incubation with dihydro-resveratrol (D-Res) , trans-resveratrol  (Res) , Dendrobium extract samples or other stilbenoid samples (compounds DR1 to DR11) , the cellular melanin contents in B16 and A375 melanocytes have been reduced as illustrated in Figures 15 to 17.
To determine the cellular tyrosinase activity, melanocytes are seeded in 12-well plates (8×104 cells/well) and stimulated with α-MSH (100 nM) for 24 hours. Post the α-MSH stimulation, cells are treated with different testing samples (5 μL) or DMSO (5 μL) for another 24 hours. At the end of experiment, the cells are washed with ice-cold phosphate buffer saline (PBS, pH 6.8) (Gibco, USA) twice and then lysed with 150 μL of PBS (pH 6.8) containing 0.1％Triton X-100 on ice. The cell lysates are centrifuged at 15,000 × g for 15 min at 4℃. An aliquot of 50 μL supernatant is mixed with 50 μL L-3, 4-dihydroxyphenylalanine (L-DOPA, Abcam, USA) solution (0.2％in PBS, pH 6.8) in 96-well plates and incubated at 37℃ for 2 hours under darkness. Optical density of each sample is measured at 475 nm. The absorbance is normalized with the protein content of each sample. The relative activity of cellular tyrosinase in melanocytes is calculated and presented as the percentage change over the DMSO-treated cells. In addition, another set of experiment is collected for Western blotting analysis of the expression levels of TRP1 and TRP2, so as their upstream regulators p-AKT and p-38.
Upon the incubation with dihydro-resveratrol (D-Res) , trans-resveratrol (Res) , Dendrobium extract samples or other stilbenoid samples (compounds DR1 to DR11) , the tyrosinase activity in B16 and A375 melanocytes has been inhibited as illustrated in Figures 18 to 20. Western blotting results are shown in Figure 21.
To determine the generation of intracellular ROS in melanocytes, B16 or A375 cells are seeded in 12-well plates (8×104 cells/well) and stimulated with α-MSH (100 nM) for 24 hours. At the end of α-MSH incubation, cells are trypsinized for detaching from the culturing plates and subjected to cellular ROS detection assay (Abcam) according to manufacturer’s instruction. In brief, cells are stained with 20 μM DCFDA for 30 min at 37℃. After staining, cells are treated with tert-butyl hydrogen peroxide (TBHP, 55 nM) for an evident level of ROS elevation prior to a 4-hour incubation with our testing extracts or compounds or ascorbic acid (AC) in a volume of 5 μL. Signals of ROS generation will be detected using a fluorescence microplate reader. DCF are excited by the 488 nm laser. Results of this assay are presented in Figure 22.
All assays are performed in triplicate and repeated for at least 3 times in  individual experiments. The results are presented as the mean ± standard deviation. Variance between two groups is evaluated by Student's t-test whereas variance among more than two groups is calculated by means of one-way analysis of variance (one-way ANOVA) . A p value of less than 0.05 is considered as statistically significant.
To determine the skin color of the individual human subjects, the initial skin conditions at the arms of the individuals are tested by the skin colorimeter MPA 5 on day 0. The individual typology angle (ITA°) is calculated by the colorimeter based on the luminance (L*) and yellow-blue component (b*) values. The greater the ITA° value, the greater the skin whiteness [S. Del Bino and F. Bernerd. Variations in skin colour and the biological consequences of ultraviolet radiation exposure. British Journal of Dermatology 2013； 169 (Suppl. 3) , 33-40] . In fact, skin colors are classified into 6 major divisions: very light, light, intermediate, tan, brown and dark as listed in Table 4. Two areas (2 cm × 2 cm each) are selected as treated areas whereas regions surrounding the treated areas on the arms are regarded as control areas. A volume of 200 μL of the testing stilbenoids (2％by weight dissolved in ethanol) is applied to the designated area twice a day, day and night. At the end of the first week (day 7) , individuals are tested with the skin colorimeter for recording the data about using the stilbenoids for 7 days. Again, at the end of the second week (day 14) , individuals are tested with the skin colorimeter for recording the data about using the stilbenoids for 14 days. The ITA° readings are presented in Table 5. Overt whitening effect from dihydro-resveratrol or trans-resveratrol is obtained from most individuals. A set of representative images taken from a human individual who uses the topical treatment is shown in Figure 23.
