Use of gallic acid derivatives for the preparation of a product for the treatment or prevention of liver diseases

Abstract

The application belongs to the technical field of biology, and discloses application of gallic acid derivatives in preparation of products for treating or preventing liver diseases, wherein the gallic acid derivatives are one or more combinations of 2-Phenylethyl 1-O-beta-D-(6'-O-galloyl)-glucopyranoside, 2,3-digalloyl-D-glucopyranose, Kaempferol 3-O-glucoside-2"-gallate and Quercetin 3-O-(6'-O-Galloyl)-beta-galactoside; the four gallic acid derivatives can better reduce AST and ALT activities in alcohol-induced HepG2 cells relative to a model group, and 2,3-digalloyl-D-glucopyranose and Quercetin 3-O-(6'-O-Galloyl)-beta-galactoside have better effects than gallic acid and positive drug Silybin (Silybin).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically the application of gallic acid derivatives in the preparation of products for the treatment or prevention of liver diseases. Background Technology

[0002] As the core metabolic and detoxification organ in the human body, liver dysfunction is a significant global health problem. The number of people suffering from liver disease is enormous, with alcoholic liver disease (ALD) being closely related to oxidative stress, inflammatory responses, lipid metabolism disorders, and gut microbiota dysbiosis. Acetaldehyde and reactive oxygen species produced during ethanol metabolism can induce hepatocyte damage and further promote the disease's progression to fatty degeneration, fibrosis, and even liver cancer through mechanisms such as disrupting the intestinal barrier, activating inflammatory pathways, and inhibiting lipid breakdown.

[0003] Gallic acid and some of its derivatives, due to their natural origin, high safety, and multi-pathway hepatoprotective activity, have become promising candidate substances for the development of liver disease treatment products. Existing technologies disclose that gallic acid and some of its derivatives have certain hepatoprotective effects. For example, existing technology CN120284948A discloses the application of gallic acid in the preparation of drugs for treating alcoholic liver disease, finding that gallic acid can significantly reduce alcohol-induced hepatocyte damage and inhibit programmed hepatocyte necrosis in both in vivo and in vitro experiments. However, gallic acid may suffer from extremely low bioavailability and poor stability, leading to unsatisfactory efficacy. Gallic acid derivatives, on the other hand, may overcome these problems effectively and exhibit better efficacy.

[0004] Golden-edged rose (Rosa chinensis cv. 'JinBian'), a unique resource used both as food and medicine, is rich in various phenolic compounds with gallic acid as the backbone. Its unique matrix may give rise to derivatives with novel structures and superior activity. Therefore, the technical problem to be solved by this invention is: how to develop gallic acid derivatives with better effects for use in the preparation and treatment or prevention of liver diseases. Summary of the Invention

[0005] The purpose of this invention is to provide the use of gallic acid derivatives in the preparation of products for the treatment or prevention of liver diseases.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The use of gallic acid derivatives in the preparation of products for the treatment or prevention of liver diseases, wherein the gallic acid derivative is one or more combinations of 2-Phenylethyl 1-O-β-D-(6'-O-galloyl)-glucopyranoside, 2,3-digalloyl-D-glucopyranose, Kaempferol 3-O-glucoside-2"-gallate, and Quercetin 3-O-(6'-O-Galloyl)-β-galactoside;

[0008] The 2-Phenylethyl 1-O-β-D-(6'-O-galloyl)-glucopyranoside has the structural formula described in Formula I;

[0009]

[0010] Formula I;

[0011] The 2,3-digalloyl-D-glucopyranose described herein has the structural formula described in Formula II;

[0012]

[0013] Formula II;

[0014] The Kaempferol 3-O-glucoside-2"-gallate described herein has the structural formula described in Formula III;

[0015]

[0016] Formula III;

[0017] The Quercetin 3-O-(6'-O-Galloyl)-β-galactoside described herein has the structural formula described in Formula IV;

[0018]

[0019] Formula IV.

[0020] Preferably, the product is a medicine for treating and / or preventing liver disease.

[0021] Preferably, the liver disease is alcoholic liver disease.

[0022] In addition, the present invention also discloses a medicament for treating and / or preventing liver disease, wherein the active ingredient of the medicament is a gallic acid derivative as described above.

[0023] Preferably, the dosage form of the drug is one of liquid, tablet, capsule, powder, or pill.

