Galloyl-structured phenolic acid compounds for reversing hepatic amyloidosis and alcoholic liver damage and their application

Abstract

The present application discloses a phenolic acid compound containing a galloyl structure for reversing hepatic amyloidosis and alcoholic liver damage and its application, and relates to the technical field of utilization of Phyllanthus emblica. The compound is selected from at least one of the following structural compounds 1 to 8: The five fractions Fr.A-Fr.E separated from Phyllanthus emblica as raw materials, the main component of which is gallic acid, have a significant restorative effect on the decreased viability of hepatocytes caused by LYSO-6 and alcohol, and show an increase in the survival rate of hepatocytes after alcohol stimulation and LYSO-6 stimulation.

Description

Technical Field

[0001] The present application relates to the technical field of utilization of Phyllanthus emblica, in particular to a phenolic acid compound containing a galloyl structure for reversing hepatic amyloidosis and alcoholic liver damage and application thereof. Background Art

[0002] Hepatic amyloidosis is part of systemic amyloid lesions. When amyloid substances are deposited in the liver, causing liver pathological changes and related clinical manifestations, it is called hepatic amyloidosis. Hepatic amyloidosis is a rare clinical disease. It can be the liver manifestation of systemic amyloidosis, and it can also be secondary to chronic infections, tuberculosis, rheumatoid arthritis and other diseases. Common clinical manifestations are liver enlargement, ascites, massive proteinuria, hypoproteinemia, which are easily misdiagnosed as fatty liver, Bossard syndrome, etc., and even cause long-term misdiagnosis. There is still no effective specific treatment for the disease, but early diagnosis, early intervention, and timely treatment of related complications can improve the patient's prognosis to a certain extent. Hepatic amyloidosis is mainly treated with drugs and generally cannot be cured. Related symptoms can be improved after active treatment.

[0003] Alcoholic liver damage is a common liver disease caused by long-term heavy drinking. It usually manifests as alcoholic fatty liver in the early stage. If it is not effectively treated, it may develop into alcoholic hepatitis, liver fibrosis, cirrhosis and even hepatocellular carcinoma. Due to the complexity of its pathogenesis, there is currently no specific clinical prevention and treatment drug. The main treatment methods are still alcohol abstinence, nutritional therapy, drug therapy and combined therapy. At present, the liver protection drugs sold on the market are mainly Western medicines, and the toxic side effects cannot be effectively controlled during long-term medication.

[0004] Existing documents such as CN202410386680.9 disclose that Yunnan polygonatum extract has a restorative effect on the decreased liver cell viability caused by LYSO-6 and alcohol, and Yunnan polygonatum extract has the function of reversing hepatic amyloidosis and alcoholic liver damage. However, this document only discloses relevant research on Yunnan polygonatum extract, and does not involve the performance of other plant extracts.

[0005] Phyllanthus emblica is a small deciduous tree of the Euphorbiaceae family, widely distributed in tropical and subtropical areas. PhyllanthusemblicaThe dried mature fruit of L. Phyllanthus emblica, also known as Yunnan olive, Amla, and oil emblica, has a history of nearly two thousand years of edible or medicinal use in my country. It is not only an important economic crop in southwest my country, but also an important Chinese medicine and multi-ethnic medicine resource. Modern plant chemistry and pharmacology studies have shown that Phyllanthus emblica contains bioactive substances such as phenolic acids, vitamins, amino acids, flavonoids, polysaccharides, and sterols. It has good anti-inflammatory, antioxidant, anti-tumor, anti-aging, liver protection, and immune regulation effects. It is one of the three health plants designated by the World Health Organization for promotion and cultivation worldwide, and is also included in the list of "medicine and food of the same origin". Since 1977, Phyllanthus emblica has been included in various editions of the "Chinese Pharmacopoeia" as a Tibetan medicinal material. It tastes sweet, sour, astringent, and cool in nature. It has the effects of clearing heat and cooling blood, digesting food and strengthening the stomach, and promoting fluid and relieving cough.

