CN110698328B - Method of use of dihydroresveratrol or its stilbene derivatives and/or chemical variants as an antimicrobial agent - Google Patents

Abstract

The present invention relates to a composition for preventing or reducing the growth of microorganisms, especially dermatophytes and/or acidic and acidogenic Gram-positive bacteria. More specifically, it concerns trans-3,5,4'-trihydroxydibenzoyl (also known as dihydroresveratrol) or its dihydrostilbene derivatives and/or chemical variants as Use of antimicrobial agents. The present invention also relates to the management of dermatophytosis, usually caused by dermatophytes, and the management of dental caries, usually mediated by acidic and acidogenic Gram-positive streptococci. Accordingly, the present invention has application in the prevention, alleviation and/or treatment of dermatophytosis and dental caries.

Description

Dihydroresveratrol or its stilbene derivatives and/or chemical variants as antimicrobial How to use the agent

technical field

The present invention relates to a composition for preventing or reducing the growth of microorganisms, especially dermatophytes and/or acidic and acidogenic Gram-positive bacteria. More specifically, it concerns trans-3,5,4'-trihydroxydibenzoyl (also known as dihydroresveratrol) or its dihydrostilbene derivatives and/or chemical variants as Use of antimicrobial agents. The invention also relates to the management of dermatophytosis, often caused by dermatophytes, and the management of dental caries, often mediated by acidic and acidic streptococci. Accordingly, the present invention has application in the prevention, alleviation and/or treatment of said dermatophytosis and dental caries.

Background technique

Ringworm, which refers to infections of the skin and nails, is a public health concern worldwide. Typically, dermatophytosis is caused by skin fungi. Tinea pedis, also known as athlete's foot, is the most common form of skin fungus that causes skin disease. The incidence of tinea pedis has been steadily increasing in recent years, especially in developed countries where up to 15-25% of the population is affected [Fay Crawford]. This fungal skin infection is often found in swimmers and runners, and is more common in older adults. Most cases of athlete's foot are caused by Trichophyton rubrum and Trichophyton mentagrophytes. It is generally accepted that occlusion is the key to enhancing dermatophytic infections of the feet, as shoes provide an enclosed environment with relatively high humidity and warmth, ideal for the growth of said dermatophytes. The invention can significantly reduce the growth of Trichophyton rubrum and Trichophyton mentagrophytes under optimal environment conditions.

The commonly used fungicides are mainly azo, which can be further divided into itraconazole, ketoconazole and fluconazole. Among them, fluconazole is considered to have the largest minimum inhibitory concentration (MIC) against various fungi. However, Trichophyton rubrum and Trichophyton mentagrophytes showed significant resistance to the above three nitrogen compounds at different levels. Therefore, higher requirements are put forward for the discovery and development of novel and highly effective antifungal agents.

Over the past few decades, much pharmacological research has focused on the utilization of drug-like small molecules as therapeutic agents derived from herbal constituents, especially polyphenolic stilbenes. In fact, stilbenes are a class of compounds consisting of a general C6-C2-C6 structure, which are often isolated as monomers and oligomers for use as active ingredients in botanical supplements or pharmaceutical formulations. There is increasing evidence that stilbenes are potent antioxidants, as most of them are produced as phytotoxins against abiotic and biotic stressors. In addition, some stilbenes have been reported to have antimicrobial activity, especially against fungal growth.

Therefore, the use of dihydro-resveratrol or its dihydrostilbene derivatives and/or chemical variants to prepare antifungal agents has become one of the objectives of the present invention.

In addition to skin mycoses, dental caries (occurrence of dental caries) is also a common public health problem worldwide. In general, dental caries is characterized by the cessation or erosion of the hard tissue of the tooth, which can lead to tooth pain, sensitive teeth, and loss of tooth vitality. Foods containing fermentable carbohydrates (primarily mono-, di-, and polysaccharides) have long been considered a major cause of dental caries because they are readily available nutrients in the oral cavity for bacteria such as Streptococcus mutans and Streptococcus distalis Source [van houte J]. In fact, these Gram-positive bacteria are acidfast (acid-resistant) and acidogenic (acid-producing); they metabolize dietary sugars (especially sucrose) to lactic acid, which leads to demineralization of enamel and dentin. At the same time, the sticky macromolecules they synthesize, such as polysaccharides, promote bacterial attachment to tooth surfaces by forming plaque and biofilm. As a result, caries forms. Furthermore, these acidfast and acidogenic streptococci largely recovered at the initial stage of culture and after established carious lesions. Inhibition of the above-mentioned Streptococci, especially Streptococcus mutans, is significantly beneficial in reducing plaque and biofilm, thereby reducing the occurrence of dental caries. The growth of Streptococcus mutans and Streptococcus farreus cultivated under optimal environmental conditions in the present invention can be significantly reduced.

Therefore, a method of using dihydroresveratrol or its dihydrostilbene derivatives and/or chemical variants as an antimicrobial agent is another object of the present invention.

Contents of the invention

Accordingly, a first aspect of the present invention relates to a composition for preventing or reducing the growth of microorganisms, in particular the growth of Trichophyton rubrum, Trichophyton mentagrophytes, Streptococcus mutans and Streptococcus distalis, in areas where microorganisms are found. More specifically, it concerns trans-3,5,4'-trihydroxydibenzoyl (also known as dihydroresveratrol) or its dihydrostilbene derivatives and/or chemical variants as Use of antimicrobial agents. The invention also relates to the management of dermatophytosis, typically caused by Trichophyton rubrum and Trichophyton mentagrophytes; and the management of dental caries, typically mediated by Streptococcus mutans and Streptococcus distalis. Therefore, the application of the present invention is in the prevention, alleviation or treatment of dermatophytosis and dental caries.

In a first embodiment of the first aspect of the present invention, there is provided an antimicrobial agent comprising a compound of formula (3):

in

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 form a cyclic group together with the carbon atom to which it is attached;

R ¹¹ is independently hydrogen or selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 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 or -N(R ¹³ )R ¹⁴ ;

R ¹³ and R ¹⁴ are each independently hydrogen or selected from hydrocarbyl and heterocyclic groups, any one of which is optionally selected from halogen, cyano, amine through 1, 2, 3, 4 or 5 Substituents of radical, hydroxyl, C _1-6 alkyl or C _1-6 alkoxy;

or its enantiomers;

or a pharmaceutically acceptable salt or prodrug thereof;

or mixtures, derivatives and/or chemical variants thereof.

In a second embodiment of the first aspect of the present invention, an antimicrobial agent is provided, wherein the compound further comprises a compound of the following formula:

二氢白藜芦醇；

Dihydroresveratrol;

In a third embodiment of the first aspect of the present invention, an antimicrobial agent is provided, wherein the compound further comprises a compound of formula (I):

wherein R independently or collectively are OC(=O)R'; R' is alkenyl.

A second aspect of the present invention relates to a method of preventing, alleviating and/or treating dermatophytosis comprising administering an antimicrobial agent of the present invention to an individual in need thereof.

A third aspect of the present invention relates to a method of preventing, alleviating and/or treating dental caries comprising administering an antimicrobial agent of the present invention to an individual in need thereof.

A fourth aspect of the present invention relates to a method of preventing or reducing the growth of microorganisms, including dermatophytes and/or acidic and acidogenic Gram-positive bacteria, the method comprising applying the antimicrobial agent of the present invention to the The dermatophytes and/or the acidic and acid-producing Gram-positive bacteria are found in said locations.

In a first embodiment, the dermatophytes include Trichophyton rubrum and Trichophyton mentagrophytes.

In a second embodiment, the acidic and acidogenic Gram-positive bacteria include Streptococcus mutans and Streptococcus distalis.

In a third embodiment, the antimicrobial agent is formulated as a topical bactericidal or bacteriostatic gel, lotion, cream, cream, paste, solution or moistening spray for preventing, alleviating and/or treating dermatophytosis fungal disease.

In a fourth embodiment, the antimicrobial agent is formulated as toothpaste, oral gel, toothbrush sanitizer, mouthwash or chewing gum for preventing, alleviating and/or treating dental caries.

In a fifth embodiment, the concentration of said antimicrobial agent in a method for preventing, alleviating and/or treating dermatophytosis is 25 μM to 100 μM.

In a sixth embodiment, the concentration of said antimicrobial agent in a method for preventing, alleviating and/or treating dental caries is 25 μΜ to 100 μΜ.

In a seventh embodiment, the concentration of the antimicrobial agent is in the range of 25 μM to 100 μM for preventing or reducing the growth of skin fungi.

In an eighth embodiment, the concentration of the antimicrobial agent is in the range of 25 μM to 100 μM for preventing or reducing the growth of acidic and acidogenic Gram-positive bacteria.

In a ninth example, dermatophytes were found in athlete's foot in an individual in need of an antimicrobial agent of the invention.

In a tenth example, acidic and acidogenic Gram-positive bacteria are found in the oral cavity of an individual in need of an antimicrobial agent of the present invention.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.

The present invention includes all such changes and modifications. The present invention also includes all the steps and features mentioned or indicated in this specification individually or collectively, and any and all combinations of any two or more of these steps or features.

Throughout this specification, unless the context dictates otherwise, the word "comprise" or variations such as "comprises" or "comprising" is understood to mean including the stated whole or whole , but does not exclude any other whole or group of wholes. It should also be noted that in this disclosure, and especially in claims and/or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have corresponding patent claims. the meaning ascribed to it in the law; for example it may mean "includes", "included", "including" and the like; and expressions such as "consisting essentially of )" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, for example, they render an element unnecessary to be explicitly enumerated, but exclude prior art discoveries or basis affecting the present invention or elements of a novel feature.

Furthermore, throughout this specification and claims, unless otherwise specified herein, the word "include" or variations such as "includes" or "including" shall be understood to mean Include the stated whole or group of wholes, but not exclude any other whole or group of wholes.

