Application of gentianopsis paludosa or extract of gentianopsis paludosa in preparation of medicine for treating alcoholic liver injury

Abstract

The invention relates to the technical field of medicine extracts, in particular to application of gentianopsis paludosa or extracts of gentianopsis paludosa in preparation of medicine for treating alcoholic liver injury, and experimental results show that compared with a normal group, the liver index of mice in a model group is increased; compared with a model group, the LI of mice in the ethyl acetate extract group is obviously reduced; compared with a model group, all extracts of the gentianopsis paludosa can reduce the content of ALT, AST, T-CHO and TG in serum and the content of MDA in the liver to different degrees and improve the activity of SOD, and after a fuzzy matter-element model based on a weight coefficient is combined with pharmacodynamics for comprehensive evaluation, it is found that the ethyl acetate extract of the gentianopsis paludosa has the best effect of protecting the alcoholic liver injury; by combining RT-PCR and Western-Blot results, compared with a model group, the positive control group and the ethyl acetate extract group can up-regulate expression of Nrf2, NQO1 and HO-1 mRNA to different degrees; the expression of the Nrf2 protein is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical extracts, and particularly to the application of Gentianopsis paludosa or its extracts in the preparation of drugs for treating alcoholic liver injury. Background Art

[0002] Alcoholic liver disease (ALD) is the most common liver disease caused by excessive intake and misuse of alcohol, and its pathological features are early fatty degeneration to alcoholic fatty liver disease (AFLD). Clinically, ballooning degeneration of hepatocytes, neutrophil infiltration, abnormal liver function indicators, etc. can be seen. In severe cases, it can lead to liver fibrosis, cirrhosis, liver failure, and even death. ALD has become the main cause of chronic liver disease worldwide. The pathogenesis of ALD is related to oxidative stress, lipid peroxidation, inflammatory cytokines, immune disorders, etc. Ethanol-induced oxidative stress leads to an increase in the production of reactive oxygen species (ROS), resulting in a large amount of lipid peroxides being generated, which in turn destroys the antioxidant system, ultimately leading to hepatocyte necrosis and / or apoptosis.

[0003] Traditional Chinese medicine extracts are highly pure active ingredients or effective parts separated from traditional Chinese medicinal materials through modern technologies. Compared with traditional Chinese medicine decoction pieces, extracts have the advantages of clear composition, controllable dosage, and convenience for modern preparation development, and are widely used in the fields of drug research and development, health products, functional foods, etc. Gentianopsis paludosa (Munro) Ma., a Tibetan medicine, is an annual herb of the genus Gentianopsis in the Gentianaceae family, and has the effects of clearing heat and detoxifying, and strengthening the spleen and stopping diarrhea. The invention patent CN106420989A discloses a composition of Gentianopsis paludosa and Agrimonia pilosa for treating colon cancer, and the invention patent CN103536803A discloses a traditional Chinese medicine composition containing Gentianopsis paludosa for treating cataracts; the invention patent CN104288497A discloses a traditional Chinese medicine composition containing Gentianopsis paludosa for treating meningitis. However, the above studies are all based on the water extracts of Gentianopsis paludosa, and there is no research on other types of extracts.

[0004] During the research process, the inventors found that when treating alcoholic liver injury, the effects of different extracts of Gentianopsis paludosa are different. Among them, the ethyl acetate extract of Gentianopsis paludosa has the best effect, providing a new research idea for guiding the clinical application of the Tibetan medicine Gentianopsis paludosa. Summary of the Invention

[0005] The primary object of the present invention is to provide the application of Gentianopsis paludosa in the preparation of drugs for treating alcoholic liver injury.

[0006] The second object of the present invention is the application of Gentianopsis paludosa extract in the preparation of a medicament for treating alcoholic liver injury.

[0007] Preferably, the Gentianopsis paludosa extract includes one or more of an aqueous extract, a 95% ethanol extract, an ethyl acetate extract, a n-butanol extract, and a petroleum ether extract.