[0006] Existing studies on the effects of emblica extract on the liver include emblica extract (emblica extract containing 35wt% to 55wt% of a mixture of Emblicanin A and Emblicanin B, 4wt% to 15wt% of Punigluconin, 10wt% to 20wt% of long-pedunculated aristolochicine, 5wt% to 15wt% of rutin and 10wt% to 30wt% of Gallo-ellagitannoids) can increase mitochondrial activity in the liver (CN201810545518.1); the substance is a tannin type, which has a large structural molecular weight and poor human absorption;

[0007] The emblica extract was extracted by ultrasonic extraction in a water bath, and then concentrated by vacuum negative pressure, rotary evaporation and vacuum freeze drying to obtain the emblica extract, which can effectively relieve liver enlargement caused by alcohol, improve the antioxidant function of the liver, and effectively prevent and relieve liver damage caused by alcohol (CN202211624986.0), but the specific active substances were not clearly stated.

[0008] However, none of the above documents discloses that the extract of Phyllanthus emblica can exert a corresponding therapeutic effect on hepatic amyloidosis.

[0009] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0010] In response to the above technical problems, the present application provides a phenolic acid compound containing a galloyl structure and its application for reversing hepatic amyloidosis and alcoholic liver damage.

[0011] The present application provides a phenolic acid compound containing a galloyl structure for reversing hepatic amyloidosis and alcoholic liver damage, which is selected from at least one of the following structural formulas 1 to 8:

[0012]

[0013] The above compounds 1 to 8 can all improve the survival rate of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 hours, showing an excellent repair effect on such damaged hepatocytes. The numbers of the compounds are shown above.

[0014] Preferably, it is compound 5 or / and 7; compound 5 or / and 7 is used for reversing alcoholic liver damage, and exhibits a relatively superior restoring effect on the activity of alcohol-stimulated liver cells.

[0015] Preferably, the preparation method comprises the following steps:

[0016] The raw materials at least include: Phyllanthus emblica, which is subjected to cold soaking, ethanol-water hot extraction, reduced pressure concentration, and extraction, and then the obtained petroleum ether extract, ethyl acetate extract, and aqueous layer extract are chromatographed to obtain compounds 1 to 8; the ethyl acetate extract and aqueous layer extract are used to reverse hepatic amyloidosis and alcoholic liver damage.

[0017] Any operation process not described in detail can be processed by common methods in the art.

[0018] Preferably, the ethyl acetate extract is eluted by normal phase column chromatography to obtain the main fractions: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E;

[0019] Fr.D was separated by reverse phase chromatography to obtain sub-fractions: D-1, D-2, D-3, and D-4;

[0020] Fr.E was separated by reverse phase chromatography to obtain sub-fractions: E-1, E-2, E-3, E-4, E-5, E-6, E-7, E-8, and E-9. The commonly used treatment methods in each elution process were all processed according to the commonly used methods in the art;

[0021] The ethyl acetate extract and the aqueous extract had a protective and restorative effect on NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h;

[0022] The ethyl acetate extract and the aqueous layer extract both had a protective and restorative effect on the decreased liver cell viability induced by 100 mM alcohol.

[0023] Preferably, the subfraction D-3 is eluted by normal phase column chromatography to obtain subfraction D-3-1 and subfraction D-3-2;

[0024] The fraction E-2 was eluted by normal phase column chromatography to obtain subfractions: E-2-1, E-2-2, E-2-3, E-2-4, E-2-5, E-2-6, E-2-7, and E-2-8;

[0025] Subfraction D-3-2 was run on a Sephadex LH-20 column to obtain fine fractions: D-3-2-1, D-3-2-2, and D-3-2-3;

[0026] Subfraction E-2-5 was subjected to Sephadex LH-20 column to obtain fine fractions: E-2-5-1 and E-2-5-2;

[0027] Subfraction E-2-7 was eluted by normal phase column chromatography to obtain fine fractions E-2-7-1, E-2-7-2, E-2-7-3, E-2-7-4 and E-2-7-5. The commonly used treatment methods in each elution process were all carried out according to the commonly used methods in the art.