Additional definitions of terms used herein can be found in and throughout the Detailed Description of the Invention. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present 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.

Description of drawings

The above and other objects and features of the present invention can be seen from the following description of the present invention in conjunction with the accompanying drawings, wherein:

Figure 1 shows increased body water in rats due to pancreatic edema induced by bombesin-induced acute pancreatitis. The resulting weights are expressed as the ratio of pancreas weight to body weight. A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Figures 2A to 2F show the changes in pancreatic tissue structure and morphology in the control group (Figure 2A), the bombesin group (Figure 2B) and the dihydro-resveratrol treatment group (D-Res) (Figure 2C to 2F), using Su Oxygenin and Eosin (H&E) staining. Images are shown with a scale bar of 50 μm.

Figures 3A to 3E show the structural and morphological changes in the lung tissue of the control group (Figure 3A), the bombesin group (Figure 3B) and the dihydro-resveratrol treatment group (D-Res) (Figure 3C to 3E). Images are shown at 200X magnification.

Figure 4A shows the MPO activity measured by colorimetric spectrophotometry, which represents the retention of neutrophils in the pancreatic tissues of the control, bombesin and dihydro-resveratrol-treated (D-Res) groups. A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Figure 4B shows the MPO activity measured by colorimetric spectrophotometry, which represents the retention of neutrophils in the lung tissues of the control, bombesin and dihydro-resveratrol-treated (D-Res) groups. A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Fig. 5A shows the detection of TNF-α levels in pancreatic tissues of the control group, the bombesin group and the dihydro-resveratrol treatment group (D-Res) by enzyme-linked immunosorbent assay (ELISA). A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Figure 5B shows the determination of TNF-α levels in the lung tissues of the control group, the bombesin group and the dihydro-resveratrol treatment group (D-Res) by enzyme-linked immunosorbent assay (ELISA). A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Fig. 6A shows the measurement of glutathione content in pancreatic tissues of the control group, the bombesin group and the dihydro-resveratrol treatment group (D-Res) by colorimetric spectrophotometry. A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Fig. 6B shows the ameliorative effects of dihydro-resveratrol (D-Res) and trans-resveratrol (Res) on reduced water content in rats with bombesin-induced acute pancreatitis and pancreatic edema. The resulting weights are expressed as the ratio of pancreas weight to body weight. A p-value of less than 0.05 was considered statistically significant. *p<0.05 compared with normal saline control group (Con); #p<0.05 compared with bombesin group.

Figure 7 shows metabolic rate measurements of MTT cell proliferation in pancreatic acinar cells treated with dihydro-resveratrol and trans-resveratrol. *p<0.05 compared to cells not treated with dihydro-resveratrol or trans-resveratrol.

Figures 8A to 8H show eight derivatives (ie Compound I to Compound VIII) from dihydroresveratrol (ie Compound 2), respectively.

Figure 8I shows the chemical structure of dihydroresveratrol (ie compound 2).

Figure 9 shows Western blot of LTC-14PSCs preincubated with TGF-β (5 ng/mL) and treated with 20 μg/mL of trans-resveratrol (Resv) or dihydroresveratrol or stilbene compound I 24 hours to VIII treatment. The control group was not treated with Resv or any stilbene drugs.

Figure 10 shows Western blot probing of α-SMA and fibronectin (FN1) in LTC-14 cells pre-incubated with TGF-β (5 ng/mL) and probed with trans-resveratrol or dihydro-resveratrol Treat at indicated concentrations.

Figure 11 shows the green fluorescent signal of fibril α-SMA in LTC-14 cells (identified by arrow marks), indicating the extent of PSC activation.

Figure 12 shows that in mice with bombesin (Cer)-induced chronic pancreatitis, there was no treatment with dihydro-resveratrol (D-Res, 20mg/kg/day) or trans-resveratrol ( Res, 20 mg/kg/day), the fluorescent signal of fibronectin (FN1) deposition in pancreatic tissue sections was used to estimate the degree of fibrosis.

Figure 13 shows the glucose levels in normal mice (control group) and mice with chronic pancreatitis (Cer) treated with or without dihydroresveratrol (D-Res, 20 mg/kg/day) in the intraperitoneal glucose tolerance test reaction. At all time points, there were significant differences between the Cer group and the Cer+D-Res group (p<0.05).

Figure 14 shows the fluorescence signal of insulin in pancreatic tissue sections for the assessment of the pancreatic insulin secreting cell (ie beta cell) region. The therapeutic effects of the control group, the chronic pancreatitis group and the chronic pancreatitis group at the dose of 20 mg/kg/day dihydroresveratrol (D-Res) were compared.

Figure 15 shows the inhibitory effects of dihydro-resveratrol (D-Res), trans-resveratrol (Res) and compounds DR1 to DR11 on melanin content in B16 melanocytes. Ascorbic acid (AC) and BHA served as positive controls. Data are presented as mean ± SEM (n=3). *p<0.05 and **p<0.01 compared to DMSO-treated cells.

Figure 16 shows the inhibitory effects of dihydro-resveratrol (D-Res), trans-resveratrol (Res) and compounds DR1 to DR11 on melanin content in A375 melanocytes. Ascorbic acid (AC) and BHA served as positive controls. Data are presented as mean ± SEM (n=3). *p<0.05 and **p<0.01 compared to DMSO-treated cells.

Figure 17 shows Dendrobium nobile extract (JCSH, 50 μg/mL), Dendrobium officinale extract (TPSH, 50 μg/mL), trans-resveratrol (Res, 25 μM) and dihydroresveratrol (D- Res, 25μM) inhibits melanin content in B16 melanocytes. Ascorbic acid (AC) was used as a positive control. Data are presented as mean ± SEM (n=3). *p<0.05 and **p<0.01 compared to DMSO-treated cells.

Figure 18 shows the inhibitory effect of dihydroresveratrol (D-Res), trans-resveratrol (Res) and compounds DR1 to DR11 on cellular tyrosinase activity in B16 melanocytes (25 μM). Ascorbic acid (AC) and BHA served as positive controls. Data are presented as mean ± SEM (n=3). *p<0.05 and **p<0.01 compared to DMSO-treated cells.

Figure 19 shows the inhibitory effect (25 μM) of dihydroresveratrol (D-Res), trans-resveratrol (Res) and compounds DR1 to DR11 on cellular tyrosinase activity in A375 melanocytes. Ascorbic acid (AC) and BHA served as positive controls. Data are presented as mean ± SEM (n=3). *p<0.05 and **p<0.01 compared to DMSO-treated cells.

Figure 20 shows Dendrobium nobile extract (JCSH, 50 μg/mL), Dendrobium officinale extract (TPSH, 50 μg/mL), trans-resveratrol (Res, 25 μM) and dihydroresveratrol (D- Res, 25 μM) inhibits cellular tyrosinase activity in B16 melanocytes. Ascorbic acid (AC) was used as a positive control. Data are presented as mean ± SEM (n=3). *p<0.05 and **p<0.01 compared to DMSO-treated cells.

Figure 21 shows Dendrobium nobile extract (JCSH, L: 10; M: 25; H: 50 μg/mL) and Dendrobium officinale extract (TPSH, L: 10; M: 25; H: 50 μg/mL), two Hydrogen-resveratrol (D-Res, L:25; H:50μM), trans-resveratrol (Res, L:25; H:50μM) and ascorbic acid (AC, H:50μg/mL) on B16 melanin Inhibition of TRP-1, TRP-2, Phospho-AKT and Phospho-P38 protein levels in cells. GAPDH was used as a loading reference.

Figure 22 shows Dendrobium nobile extract (JCSH, high: 50 μg/mL; low: 25 μg/mL), Dendrobium officinale extract (TPSH, high: 50 μg/mL; low: 25 μg/mL), trans-resveratrol Inhibitory effects of alcohol (Res, high: 50 μM; low: 25 μM), dihydroresveratrol (d-R, high: 50 μM; low: 25 μM) and ascorbic acid (AC, 50 μM) on ROS generation in B16 melanocytes. Fluorescent signal was measured at Ex488nm. Data are presented as mean ± SEM (n=3). *p<0.05 compared to TBHP treatment alone (ie no Dendrobium extract or compound).

Figure 23 shows the whitening effect of stilbene solutions containing 2% trans-resveratrol (Res) or 2% dihydroresveratrol (D-Res) on individual arms on day 7 and day 14.

Figure 24 shows tumors obtained from tumor-bearing mice treated with vehicle solution, 5-FU or D-Res for 21 days at the end of the experiment.

Figure 25 shows tumor size in tumor-bearing mice treated with vehicle solution 5-FU or D-Res. Tumor size was monitored by measuring two perpendicular diameters with calipers and expressed as volume. Tumor volume (V) was calculated according to the equation: V=0.5*( ^LxW2 ), where L is the length and W is the width in millimeters.

Figure 26 shows tumor weights in tumor-bearing nude mice treated with vehicle solution, 5-FU or D-Res for 21 days.

Figure 27 shows the body weight of tumor-bearing nude mice treated with vehicle solution, 5-FU or D-Res for 21 days.

Figure 28 shows the inhibitory effect of D-Res on cell migration of human melanoma A375 cells and human pancreatic ductal adenocarcinoma PANC-1 cells after 24 hours of treatment. DMSO was used as an experimental control.

Figure 29 shows the percent change in denuded area in PANC-1 cells after treatment with DMSO or D-Res in a cell migration assay.

Figure 30 shows the growth inhibitory effect of dihydroresveratrol (100 μΜ) and DR8 (100 μΜ) on T. mentagroscens after one week of culture. The negative control group did not contain fungi, and the experimental control group did not contain test compounds.