[0008] Preferably, the preparation method of the Gentianopsis paludosa extract is as follows: Weigh the medicinal material Gentianopsis paludosa, crush it, soak it with 5 times the amount of water, 95% ethanol, ethyl acetate, n-butanol, and petroleum ether respectively, and then reflux and extract. The extract is recovered by a rotary evaporator, and then dried under reduced pressure to obtain a dry extract, which is ground and reserved for use.

[0009] Preferably, the Gentianopsis paludosa extract is an ethyl acetate extract.

[0010] The third object of the present invention is to provide a pharmaceutical composition for treating alcoholic liver injury, which includes Gentianopsis paludosa extract and excipients.

[0011] Preferably, the pharmaceutical composition is prepared into powder, decoction, pill, capsule, tablet, granule, and oral liquid by adding pharmaceutically acceptable excipients.

[0012] The fourth object of the present invention is to provide the application of the pharmaceutical composition in the preparation of a medicament for treating alcoholic liver injury.

[0013] The beneficial effects of the present invention are as follows: The present invention provides the application of Gentianopsis paludosa in the preparation of a medicament for treating alcoholic liver injury, and details the effects of different solvent extracts of Gentianopsis paludosa in treating alcoholic liver injury. The experimental results show that before modeling, the fur color of each group of mice was smooth, and their diet and activities were normal; after modeling, except for the normal group, the food intake of each group decreased significantly, the spontaneous activities decreased significantly, the movement was slow, and the gait was unstable. Compared with the normal group, the liver index of the model group mice increased, and the result was statistically significant (P<0.05); compared with the model group, the LI of the GEE group mice decreased significantly (P<0.05); the LI of the other groups of mice did not decrease significantly. Compared with the model group, each extract of Gentianopsis paludosa could reduce the contents of ALT, AST, T-CHO, TG in serum and MDA in the liver to varying degrees, and improve the SOD activity. After comprehensive evaluation using the fuzzy matter-element model based on the weight coefficient combined with pharmacodynamics, it was found that the ethyl acetate extract of Gentianopsis paludosa had the best effect in protecting against alcoholic liver injury. Combining the results of RT-PCR and Western-Blot, compared with the model group, the positive control group and the ethyl acetate extract group could up-regulate the expression of Nrf2, NQO1, and HO-1mRNA to varying degrees (P<0.05); and promote the expression of Nrf2 protein (P<0.05). It has a good therapeutic effect on alcoholic liver injury and has broad application prospects. Brief Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained according to the provided accompanying drawings.

[0015] Figure 1 Expression of Nrf2 protein in mouse liver tissue Detailed Description of the Embodiments

[0016] The following will describe in detail the embodiments of the present invention. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by referring to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0017] For those technical or conditions not specified in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. For the reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0018] Example 1

[0019] 1. Materials

[0020] 1.1 Animals

[0021] 80 Kunming SPF grade male mice, weighing 20 ± 2 g, were provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Animal Qualification Certificate No.: SYXK (Gan) 2015-0005). The mice were raised under specific pathogen-free conditions, with a temperature of 22 ± 2 °C and a humidity of 50-60%, and free access to food and water.

[0022] 1.2 Drugs and Reagents

[0023] The medicinal materials of Gentianopsis paludosa were collected from Yeliguan Town, Lintan County, Gansu Province, and identified as Gentianopsis paludosa (Munro) Ma. of the Gentianaceae family by Professor Chen Yuan of Gansu Agricultural University. Reduced glutathione, Shandong Lukang Pharmaceutical Co., Ltd., batch number 181204505; 56-degree Red Star Erguotou liquor, Beijing Red Star Co., Ltd., batch number 08201637105; alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (T-CHO), superoxide dismutase (SOD), malondialdehyde (MDA), total protein quantification (Coomassie brilliant blue method) kits, Nanjing Jiancheng Bioengineering Institute, batch numbers 20190422, 20190422, 20180424, 20190506, 20190428, 20190425, 20190417; RNA extraction kit, cDNA reverse transcription kit, real-time fluorescence quantitative PCR detection kit, QIAGEN Company of Germany, batch numbers 204574, 205311, 204154; Nrf2, NQO1, HO-1 primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 1; rabbit anti-Nrf2 polyclonal antibody, Genetex Company of the United States, batch number GTX103322; BCA protein content determination kit, Shanghai Beyotime Biotechnology Co., Ltd., batch number P0012; Tris, glycine, SDS, Sigma Company of the United States; 95% ethanol, ethyl acetate, n-butanol, petroleum ether, Tianjin Fuyu Fine Chemical Co., Ltd.