Table 4. Skin classification based on ITA°:
Figure PCTCN2017107798-appb-000053
Table 5. ITA° measurements of individual human subjects on the indicated time-points:
Figure PCTCN2017107798-appb-000054
Figure PCTCN2017107798-appb-000055
General Synthetic Route of DR1 to DR3
Figure PCTCN2017107798-appb-000056
To a mixture of dihydro-resveratrol (0.2 mmol) and an alkyl bromide (RBr) (1.2 mmol) in dimethylformamide (DMF, 2 mL) , K2CO3 (1.2 mmol) was added. The resulting mixture was stirred at room temperature until the starting material disappeared on thin-layer chromatography (TLC) . The mixture was diluted with H2O (10 mL) and washed with dichloromethane (DCM, 10 mL) three times. The combined organic layer was washed with saturated sodium chloride (NaCl) twice, dried over anhydrous Na2SO4, concentrated in vacuum and purified by preparative (prep-) TLC (PE/EA ＝ 5/1 or 3/1) to afford the desired compound (s) . The R group in RBr can be OR11, -OCH2R11 or -OCH2C (O) OR11, where R11 is hydrocarbyl.
DR1:
Figure PCTCN2017107798-appb-000057
High-resolution mass spectrometry (HRMS) : 511.1997 [M+Na] +
Proton nuclear magnetic resonance (1H NMR, 400 MHz, CDCl3) δ 1.31 (9H, t, J ＝ 7.1 Hz) , 2.82 (4H, s) , 4.22-4.32 (6H, m) , 4.56 (4H, s) , 4.60 (2H, s) , 6.29-6.41 (3H, m) , 6.80-6.86 (2H, m) , 7.04-7.13 (2H, m) .
DR2:
Figure PCTCN2017107798-appb-000058
HRMS: 373.1770 [M+Na] +.
1H NMR (400 MHz, CDCl3) δ 2.84 (4H, d, J ＝ 2.1 Hz) , 4.49 (4H, dt, J ＝ 5.3, 1.4 Hz) , 4.51-4.53 (2H, m) , 5.25-5.35 (3H, m) , 5.37-5.48 (3H, m) , 5.99-6.13 (3H, m) , 6.36 (3H, s) , 6.81-6.90 (2H, m) , 7.05-7.15 (2H, m) .
DR3:
Figure PCTCN2017107798-appb-000059
HRMS: 379.2220 [M+Na] +.
1H NMR (400 MHz, CDCl3) δ 0.98-1.08 (9H, m) , 1.74-1.90 (6H, m) , 2.75-2.90 (4H, m) , 3.85-3.95 (6H, m) , 6.29-6.39 (3H, m) , 6.81-6.87 (2H, m) , 7.07-7.15 (2H, m) .
General Synthetic Route of DR4 to DR11
Figure PCTCN2017107798-appb-000060
To a mixture of dihydro-resveratrol (0.2 mmol) and acyl chloride (RCOCl) (1.2 mmol) in DCM (2 mL) , Et3N (1.2 mmol) at 0 ℃ was added. The resulting mixture was warmed to room temperature and stirred until the starting material disappeared on TLC. The mixture was diluted with H2O (10 mL) and washed with DCM (10 mL) three times. The combined organic layer was washed with saturated NaCl twice, dried over anhydrous Na2SO4, concentrated in vacuum and purified by prep-TLC (PE/EA ＝ 5/1 or 3/1) to afford the desired compound (s) . The R group in RCOCl can be -R11 or -CH2R11, where R11 is hydrocarbyl, which is optionally substituted with 1, 2, 3, 4 or 5 R12, and where R12 is halogen or -OR13, and where R13 is hydrogen or hydrocarbyl.
DR4:
Figure PCTCN2017107798-appb-000061
HRMS: 463.2044 [M+Na+] .
1H NMR (400 MHz, CDCl3) δ δ 0.97-1.10 (9H, m) , 1.72-1.89 (6H, m) , 2.47-2.58 (6H, m) , 2.91 (4H, s) , 6.74-6.84 (3H, m) , 6.94-7.04 (2H, m) , 7.13-7.21 (2H, m) .
DR5:
Figure PCTCN2017107798-appb-000062
HRMS: 667.0427 and 669.0408 [M+Na+] .
1H NMR (400 MHz, CDCl3) δ 3.01 (4H, s) , 6.99-7.06 (3H, m) , 7.10-7.17 (2H, m) , 7.22-7.27 (2H, m) , 7.46-7.55 (6H, m) , 8.09-8.18 (6H, m) .
DR6:
Figure PCTCN2017107798-appb-000063
HRMS: 655.1964 [M+Na+] .
1H NMR (400 MHz, CDCl3) δ 3.00 (4H, s) , 3.89-3.92 (9H, m) , 6.97-7.04 (9H, m) , 7.11-7.16 (2H, m) , 7.23-7.27 (2H, m) , 8.13-8.18 (6H, m) .
