Compound ethyl gallate extracted from Bupleurum chinense and its extraction method and application

Abstract

The present invention relates to ethyl gallate, a compound extracted from Bupleurum chinense, and an extraction method and application thereof, which can effectively solve the extraction and application problems of Bupleurum chinense. The technical solution is as follows: the chemical structural formula of the ethyl gallate is as follows: the present invention discovers and separates the compound ethyl gallate (EG) from Bupleurum chinense. The results of relevant in vivo activity experiments show that ethyl gallate can significantly improve liver damage in mice induced by lipopolysaccharide (LPS) combined with D-galactosamine (D-GalN), has obvious anti-liver damage improvement effects, can be effectively used to prepare acute liver damage drugs, and expands the medicinal value of Bupleurum chinense.

Description

1. Technical Field

[0001] The present invention relates to the field of drug extraction, in particular to ethyl gallate, a compound extracted from Bupleurum chinense, and an extraction method and application thereof. 2. Background Technology

[0002] The liver is the body's largest metabolic organ, responsible for numerous vital functions, including synthesis, detoxification, and immunity. Drug abuse, food poisoning, and alcohol intoxication can all lead to acute liver damage. Acute liver injury is a common and serious clinical condition. In its advanced stages, it manifests as acute liver failure, with a high incidence and mortality rate. Lipopolysaccharides (LPS), lipid and polysaccharide compounds found on the cell walls of Gram-negative bacteria, trigger immune cell activation and inflammatory responses, leading to cell apoptosis and necrosis. D-galactosamine is a selective hepatotoxic drug that, by competing for the production of uridine diphosphate (DDP), depletes uridine phosphate, leading to structural and functional abnormalities in hepatocytes and exacerbating the damage caused by LPS. Currently, acute liver injury is difficult to identify and treat clinically, both domestically and internationally. Antibiotics are often used, but these antibiotics are prone to resistance, are costly, and have significant side effects. Therefore, new therapies and therapeutic targets are urgently needed to better manage the symptoms and progression of acute liver injury.

[0003] Bupleurum chinense is the dried root of Gypsophila oldhamiana Mip and Gypsophila licentiana Hand-Hazz, both of the Caryophyllaceae family. It is listed in the Henan Provincial Medicinal Materials Standard and has the properties of clearing heat and cooling the blood. It is used for hepatitis, asthenia due to bone steaming, yin deficiency and chronic malaria, and infantile malnutrition. While Bupleurum chinense has a long history of folk medicine use, there are currently no reports on the extraction of ethyl gallate from this plant and its specific medicinal value. 3. Summary of the Invention

[0004] In view of the above situation, in order to solve the defects of the existing technology, the purpose of the present invention is to provide a compound ethyl gallate extracted from Bupleurum chinense and its extraction method and application, which can effectively solve the extraction and application problems of Bupleurum chinense.

[0005] The technical solution provided by the present invention is that the chemical structural formula of ethyl gallate is as follows:

[0006]

[0007] The extraction method of ethyl gallate comprises the following steps:

[0008] 1) The roots of the fine-leaved stone flower (with the above-ground residual stems) were broken into coarse segments and extracted with 70%-80% ethanol under reflux three times for 1-3 hours each time, with a solvent volume of 6-10 times. The extract was concentrated to 40-60 L by rapid centrifugation and concentrated under reduced pressure until there was no alcohol smell. Equal volumes of petroleum ether, ethyl acetate, and n-butanol were added sequentially and extracted repeatedly. After concentration under reduced pressure, the extract was transferred to an evaporating dish and dried to obtain extracts from each part;

[0009] 2) The ethyl acetate extract of the fine-leaved stone flower obtained in step 1) was purified by macroporous resin (DM-130), activated in 95% ethanol for 24 hours before use to wash away impurities in the filler, suspended in 95% ethanol, wet-packed on a column, 1BV=20L, and then rinsed with 95% ethanol. When the washed ethanol was mixed with an equal amount of water and remained turbid, the column was transitioned from 95% ethanol to 10% ethanol in sequence, the ethyl acetate extract was dissolved in 10% ethanol, and slowly added to the column bed. The lower end opening of the column was closed or reduced to allow the sample to slowly adsorb on the macroporous resin, and gradient elution was performed using 10%, 30%, 50%, 70%, and 95% ethanol solutions. Each gradient rinse was four column volumes, and each gradient eluate was collected and concentrated under reduced pressure to obtain five components Fr1-Fr5;