[0024] Table 1 Primer Sequences and Product Lengths

[0025]

[0026] 1.3 Instruments

[0027] iMark multi-functional microplate reader, Bio Rad Company of the United States; NanoDrop nucleic acid concentration measuring instrument, Thermo Company of the United States; DM7500 real-time quantitative PCR instrument, Agilent Company of the United States; ImageQuant 400 gel imaging system, GE Healthcare Company of the United Kingdom; M8288 micro vortex mixer, IKA Company of Germany; F6 / 10-10G ultra-fine homogenizer, Shanghai Fluke Company; TD6 tabletop low-speed centrifuge, Huxi Instrument and Equipment Co., Ltd. of Hunan; DZF-6050 vacuum drying oven, Shanghai Jinghong Experimental Equipment Co., Ltd.; EV400 rotary evaporator, Beijing Labtech Instrument Co., Ltd.

[0028] 2. Methods

[0029] 2.1 Drug Preparation

[0030] Weigh 500 g of Gentianopsis paludosa, a medicinal material, pulverize it, and soak it separately with 5 times the amount of water, 95% ethanol, ethyl acetate, n-butanol, and petroleum ether for 22 h, and then reflux extract for 2 h. After the extract is recovered by a rotary evaporator and dried under reduced pressure, a dry extract is obtained, which is ground and reserved for use. The yields are 16.92%, 15.56%, 5.57%, 11.66%, and 1.65% respectively. Prepare a 0.5% sodium carboxymethyl cellulose suspension before administration.

[0031] 2.2 Replication, grouping and administration of animal models

[0032] 80 SPF-grade male Kunming mice, weighing 20 ± 2 g, after adaptive feeding, are randomly divided into 8 groups according to body weight, with 10 mice in each group, namely the normal group (Normal), the model group (Control), the positive control group (Positive), the water extract group (GAE), the alcohol extract group (GCE), the ethyl acetate extract group (GEE), the n-butanol extract group (GNE), and the petroleum ether extract group (GPE); the normal group and the model group are given an equal volume of normal saline, and each administration group is given a dose of 4.2 g·kg -1 by gavage, which is 30 times the adult clinical dose; the positive control group is gavaged with 0.08 g·kg -1 reduced glutathione; the gavage volume is 10 mL·kg -1 for 12 consecutive days by gavage. Starting from the 13th day, except for the normal group which is gavaged with distilled water, the other groups of mice are gavaged with 56-degree Hongxing Erguotou liquor at 8:00 am every day, and the gavage dose is 16 g·kg -1 In the afternoon, the normal administration is carried out for 3 consecutive days. During the experiment, the mice in each group are routinely bred and allowed to eat and drink freely. Observe the general activities and mental status of the animals in each group during the experiment; record the body weight changes with a cycle of 3 days. After the last gavage, fast the mice for 12 h without water, take blood from the orbital venous plexus, decapitate the mice by cervical dislocation, take liver tissues, weigh them, and freeze them in a -80 °C refrigerator for later use.

[0033] 2.3 Detection of biochemical indexes in serum and liver tissues

[0034] Let the plasma stand at room temperature for 30 min, centrifuge at 3500 r·min -1 for 10 min to separate the serum, and store it at -20 °C. Detect the contents of AST, ALT, T-CHO, and TG in the serum according to the kit instructions. Take a part of the liver tissue, add an appropriate amount of precooled normal saline to make a 10% liver homogenate, centrifuge and take the supernatant, and detect the contents of MDA, SOD, and total protein in the liver homogenate according to the kit instructions.