[0028] Preferably, the sub-fraction D-3-2-2 is separated by semi-preparative liquid chromatography to obtain compounds 5, 6, 7 and 8. The commonly used treatment methods in each purification process are all carried out according to the commonly used methods in the art.

[0029] Preferably, the subfraction E-2-5-2 is separated by semi-preparative liquid chromatography to obtain compound 4;

[0030] The sub-fraction E-2-7-5 was separated by semi-preparative liquid chromatography to obtain compounds 1, 2 and 3. The commonly used treatment methods in each elution process were all carried out according to the commonly used methods in the art.

[0031] Another aspect of the present application provides a use of the galloyl-structured phenolic acid compound for reversing hepatic amyloidosis and alcoholic liver damage in liver protection. The above compound can effectively protect liver cells and improve the survival rate of damaged cells.

[0032] Another aspect of the present application provides a preparation for preventing and / or treating hepatic amyloidosis, comprising at least 10-50 μg / ml of the above-mentioned phenolic acid compound containing a galloyl structure for reversing hepatic amyloidosis and alcoholic liver damage.

[0033] The preparation can be a dosage form corresponding to a drug, such as a lozenge, granule, capsule, tablet, injection, or patch. When preparing the corresponding dosage form, corresponding auxiliary materials can be added according to the requirements of the art. The preparation process is also carried out according to the existing process.

[0034] Another aspect of the present application also provides a preparation of a drug for preventing and / or treating alcoholic liver damage, comprising at least 10-50 μg / ml of the above-mentioned phenolic acid compound containing a galloyl structure for reversing hepatic amyloidosis and alcoholic liver damage.

[0035] The preparation can be a dosage form corresponding to a drug, such as a lozenge, granule, capsule, tablet, injection, or patch. When preparing the corresponding dosage form, corresponding auxiliary materials can be added according to the requirements of the art. The preparation process is also carried out according to the existing process.

[0036] The beneficial effects of this application include:

[0037] 1) The galloyl-structured phenolic acid compounds and their applications for reversing hepatic amyloidosis and alcoholic liver damage provided in this application, the five fractions Fr.A-Fr.E separated from Phyllanthus emblica as raw materials, the main components of which are gallic acid, have a significant restorative effect on the decreased viability of liver cells caused by LYSO-6 and alcohol, and show an increase in the survival rate of liver cells after alcohol stimulation and LYSO-6 stimulation. The galloyl-structured phenolic acid compounds are separated from Phyllanthus emblica, which is a rich plant resource and easy to obtain raw materials. The isolation method of this compound is simple and easy to operate, the structure is novel and unique, and it has liver-protecting activity.

[0038] 2) The present application provides phenolic acid compounds containing a galloyl structure for reversing hepatic amyloidosis and alcoholic liver damage and their applications, and the use of phenolic acid compounds containing a galloyl structure obtained by separating Phyllanthus emblica as a raw material in the preparation of liver protection drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of the process for extracting phenolic acid compounds containing galloyl structures in the embodiments provided in this application;

[0040] Figure 2 Schematic diagram of the effects of the extracts of each layer of 10 μg / ml Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h in the examples provided in this application; (Note: n≧3, ns P >0.05,*** P <0.001,** P <0.01,* P <0.05)

[0041] Figure 3 Schematic diagram of the effects of the extracts of each layer of 20 μg / ml Phyllanthus emblica extract in the examples provided in this application on the viability of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 h; (Note: n≧3, ns P>0.05,*** P <0.001,** P <0.01,* P <0.05)

[0042] Figure 4 Schematic diagram of the effects of each extraction layer product of 50 μg / ml Phyllanthus emblica extract in the examples provided in this application on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h; (Note: n≧3, ns P >0.05,*** P <0.001,** P <0.01,* P <0.05)

[0043] Figure 5 This is a schematic diagram of the effects of 10 μg / ml of each extract layer of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h in the examples provided in this application. (Note: n≧3, ns P >0.05,*** P <0.001,** P <0.01,* P <0.05)