Figure 31 shows the growth inhibition of S. mutans by dihydroresveratrol (100 μΜ) and DR8 (100 μΜ) after 10 hours of incubation. The negative control group did not contain bacteria, and the experimental control group did not contain test compounds.

detailed description

The present invention is not limited in scope by any particular examples described herein. The following examples are for illustration only.

definition

Hydrocarbyl

The term "hydrocarbyl" as used herein includes reference to moieties consisting solely of hydrogen and carbon atoms; such moieties may contain aliphatic and/or aromatic moieties. 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 C _1-6 alkyl groups (such as C ₁ , C ₂ , C ₃ or C ₄ alkyl groups, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl C _1-6 alkyl substituted by aryl (such as benzyl) or cycloalkyl (such as cyclopropylmethyl); cycloalkyl (such as cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl ); aryl (eg phenyl, naphthyl or cyclohexyl) and the like.

alkyl

The term "alkyl" as used herein includes reference to straight-chain or branched-chain alkyl moiety of carbon atoms. Examples of the alkyl group include "C _1-6 alkyl" and "C _2-10 alkyl". The term "C _1-6 alkyl" as used herein includes reference to straight or branched chain alkyl moieties having 1, 2, 3, 4, 5 or 6 carbon atoms. The term "C _2-10 alkyl" as used herein includes reference to straight chain or branched alkyl moieties. This term includes references to groups such as methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl or tert-butyl), pentyl, hexyl and the like. Specifically, the alkyl moiety can have 1, 2, 3, 4, 5 or 6 carbon atoms.

Alkenyl

The terms "alkenyl" and "C _2-6 alkenyl" as used herein include reference to compounds having 2, 3, 4, 5, or 6 carbon atoms and, where applicable, at least one E or Z stereochemical doublet. The straight or branched chain alkyl portion of the bond. The term includes references to compounds such as vinyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, Groups such as 1-hexenyl, 2-hexenyl and 3-hexenyl.

Alkynyl

The terms "alkynyl" and "C _alkynyl " as used herein include reference to straight or branched chain alkanes having 2, 3, 4, 5 or 6 carbon atoms and additionally having at least one triple bond. base part. This term includes references to compounds 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 groups.

Alkoxy

The terms "alkoxy" and "C _1-6 alkoxy" include references to -O-alkyl, wherein the alkyl is straight or branched, comprising 1, 2, 3, 4, 5 1 or 6 carbon atoms. In one class of embodiments, the alkoxy group has 1, 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

alkyl

The term "cycloalkyl" as used herein includes reference to alicyclic moieties having 3, 4, 5, 6, 7 or 8 carbon atoms. The group can be a bridged or multi-ring system. Typically cycloalkyl groups are monocyclic. The term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norethylene glycol, bicyclo[2.2.2]octyl, and the like.

Aryl

The term "aryl" as used herein includes reference to aromatic ring systems containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms . Aryl is usually phenyl, but can be a polycyclic system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, cyanyl, indenyl, anthracenyl, and the like.

ring group

"Ring group" means a ring or ring system which may be unsaturated or partially unsaturated but which is usually saturated, usually containing 5 to 13 ring-forming atoms, eg, a 5- or 6-membered ring. A ring or ring system may be substituted with one or more hydrocarbyl groups. Cyclic groups include carbocyclyl and heterocyclyl molecules.

carbon ring

The term "carbocyclyl" as used herein includes references to Or a saturated (eg cycloalkyl) or unsaturated (eg aryl) ring moiety of 16 carbon ring atoms. In particular, carbocyclyl includes a 3-10 membered ring or ring system, especially a 5-6 membered ring, which may be saturated or unsaturated. A ring or ring system may be substituted with one or more hydrocarbyl groups. The carbocyclic moiety is for example selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo[2.2.2]octyl, phenyl, naphthyl, fluorenyl, aztenyl, indene base, anthracenyl, etc.

Heterocyclyl

The term "heterocyclyl" as used herein includes references to or a saturated (eg heterocycloalkyl) or unsaturated (eg heteroaryl) heterocyclic moiety of 16 ring atoms, wherein at least one ring atom is selected from nitrogen, oxygen, phosphorus, silicon and sulfur. Specifically, heterocyclyl includes 3 to 10 membered rings or ring systems, more particularly 5 or 6 membered rings, which may be saturated or unsaturated. A ring or ring system may be substituted with one or more hydrocarbyl groups.

The heterocyclic moiety is, for example, selected from oxiranyl, azido, 1,2-oxazolylsulfanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thienyl, Isobenzofuryl, furyl benzyl, chromenyl, 2H pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizinyl, imidazolyl, imidazolyl, benzimidazolyl, pyrazolyl, Pyrazinyl, pyrrole imidazolinyl, thiazolyl, isothiazolyl, bithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidinyl, piperazinyl, pyridazinyl , morpholinyl, thiomorpholinyl, especially thiomorpholinyl, indolyl, isoindolyl, 3H indolyl, indolyl, benzimidazolyl, heteroaryl, indolyl, triazole Base, tetrazolyl, purinyl, 4/V-quinolyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl Base, benzofuryl, dibenzofuryl, benzothienyl, dibenzothienyl, phthalazinyl, cycloalkyl, quinoxalinyl, quinazolinyl, quinazolinyl, cinnamyl alcohol , pteridinyl, carbazolyl, β-carbene, phenanthridine, acridinyl, cyclopyrimidinyl, phenanthralinyl, furazepinyl, phenylalanine nazidyl, phenothiazinyl, Phenoxazinyl, chromenyl, isotryptamine, tryptamine, etc.

Heterocycloalkyl

The term "heterocycloalkyl" as used herein includes reference to ring carbon atoms having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 3, A saturated heterocyclic moiety of 4 or 5 ring heteroatoms. This group may be a polycyclic system, but more often it is a monocyclic system. The term includes references to compounds such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolidinyl, indolyl, piperazinyl, thiazolidinyl, Morpholinyl, thiomorpholinyl, quinolinyl and other groups. A ring or ring system may be substituted with one or more hydrocarbyl groups.

Heteroaryl

The term "heteroaryl" as used herein includes reference to any group having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms. Aromatic heterocyclic ring systems in which at least one ring atom is selected from nitrogen, oxygen and sulfur. The group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but more usually monocyclic. A ring or ring system may be substituted with one or more hydrocarbyl groups. The term includes references such as pyrimidinyl, furyl, benzo[b]thiophenyl, thiphenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridyl, benzo[b]furyl, pyrazinyl, Purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, Isoindolyl, indazolyl, purinyl, isoquinyl and other groups. o-formyl, quinazolinyl, pteridinyl, etc.

halogen

The term "halogen" as used herein includes reference to compounds having F, Cl, Br or I.

Halogenated part

The expression "halogen-containing moiety" as used herein includes reference to a moiety comprising 1 to 30 polyvalent atoms selected from carbon, nitrogen, oxygen and sulfur, which moiety includes at least one halogen. The moiety may be hydrocarbyl (eg C _1-6 alkyl or C _1-6 alkoxy) or carbocyclyl (eg aryl).

replaced

The term "substituted" as used herein in reference to a moiety means that one or more hydrogen atoms (especially up to 5, more especially 1, 2 or 3) of said moiety are replaced by said substituent The corresponding quantities of independently replace each other. The term "optionally substituted" as used herein means substituted or unsubstituted. Of course, it is understood that substituents are only at their chemically possible positions, and one skilled in the art will be able to determine without undue effort (experimentally or theoretically) whether a particular substitution is possible.

Enantiomers

The term "enantiomer" as used herein means one of two stereoisomers that are mirror images of each other.

Racemate

The term "racemate" as used herein means a mixture of equal amounts of enantiomers of a chiral molecule.

diastereoisomers

The term "diastereoisomer" as used herein means one of a class of stereoisomers that are not enantiomers, but have different configurations at one or more equivalent chiral centers. Examples of diastereomers are epimers that differ in the configuration of only one chiral center.

Prodrug

A prodrug is a drug administered as an inactive (or less than fully active) chemical derivative that is subsequently converted to the active drug in the body, usually by 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 those moieties may be the same or different. Thus, the identity of each part is independent of the identity of one or more other parts. .

Examples of the present invention are described below. The preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. Furthermore, it should be understood that the features specified in each embodiment can be combined with other specified features to provide other embodiments.

Description of the previously claimed invention

In several established animal models, repetitive intraperitoneal injections of the cholecystokinin-secreting drug, bombesin, are the most widely used and highly reproducible method for experimental acute pancreatitis. Followed by a single injection of lipopolysaccharide (LPS), lung injury, characterized by neutrophil retention in lung tissue and increased permeability of the alveolar membrane barrier, is often considered a complication associated with acute pancreatitis. To diagnose the onset of acute pancreatitis, a large amount of digestive enzyme, ie, α-amylase, leaked into the blood as the main pathological parameter. To assess the severity of acute pancreatitis and associated lung injury, morphological changes in organ architecture including interstitial edema, cellular damage, leukocyte infiltration, and hemorrhage were described as histological and/or pathological parameters. In addition to histological examination, abnormal MPO activity is often used to assess the severity of a neutrophil-mediated inflammatory state. Local and systemic inflammatory responses were further evidenced by high levels of pro-inflammatory cytokines in homogenates of affected tissues. Furthermore, glutathione depletion, a defense mechanism, is one of the most common parameters for assessing the severity of tissue damage.

The subject to be treated by the methods of the present invention can be a human or an animal. The previously claimed invention is applicable to all forms of acute pancreatitis and in particular to systemic complications associated with acute pancreatitis, including lung injury.

Dihydroresveratrol, also known as trans-3,5,4'-trihydroxydibenzoyl, is a polyphenol derivative belonging to the stilbene family, usually obtained from plant extracts. In fact, dihydroresveratrol is a plant defensin produced by various plant species such as orchidaceae and cannabis against abiotic and biotic challenges, especially in the context of fungal infections, as described by Fritzemeier, K.H., Kindl, H reported in 1983 (9,10-dihydrophenanthrenes asphytoalexins of Orchidaceae.Biosynthetic studies in vitro and in vivo proving the route from L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene and dihydrophenanthrenes ("9,10-dihydrophenanthrenes asphytoalexins of Orchidaceae Plant defensins of the family Plant. In vitro and in vivo biosynthetic studies demonstrate pathways from L-phenylalanine to dihydro-m-coumaric acid, dihydrostilbene and dihydrophenanthrene"). Eur JBiochem 133, 545-550 ).