[0035] 2.4 Evaluation of the protective effect of different solvent extracts of Gentianopsis paludosa on alcoholic liver injury by the fuzzy matter-element model

[0036] Reference method to establish an n-dimensional composite fuzzy matter-element model R for evaluating the protection of different solvent extracts of Gentianopsis paludosa against alcoholic liver injury 8-7 There were 8 samples (M1, M2, …, M8), and 7 indices (C1-LI, C2-AST, C3-ALT, C4-T-CHO, C5-TG, C6-SOD, C7-MDA) were calculated for each sample.

[0037] 2.5 Detection of Nrf2, NQO1, and HO-1 mRNA expression by real-time fluorescence quantitative-polymerase chain reaction (RT-PCR)

[0038] Take about 100 mg of liver tissue, add liquid nitrogen and grind it in an ice bath to make a homogenate. Extract total RNA according to the instructions of the RNA extraction kit. Measure the absorbance ratio at 260 nm / 280 nm with a multifunctional microplate reader and calculate the RNA purity. Reverse transcribe according to the instructions of the cDNA reverse transcription kit to synthesize cDNA. Dissolve 10 ng of sample cDNA, 5 μM primers, and 10 μL Power GREEN PCRMASTER MIX in DEPC water, and place the total volume of 20 μL in a PCR tube for reaction. The reaction conditions were pre-denaturation at 95 °C for 10 min, denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, and extension at 72 °C for 5 min, for a total of 40 cycles. Apply 2 -ΔΔCT methods to calculate the relative content of each gene, and each experiment was repeated four times.

[0039] 2.6 Detection of the expression of Nrf2 protein in liver tissue by Western Blot

[0040] Weigh 30 mg of liver tissue, cut it into pieces and add 0.3 mL of protein lysate to homogenize and extract proteins. Centrifuge at 14000 r·min -1 , 4 °C for 10 min, and take the supernatant. Detect the protein concentration by the BCA method, and adjust the protein concentration of all samples to 4 μg·μL -1 with protein lysate. Store at -80 °C after heating and denaturation. Prepare the gel, load 10 μL of sample, transfer the membrane by wet transfer method after electrophoresis, block with 5% skim milk powder for 1.5 h, incubate with the primary antibody at 1:1000 at 4 °C overnight, incubate with the secondary antibody at 1:3000 for 1 h, add the ECL chemiluminescent solution (mix solution A and solution B at 1:1), and immediately acquire images with Image Quant 400. Use β-actin as an internal reference, and calculate the gray value with Image J.

[0041] 2.7 Statistical processing

[0042] Analyze using SPSS 17.0 statistical software. Measurement data are expressed as mean ± standard deviation Presentation. One-way ANOVA was used for inter-group comparison. For homogeneous variances, the LSD method was used; for heterogeneous variances, the Games-Howell method was used for testing. P < 0.05 was considered statistically significant.

[0043] 3. Results

[0044] 3.1 General observation of mice and changes in liver index (LI)

[0045] Before modeling, the fur color of mice in each group was smooth and shiny, and their diet and activities were normal; after modeling, except for the normal group, the food intake of each group decreased significantly, spontaneous activities were significantly reduced, movement was slow, and gait was unsteady. Compared with the normal group, the liver index of mice in the model group increased, and the result was statistically significant (P < 0.05); compared with the model group, the LI of mice in the GEE group decreased significantly (P < 0.05); the decrease in LI of mice in other groups was not obvious. The results are shown in Table 2.

[0046] Table 2 Changes in body weight and liver index of mice ( n = 10)

[0047]

[0048] Note: vs normal, * P < 0.05; vs control, # P < 0.05.