[0010] 3) Purify the fraction Fr2 obtained in step 2) by MCI column chromatography: wet packing, dry loading, and gradient elution using 10%, 30%, 50%, 70%, and 95% methanol in water; 1 BV = 400 mL, 1 / 2 of the fraction was collected once, resulting in a total of 41 fractions, which were combined for TLC analysis to obtain Fr2-1 to Fr2-6;

[0011] 4) Purifying the fraction Fr2-4 obtained in step 3) by ODS column chromatography: wet packing, dry loading, and isocratic elution with 72% methanol to obtain a total of four fractions Fr2-4-1 to Fr2-4-4;

[0012] 5) The fraction Fr2-4-4 obtained in step 4) was semi-preparatively purified and isocratically eluted with 38% methanol in water at a flow rate of 3 mL / min and dual-channel wavelengths of 210 nm and 254 nm to obtain ethyl gallate.

[0013] The invention discloses an application of ethyl gallate, a compound extracted from Bupleurum chinense, in preparing a medicine for acute liver injury.

[0014] The present invention discovered and separated the compound ethyl gallate (EG) from Bupleurum chinense. The results of relevant in vivo activity experiments showed that ethyl gallate can significantly improve the liver damage of mice induced by lipopolysaccharide (LPS) combined with D-galactosamine (D-GalN), has obvious anti-liver damage improvement effect, can be effectively used to prepare acute liver injury drugs, and expand the medicinal value of Bupleurum chinense. IV. Description of the Figures

[0015] Figure 1 It is the molecular structural formula of the compound ethyl gallate of the present invention.

[0016] Figure 2 1H-NMR spectrum of ethyl gallate, a compound of the present invention.

[0017] Figure 3 For the compound of the present invention, ethyl gallate 13 C-NMR spectrum.

[0018] Figure 4 The results are the detection results of EG of the present invention on the levels of ALT, AST and LDH in the blood of mice with acute liver injury induced by LPS combined with D-GalN.

[0019] Figure 5 This is the detection result of EG of the present invention on the levels of F4 / 80 and LY6G in the liver tissue of mice with acute liver injury induced by LPS combined with D-GalN.

[0020] Figure 6 This is the result of the interaction between EG and RAS in liver tissue of mice with acute liver injury induced by LPS combined with D-GalN.

[0021] Figure 7 This is the effect of EG of the present invention on RAS / Raf / ERK pathway-related proteins in liver tissue of mice with acute liver injury induced by LPS combined with D-GalN. V. Specific Implementation Methods

[0022] The specific implementation of the present invention is further described in detail below with reference to the accompanying drawings and examples.

[0023] Example 1

[0024] When the present invention is specifically implemented, the method for extracting ethyl gallate comprises the following steps:

[0025] 1) 20 kg of roots of Schizonepeta tenuifolia (with above-ground residual stems) were broken into coarse segments and extracted with 70% ethanol under reflux three times for 2 h each time, with the amount of solvent increased by 10, 8, and 6 times, respectively. The extract was concentrated to 50 L by rapid centrifugation and concentrated under reduced pressure until there was no alcohol smell. Equal volumes of petroleum ether, ethyl acetate, and n-butanol were added sequentially and extracted repeatedly. After concentration under reduced pressure, the extract was transferred to an evaporating dish and dried to obtain extracts from each part.

[0026] 2) The ethyl acetate extract of the fine-leaved stone flower obtained in step 1) is purified by macroporous resin (DM-130), activated in 95% ethanol for 24 hours before use to wash away impurities in the filler, suspended in 95% ethanol, wet-packed on a column, 1BV=20L, and then rinsed with 95% ethanol. When the washed ethanol is mixed with an equal amount of water and remains turbid, it is transitioned from 95% ethanol to 10% ethanol in sequence, the ethyl acetate extract is dissolved in 10% ethanol, slowly added to the column bed, the lower end opening of the column is closed or reduced, and the sample is slowly adsorbed on the macroporous resin. Gradient elution is performed using 10%, 30%, 50%, 70%, and 95% ethanol solutions, with each gradient washing four column volumes. The gradient eluates are collected and concentrated under reduced pressure to obtain five components Fr1-Fr5.