The previously claimed invention relates to the use of polyphenol derivatives of the stilbene family having the formula 1:

Wherein, R ₁ , R ₂ and R ₃ are each independently selected from alkyl groups. The term "alkyl" alone or in combination with other groups includes reference to straight chain alkyl moieties having 1, 2, 3, 4, 5 or 6 carbon atoms. The term is further exemplified by groups such as methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl and tert-butyl), pentyl, hexyl, etc., to Improves tissue damage in the pancreas and lungs.

The previously claimed invention also relates to the use of stilbene compounds containing trans-3,5,4'-trihydroxydibenzoyl (also known as dihydroresveratrol), see compound 2:

Improve pancreas and lung tissue damage. In the previously claimed invention, this particular stilbene derivative was obtained in the form of a white powder by hydrogenation of trans-resveratrol.

Furthermore, the previously claimed invention relates to a process for the manufacture of the above-mentioned compound, a pharmaceutical preparation containing the same and the use of the compound for the preparation of a pharmaceutical preparation.

Oral administration of a sufficient amount of dihydroresveratrol (dose not less than 20 mg/kg) can significantly improve the severity of acute pancreatitis and related lung injury in bombesin-treated rats. In terms of pathological parameters, rats with acute pancreatitis had reduced pancreatic water content (Fig. 1), decreased plasma α-amylase levels (Table 1), more complete acinar morphology (Fig. 2C to 2F) and a reduction in alveolar wall thickening and hemorrhage (Fig. 3C to 3E).

Table 1: Plasma alpha-amylase activity expressed in U/μl/min. P values less than 0.05 were considered statistically significant, and S.D. represents standard deviation. *p<0.05 compared with the control group; #p<0.05 compared with the bombesin group.

Bombesin-induced increased levels of neutrophil sequestration (quantified as MPO activity) were significantly inhibited in pancreatic and lung tissues by administration of dihydroresveratrol (Fig. 4A and 4B). Injection of dihydroresveratrol (Fig. 5A and 5B) significantly inhibited bombesin-induced increase in TNF-α levels in pancreas and lung.

Glutathione deficiency is a clear sign of tissue damage. The bombesin-induced decrease in glutathione levels in the pancreas was significantly restored by administration of dihydro-resveratrol ( FIG. 6A ).

In the previously patented invention, dihydro-resveratrol was completely and readily soluble in 0.5% (weight/volume, w/v) methanol, while trans-resveratrol was dissolved in 2.5% (w/v) under vigorous shaking. /v) in methanol (Table 2). Therefore, dihydro-resveratrol is at least 5 times more soluble than trans-resveratrol. Compared with trans-resveratrol, the improving effect of dihydro-resveratrol was more likely to reduce the water content caused by pancreatic edema in rats with bombesin-induced acute pancreatitis (Fig. 6B).

Table 2: Solubility of dihydro-resveratrol and trans-resveratrol in methanol.

trans-resveratrol dihydroresveratrol Required Methanol (w/v) 2.5% 0.5%

Assessment of mitochondrial metabolic rate by MTT assay determined that the cytotoxicity of dihydro-resveratrol in pancreatic acinar cells was approximately 500 μM and that of trans-resveratrol was approximately 250 μM (Figure 7). Therefore, dihydro-resveratrol is 50% less cytotoxic than trans-resveratrol.

experiment

Preparation and structure identification of dihydroresveratrol. Using trans-resveratrol as raw material, hydrogenation can give white powder C ₁₄ H ₁₄ O ₃ . A solution of trans-resveratrol (10 g, 43.8 mmol) in absolute ethanol (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 the catalyst. The filtrate was evaporated in vacuo, and the residue was chromatographed on silicone oil, eluting with petroleum ether and ethyl acetate (1:1), to give dihydroresveratrol as a white amorphous powder (9.6 g, 95% yield): H -ESIMS ([M+1]+m/z 231.1026, calcd of C ₁₄ H ₁₄ O ₃ 231.1016); ¹ H NMR (methanol-d4, 400MHz) δ 6.96 (2H, ABd, J=8.3Hz), 6.67 ( 2H,ABd,J=8.4Hz),6.13(2H,brd,J=2.2Hz),6.09(1H,brt,J=2.2Hz),2.74(2H,brdd,J=8.5,5.6),2.67(2H , brdd, J = 8.3, 5.2); ¹³ C NMR (methanol-d4, 100 MHz) 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).

Biological activity evaluation. 28-day-old Sprague-Dawley rats weighing 70 to 90 g were randomly divided into 6 groups, with 6 to 8 rats in each group. Rats were placed in an ambient temperature of 23±2°C, a relative humidity of 60% to 80%, and a 12-hour light/dark cycle. Rats were starved overnight prior to experiments but allowed free access to water. Rats with experimental acute pancreatitis were injected with bombesin (50 μg/kg) every 6 hours under the supermaximum stimulating dose, and 1 hour after the last injection of bombesin, a single dose of LPS (7.5 mg/kg) was injected. This group of rats was designated as the bombesin group. The control group was injected with 0.9% saline instead of bombesin in the same volume and at the same time interval. Treatment groups were given bombesin and oral doses of dihydroresveratrol (10, 20 or 50 mg/kg) designated as bombesin + D-Res 10 or 20 or 50 mg/kg. The therapeutic intervention was performed 30 minutes after the first bombesin injection for 3 consecutive hours. Immediately after sacrifice, the pancreas was removed, weighed, trimmed of fat, and fixed in 4% paraformaldehyde in phosphate-buffered saline overnight at 4°C. Samples were then processed, embedded in solid paraffin, sectioned and H&E stained. TNF-α levels in pancreas and lung specimens were measured using a commercial enzyme-linked immunosorbent assay kit. Tissue homogenates were subjected to biochemical analysis to assess MPO activity and glutathione content.

Functionally intact acini were isolated from pancreatic tissue by collagenase digestion and mild shearing force. Add 5% fetal bovine serum (GiBCO) and 1% penicillin streptomycin (GIBCO) to the modified Dulbecco's Eagle's medium (GIBCO), inoculate at 37°C in 5% CO2, 95% humidified air LTC-14 cells were seeded in 96-well plates at a density of 1×10 ⁴ /well, and cultured with different concentrations of dihydro-resveratrol or trans-resveratrol (dissolved in DMSO) for 24 hours. At the end of the 24 h treatment, the MTT reagent was added to the cells. After a reaction time of 3 hours, the MTT product was dissolved in DMSO and the absorbance was measured at 570 nm.

result

After bombesin induction, due to the occurrence of pancreatic edema, the ratio of pancreas weight to body weight in rats with acute pancreatitis was significantly increased by about 60% compared with the non-bombesin-induced control group. Oral administration of dihydroresveratrol at a dose of not less than 20 mg/kg significantly reduces pancreatic edema, a phenomenon reflected in a significantly lower pancreas-to-body weight ratio. Compared with trans-resveratrol, a well-recognized antioxidant, dihydro-resveratrol is more promising in reducing pancreatic edema. Dose translation according to the standard: Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited, Journal of the Federation of American Societies for Experimental Biology (FASEB J) "22(3):659-661, the comparable dose is 3.24mg/kg.

After oral administration of dihydroresveratrol, focal dilation of parenchymal interlobular septa, cytoplasmic contraction, and leukocyte infiltration were significantly reduced in rats with pancreatitis, while lung wall thickening and pulmonary tissue hemorrhage in rats with bombesin-induced acute pancreatitis were significantly improved .

To alleviate the inflammatory conditions of the pancreas and lungs, oral administration of dihydroresveratrol significantly decreased the activities of the pro-inflammatory cytokines TNF-α and MPO in the pancreas and lung tissues.

Compared with the control group untreated with bombesin, the content of glutathione in pancreatic tissue induced by bombesin decreased sharply by more than 50%. Oral administration of dihydroresveratrol significantly inhibits bombesin-induced depletion of pancreatic glutathione.

The solubility of dihydro-resveratrol in methanol solvent is at least 5 times that of trans-resveratrol. By evaluating the metabolic rate of acinar mitochondria, it was found that the cytotoxicity of dihydro-resveratrol was about 500 μM, while that of trans-resveratrol was about 250 μM. Therefore, the cytotoxicity of dihydro-resveratrol was higher than that of 50% lower in trans-resveratrol.

In a first embodiment of the first aspect of the previously claimed invention, there is provided a method of treating acute inflammatory states of the pancreas and associated systemic complications by administering to an individual in need thereof a drug comprising an effective amount of a composition of stilbene derivatives, said compound comprising a compound of formula (I),

Wherein, R ₁ , R ₂ and R ₃ are each independently selected from alkyl groups. The term "alkyl" alone or in combination with other groups includes reference to straight chain alkyl moieties having 1, 2, 3, 4, 5 or 6 carbon atoms. The term is further exemplified by methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl and tert-butyl), pentyl, hexyl, etc. and their derivatives or chemical variants; or mixtures of said compounds, derivatives and/or chemical variants thereof.

In a second embodiment of the first aspect of the previously claimed invention, there is provided a method of treating acute inflammatory states of the pancreas and associated systemic complications wherein the stilbene derivative is trans-3,5, 4'-trihydroxydibenzoyl or dihydroresveratrol, which is a compound of formula (2):

and derivatives or chemical variants thereof; or mixtures of said compounds, derivatives and/or chemical variants thereof.

In a third embodiment of the first aspect of the previously claimed invention, there is provided a method of treating an acute inflammatory state of the pancreas and associated systemic complications, wherein the individual is a human or an animal.