DR7:
Figure PCTCN2017107798-appb-000064
HRMS: 565.1600 [M+Na] +.
1H NMR (400 MHz, CDCl3) δ 3.02 (4H, s) , 7.02-7.08 (3H, m) , 7.12-7.20 (2H, m) , 7.25-7.29 (2H, m) , 7.50-7.57 (6H, m) , 7.60-7.70 (3H, m) , 8.17 -8.28 (6H, m)
DR8:
Figure PCTCN2017107798-appb-000065
HRMS: 415.1140 [M+Na] +
1H NMR (400 MHz, CDCl3) δ 2.94 (4H, s) , 5.98-6.08 (3H, m) , 6.23-6.43 (3H, m) , 6.58-6.70 (3H, m) , 6.84-6.97 (3H, m) , 7.01-7.13 (2H, m) , 7.16-7.26 (2H, m) .
DR9:
Figure PCTCN2017107798-appb-000066
HRMS: 514.2550 [M+Na] +.
1H NMR (400 MHz, CDCl3) δ 1.61-1.79 (12H, m) , 1.88-2.09 (12H, m) , 2.82-3.08 (7H, m) , 6.72-6.83 (3H, m) , 6.96-7.03 (2H, m) , 7.15-7.20 (2H, m) .
DR10:
Figure PCTCN2017107798-appb-000067
HRMS: 607.2070 [M+Na] +.
1H NMR (400 MHz, CDCl3) δ 2.91 (4H, s) , 5.27 (6H, s) , 6.91-6.98 (3H, m) , 7.08-7.12 (2H, m) , 7.14-7.19 (2H, m) , 7.38-7.46 (15H, m) .
DR11:
Figure PCTCN2017107798-appb-000068
HRMS: 505.2550 [M+Na] +.
1H NMR (400 MHz, CDCl3) ·1.34-1.38 (27H, m) , 2.86-2.94 (4H, m) , 6.77 (3H, d, J ＝ 2.1 Hz) , 6.93-7.02 (2H, m) , 7.14-7.21 (2H, m)
INDUSTRIAL APPLICABILITY
The present invention relates to a skin-protection composition that comprises stilbenoid (s) and/or stilbenoid-containing extract (s) obtained from Dendrobium plants, such as Dendrobium officinale and Dendrobium nobile for the management of melanogenesis, skin-darkening and skin-aging. More particularly, it relates to the usage of Dendrobium ingredients and stilbenoids to reduce the formation of melanin in melanocytes. It also relates to the usage of Dendrobium ingredients and stilbenoids to reduce the generation of reactive oxygen species and oxidative free radicals. This invention relates to the use Dendrobium-derived extracts or ingredients or stilbenoids in the formulation of skin-protection, skin-whitening and/or anti-skin aging products.

Claims (15)

  1. A compound of formula (4) :
    Figure PCTCN2017107798-appb-100001
    wherein
    R2, R4, and R8 are each independently selected from -OR11, -OCH2R12, -OC (O) R11, -OCH2C (O) OR12 and -OC (O) CH2R12;
    R1, R3, R5, R6, R7, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR12 and -OC (O) R12; or R2 and R3, or R7 and R8 are taken together with the carbon atoms to which they are attached to form a cyclic group;
    R11 is independently selected from - (CH2) -hydrocarbyl, C2-10 alkyl, alkenyl, cycloalkyl, aryl and heterocyclyl, each of these groups is optionally substituted with 1, 2, 3, 4 or 5 R13;
    R12 is independently selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R13;
    R13 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR14, -C (O) R15, -C (O) N (R14) R15, -C (O) OR14, -OC (O) R15, -S (O) 2R14, -S (O) 2N (R14) R15, -N (R14) R15;
    R14 and R15 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy;
    or a pharmaceutically acceptable salt or prodrug thereof.
  2. The compound of claim 1, wherein said compound is an optically pure stereoisomer, an enantiomer, a racemate, or a diastereomer thereof.
  3. The compound of claim 1, wherein said compound is selected from DR1, DR2, DR3, DR4, DR5, DR6, DR7, DR8, DR9, DR10 or DR11 with the following formulae respectively:
    Figure PCTCN2017107798-appb-100002
    Figure PCTCN2017107798-appb-100003
    Figure PCTCN2017107798-appb-100004
    Figure PCTCN2017107798-appb-100005
    or a pharmaceutically acceptable salt or prodrug thereof.
  4. A composition for treating, preventing and/or delaying progress of skin darkening comprising an effective amount of the compound of any one of claims 1 to 3 and a pharmaceutically acceptable carrier thereof.