[0027] 3) Purify the fraction Fr2 obtained in step 2) by MCI column chromatography: wet packing, dry loading, and gradient elution using 10%, 30%, 50%, 70%, and 95% methanol in water; 1 BV = 400 mL, 1 / 2 of the fraction was collected once, resulting in a total of 41 fractions, which were combined for TLC analysis to obtain Fr2-1 to Fr2-6;

[0028] 4) Purifying the fraction Fr2-4 obtained in step 3) by ODS column chromatography: wet packing, dry loading, and isocratic elution with 72% methanol to obtain four fractions Fr2-4-1 to Fr2-4-4;

[0029] 5) The fraction Fr2-4-4 obtained in step 4) was semi-preparatively purified by isocratic elution with 38% methanol in water at a flow rate of 3 mL / min and dual-channel wavelengths of 210 nm and 254 nm to obtain ethyl gallate (tR = 19 min).

[0030] The compound extracted from the present invention is identified as a new compound ethyl gallate after determination, and the activity of the compound ethyl gallate is studied. The results show that it can significantly improve the liver damage of mice induced by lipopolysaccharide (LPS) combined with D-galactosamine (D-GalN), and has obvious anti-liver damage improvement effect. The relevant specific determination and experimental data are as follows: 1 Instruments, reagents and animals

[0031] Nuclear magnetic resonance was performed using a Bruker AVANCE III 500 NMR instrument (TMS internal standard) (Bruker), infrared spectroscopy was performed using a Nicolet is 10 Microscope Spectrometer (Thermo Scientific, USA), ultrahigh performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry was performed using an Agilent 6546 UPLC-Q-TOF-MS system (Agilent, USA), and ultraviolet spectroscopy was performed using a Shimadzu UV-2401PC apparatus, FDU-2110 freeze dryer (Shanghai Ailang Instrument Co., Ltd.), carbon dioxide incubator (Thermo Fisher Scientific Inc.), 3020 multifunctional microplate reader (Thermo Fisher Scientific Inc.), AE2000 inverted microscope (Motic Industrial Group Co., Ltd.), superconducting nuclear magnetic resonance instrument (Bruker, Switzerland, DPX-500), semi-preparative high performance liquid chromatography (Beijing Cypress Technology Co., Ltd., LC-52), fully preparative high performance liquid chromatography (Shanghai Wufeng Science Co., Ltd., LC100), rotary evaporator (EYELA Tokyo Rika, Japan, N-1000, BSA124S-CW), condensed water circulation device (EYELA Tokyo Rika, Japan, N-1111), Agilent high performance liquid chromatograph (Agilent Technologies, USA, Agilent 1260 Infinity II).

[0032] Alanine aminotransferase (ALT) (Cat. No. C009-2-1), aspartate aminotransferase (AST) (Cat. No. C010-2-1), and lactate dehydrogenase (LDH) (Cat. No. A020-2-2) were purchased from Nanjing Jiancheng Bioengineering Research Institute. D-galactosamine hydrochloride (Cat. No. G115554) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and lipopolysaccharide (Batch No. 0000081275) was purchased from SIGMA. Silybin capsules (Approval No.: National Medicine Standard H20040299) were purchased from Tianjin Tasly Saint Pharmaceutical Co., Ltd. Bupleurum chinense was collected in Tianshui County, Gansu Province in August 2022 and identified as the underground root of Gypsophila licentiana Hand.-Mazz. by Professor Chen Suiqing of Henan University of Traditional Chinese Medicine. It is preserved in the Henan Key Laboratory of Traditional Chinese Medicine Resources and Chinese Medicinal Chemistry.

[0033] Forty SPF-grade male BALB / c mice weighing 18–22 g were purchased from Zhejiang Weitonglihua Experimental Technology Co., Ltd., license number SCXK(Zhe)2024-0001. The animal husbandry and experimental procedures performed in this study were approved by the Experimental Animal Ethics Committee of Henan University of Traditional Chinese Medicine and adhered to animal protection, welfare, and ethical principles. The ethical review approval number is IACUC-202402006. Animals were housed at the Animal Experimental Center of Henan University of Traditional Chinese Medicine under a 12-h light cycle and a constant temperature of 24°C. They had free access to food and water.

[0034] 2 Structural feature identification

[0035] Ethyl gallate: white needle crystal, after nuclear magnetic resonance ( 1 H-NMR and 13 C-NMR) technology was used to compare the data of compound 3 in the literature (Yu Zhibin, Yang Guangyi, Wu Xia, et al. Study on the chemical components of Jiuxincao [J]. Natural Product Research and Development, 2007, (01): 67-69.). The two data were basically consistent, and its chemical structure was identified as:

[0036]

[0037] Table 1 NMR data assignment table of compound ethyl gallate

[0038]

[0039] 3 In vivo experimental methods

[0040] 3.1 Animal experiments

[0041] After 7 days of adaptive feeding, 40 male BALB / c mice were randomly divided into a normal group (CON), a model group (M), a positive drug silibinin group (Y, 48 mg / kg), a low-dose ethyl gallate group (EG-L, 20 mg / kg), and a high-dose ethyl gallate group (EG-H, 40 mg / kg), with 8 mice in each group. Except for the normal group, which received an intraperitoneal injection of the same dose of normal saline, the other four groups received intraperitoneal injections of 10 μg / kg lipopolysaccharide and 700 mg / kg D-galactosamine to establish the model. Six hours after modeling, samples were collected and analyzed. Samples were stored at -80°C until further use.