[0044] Figure 6 This is a schematic diagram of the effects of 20 μg / ml of each extract layer of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h in the examples provided in this application. (Note: n≧3, ns P >0.05,*** P <0.001,** P <0.01,* P <0.05)

[0045] Figure 7 This is a schematic diagram of the effects of 50 μg / ml of each extract layer of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h in the examples provided in this application. (Note: n≧3, ns P >0.05,*** P <0.001,** P <0.01,* P <0.05)

[0046] Figure 8The results of the effect of the gallic acid components of Phyllanthus emblica on the viability of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 hours in the examples provided in this application; (Note: #### P<0.0001 compared with the blank group, **** P<0.0001, *** P<0.001, ** P<0.01, * P<0.05 compared with the model group, ns means no significant difference)

[0047] Fig. 9 Schematic diagram of the effect of emblica gallic acid components on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h in the examples provided in this application; (Note: #### P<0.0001 compared with the blank group, **** P<0.0001, *** P<0.001, * P<0.05 compared with the model group, ns means no significant difference) DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the protection scope of the present invention.

[0049] Example

[0050] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0051] Example 1 Preparation of Compounds 1 to 8

[0052] As Figure 1 The method for preparing compounds 1 to 8 comprises the following steps:

[0053] 1) Dried Phyllanthus emblica (2.9 kg) was crushed into 90 mesh, cold-soaked with acetone three times, each time for three days, and hot-extracted with 75% ethanol water four times, each time for three hours, and the filtrates were combined to obtain the total extract of Phyllanthus emblica;

[0054] 2) concentrating the total extract of Phyllanthus emblica under reduced pressure at 55° C. to obtain a total extract of Phyllanthus emblica (1.5 kg);

[0055] 3) The total extract of Phyllanthus emblica was uniformly suspended in distilled water (total extract of Phyllanthus emblica and distilled water = 1:1.5, V / V), and extracted with petroleum ether and ethyl acetate in turn to obtain petroleum ether extraction layer material (27.2 g) and ethyl acetate extraction layer material (144.5 g), respectively;

[0056] 4) Take the petroleum ether extract layer (Zgl-fPE), ethyl acetate extract layer (Zgl-fEA) and water extract layer (Zgl-fW) to test the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h and 100 mM alcohol for 24 h as described in 4.1 to 4.2 below, and determine that the ethyl acetate extract layer is the active site for protecting the liver;

[0057] 5) The ethyl acetate extraction layer was loaded onto a normal phase silica gel column and gradient eluted with petroleum ether-acetone (100:1-1:1, V / V), dichloromethane-methanol (5:1-1:1, V / V), and methanol (concentration 100%) to obtain five main fractions: Fr.A (18 g), Fr.B (3 g), Fr.C (6 g), Fr.D (20 g), and Fr.E (40 g);

[0058] The main fraction Fr.D was separated by reverse phase chromatography to obtain 4 sub-fractions: D-1 (8 g), D-2 (7.6 g), D-3 (3 g), and D-4 (1 g);

[0059] The main fraction Fr.E was separated by reverse phase chromatography to obtain 9 sub-fractions: E-1 (4 g), E-2 (20 g), E-3 (3.2 g), E-4 (5.6 g), E-5 (0.9 g), E-6 (2.9 g), E-7 (1.2 g), E-8 (1 g), and E-9 (1 g);

[0060] 6) The sub-fraction D-3 was eluted by normal phase column chromatography using an eluent having a volume ratio of dichloromethane:methanol:formic acid = 39:1:0.1 to obtain sub-fractions D-3-1 and D-3-2;

[0061] The subfraction E-2 was eluted by normal phase column chromatography using an eluent of dichloromethane-methanol-formic acid in a volume ratio of 35:1:0.1 to obtain subfractions E-2-1, E-2-2, E-2-3, E-2-4, E-2-5, E-2-6, E-2-7, and E-2-8;