  5. Use of the compound of any one of claims 1 to 3 and/or the derivatives or chemical variants thereof alone and/or in combination with one or more other pharmaceutically acceptable skin-whitening and skin-protection agent (s) in preparation of a composition for treatment, prevention or delay of progression of a skin darkening in a subject in need thereof.
  6. The use of claim 5, wherein said composition is applied topically to said subject.
  7. The use of claim 5 or 6, wherein said subject is human.
  8. A skin whitening and skin protection against UV exposure, skin damage and aging compound of formula (5) :
    Figure PCTCN2017107798-appb-100006
    wherein
    R2, R4, and R8 are each independently selected from -OR11, -OCH2R11, -OC (O) R11, -OCH2C (O) OR11 and -OC (O) CH2R11;
    R1, R3, R5, R6, R7, R9 and R10 are each independently selected from hydrogen, halogen, trifluoromethyl, -OR11 and -OC (O) R11; or R2 and R3, or R7 and R8 are taken together with the carbon atoms to which they are attached to form a cyclic group;
    R11 is independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 R12;
    R12 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, -OR13, -C (O) R14, -C (O) N (R13) R14, -C (O) OR13, -OC (O) R14, -S (O) 2R13, -S (O) 2N (R13) R14, -N (R13) R14;
    R13 and R14 are each independently hydrogen or selected from hydrocarbyl and heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C1-6 alkyl and C1-6 alkoxy; or an enantiomer, optically pure stereoisomer, racemate or diastereomer thereof; or a pharmaceutically acceptable salt or prodrug thereof.
  9. The skin whitening and skin protection against UV exposure, skin damage and aging compound according to claim 8, wherein the compound has a formula of:
    Figure PCTCN2017107798-appb-100007
    Figure PCTCN2017107798-appb-100008
    Figure PCTCN2017107798-appb-100009
    Figure PCTCN2017107798-appb-100010
    Figure PCTCN2017107798-appb-100011
    or an enantiomer thereof;
    or a pharmaceutically acceptable salt or prodrug thereof.
  10. A composition for skin whitening and skin protection against UV exposure, skin damage and aging comprising an effective amount of the skin whitening and skin  protection compound of claim 8 or 9 and a pharmaceutically acceptable carrier thereof.
  11. Use of the compound of claim 8 or 9 in preparation of a topical formulation for skin whitening and skin protection against UV exposure, skin damage and aging in a subject in need thereof.
  12. The use of claim 11, wherein said topical formulation is in a form of a paste, solid, powder, particle, emulsion, day cream, night cream, face lotion, body lotion, body butter, skin peel, mask, shower gel, sun cream, sun lotion, after sun cream, after sun lotion, lip balm, or lip gloss, or alike.
  13. The use of claim 11 or 12, wherein said subject is human.
  14. The composition of claim 4 or 10, wherein the composition is in the form of a paste, solid, powder, particle, emulsion, day cream, night cream, face lotion, body lotion, body butter, skin peel, mask, shower gel, sun cream, sun lotion, after sun cream, or after sun lotion.
  15. A method of synthesizing the compound of claim 3 or 9 from dihydro-resveratrol comprising the following two synthesis schemes:
    Scheme 1 for said DR1 to DR3:
    Figure PCTCN2017107798-appb-100012
    wherein
    R is -OR11, -OCH2R11 or -OCH2C (O) OR11;
    R11 is hydrocarbyl;
    and
    Scheme 2 for said DR4 to DR11:
    Figure PCTCN2017107798-appb-100013
    wherein
    R is -R11 or -CH2R11;
    R11 is hydrocarbyl, which is optionally substituted with 1, 2, 3, 4 or 5 R12;
    R12 is halogen or -OR13; and
    R13 is hydrogen or hydrocarbyl.
PCT/CN2017/107798 2016-11-15 2017-10-26 Skin-protection composition containing dendrobium-based ingredients Ceased WO2018090805A1 (en)

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CN201780069402.9A CN109952286B (en) 2016-11-15 2017-10-26 Dendrobium-based skin protection composition
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US15/351,636 US9738581B2 (en) 2015-06-16 2016-11-15 Method of using dihydro-resveratrol or its stilbenoid derivatives and/or chemical variants in treatments of fibrotic and diabetic conditions
US15/352,903 2016-11-16
US15/352,903 US9956152B2 (en) 2015-06-16 2016-11-16 Skin-protection composition containing dendrobium-based ingredients
US15/633,780 2017-06-27
US15/633,780 US9877931B2 (en) 2015-06-16 2017-06-27 Method of using dihydro-resveratrol or its stilbenoid derivatives and/or chemical variants in treatments of fibrotic and diabetic conditions

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