In a fourth embodiment of the first aspect of the previously claimed invention, there is provided a method of treating acute inflammatory states of the pancreas and associated systemic complications, wherein said composition is administered orally.

In a fifth embodiment of the first aspect of the previously claimed invention there is provided a method of treating acute inflammatory states of the pancreas including all forms of acute pancreatitis and associated systemic complications, Associated systemic complications include lung injury.

In a sixth embodiment of the first aspect of the previously claimed invention, there is provided a method of treating acute inflammatory states of the pancreas and associated systemic complications, wherein said composition is administered at a dose of not less than 20 mg/kg The dose is administered to the subject no less than 3 times per day.

In a seventh embodiment of the first aspect of the previously claimed invention, there is provided a method of treating acute inflammatory states of the pancreas and associated systemic complications, wherein said composition is present in an amount of not less than 3.24 mg/kg The dose of is administered to the individual not less than 3 times a day.

In a first embodiment of the second aspect of the previously claimed invention there is provided a process for the preparation of compounds of formula C ₁₄ H ₁₄ O ₃ and formula (2),

It is a stilbene derivative, and its chemical name is trans-3,5,4'-trihydroxydibenzoyl after hydrogenation of trans-resveratrol.

In a second embodiment of the second aspect of the previously claimed invention, there is provided a process for the preparation of compounds of formula C ₁₄ H ₁₄ O ₃ and formula (2), wherein the hydrogenation of trans-resveratrol comprises the steps :

- Dissolve trans-resveratrol in absolute ethanol, room temperature 5atm H ₂ pressure, 10% Pd/c and stir for 8 hours;

- Filtration of the catalyst from the stirred solution;

- Evaporate the filtrate in vacuo to produce a residue;

- Chromatography on silicone oil, eluting the residue with petroleum ether and ethyl acetate (1:1), affords dihydroresveratrol.

Other Embodiments of Previously Claimed Inventions

TGF-β has been reported by some previous studies as a potent inducer of PSC activation, in which a series of fibrotic mediators including fibronectin (FN1) are upregulated. In rat immortalized PSC LTC-14 cells, exogenous addition of recombinant TGF-β (5 ng/mL) significantly increased the expression levels of fibril α-SMA and ECM protein fibronectin (FN1). In another embodiment of the previously claimed invention, the inventors found that administration of dihydroresveratrol significantly reduced the expression of α-SMA and fibronectin (FN1) in rat PSCs under challenge with TGF-β The expression level. Dihydro-resveratrol derivatives have similar inhibitory effects on PSC. The inhibitory effect of dihydro-resveratrol was more pronounced than that of trans-resveratrol, a known antioxidant. Among these examined stilbenes, dihydroresveratrol had the most potent anti-fibrotic effect in PSCs despite modest structural differences.

The previously claimed invention provides a compound for the inhibition of fibrotic mediators of stellate cells present in the internal organs of an individual in need of treatment, having the chemical formula

in,

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 form a cyclic group together with the carbon atom to which they are attached;

R ¹¹ is independently hydrogen or selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 1, 2, 3, 4 or 5 R ¹² ;

R ¹² is independently selected from halogen, trifluoromethyl, cyano, nitro, pendant oxy, -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 heterocyclic groups, any of which is optionally selected from halogen, cyano, amine through 1, 2, 3, 4 or 5 independently Substituents of radical, hydroxyl, C _1-6 alkyl and C _1-6 alkoxy;

or its enantiomers;

or a pharmaceutically acceptable salt or prodrug thereof;

Or mixtures of said compounds, derivatives and/or chemical variants thereof.

The previously claimed invention further provides nine embodiments of compounds of the formula:

Internal organs can be, for example, the individual's pancreas, liver, kidneys, and lungs. An individual may be a human individual.

experiment

In a humidified environment of 95% air and 5% carbon dioxide, LTC-14 cells were cultured at 37° C. in IMDM supplemented with 10% fetal bovine serum (FBS). Cells used in all experiments were between passage 9 and 25. LTC-14 cells were seeded in a 12-well plate at a density of 1×10 ⁵ /well and treated with 5 ng/mL recombinant TGF-β with 0, 1, 5, 10 and 20 μg/mL dihydro-resveratrol Incubate for 24 hours in IMDM supplemented with 0.2% FBS. Cells are then harvested for protein extraction and western blot analysis or immunofluorescent staining.

The total protein of LTC-14 cells was extracted with RIPA lysis buffer containing protease inhibitors. Cell lysates were loaded and separated by SDS polyacrylamide gel electrophoresis. After wet electroblotting, proteins were transferred to PVDF membranes (Bio-rad), blocked with 5% nonfat dry milk, probed with antibodies, and visualized using an ECL kit (GE Healthcare).

For immunofluorescent staining of α-SMA, LTC-14 cells were seeded onto poly-L-lysine-coated coverslips at a density of 1 × 105 in a 24-well plate and treated with ⁵ ng/ml TGF- β was incubated with 0 and 10 μg/ml dihydroresveratrol for 24 hours in IMDM supplemented with 0.2% FBS. Cells were then fixed, blocked with 3% BSA, probed with antibodies and mounted with fluorescent mounting medium containing 4',6-diamino-2-phenylindole (DAPI). Images were captured using a Nikon microscope and analyzed using SPOT advanced software.

Biological activity evaluation. C57/BL6 mice aged 28 days and weighing 20-25 g were randomly divided into 4 groups with 6-8 mice in each group. Mice were housed in an ambient temperature of 23±2°C, a relative humidity of 60% to 80%, and a 12-hour light/dark cycle. Mice were starved overnight but allowed free access to water before performing the glucose tolerance test. Experimental chronic pancreatitis in mice was induced by intravenous injection of bombesin (50 μg/kg) 4 hours a day, 3 days a week for 6 weeks, at a supramaximal stimulating dose (50 μg/kg). The control group was injected with 0.9% saline instead of bombesin in the same volume and at the same time interval. The treatment group given bombesin and an oral dose of dihydroresveratrol (20 mg/kg/day) was designated as bombesin+D-Res. Dihydroresveratrol at a dose of 50 mg/kg/day was also tried during treatment, but there was no statistically significant difference in fibrosis formation at a dose of 20 mg/kg/day. However, this high dose produced no adverse effects in in vivo trials. Therefore, it was concluded that the effective dose of dihydroresveratrol is at least 20 mg/kg/day. The group using trans-resveratrol was designated as bombesin+RES. The drug intervention of the two compounds was from the first day of the 4th week to the end of the experiment, ie a total of 3 weeks. At the end of the 6-week experiment, mice were subjected to an intraperitoneal glucose tolerance test (IPGTT). Prior to the IPGTT, mice had been starved for 14 hours with a 15% (w/v) glucose solution injected to individual animals at 1.5 g glucose per kg body weight. Approximately 1 μL of blood was obtained from the tail vein, and blood glucose levels were monitored using a glucometer (Medisign, Korea) 30 min before glucose injection (i.e., fasting level) and 10, 20, 30, and 60 min after glucose injection. Immediately after sacrifice, the pancreas was removed, weighed, trimmed of fat, and fixed in 4% paraformaldehyde in phosphate-buffered saline overnight at 4°C. Samples were then processed, embedded in solid paraffin, sectioned and immunostained.

According to the dose conversion formula, human equivalent dose (mg/kg) = animal dose (mg/kg) multiplied by animal Km/human Km, wherein the mouse Km number is 3, and the human Km number is 37 (Guidance for Industry Estimating the Maximum SafeStarting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers), the human equivalent effective dose of dihydroresveratrol of the previously claimed invention is at least 1.622mg/kg/day.

In another embodiment of the previously claimed invention, 8 other dihydroresveratrol derivatives (compounds I to VIII) and dihydroresveratrol (compound 2) are shown in Figures 8A-8I. In this example, each compound was tested as follows:

EXAMPLES OF THE COMPOUNDS IN EXPERIMENTAL Figures 8A-8I

LTC-14 cells were pre-incubated with TGF-β (5 ng/mL) and treated with trans-resveratrol (Resv), dihydroresveratrol (D-Res) or stilbene compounds i-viii at 20 μg/mL Process for 24 hours. The control group was not treated with Resv or any stilbene drugs. Total protein was extracted and analyzed by Western blotting. As shown in Figure 9.

LTC-14 cells are pancreatic stellate cells. α-SMA is an essential marker of liver fibrosis, while β-actin serves as a loading reference. Therefore, the expression level of α-SMA indicates the degree of activation of PSCs. TGF-β was added since it is considered a potent inducer of fibrosis. The inhibitory effect on α-SMA expression was tested between dihydroresveratrol and compounds i to viii relative to trans-resveratrol (Resv). All tested compounds exerted a suppressive effect on α-SMA expression.

LTC-14 cells were pre-cultured with TGF-β (5 ng/mL) and treated with trans-resveratrol or dihydro-resveratrol at the indicated concentrations. Total protein was extracted and analyzed by Western blotting. As shown in Figure 10.

Fibronectin (FN1) is a major extracellular matrix protein that is produced upon fibrosis or activation of pancreatic stellate cells. Its expression level indicates the degree of fibrosis. The inhibitory effects on fibronectin (FN1) and α-SMA levels were tested between dihydroresveratrol (ie compound 2) and trans-resveratrol.

LTC-14 cells were pre-cultured with TGF-β (5 ng/mL), and treated with 20 μg/mL dihydro-resveratrol for 24 hours before immunofluorescence staining.

LTC-14 cells are pancreatic stellate cells. α-SMA is an essential marker of liver fibrosis, while β-actin serves as a loading reference. Therefore, the green fluorescent signal of α-SMA (identified by the arrow mark in Figure 11) indicates the degree of activation of the PSC. TGF-β was added since it is considered a potent inducer of fibrosis. The inhibitory effect of dihydroresveratrol on the expression level of α-SMA was detected.