[0049] 3.2 Changes in the contents of AST, ALT, TG, and T-CHO in the serum of mice

[0050] Under normal circumstances, the contents of AST and ALT in the serum are very low. When the liver is damaged, AST and ALT in hepatocytes will be released into the blood. Detecting the levels of AST and ALT in the serum can accurately reflect the degree of early liver injury. When alcohol enters the body, it can promote the synthesis of TG from lipid substances in the liver, and at the same time, it can block the synthesis and secretion of lipoproteins by the liver, affecting the transport of TG out of the liver, and then leading to the accumulation of excessive lipid substances in the liver, causing fatty liver. Compared with the normal group, the contents of AST, ALT, TG, and T-CHO in the serum of mice in the model group increased significantly (P < 0.05). Compared with the model group, the contents of AST, ALT, TG, and T-CHO in the serum of mice in the positive control group and the GEE group decreased significantly (P < 0.05); the serum ALT level of mice in the GCE group decreased significantly (P < 0.05); the serum TG content of mice in the GNE group decreased significantly (P < 0.05); the activities of ALT and AST and the contents of T-CHO and TG in other drug-administered groups decreased slightly, but the results were not statistically significant. The results are shown in Table 3.

[0051] Table 3 Changes in the contents of AST, ALT, TG, and T-CHO in the serum of mice ( n = 10)

[0052]

[0053] Note: vs normal, * P < 0.05, ** P < 0.01; vs control, # P < 0.05, ## P < 0.01.

[0054] 3.3 Changes in SOD activity and MDA content in mouse liver tissue

[0055] SOD is an important antioxidant enzyme in the body, which can efficiently scavenge various reactive oxygen free radicals, counteract, block and timely repair the damage caused by oxygen free radicals to cells. When its activity decreases, a large amount of free radicals accumulate, further aggravating the degree of liver damage. MDA is the final toxic product produced by the attack of oxygen free radicals on lipids in the body, which can further form protein adducts and stimulate the body to produce antibody-mediated immune damage. The content of MDA is often used to reflect the degree of lipid peroxidation. As can be seen from the results in Table 4, compared with normal group mice, the SOD activity in the liver tissue of model group mice decreased significantly (P < 0.05), and the MDA content increased significantly (P < 0.01). Compared with the model group, the SOD activity in the liver tissue of the GEE group increased significantly (P < 0.05), and the SOD activity in the positive control group increased, but the result was not statistically significant. Compared with the model group, the MDA contents in the positive control group, GCE group, GEE group and GNE group all decreased significantly (P < 0.05).

[0056] Table 4 Changes in SOD activity and MDA content in mouse liver tissue( n = 10)

[0057]

[0058] Note: vs normal, * P < 0.05, ** P < 0.01; vs control, # P < 0.05, ## P < 0.01.

[0059] 3.4 Evaluation of the protective effect of different solvent extracts of Gentianopsis paludosa on alcoholic liver injury by the fuzzy matter-element model

[0060] The composite fuzzy matter-element model established from Tables 2 - 4 is as follows:

[0061]

[0062] For the type where the smaller the better, therefore, the membership degree fuzzy matter-element from the optimal is established as follows:

[0063]

[0064]

[0065]

[0066] Calculate the mean value of each evaluation index according to Table 2-4 Calculate the mean square deviation of each evaluation index: Di = 1.00, 45.65, 21.05, 20.09, 2.60, 113.64, 0.43; the coefficient of variation δi = 0.24, 0.95, 0.82, 4.94, 2.11, 0.89, 0.34; the weight Wi = 0.02, 0.09, 0.08, 0.48, 0.20, 0.09, 0.03. Finally, according to Wi and R △ Obtain the closeness composite fuzzy matter element:

[0067]

[0068] The calculated closeness values are sorted from largest to smallest as: M1>M6>M3>M7>M5>M4>M8>M2, that is, normal group > ethyl acetate extract group > positive control group > n-butanol extract group > 95% ethanol extract group > water extract group > petroleum ether extract group > model group.