[0062] 7) Subfraction D-3-2 was loaded onto a Sephadex LH-20 column and eluted with dichloromethane-methanol (1:1, V / V) to obtain subfractions D-3-2-1, D-3-2-2, and D-3-2-3;

[0063] Subfraction E-2-5 was loaded onto a Sephadex LH-20 column and eluted with 95% methanol to obtain fine fractions E-2-5-1 and E-2-5-2;

[0064] Subfraction E-2-7 was eluted by normal phase column chromatography using an eluent of dichloromethane-methanol-formic acid in a volume ratio of 9:1:0.1 to obtain subfractions E-2-7-1, E-2-7-2, E-2-7-3, E-2-7-4, and E-2-7-5;

[0065] 8) The sub-fraction D-3-2-2 was separated and purified by semi-preparative liquid chromatography. The chromatographic column of the semi-preparative liquid chromatography was Waters X-Select HSS T3 column: C-18, 5 μm, 250×10 mm, 5 μm; mobile phase: 9% acetonitrile, absorption wavelength: 210, 270 nm, flow rate: 3 mL / min, to obtain compounds 5 (19 mg), 6 (3.4 mg), 7 (13.8 mg) and 8 (20.7 mg);

[0066] The sub-fraction fraction E-2-5-2 was separated and purified by semi-preparative liquid chromatography. The column for semi-preparative liquid chromatography was Waters X-Select HSS T3 column: C-18, 5 μm, 250×10 mm, 5 μm; mobile phase: 13% acetonitrile, absorption wavelength: 210, 270 nm, flow rate: 3 mL / min, to obtain compound 4 (9.5 mg);

[0067] The subfraction E-2-7-5 was separated and purified by semi-preparative liquid chromatography. The column for semi-preparative liquid chromatography was Waters X-Select HSS T3 column: C-18, 5 μm, 250×10 mm, 5 μm; mobile phase: 22% methanol, absorption wavelength: 210, 270 nm, flow rate: 3 mL / min, to obtain compounds 1 (4.1 mg), 2 (5 mg) and 3 (5 mg);

[0068] Example 2 Test compounds 1 to 8

[0069] Compound 4 is a pale yellow amorphous powder, HR-ESI-MS m / z 541.0841 [M-H] － The molecular formula is C 22 H 22 O 16 The compound 1 H-NMR (CD3COCD3, 500 MHz) and 13 C-NMR (CD3COCD3, 500 MHz) data are shown in Table 1:

[0070] Table 1

[0071]

[0072] Compound 5 is a pale yellow amorphous powder, HR-ESI-MS m / z 299.0405 [M-H] － The molecular formula is C 12 H 12 O9. The compound 1 H-NMR (CD3COCD3, 500 MHz) and 13 C-NMR (CD3COCD3, 500 MHz) data are shown in Table 2:

[0073] Table 2

[0074]

[0075] Compound 6 is a pale yellow amorphous powder, HR-ESI-MS m / z 371.0624 [M-H] － The molecular formula is C 15 H 16 O 11 The compound 1 H-NMR (CD3COCD3, 500 MHz) and 13 C-NMR (CD3COCD3, 500 MHz) data are shown in Table 3:

[0076] Table 3

[0077]

[0078] Compound 7 is a pale yellow amorphous powder, HR-ESI-MS m / z 299.0415 [M-H] － The molecular formula is C 12 H 12 O9. The compound 1 H-NMR (CD3COCD3, 500 MHz) and 13 C-NMR (CD3COCD3, 500 MHz) data are shown in Table 4:

[0079] Table 4

[0080]

[0081] Compounds 1, 2, 3, and 8 were compared with the following literature and were Corilagin [1] Mallonin [2] 、1,6-Di- O -galloyl- β -D-glucose [3] ,Mucic Acid 6-Methyl Ester 2- O-Gallate [4] Since the properties of this part of the material are already known, no subsequent performance research will be conducted.

[0082] 1. Thitilertdecha N, Teerawutgulrag A, Kilburn JD, et al. Identification of majorphenolic compounds from Nephelium lappaceum L. and their antioxidant activities. Molecules , 2010, 15(3): 1453-1465.