Pancreatic tissue sections were stained using an anti-fibronectin (FN1 ) antibody; thus, the immunostaining signal indicated the extent of fibronectin (FN1 ) deposition in the parenchyma. The use of dihydro-resveratrol 20mg/kg/day (bombesin + D-RES) can reduce such deposition in chronic pancreatitis, compared with trans-resveratrol (bombesin + RES ) is more significant. As shown in Figure 12.

Biological activity evaluation. C57/BL6 mice aged 28 days and weighing 20-25 g were randomly divided into 4 groups with 6-8 mice in each group. Mice were housed in an ambient temperature of 23±2°C, a relative humidity of 60% to 80%, and a 12-hour light/dark cycle. After oral administration of dihydroresveratrol (20 mg/kg/day), the fasting blood glucose level of mice with chronic pancreatitis (Cer) was significantly higher than that of the control group, indicating that these mice with chronic pancreatitis developed hyperglycemia, which is a symptom of diabetes obvious features. Importantly, after 3 weeks of dihydroresveratrol treatment (bombesin + D-Res), their impaired glucose tolerance has been significantly corrected. As a result, the hyperglycemic state of mice with chronic pancreatitis was improved. As shown in Figure 13.

When dihydroresveratrol (bombesin + D-Res) was administered orally at a dose of 20 mg/kg/day, the severity of pancreatitis and the shrinkage and destruction of islets (especially β cells) were significantly improved. As shown in Figure 14, pancreatic beta-cell regions were visualized by immunofluorescence insulin signal. A decrease in β-cell area or mass is associated with the development of glucose intolerance or diabetes. Therefore, restoration of β-cell area by dihydro-resveratrol treatment suggests that this stilbene drug is beneficial in the treatment of pancreatic-derived diabetes (ie, type 3C diabetes).

According to the dose conversion formula, human equivalent dose (mg/kg) = animal dose (mg/kg) multiplied by animal Km/human Km, wherein the mouse Km is 3, and the human Km is 37 (Guidance for Industry Estimating the Maximum SafeStarting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers) ("therapeutic agent initial clinical trial maximum safety starting dose assessment industry guideline in adult healthy volunteers"), the effective human equivalent dose of dihydroresveratrol of the present invention is at least 1.622mg /kg/day.

Another further embodiment of the previously claimed invention

Plants of the genus Dendrobium, commonly known as "Dendrobium", are widely used in the system of traditional Chinese medicine as well as folk medicine for the treatment of various diseases, such as chronic atrophic gastritis, diabetes, cardiovascular diseases, etc. Dendrobium extracts or components contain a large amount of various stilbene compounds, such as trans-resveratrol and dihydro-resveratrol, which are potential compounds against oxidative stress in humans. However, the use of these compounds for skin protection or whitening has not been disclosed.

Since oriental cosmetics prefer botanical ingredients, the previously claimed invention relates to dendrobium-derived stilbenes, in particular trans-resveratrol, dihydro-resveratrol or dihydro-resveratrol derivatives in the reduction of melanin Use in the formation, which has the potential to inhibit the generation of oxidative free radicals and active oxygen. The compositions of the present invention are applied to an individual in need thereof by topical application. The composition of the present invention is day cream, night cream, face lotion, body lotion, body oil, cuticle softening essence, facial mask, shower gel, sunscreen, sunscreen lotion, after-sun cream or after-sun lotion.

The composition of the present invention comprises one or more extracts extracted from the Dendrobium plant.

The compositions of the present invention comprise one or more stilbenes of the formula:

in

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 form a cyclic group together with the carbon atom to which it is attached;

R ¹¹ is independently hydrogen or selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 1, 2, 3, 4 or 5 R ¹² ;

R ¹² is independently selected from halogen, trifluoromethyl, cyano, nitro, pendant oxy, -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 heterocyclic groups, any of which is optionally selected from halogen, cyano, amine through 1, 2, 3, 4 or 5 independently Substituents of radical, hydroxyl, C _1-6 alkyl and C _1-6 alkoxy;

or its enantiomers;

or a pharmaceutically acceptable salt or prodrug thereof;

or mixtures, derivatives and/or chemical variants thereof.

Compositions of the present invention include stilbenes having the formula:

It is dihydroresveratrol and its derivatives or chemical variants; or a mixture of such compounds, its derivatives and/or chemical variants, or has the following formula:

The ingredients of the present invention for skin lightening and skin protection include stilbenes having the following chemical formula:

in

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 form a cyclic group together with the carbon atom to which it is attached;

R ¹¹ is independently hydrogen or selected from hydrocarbyl and heterocyclyl, any of which is optionally substituted by 1, 2, 3, 4 or 5 R ¹² ;

R ¹² is independently selected from halogen, trifluoromethyl, cyano, nitro, pendant oxy, -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 heterocyclic groups, any of which is optionally selected from halogen, cyano, amine through 1, 2, 3, 4 or 5 independently Substituents of radical, hydroxyl, C _1-6 alkyl and C _1-6 alkoxy;

or its enantiomers;

or a pharmaceutically acceptable salt or prodrug thereof;

or mixtures, derivatives and/or chemical variants thereof.

For the determination of antioxidant capacity, spectrophotometric decolorization assays were used and single-action with preformed free radicals of 2,2'-azine ratio (3-ethylbenzothiazoline-6-sulfonic acid) (also known as ABTS ) are complementary. In this test, ABTS (Abcam, USA) was dissolved in water at a concentration of 7 mM, ABTS radical cations were generated by reacting ABTS stock solution with 2.45 mM ammonium persulfate (Sigma-Aldrich, USA), and allowing the mixture to dry before use. Incubate at room temperature in the dark for 16 hours. When testing Dendrobium extract samples or stilbene samples, dilute the ABTS radical solution with ethanol to make the absorbance at 734 nm 0.70, and equilibrate at 30 °C. Dilutions of test samples (0.1 mL) or DMSO (0.1 mL) were incubated with ABTS radical solution (0.9 mL) for 15 minutes prior to absorbance at 734 nm. DMSO was a vehicle treatment, while Trolox (Abcam, USA) was a well-known vitamin E derivative ranging from 0.001 to 0.05 mg/mL as a positive drug reference. According to the Trolox standard curve, the antioxidant capacity of the test sample is expressed as the amount equivalent to Trolox, and the unit is milligram (mg).

Pre-formed ABTS free radicals were scavenged after incubation with dihydro-resveratrol (D-Res), trans-resveratrol (Res), Dendrobium extract samples, or other stilbene samples (compounds DR1 to DR11) , and normalized its antioxidant capacity to the TRolox positive standard, as shown in Table 3.

table 3. Antioxidant capacity of stilbenes equivalent to Trolox amount (mg)

compound Trolox amount (mg) standard deviation D-Res 4969.611 333.841 Res 4296.325 83.132 DR1 105.736 24.978 DR2 30.178 52.270 DR3 119.325 90.071 DR4 94.277 84.519 DR5 0 0 DR6 0 0 DR7 0 0 DR8 15.893 27.527 DR9 0 0 DR10 0 0 DR11 0 0

Cellular melanin content and tyrosinase activity were measured in cultured B16 and A375 melanocytes. In fact, melanocytes are cells that produce melanin, a group of endogenous pigments that give rise to a variety of skin colors. B16 cells (Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, China) and A375 cells (American Type Culture Collection, USA) were routinely grown in DMEM medium (Gibco, USA) supplemented with 10% heat-activated fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA), grown at 37°C in a humidified environment of 95% air and 5% carbon dioxide.

To measure cellular melanin content, melanocytes were seeded in 12-well plates (8×10 ⁴ cells/well) and stimulated with α-melanocyte stimulating hormone (α-MSH, 100 nM) for 24 hours. The purpose of this stimulation is to accelerate the cellular formation of melanin in melanocytes so that the ability of the test compound or extract to reduce melanin formation becomes more apparent. Next, cells were treated with different Dendrobium extract samples or stilbene samples (5 μL) or DMSO (5 μL, Sigma-Aldrich, USA) for 24 hours under α-MSH stimulation. DMSO is a medium treatment. After the experiment, cells were trypsinized to detach from the culture plate. After centrifugation, the melanin granules of each sample were incubated with 100 μL of 1N sodium hydroxide solution (Sigma-Aldrich, USA) at 70° C. for 1.5 hours. After cooling to room temperature, centrifuge at 15,000 x g for 10 minutes. The supernatant (100 μL) of each sample was transferred to a 96-well plate to read the absorbance. Read absorbance at 405 nm. Relative melanin content of each sample was normalized to protein content and presented as percent change compared to DMSO-treated cells.

After incubation with dihydro-resveratrol (D-Res), trans-resveratrol (Res), Dendrobium extract samples or other stilbene samples (compounds DR1 to DR11), as shown in Figures 15 to 17, Cellular melanin content has been reduced in B16 and A375 melanocytes.

To measure cellular tyrosinase activity, melanocytes were seeded in 12-well plates (8×10 ⁴ cells/well) and stimulated with α-MSH (100 nM) for 24 hours. After α-MSH stimulation, cells were treated with different test samples (5 μL) or DMSO (5 μL) for 24 hours. At the end of the experiment, the cells were washed twice with ice-cold phosphate buffered saline (PBS, pH 6.8) (Gibco, USA), and then dissolved on ice with 150 μL PBS (pH 6.8) containing 0.1% Triton X-100. Cell lysates were centrifuged at 15000 xg for 15 minutes at 4°C. A fraction of 50 μL supernatant was mixed with 50 μL L-3,4-dihydroxyphenylalanine (L-DOPA, Abcam, USA) solution (0.2%, pH6.8) in a 96-well plate at 37 Incubate in the dark for 2 hours at °C. The optical density of each sample was measured at 475 nm. Absorbance was normalized to the protein content of each sample. The relative activity of cellular tyrosinase in melanocytes was calculated and expressed as a percent change compared to DMSO-treated cells. In addition, we collected another set of experiments for Western blot analysis of the expression levels of TRP1 and TRP2, and their upstream regulators P-AKT and P-38.