[0042] 3.2 Detection of mouse liver index

[0043] After modeling, each mouse was weighed, blood was collected from the eyeball, and liver tissue was removed to calculate the liver index, which was calculated as follows: liver index = liver mass (g) / body mass (g) × 100%.

[0044] 3.3 Pathological observation of mouse liver tissue

[0045] The blood stains on the surface of the liver tissue were washed with normal saline, and the same part of the liver tissue of each group of mice was taken and immersed in 4% paraformaldehyde fixative for 24 hours. The tissue was dehydrated with gradient ethanol, transparentized with xylene, embedded in paraffin, cut into 5 μm thin sections, dewaxed with xylene, eluted with gradient ethanol, and stained with HE.

[0046] 3.4 Determination of liver tissue damage indicators in mice

[0047] After blood was collected from the mouse eyeball, it was placed in a 4°C refrigerator for 2 hours and centrifuged at 3000 rpm for 10 minutes. The supernatant was taken to determine the levels of ALT, AST, and LDH. The operation steps were carried out according to the kit instructions.

[0048] 3.5 Detection of F4 / 80 and LY-6G protein content in mouse liver tissue

[0049] After dehydration, paraffin-embedded liver tissue sections were immersed in EDTA fixative and microwave-fixed for 20 minutes. After cooling, sections were washed three times with PBS for 5 minutes each. Sections were then placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 minutes. Slides were then washed three times with PBS (pH 7.4) for 5 minutes each on a decolorizing shaker. Blocking with bovine serum albumin was performed for 1 hour at room temperature. The blocking solution was gently shaken off, and primary antibodies (F4 / 80 and Ly6G) prepared in a specific ratio in PBS were added dropwise to the sections and incubated overnight at 4°C. Sections were then washed three times with PBS for 5 minutes each. Secondary antibodies (HRP-labeled) were added and covered, and the sections were incubated at room temperature for 50 minutes. DAB colorimetric solution was added dropwise to the sections, and the sections were counterstained with hematoxylin for 3 minutes. After decolorization with water to blue, the sections were rinsed with running water, and mounted with neutral gum. Images of the sections were acquired, and the absorbance of all images collected was measured to analyze the expression of F4 / 80 and Ly6G.

[0050] 3.6DIA proteomics observation of differentially expressed proteins in mouse liver tissue

[0051] All samples were removed from the frozen state and transferred to a grinding tube. An appropriate amount of protein lysis buffer (8 M urea + 1% SDS, containing protease inhibitors) was added. The samples were shaken three times using a freeze grinder for 40 seconds each time and lysed on ice for 30 minutes. During this period, vortex mixing was performed for 5-10 seconds every 5 minutes. The samples were centrifuged at 16000 g for 30 minutes at 4°C. The supernatant was collected and the BCA reagent instructions were followed. 100 μg of protein solution of each sample to be tested was taken and triethylammonium bicarbonate buffer (TEBA) was added to the tube. The supernatant was stirred for 2 minutes. The final concentration of EAB was 100 mM. Tris(2-carboxyethyl)phosphine (TCEP) was added to a final TCEP concentration of 10 mM. The mixture was reacted at 37°C for 60 min. Iodoacetamide (IAM) was added to a final IAM concentration of 40 mM. The mixture was reacted at room temperature in the dark for 40 min. The mixture was centrifuged at 10,000 g for 20 min. The precipitate was taken and the sample was fully dissolved with 100 μl of 100 mM TEAB. Trypsin was added at an enzyme:protein (m / m) ratio of 1:50 and enzymatic hydrolysis was carried out at 37°C overnight. After trypsin digestion, equal amounts of samples were collected and dried using a vacuum centrifugal concentrator to extract peptides. The peptides were re-dissolved with 0.1% TFA, desalted with HLB, and dried using a vacuum concentrator. Peptide quantification was performed using a NANODROPONE (Thermo Scientific) using UV spectrophotometry. Equal amounts of peptides were dissolved in mass spectrometry loading buffer for DIA detection and analysis. The data acquisition software was Thermo Xcalibur 4.7 (Thermo, USA). The samples separated by nanoliter high-performance liquid chromatography were subjected to DIA mass spectrometry analysis using a mass spectrometer.