[0069] 3.5 Expression of Nrf2, NQO1, and HO-1 mRNA in mouse liver tissue

[0070] As shown in Table 5, compared with the normal group, the mRNA expression levels of Nrf2, NQO1, and HO-1 genes in the liver tissue of mice in the model group were significantly decreased (P<0.05); compared with the model group, the positive control group and the GEE group could up-regulate the expression of Nrf2, NQO1, and HO-1 genes to varying degrees (P<0.05); the GAE group, GCE group, GNE group, and GPE group significantly up-regulated the expression of the HO-1 gene compared with the model group (P<0.05).

[0071] Table 5 Expression of Nrf2, NQO1, and HO-1 mRNA and Nrf2 protein in mouse liver tissue( n = 3)

[0072]

[0073] Note: vs normal, * P<0.05, ** P<0.01; vs control, # P<0.05, ## P<0.01.

[0074] 3.6 Expression of Nrf2 protein in mouse liver tissue

[0075] As Figure 1 shown, compared with the normal group, the expression level of Nrf2 protein in the liver tissue of mice in the model group was significantly decreased (P<0.05); compared with the model group, the positive control group and the GEE group significantly up-regulated the expression of Nrf2 protein (P<0.05), and the changes in other groups were not obvious.

[0076] In summary, the present invention provides the application of Gentianopsis paludosa in the preparation of drugs for treating alcoholic liver injury, and details the effects of different solvent extracts of Gentianopsis paludosa in the preparation of drugs for treating alcoholic liver injury. The experimental results show that before modeling, the fur color of mice in each group was smooth and their diet and activities were normal; after modeling, except for the normal group, the food intake of each group decreased significantly, the spontaneous activity decreased significantly, the movement was slow, and the gait was unsteady. Compared with the normal group, the liver index of mice in the model group increased, and the result was statistically significant (P<0.05); compared with the model group, the LI of mice in the GEE group decreased significantly (P<0.05); the decrease in LI of mice in other groups was not obvious. Compared with the model group, each extract of Gentianopsis paludosa could reduce the contents of ALT, AST, T-CHO, TG in serum and MDA in the liver to varying degrees, and increase the SOD activity. After comprehensive evaluation using the fuzzy matter-element model based on the weight coefficient combined with pharmacodynamics, it was found that the ethyl acetate extract of Gentianopsis paludosa had the best effect on protecting alcoholic liver injury. Combining the results of RT-PCR and Western-Blot, compared with the model group, the positive control group and the ethyl acetate extract group could up-regulate the expression of Nrf2, NQO1, HO-1mRNA to varying degrees (P<0.05); and promote the expression of Nrf2 protein (P<0.05). It has a good therapeutic effect on alcoholic liver injury and has broad application prospects.

Claims

1. Use of Gentianopsis paludosa in the preparation of a drug for treating alcoholic liver injury.

2. Use of an extract of Gentianopsis paludosa in the preparation of a drug for treating alcoholic liver injury.

3. The application according to claim 2, wherein, The extract of Gentianopsis paludosa includes one or more of an aqueous extract, a 95% ethanol extract, an ethyl acetate extract, a n-butanol extract, and a petroleum ether extract.

4. The application according to claim 3, wherein The preparation method of the extract of Gentianopsis paludosa is as follows: Weigh the medicinal material Gentianopsis paludosa, crush it, soak it with 5 times the amount of water, 95% ethanol, ethyl acetate, n-butanol, and petroleum ether respectively, and then reflux and extract. After the extract is recovered by a rotary evaporator and dried under reduced pressure, a dry extract is obtained and ground for standby.

5. The application according to claim 4, characterized in that, The extract of Gentianopsis paludosa is an ethyl acetate extract.

6. A pharmaceutical composition for treating alcoholic liver injury, characterized in that, The pharmaceutical composition includes an extract of Gentianopsis paludosa and excipients.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is prepared into a powder, decoction, pill, capsule, tablet, granule, and oral liquid by adding pharmaceutically acceptable excipients.

8. Use of the pharmaceutical composition according to claim 6 or 7 in the preparation of a drug for treating alcoholic liver injury.

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