[0024] Preferably, the liver disease is alcoholic liver disease.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention discloses four gallic acids and their derivatives, 2-Phenylethyl1-O-β-D-(6'-O-galloyl)-glucopyranoside, 2,3-digalloyl-D-glucopyranose, Kaempferol3-O-glucoside-2"-gallate, and Quercetin 3-O-(6'-O-Galloyl)-β-galactoside, isolated and purified from Rosa simonii. In alcohol-induced HepG2 cells, compared to the control group, these derivatives significantly reduced intracellular AST and ALT activity, exhibiting superior hepatoprotective effects. Among them, 2,3-digalloyl-D-glucopyranose and Quercetin 3-O-(6'-O-Galloyl)-β-galactoside showed better efficacy than gallic acid and the positive control drug silybin. Attached Figure Description

[0027] Figure 1 Mass spectra of compounds 1-7;

[0028] Figure 2 For the toxicity test of compounds 1-7 on HepG2 cells;

[0029] Figure 3 The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of compounds 1–7 were measured in alcohol-induced HepG2 cells; the figure shows… ** p<0.01 indicates a comparison between the model group (EtOH) and the blank group; ## p<0.01 indicates a comparison between the sample group and the model group (EtOH). Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the instruments, reagents, and materials involved in the following embodiments are all conventional instruments, reagents, and materials already existing in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods involved in the following examples are all conventional experimental methods and detection methods already existing in the prior art.

[0031] Example 1: Isolation and purification of gallic acid derivatives

[0032] 1. After air-drying, 5 kg of rose petals from the golden-edged rose (Rosa chinensis cv. 'JinBian') collected from Anning, Yunnan Province were crushed using a pulverizer. The crushed petals were then ultrasonically extracted with a methanol aqueous solution (90%) at a ratio of 1:8 (m / v, g / mL) for 0.5 hours each time, for a total of three extractions. All extracts were collected, the solvent was removed by rotary evaporation, and the extract was freeze-dried to obtain 0.5 kg of crude extract, which was stored at 4℃.

[0033] 2. Dissolve 0.5 kg of crude extract completely in 4 L of pure water, and then extract with dichloromethane, ethyl acetate, and n-butanol in a volume ratio of 1:1 (aqueous phase: organic phase), four times each. Collect the organic phase extracts and concentrate them under reduced pressure in a rotary evaporator to obtain dichloromethane phase (46.75 g), ethyl acetate phase (160.38 g), n-butanol phase (132.54 g), and aqueous phase (151.93 g).

[0034] 3. The ethyl acetate phase rich in VM was chromatographically analyzed using a medium-pressure column loaded with RP-C18 packing material. Eluents were obtained using methanol-water solutions of different concentrations (10%~100%), with 6 column volumes eluted in each solvent system. Fractions containing the same compounds were combined by thin-layer chromatography to obtain a total of 6 fractions (JF1~JF6).

[0035] 4. Component JF2 (20 g) was separated and purified by medium-pressure preparative chromatography, using a gradient elution of 10 vol%, 30 vol%, 50 vol%, 70 vol%, and 90 vol% methanol-water solutions. The eluent was collected and rotary evaporated to obtain five components (Fr. A~E). JF2-Fr. C (8.2 g) was separated by a polyamide column and eluted with chloroform, methanol, and acetone (10:1:1, 5:1:1, and 2:1:1) to obtain compounds 1 (gallic acid, 1.5 g), 2 (methyl gallate, 35 mg), 4 (2-Phenylethyl1-O-β-D-(6'-O-galloyl)-glucopyranoside, 20 mg), and 6 (Quercetin 3-O-(6'-O-Galloyl)-β-galactoside, 113 mg). Component JF4 (10 g) was separated and purified by medium-pressure preparative chromatography, using a gradient elution of 10 vol%, 30 vol%, 50 vol%, 70 vol%, and 90 vol% methanol-water solutions. The eluent was collected and rotary evaporated to obtain five components (JF4-Fr. AE). JF4-Fr. B (2.3 g) was separated by a polyamide column and eluted with chloroform, methanol, and acetone (10:1:1, 6:1:1, and 4:1:1) to obtain compounds 3 (ethyl gallate, 95 mg), 5 (2,3-digalloyl-D-glucopyranose, 10 mg), and 7 (Kaempferol 3-O-glucoside-2"-gallat, 60 mg).

[0036] Compound 1 is gallic acid;

[0037] Compound 2 is methyl gallate;

[0038] Compound 3 is ethyl gallate;

[0039] Compound 4 is 2-Phenylethyl1-O-β-D-(6'-O-galloyl)-glucopyranoside;

[0040] Compound 5 is 2,3-digalloyl-D-glucopyranose;

[0041] Compound 6 is Quercetin 3-O-(6'-O-Galloyl)-β-galactoside;

[0042] Compound 7 is Kaempferol 3-O-glucoside-2"-gallate;

[0043] Mass spectrometry data of compounds 1-7 in negative ion mode are as follows: Figure 1 As shown in the figure; A to F correspond to compounds 1 to 7, respectively.