[0083] 2. SaijoR, NonakaG, Nishioka I. Tannins and related compounds.Lxxxiv.: Isolation and characterization of five new hydrolyzable tannins from the barkof Mallotus japonicus . Chemical and Pharmaceutical Bulletin , 1989, 37(8): 2063-2070.

[0084] 3. Nonaka G, NisiokaI. Tannins and related compounds. Chemical and Pharmaceutical Bulletin , 1983, 31(5): 1652-1658.

[0085] 4. Zhang YJ, Tanaka T, Yang CR, et al. New phenolic constituents from the fruit juice of Phyllanthusemblica . Chemical and Pharmaceutical Bulletin ,2001, 49(5): 537-540.

[0086] Example 3 Determination of the restorative effect of different extracts of Phyllanthus emblica on the decreased liver cell viability caused by LYSO-6 and alcohol

[0087] (1) Preparation of LYSO (serum lysozyme LYSO-6)

[0088] The LYSO (serum lysozyme) incubation solution was placed in a constant temperature oscillator at 65°C and 50 rpm for 6 consecutive days.

[0089] (2) Cell culture

[0090] NCTC1469 mouse normal liver cell line was cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 100 U / ml penicillin, 100 g / ml streptomycin and 0.2 mg / mL G-418, and cells in the logarithmic growth phase were used for experiments.

[0091] (3) Cell seeding plate

[0092] When the cell density increased to 80-90%, the cells were subcultured and diluted with high-glucose medium (commercially available) containing 3% fetal bovine serum to a concentration of 1×10 5 The cells were inoculated at a density of 100 / well in a 96-well plate, with 5 replicate wells in each group, and allowed to adhere for 24 h.

[0093] (4) Activity screening

[0094] 4.1 Effects of different extraction layers of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h

[0095] Cells were plated and a control group and a LYSO-6 group (1×10 -5 mol / L), LYSO-6+Zgl-fEA (ethyl acetate layer of Phyllanthus emblica extract), LYSO-6+Zgl-fPE (petroleum ether layer of Phyllanthus emblica extract), LYSO-6+Zgl-fW (water layer of Phyllanthus emblica extract). The final concentrations of the products of different extraction layers of Phyllanthus emblica extract were 10 μg / ml, 20 μg / ml, and 50 μg / ml. LYSO-6 was stimulated for 24 h 2 h before administration. After 24 h, the culture medium was removed, CCK-8 reagent was added, and the cells were incubated in a 37 ℃ incubator for 2-3 h. The absorbance (OD value) at 450 nm was detected by an enzyme marker.

[0096] The results are shown in Figures 2~4It can be seen that the survival rate of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 h after treatment with 10-50 μg / ml emblica extract was lower than that of the control group but higher than that of the LYSO-6 group, indicating that the substance has a positive effect on the recovery of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 h and can promote the recovery of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 h. In addition to the Zgl-fPE segment extract.

[0097] 4.2 Effects of different extraction layers of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h

[0098] Cell plates were set up with control group, model group, 100mM alcohol + Zgl-fEA (ethyl acetate layer of Phyllanthus emblica extract), 100mM alcohol + Zgl-fPE (petroleum ether layer of Phyllanthus emblica extract), and 100mM alcohol + Zgl-fW (water layer of Phyllanthus emblica extract). The final concentrations of the products of different extraction layers of Phyllanthus emblica extract were 10 μg / ml, 20 μg / ml, and 50 μg / ml. LYSO-6 was stimulated for 24 h 2 h before administration. After 24 h, the culture medium was removed, CCK-8 reagent was added, and the cells were incubated in a 37 ℃ incubator for 2-3 h. The absorbance (OD value) at 450 nm was detected by an enzyme marker.

[0099] 4.3 Effects of compounds 1–8 on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h

[0100] The difference from 4.1 is that the products of different extraction layers of Phyllanthus emblica extract are replaced by compounds 1-8, and the rest of the operations are the same as 4.1.