After incubation with dihydro-resveratrol (D-Res), trans-resveratrol (Res), Dendrobium extract samples or other stilbene samples (compounds DR1 to DR11), as shown in Figures 18 to 20, Has inhibited tyrosinase activity in B16 and A375 melanocytes. The Western blot results are shown in FIG. 21 .

To measure ROS production in melanocytes, B16 or A375 cells were seeded in 12-well plates (8×10 ⁴ cells/well) and stimulated with α-MSH (100 nM) for 24 hours. At the end of the α-MSH culture, cells were detached from the plate and subjected to a cellular ROS assay (Abcam) according to the manufacturer's instructions. Briefly, cells were stained with 20 μM DCFDA for 30 min at 37°C. After staining, cells were treated with tert-butyl hydroperoxide (TBHP, 55 nM) to induce a marked increase in ROS before incubation with our test extract or compound or ascorbic acid (AC) in a volume of 5 μL for 4 hr. Signals generated by ROS will be detected using a fluorescent microplate reader. DCF is excited by a 488nm laser. The results of this analysis are shown in FIG. 22 .

All analyzes were performed in triplicate and repeated at least three times in individual experiments. Results are shown as mean ± standard deviation. Variance between two groups was assessed using Student's t-test, while variance between more than two groups was calculated using one-way ANOVA. A p-value of less than 0.05 was considered statistically significant.

To determine the individual's skin color, the initial skin condition of the individual's arm was tested on day 0 with a skin colorimeter MPA5. From the lightness (L*) and yellow-blue component (b*) values, the individual type angle (ITA°) was calculated with a colorimeter. The greater the ITA° value, the greater the whiteness of the skin [S.Del Bino and F.Bernerd.Variations in skin color and the biological consequences of ultraviolet radiation exposure. British Journal of Dermatology 2013; 169(Suppl.3), 33-40]. In fact, skin color is divided into 6 categories: very light, light, medium, tan, brown and black as shown in Table 4. Two areas (2 cm x 2 cm each) were selected as the treatment area, while the area around the treated area on the arm was used as the control area. 200 [mu]L of test stilbene (2% by weight in ethanol) was applied to designated areas twice a day, day and night. At the end of the first week (Day 7), individuals were tested using a skin colorimeter and data were recorded for 7 days of stilbene use. Likewise, at the end of the second week (Day 14), individuals were tested using a skin colorimeter to record data on 14 days of stilbene use. The ITA° readings are shown in Table 5. Dihydro-resveratrol or trans-resveratrol has obvious whitening effect. Figure 23 shows a representative set of images taken from a person using local processing.

Table 4. ITA-based skin classification

Individual Type Angle (ITA°) skin classification ITA°>55° very light 41°<ITA°<55° light 28°<ITA°<41° medium 10°<ITA°<28° Brown -30°<ITA°<10° brown ITA°<-30° black

table 5. ITA° measurements in individual human subjects at indicated time points

General synthetic pathway of DR1～DR3

To a mixture of dihydroresveratrol (0.2 mmol) and bromoalkane (RBr) (1.2 mmol) in dimethylformamide (DMF, ₂ mL) was added K2CO3 ₍ 1.2 mmol). 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), washed three times with dichloromethane (DCM, 10 mL). The combined organic layers were washed twice with saturated sodium chloride (NaCl), dried _over anhydrous _Na2SO4 , concentrated in vacuo, and purified by prep-TLC (PE/EA = 5/1 or 3/1) , to obtain the desired compound.

DR1:

High Resolution Mass Spectrum (HRMS): 511.1997[M+Na] ⁺

Proton NMR ( ¹ H NMR, 400MHz, CDCl ₃ ) δ1.31(9H,t,J=7.1Hz),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:

High resolution mass spectrum (HRMS): 373.1770[M+Na]+.

¹ H NMR (400MHz, CDCl ₃ ) δ2.84 (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:

High resolution mass spectrum (HRMS): 379.2220[M+Na] ⁺ .

¹ H NMR (400MHz, CDCl ₃ )δ0.98-1.08(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 pathway of DR4～DR11

To a mixture of dihydroresveratrol (0.2 mmol) and acid chloride (RCOCl) (1.2 mmol) in DCM (2 mL) was added _ET3N (1.2 mmol) at 0 °C. The resulting mixture was warmed to room temperature and stirred until the starting material disappeared on TLC. The mixture was diluted with H ₂ O (10 mL), washed three times with DCM (10 mL). The combined organic layers were washed twice with saturated NaCl, washed over anhydrous Na ₂ SO ₄ Drying, concentration in vacuo, and purification by preparative TLC (PE/EA = 5/1 or 3/1) afforded the desired compound.

DR4:

High resolution mass spectrum (HRMS): 463.2044[M+Na ⁺ ].

¹ H NMR (400MHz, CDCl ₃ )δδ0.97-1.10(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:

High resolution mass spectrum (HRMS): 667.0427and 669.0408[M+Na ⁺ ].

¹ H NMR (400MHz, CDCl ₃ ) δ3.01 (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:

HRMS:655.1964[M+Na ⁺ ].

¹ H NMR (400 MHz, CDCl ₃ ) δ3.00 (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:

HRMS:565.1600[M+Na]+.

¹ H NMR (400 MHz, CDCl ₃ ) δ3.02 (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:

HRMS: 415.1140[M+Na]+

¹ H NMR (400 MHz, CDCl3) δ2.94 (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:

HRMS:514.2550[M+Na] ⁺ .

¹ H NMR (400 MHz, CDCl3) δ1.61-1.79 (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:

HRMS:607.2070[M+Na]+.

¹ H NMR (400 MHz, CDCl ₃ ) δ2.91 (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:

HRMS:505.2550[M+Na] ⁺ .

¹ H NMR (400MHz, CDCl ₃ ) 1.34-1.38 (27H, m), 2.86-2.94 (4H, m), 6.77 (3H, d, J=2.1Hz), 6.93-7.02 (2H, m), 7.14- 7.21(2H,m)

Another embodiment of the previously claimed invention

Dihydro-resveratrol inhibited the growth of xenograft tumors in terms of weight and volume, and showed no adverse side effects in experimental animals. Despite the remarkable structural similarity of dihydro-resveratrol to trans-resveratrol, no further metabolic breakdown occurs in the human gut and exists as colonic metabolites after microbial transformation. Therefore, dihydroresveratrol may serve as an alternative to trans-resveratrol in microbiologically restricted patients with no response.

experimental animals

All animal studies were approved and performed in accordance with the Animal Care and Use Guidelines of the Animal Ethics Committee of the Hong Kong Baptist University. BALB/c nude mice, SPF grade, male, 6-8 weeks old, were purchased from Taiwan BioLASCO. Mice were acclimatized under SPF-grade laboratory conditions for one week prior to xenograft experiments.

Cell Culture and Cell Viability Assays

Human colorectal cancer HCT-116 cell line, human pancreatic ductal adenocarcinoma PANC-1 cell line, human melanoma A375 cell line, and human embryonic kidney HEK293 cell line were purchased from the American Type Culture Collection. Cells were cultured in high glucose DMEM medium containing 10% fetal bovine serum, penicillin and streptomycin (100U) at 37°C in 95% air and 5% carbon dioxide. Dihydro-resveratrol (D-Res) was added to HCT-116, PANC-1, A375 or HEK293 cells and seeded in a 96-well plate at a density of 8000 cells per well. Adopt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) reduction test to measure the viability of test cells after 24 hours, and express ₅₀ % growth inhibition (GI50, Table 6).

Table 6: GI ₅₀ of dihydroresveratrol in various cancer cells

cell line Human cell type GI₅₀ A375 melanoma 106.59±3.37μM HCT-116 colorectal cancer 102.04±5.21μM HEK293 Embryonic kidney (used as normal control) 244.11±0.96μM PANC-1 pancreatic ductal adenocarcinoma 115.69±3.09μM

Establishment of xenograft model in nude mice

HCT-116 cells in logarithmic growth phase were collected by trypsinization method. After centrifugation and washing, cells were resuspended in serum-free DMEM medium at 3 x ¹⁰⁷ cells/mL. Nude mice were sterilized with 75% medical ethanol, and 100 μL of HCT-116 cell suspension was subcutaneously injected to the left side with a 25-G syringe. The same procedure was repeated for tumor cell injection to the right side.

animal grouping

Seven days after tumor cell injection, 24 mice with tumor volumes ranging from 32 mm ³ to 178 mm ³ were randomly divided into 3 groups, and the average initial tumor volume of mice in each group was 78.04±7.76 mm ³ (vehicle, 8 mice), 78.76±9.40 mm ³ (5-FU, 8 mice), 78.01±7.75 mm ³ (D-Res, 8 mice).

Dosing regimen and preparation of vehicle

Medium solution: 2.5% ethanol and 1:1 v/v Cremphor EL dissolved in normal saline.

5-FU solution: Dissolve 5-FU powder in 2.5% ethanol and Cremphor EL at a concentration of 5 mg/mL.

D-Res solution: Dissolve dihydro-resveratrol powder in 2.5% ethanol and Cremphor EL solution at a concentration of 20 mg/mL.

Dosing regimen

Vehicle group (n=8): 2.5% ethanol and Cremphor EL solution were injected intraperitoneally every other day for 21 days.

5-FU group (n=8): intraperitoneally inject 25 mg/kg mouse 5-FU solution every other day for 21 days.

D-Res group (n=8): intraperitoneal injection of 100 mg/kg D-Res solution every other day for 21 days. This corresponds to a dose of not less than 8.13 mg/kg of body weight.