[0052] 3.7 Detection of Ras / Raf / ERK pathway-related protein levels in mouse liver tissue

[0053] Mouse liver tissue the size of a mung bean was taken from the same position, and the total liver protein was extracted according to the instructions of the total protein extraction kit. The protein concentration was determined using the instructions of the BCA protein quantification kit. The protein was separated into different bands according to the relative molecular mass by SDS-PAGE. The internal reference protein and the target protein were transferred to a PVDF membrane and blocked with a blocking solution containing 5% BSA for 1.5 hours. The blocking solution was recovered and the primary antibodies Ras, Raf, p-Raf, ERK, p-ERK, MEK, and p-MEK were added respectively and incubated at room temperature for 2.5 hours. The membrane was washed with PBST 5 times, 5 minutes each time, and the secondary antibody (1:2000) was added and incubated at room temperature for 1 hour. The membrane was washed again with PBST 4 times and PBS once. ECL luminescence developer was added and detected in a gel imaging system.

[0054] 4 Experimental results

[0055] Effects of EG on liver function in mice with acute liver injury induced by LPS and D-GalN

[0056] The liver tissue of mice in the CON group was rosy and shiny, while the liver tissue of mice in the M group was more severely bleeding and dark red with increased liver coefficient. After EG intervention, these changes in liver tissue were alleviated and the liver coefficient was reduced (see Figure 4 A and Figure 4 C); Pathological histological analysis showed that mice in group M had inflammatory cell infiltration and cell nucleus shrinkage, and EG intervention could significantly improve the above situation (see Figure 4 B); When liver tissue is damaged or destroyed, transaminases in liver cells enter the blood, and the levels of ALT, AST, and LDH in the blood increase, indicating that liver disease has occurred. Therefore, the levels of ALT, AST, and LDH in serum are tested. EG intervention can significantly reduce the expression of these levels (see Figure 4 B).

[0057] Note: Figure 4 In the figure, A: gross image of liver tissue; B: HE staining of liver tissue; the black arrows indicate the lesion site; C: liver tissue coefficient; DF: serum ALT, AST, and LDH levels, respectively.

[0058] Note: Compared with the CON group, ## P<0.01, compared with group M, * P < 0.01, ** P<0.01.

[0059] Effects of 4.2EG on liver function in mice with acute liver injury induced by LPS and D-GalN

[0060] To further evaluate the effect of EG on liver tissue damage and inflammation in mice with acute liver injury, the present invention detected the levels of F4 / 80 and LY6G in the liver tissue of mice. Compared with the CON group, the levels of F4 / 80 and LY6G in the liver tissue of mice in the M group were significantly increased, while EG could significantly reduce the levels of F4 / 80 and LY6G in the liver tissue. Figure 5 .

[0061] Note: Figure 5 Middle, A: F4 / 80 and LY6G staining of liver tissue; BC: F4 / 80 and LY6G staining scores of liver tissue.

[0062] Note: Compared with the CON group, ## P<0.01, compared with group M, * P < 0.01, ** P<0.01.

[0063] Effect of 4.3EG on liver tissue proteomics in mice with acute liver injury induced by LPS and D-GalN

[0064] After the EG intervention, the sample points were significantly separated from the M group and showed a trend of callback to the CON group (see Figure 6 A); Compared with the CON group, there were 233 significantly decreased proteins and 559 significantly increased proteins in the M group. Compared with the M group, there were 63 significantly decreased proteins and 57 significantly increased proteins in the EG group. 44 proteins were increased in M ​​and could be decreased by EG, and 37 proteins were decreased in M ​​and could be increased by EG (see Figure 6 B); EG significantly reduced some of the proteins that were altered in the model (see Figure 6 C); KEGG pathway enrichment analysis was performed on these proteins that changed and could be called back by EG, including RAS pathway, hepatitis B, hepatitis C and other related pathways (see Figure 6 D); To further study the intermolecular interaction between EG and RAS, molecular docking was performed on EG and RAS, and the result was -5.9 kcal / mol, indicating that there is good binding activity between the two (see Figure 6 E).