[0101] 4.4 Effects of compounds 1–8 on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h

[0102] The difference from 4.2 is that the products of different extraction layers of Phyllanthus emblica extract are replaced by compounds 1-8, and the rest of the operations are the same as 4.2.

[0103] (5) Analysis of activity screening results

[0104] 5.1. Effects of different extracts of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h

[0105] The results of Experiment 4.1 are as follows Figures 2~4As shown in the data, when the concentrations were 10 μg / ml, 20 μg / ml, and 50 μg / ml, the emblica extracts Zgl-fEA (ethyl acetate layer of emblica extract) and Zgl-fW (water layer of emblica extract) had a certain restoring effect on the decreased liver cell viability caused by LYSO-6, indicating that Zgl-fEA (ethyl acetate layer of emblica extract) and Zgl-fW (water layer of emblica extract) had a protective effect on NCTC1469 liver cells stimulated with LYSO-6 for 24 h, and Zgl-fPE (petroleum ether layer of emblica extract) had no restoring effect on the decreased liver cell viability caused by 100 mM alcohol.

[0106] 5.2. Effects of different extracts of Phyllanthus emblica extract on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h

[0107] The results of Experiment 4.2 are as follows Figures 5 to 7 As shown in the data, when the concentration was 20 μg / ml and 40 μg / ml, the emblica extracts Zgl-fEA (ethyl acetate layer of emblica extract) and Zgl-fW (water layer of emblica extract) had a certain restoring effect on the decreased liver cell viability caused by 100 mM alcohol, indicating that the emblica extracts Zgl-fEA (ethyl acetate layer of emblica extract) and Zgl-fW (water layer of emblica extract) had a protective effect on NCTC1469 liver cells stimulated with 100 mM alcohol for 24 h, and Zgl-fPE (petroleum ether layer of emblica extract) had no restoring effect on the decreased liver cell viability caused by 100 mM alcohol.

[0108] 5.3. Effects of compounds 1-8 on the viability of NCTC1469 hepatocytes stimulated with LYSO-6 for 24 h

[0109] The results of Experiment 4.3 are as follows Figure 8 As shown in the results, compounds 1-8 can effectively improve the survival rate of NCTC1469 hepatocytes stimulated by LYSO-6 for 24 h. The improvement effect of these phenolic acid components with galloyl groups on NCTC1469 hepatocytes may be related to the concentration, molecular structure, molecular size, the ability of electron delocalization to form large π bonds, and the ability to be oxidized.

[0110] 5.4. Effects of compounds 1-8 on the viability of NCTC1469 hepatocytes stimulated with 100 mM alcohol for 24 h

[0111] The results of Experiment 4.4 are as follows Fig. 9As shown, compounds 1-8 have significant restorative effects on alcohol-stimulated liver cell viability, among which compounds 5 and 7 show relatively better restorative effects on alcohol-stimulated liver cell viability. In addition, compounds 5 and 7 differ in structure only in the connection site between the gallic acid nucleus and the carbon on methyl succinate. It is speculated that the different positions of the substituents may affect the activity.

[0112] The cell survival rates were higher than those in the control group.

[0113] Example 4 Tablets

[0114] Add 3 parts by mass of dextrin and 3 parts by mass of soluble starch to compound 5 (10 parts by mass), mix well, granulate, dry and granulate; add part by mass of magnesium stearate, mix well, and press into tablets. The weight of each plain tablet is 0.45 g to obtain tablets.