[0044] Example 2: Hepatoprotective effect of gallic acid and its derivatives in alcohol-induced HepG2 cells

[0045] (1) Cell culture

[0046] The experiment was conducted using the human liver-derived cell line (HepG2, purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences). The cells were cultured in DMEM medium containing 10 vol% fetal bovine serum (FBS) and 1 vol% penicillin / streptomycin (Gibco, Big Island, New York, USA) and incubated at 37°C in a 5 vol% CO2 incubator.

[0047] (2) Cytotoxicity evaluation

[0048] 200 μL of HepG2 cells were seeded into 96-well cell culture plates (2 × 10⁶ cells per well). 4 Cells were incubated at 37°C for 24 h, and then 200 μL of seven gallic acids and their derivatives (20 and 40 μM) at different concentrations were added to 96-well plates. After 24 h of incubation, the culture medium was removed, and 200 μL of MTT (0.5 mg / mL) dissolved in PBS was added to the 96-well cell culture plate. The plates were then incubated at 37°C in a 5 vol% CO2 incubator for another 4 hours. 200 μL of DMSO was added and reacted for 10 min. The absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated. The toxicity of the seven gallic acids and their derivatives (compounds 1-7) to HepG2 cells was evaluated by the MTT assay. Cell viability (%) = (A... 样品 / A 空白 ) × 100%, where A 样品 The absorbance values ​​are shown for the experimental groups with the addition of 6 gallic acids and their derivatives; Blank A is the absorbance value of the blank control group without the addition of the compounds; the results are shown in […]. Figure 2 The results in the figure show that among the seven gallic acids and their derivatives, only compound 7 exhibited toxicity to HepG2 cells at 40 μM within the concentration range of 20 and 40 μM. Therefore, 20 μM was selected for subsequent experiments.

[0049] (3) Evaluation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels

[0050] HepG2 cells in the logarithmic growth phase were seeded into 6-well cell culture plates (4 × 10⁶ cells per well). 5Cells were cultured at 37°C for 24 hours (2 mL per cell), and then compounds 1-7 (20 μM) and silybin (20 μM) were added to the culture medium. The cells were then co-incubated with 800 mM ethanol (EtOH) for another 24 hours. Cells were washed with PBS, and complete culture medium containing 800 mM ethanol was added. AST and ALT accumulation in the cells was measured using an AST and ALT assay kit.

[0051] See results Figure 3 Compounds 4-7 showed significant inhibitory effects on the accumulation of AST and ALT at 20 μM, with compound 5 showing the best effect, and its inhibitory effect on AST accumulation was better than that of compounds 1-3. In addition, compounds 5 and 6 were superior to the positive control drug silymarin, showing good inhibitory activity. Furthermore, these two gallic acid derivatives had a better protective effect against alcohol-induced HepG2 cell damage than gallic acid itself (compound 1).

[0052] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. The application of gallic acid derivatives in the preparation of products for the treatment or prevention of liver diseases, characterized in that, The gallic acid derivatives mentioned are one or more combinations of 2-Phenylethyl1-O-β-D-(6'-O-galloyl)-glucopyranoside, 2,3-digalloyl-D-glucopyranose, Kaempferol 3-O-glucoside-2"-gallate, and Quercetin 3-O-(6'-O-Galloyl)-β-galactoside; The 2-Phenylethyl 1-O-β-D-(6'-O-galloyl)-glucopyranoside has the structural formula described in Formula I; Formula I; The 2,3-digalloyl-D-glucopyranose described herein has the structural formula described in Formula II; Formula II; The Kaempferol 3-O-glucoside-2"-gallate described herein has the structural formula described in Formula III; Formula III; The Quercetin 3-O-(6'-O-Galloyl)-β-galactoside described herein has the structural formula described in Formula IV; Formula IV.

2. The application according to claim 1, characterized in that, The product described is a medicine for the treatment and / or prevention of liver disease.

3. The application according to claim 1, characterized in that, The liver disease mentioned is alcoholic liver disease.

4. A medicine for treating and / or preventing liver disease, characterized in that, The active ingredient of the drug is the gallic acid derivative as described in claim 1.

5. The drug according to claim 4, characterized in that, The dosage form of the drug is one of the following: liquid, tablet, capsule, powder, or pill.

6. The drug according to claim 4, characterized in that, The liver disease mentioned is alcoholic liver disease.

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