[0065] Note: Figure 6 Middle, A: PCA diagram of each group; B: Venn diagram of differential proteins between groups; C: heat map of differential proteins between groups; D: pathway enrichment analysis of differential proteins in liver tissue of mice in each group; E: binding conformation of EG and RAS.

[0066] Effects of 4.4EG on RAS / Raf / ERK pathway-related proteins in liver tissue of mice with acute liver injury induced by LPS and D-GalN

[0067] In order to further explore the interaction between EG and RAS, in the present invention, the effect of EG on RAS activation in liver tissue of mice with acute liver injury and its downstream related effector molecules, including the expression levels of Raf, p-Raf, ERK, p-ERK, MEK, and p-MEK, was studied. The results showed that compared with the CON group, RAS in the liver tissue of mice in the M group was activated, and the levels of p-Raf / Raf, p-ERK / ERK, and p-MEK / MEK were significantly increased; while EG intervention significantly inhibited the activation of RAS in the liver tissue of mice, and the levels of p-Raf / Raf also tended to decrease, and the levels of p-ERK / ERK and p-MEK / MEK also decreased significantly (see Figure 7 AE).

[0068] Note: Figure 7 Middle, AE: Immunoblot images and quantification results of RAS, p-Raf / Raf, p-ERK / ERK, and p-MEK / MEK in liver tissues.

[0069] Note: Compared with the CON group, ## P<0.01, compared with group M, *P < 0.01, ** P<0.01.

[0070] 5 Conclusion

[0071] In summary, it can be seen that the raw materials of the present invention are rich and the extraction method is easy to operate. The compound ethyl gallate (EG) was discovered and separated from Bupleurum chinense. The results of relevant in vivo activity experiments showed that ethyl gallate can significantly improve the liver damage of mice induced by lipopolysaccharide (LPS) combined with D-galactosamine (D-GalN), and has obvious anti-liver damage improvement effect. It can be effectively used to prepare acute liver injury drugs, providing experimental basis for the pharmacological research of Bupleurum chinense and the development of drugs against acute liver injury, opening up the medicinal value of Bupleurum chinense, and has huge development and application prospects, with huge economic and social benefits.

[0072] It should be pointed out that the above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any technician familiar with this profession can make changes or modify the technical content disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention, and all of them fall within the scope of protection of the present invention.

Claims

1. A method for extracting ethyl gallate from Bupleurum chinense, characterized in that: The following steps are involved: 1) Break the roots of the fine-leaved stone flower into coarse segments and extract them with 70%-80% ethanol under reflux for three times, each time for 1-3 hours, using a 6-10-fold volume of solvent. Concentrate the extract to 40-60 L by rapid centrifugation, and concentrate under reduced pressure until there is no alcohol smell. Repeatedly extract with equal volumes of petroleum ether, ethyl acetate, and n-butanol, and concentrate under reduced pressure. After concentration, transfer the extract to an evaporating dish and dry it to obtain the extract of each part. 2) The ethyl acetate extract of the fine-leaved stone flower obtained in step 1) was purified by macroporous resin DM-130. Before use, it was activated in 95% ethanol for 24 hours to wash away impurities in the filler, suspended in 95% ethanol, wet-packed on the column, 1BV=20L, and then rinsed with 95% ethanol. When the washed ethanol was mixed with an equal amount of water and remained turbid, the column was transitioned from 95% ethanol to 10% ethanol in sequence. The ethyl acetate extract was dissolved in 10% ethanol and slowly added to the column bed. The lower end opening of the column was closed or reduced to allow the sample to slowly adsorb on the macroporous resin. Gradient elution was performed using 10%, 30%, 50%, 70%, and 95% ethanol solutions, with each gradient washing four column volumes. The gradient eluates were collected and concentrated under reduced pressure to obtain five components Fr1-Fr5; 3) Purify the fraction Fr2 obtained in step 2) by MCI column chromatography: wet packing, dry loading, and gradient elution using 10%, 30%, 50%, 70%, and 95% methanol in water; 1 BV = 400 mL, with 1 / 2 fraction collected once, yielding a total of 41 fractions, which were combined for TLC analysis to yield Fr2-1 through Fr2-6; 4) Purify the fraction Fr2-4 obtained in step 3) by ODS column chromatography: wet packing, dry loading, and isocratic elution with 72% methanol to obtain four fractions Fr2-4-1 to Fr2-4-4; 5) The component Fr2-4-4 obtained in step 4) was semi-preparatively purified using isocratic elution with 38% methanol in water at a flow rate of 3 mL / min and dual-channel wavelengths of 210 nm and 254 nm to obtain ethyl gallate.

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