[0115] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a galloyl structure phenolic acid compound, characterized in that: The following steps are involved: 1) 2.9 kg of dried Phyllanthus emblica was crushed into 90 mesh, cold-soaked with acetone three times, each time for three days, and hot-extracted with 75% ethanol water four times, each time for three hours, and the filtrates were combined to obtain the total extract of Phyllanthus emblica; 2) concentrating the total extract of Phyllanthus emblica under reduced pressure at 55° C. to obtain 1.5 kg of total extract of Phyllanthus emblica; 3) The total extract of Phyllanthus emblica was uniformly suspended in distilled water, the volume ratio of the total extract of Phyllanthus emblica to distilled water was 1:1.5, and extracted with petroleum ether and ethyl acetate in sequence to obtain 27.2 g of petroleum ether extraction layer material and 144.5 g of ethyl acetate extraction layer material, respectively; 4) The ethyl acetate extraction layer was loaded onto a normal phase silica gel column and gradient eluted with petroleum ether-acetone at a volume ratio of 100:1 to 1:1, dichloromethane-methanol at a volume ratio of 5:1-1:1, and 100% methanol to obtain five main fractions: 18 g Fr.A, 3 g Fr.B, 6 g Fr.C, 20 g Fr.D, and 40 g Fr.E; The main fraction Fr.D was separated by reverse phase chromatography to obtain 4 sub-fractions: 8 g D-1, 7.6 g D-2, 3 g D-3, and 1 g D-4; The main fraction Fr.E was separated by reverse phase chromatography to obtain 9 sub-fractions: 4 g E-1, 20 g E-2, 3.2 g E-3, 5.6 g E-4, 0.9 g E-5, 2.9 g E-6, 1.2 g E-7, 1 g E-8, 1 g E-9; 5) The sub-fraction D-3 was eluted by normal phase column chromatography using an eluent having a volume ratio of dichloromethane:methanol:formic acid = 39:1:0.1 to obtain sub-fractions D-3-1 and D-3-2; The subfraction E-2 was eluted by normal phase column chromatography using an eluent of dichloromethane-methanol-formic acid in a volume ratio of 35:1:0.1 to obtain subfractions E-2-1, E-2-2, E-2-3, E-2-4, E-2-5, E-2-6, E-2-7, and E-2-8; 6) Subfraction D-3-2 was loaded onto a Sephadex LH-20 column and eluted with dichloromethane-methanol, wherein the eluent was a mixture of dichloromethane and methanol in a volume ratio of 1:1, to obtain subfractions D-3-2-1, D-3-2-2, and D-3-2-3; Subfraction E-2-5 was loaded onto a Sephadex LH-20 column and eluted with 95% methanol to obtain fine fractions E-2-5-1 and E-2-5-2; Subfraction E-2-7 was eluted by normal phase column chromatography using an eluent of dichloromethane-methanol-formic acid in a volume ratio of 9:1:0.1 to obtain subfractions E-2-7-1, E-2-7-2, E-2-7-3, E-2-7-4, and E-2-7-5; 7) The fine fraction D-3-2-2 was separated and purified by semi-preparative liquid chromatography. The chromatographic column for semi-preparative liquid chromatography was Waters X-Select HSS T3 column: C-18, 5 μm, 250×10 mm, 5 μm; Mobile phase: 9% acetonitrile, absorption wavelength: 210, 270 nm, flow rate: 3 mL / min, to obtain 19 mg of compound 5, 3.4 mg of compound 6, 13.8 mg of compound 7 and 20.7 mg of compound 8; The fine fraction E-2-5-2 was separated and purified by semi-preparative liquid chromatography. The chromatographic column for semi-preparative liquid chromatography was Waters X-Select HSS T3 column: C-18, 5 μm, 250 × 10 mm, 5 μm; Mobile phase: 13% acetonitrile, absorption wavelength: 210, 270 nm, flow rate: 3 mL / min, to obtain 9.5 mg of compound 4; The fine fraction E-2-7-5 was separated and purified by semi-preparative liquid chromatography. The chromatographic column of the semi-preparative liquid chromatography was Waters X-Select HSS T3 column: C-18, 5 μm, 250×10 mm, 5 μm; mobile phase: 22% methanol, absorption wavelength: 210, 270 nm, flow rate: 3 mL / min, to obtain 4.1 mg of compound 1, 5 mg of compound 2 and 5 mg of compound 3; The galloyl structure phenolic acid compounds are compounds 1 to 8, wherein the structural formulas of compounds 1 to 8 are: 。

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