Xenograft tumors derived from HCT-116 human colorectal cancer cells compared with 5-FU, the leading anticancer drug, in terms of tumor size (Fig. 24), tumor volume (Fig. The growth of was sufficiently inhibited by 21-day administration of D-Res. The numerical data and statistical data of the tumor volume, tumor weight and body weight of the tumor-bearing mice are shown in Tables 7, 8 and 9, respectively. Notably, 21 days of D-Res administration did not result in significant weight loss (Fig. 27). This result means that D-Res is not a toxic substance that can cause serious adverse effects in animals. In addition to in vivo experiments, the application of D-Res (25 μM) did significantly reduce the migration ability of A375 human melanoma cells and PANC-1 human pancreatic ductal adenocarcinoma cells in vitro ( FIG. 28 ). Figure 29 shows the changes in the percent denudation area of PANC-1 cells indicating cell migration ability after 24 hours of treatment with D-Res or DMSO (ie, the experimental control group). Furthermore, the GI ₅₀ values in Table 6 indicate that D-Res is selective for cancer cells.

The effective dose of D-Res is 20 mg/kg (body weight) to 100 mg/kg (body weight) per day. According to the dose conversion formula (Nair AB, Jacob S.A simple practice guide for dose conversion between animals and human (“Simple practice guide for dose conversion between animals and humans”). J Basic Clin Pharma 2016; 7:27-31) equation (2 ), the effective converted human dose of D-res of the present invention ranges from 1.62 mg/kg (body weight) to 8.13 mg/kg (body weight) per day.

Table 7 Tumor volume (mm ³ ) of HCT-116 human colorectal cancer nude mice treated with medium solution, fluorouracil (5-FU) and dihydroresveratrol (D-Res) for 21 days:

There was a statistically significant difference in tumor volume between the dihydroresveratrol group and the medium group, and a statistically significant difference in the tumor volume between the 5-FU group and the medium group, P<0.001.

Table 8 Tumor weight (g) of HCT-116 human colorectal cancer nude mice treated with medium solution, fluorouracil (5-FU) and dihydroresveratrol (D-Res) for 21 days

The difference in tumor weight between the dihydroresveratrol group and the medium group was statistically significant, while the difference in the tumor weight between the 5-FU group and the medium group was statistically significant, P<0.05.

Table 9 Body weight (g) of HCT-116 human colorectal cancer nude mice treated with vehicle solution, fluorouracil (5-FU) and dihydroresveratrol (D-Res) for 21 days.

Administration of dihydroresveratrol did not significantly reduce the body weight of experimental nude mice.

Further embodiments of the invention

Another embodiment of the present invention is to provide a method of using dihydroresveratrol or its dihydrostilbene derivatives and/or chemical variants as an antimicrobial agent in an individual.

Antimicrobial agent of the present invention also includes formula (I) compound:

wherein R independently or collectively are OC(=O)R'; R' is alkenyl.

experiment

Evaluation of antifungal activity. In a single well of a 96-well microplate, add 10 μL of the test compound to 190 μL of growth medium (Difco ^™ YM) containing Trichophyton rubrum or Physcomitrella barbadensis at a density of approximately ⁵ x 105 cFU overnight culture of bacteria. Test compounds included dihydroresveratrol and DR1 to DR11. Wells incubated with 10 μL of 0.5% DMSO only (ie, no test compound) were used as experimental controls for fungal growth, while wells incubated with media alone (ie, no fungus) were used as negative controls. In order to evaluate the efficiency of inhibiting the growth of skin fungi, the above two fungi were incubated with or without the test compound for one week at 30°C, and then the OD was read at 510 nm; trans-resveratrol was used as the reference drug. Table 10 shows the percent growth inhibition of Trichophyton mentagrophytes at different concentrations of the test compounds, while the percent growth inhibition of Trichophyton rubrum is shown in Table 11. Images of Physcomitarum mentagrota cultures were taken after treatment with dihydroresveratrol (100 μM) and DR8 (100 μM) compared to the growth of the experimental and negative control groups ( FIG. 30 ).

Evaluation of antibacterial activity. In a single well of a 96-well microplate, add 10 µL of the test compound to 190 µL of cultured growth medium (BBL ^™ brain-heart perfusion) containing deformed chains at a concentration of approximately ⁵ x 105 CFU Overnight cultures of cocci or streptococci. Test compounds included dihydroresveratrol and DR1 to DR11. Wells incubated with 10 μL of 0.5% DMSO only (ie, no test compound) were used as experimental controls for bacterial growth, while wells incubated with culture only (ie, no bacteria) were used as negative controls. To evaluate the efficiency of inhibiting the growth of cariogenic Streptococcus, both bacteria were incubated with the test compound at 30°C for 10 hours, and then the OD value was read at 600 nm; trans-resveratrol was used as the reference drug. Table 12 shows the growth inhibition rates of different concentrations of the tested compounds on Streptococcus mutans, while the growth inhibition rates on Streptococcus distalis are shown in Table 13. Images of S. mutans were taken after treatment with dihydroresveratrol (100 μM) and DR8 (100 μM) compared to the growth of the experimental and negative control groups ( FIG. 31 ).

The compound is used topically in the treatment of dermatophytosis and dental caries.

The compound is a gel, lotion, cream, emulsion, paste, solution or moist spray for the treatment of dermatophytosis.

The compound is toothpaste, oral gel, toothbrush sanitizer, mouthwash or chewing gum for the management of dental caries.

result

Table 10 shows the inhibitory effects of dihydro-resveratrol and DR1 to DR11 on the growth of Physcomitrella mentagrota after one week of culture. Among the tested compounds, DR8 had the strongest inhibitory effect on Physcomitrella mentagrophyllum.

Table 10 The inhibitory effect of different concentrations of compounds on the growth of Physcomitrella mentagrophyllum.

Table 11 shows the inhibitory effects of dihydroresveratrol and DR1 to DR11 on the growth of Trichophyton rubrum after one week of culture. Among the test compounds, DR8 showed the strongest inhibitory effect on Trichophyton rubrum.

The inhibitory effect of table 11 different concentration compounds on the growth of Trichophyton rubrum

Table 12 shows the inhibitory effects of dihydroresveratrol and DR1 to DR11 on the growth of Streptococcus mutans after 10 hours of incubation. Among the test compounds, DR8 had the strongest inhibitory effect on Streptococcus mutans.

Table 12. Inhibitory Effects of Different Concentration Compounds on the Growth of Streptococcus mutans

Table 13 shows the inhibitory effects of dihydroresveratrol and DR1 to DR11 on the growth of Streptococcus distalis after 10 hours of incubation. Among the test compounds, DR8 had the strongest inhibitory effect on Streptococcus aureus.

Table 13. The Inhibitory Effects of Different Concentration Compounds on the Growth of Streptococcus aureus

Industrial applicability

The present invention relates to antibacterial compositions comprising trans-3,5,4'-trihydroxydibenzoyl (also known as dihydroresveratrol) or its dihydrostilbene derivatives and/or chemical variants. More specifically, it relates to the use of dihydroresveratrol or its derivatives to reduce the microbial growth of Trichophyton rubrum and Trichophyton mentagrophytes, Streptococcus mutans and Streptococcus distalis. The present invention relates to the use of said composition in topical bactericidal or bacteriostatic gels, lotions, creams, emulsions, pastes, solutions or moist spray formulations for the treatment of dermatophytosis. The invention also relates to the use of said composition in toothpaste, oral gel, toothbrush sanitizer, mouthwash or chewing gum formulations for the management of dental caries.

Claims (17)

1. An antimicrobial agent comprising a compound of formula (3):

in 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 hydrogen; R ¹¹ is independently hydrogen or selected from C _1-6 alkyl, C _2-6 alkenyl, cycloalkyl having 5 to 6 carbons and phenyl, wherein phenyl is optionally substituted by one R ¹² ; R ¹² is independently selected from halogen and -OCH ₃ ; or a pharmaceutically acceptable salt or a mixture thereof. 2. The antimicrobial agent according to claim 1, wherein the compound comprises a compound of the following formula:

3. The antimicrobial agent according to claim 1, wherein said compound further comprises a compound of formula (I):

wherein R independently or collectively are OC(=O)R';R' is C _2-6 alkenyl. 4. The use of the antimicrobial agent according to claims 1-3 in the preparation of medicines for preventing, alleviating and/or treating dermatophytosis. 5. The use of the antimicrobial agent according to claims 1-3 in the preparation of medicines for preventing, alleviating and/or treating dental caries. 6. Use of the antimicrobial agent of claim 1 in the preparation of a medicament for preventing or reducing microbial growth. 7. Use according to claim 6, wherein the microorganisms comprise dermatophytes and/or acidic and acidogenic Gram-positive bacteria. 8. The use according to claim 7, wherein the dermatophyte comprises Trichophyton rubrum and Trichophyton mentagrophytes. 9. The use according to claim 7, wherein the acidic and acidogenic Gram-positive bacteria comprise Streptococcus mutans and Streptococcus farnus. 10. The use of claim 8, wherein the microorganism is found in tinea pedis of the individual administered the antimicrobial agent. 11. The use of claim 9, wherein the microorganism is found in the oral cavity of the individual administered the antimicrobial agent. 12. The use according to claim 4, wherein the antimicrobial agent is formulated as a topical fungicidal or bacteriostatic gel, lotion, cream, emulsion, paste, solution or moisturizing spray. 13. The use according to claim 5, wherein the antimicrobial agent is formulated as toothpaste, oral gel, toothbrush sanitizer, mouthwash or chewing gum. 14. The use according to claim 4, wherein the concentration of the antimicrobial agent is between 25 μM and 100 μM. 15. The use according to claim 5, wherein the concentration of the antimicrobial agent is between 25 μM and 100 μM. 16. The use according to claim 8, wherein the concentration of the antimicrobial agent is between 25 μM and 100 μM. 17. The use according to claim 9, wherein the concentration of the antimicrobial agent is between 25 μM and 100 μM.

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