CN112274588B

CN112274588B - Preparation method and application of extract of plant of genus Convolvulus

Info

Publication number: CN112274588B
Application number: CN201910672468.8A
Authority: CN
Inventors: 吉腾飞; 孙华; 王佳佳; 张凡; 索鑫玥; 姜琳
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-07-24
Filing date: 2019-07-24
Publication date: 2022-07-22
Anticipated expiration: 2039-07-24
Also published as: CN112274588A

Abstract

The invention belongs to the technical field of medicines, and relates to a preparation method and application of a pig dentate plant extract, in particular to an extract which is extracted from a pig dentate plant (Erythronium) and has the effects of protecting liver and treating non-alcoholic fatty liver and liver fibrosis, a preparation method of the extract, a pharmaceutical composition containing the pig dentate plant (Erythronium) or the extract, and application of the pig dentate plant (Erythronium) or the extract and the pharmaceutical composition thereof as medicines, especially application in preparing medicines for preventing and/or treating liver diseases.

Description

Preparation method and application of extract of plant of genus Convolvulus

Technical Field

The invention belongs to the technical field of medicines. Relates to a preparation method and application of a Chinese pig tooth plant (Erythronium) extract, in particular to an extract obtained by extracting and separating a bulb or an overground part of a pig tooth plant, and application of the extract and a pharmaceutical composition of the extract in treating liver diseases, especially liver protection, and non-alcoholic fatty liver and liver fibrosis.

Background

Plants of the genus liliaceae, the genus hyorhinis (Erythronium) have two types in China: respectively, Erythronium japonicum and Xinjiang Erythronium sibiricum. The south of Jilin (Linjiang) produced by Adenophora erythrosa (Erythronium japonicum) is distributed in the northeast of China, and the raw forest is used for moistening the wetland; japan and Korea are also distributed, so it is also called Japanese pig tooth. The Erythronium sibiricum (also called drumstick ginseng, Xinjiang) is produced in northern Xinjiang (from Tianshan area to Altai and Fuhai), also called Siberian Erythrochan, grows under forest, under bush and on subhigh mountain grassland, has the elevation of 1100 plus 2500 m, grows herbaceous plants for many years, has the whole plant height of 10-45 cm, has single, nearly upright, slender and cylindrical bulb, has the bulb length of 3-10 cm and the diameter of 1-2.5 cm, and is externally coated with brown bulb skin. The Xinjiang odontobutis is one of the long-history edible plants of Kazakh nationality. Its bulb and tender leaf can be eaten, and its nutrient value is high, and the bulb is rich in starch, and its trace element content is also high, in which the Fe content is highest. Bulbs, leaves and flowers are all edible. The bulb can be boiled in water, stewed with milk, fried and stewed in soup, or dried for storage. The Buick or Buick ginseng mentioned in the Kazakhstan medicine formula is the perennial bulb of the pig tooth flower in Xinjiang. The "Buick ginseng" has the health-care functions of nourishing, building up body and strengthening body. Research shows that the ethanol extract of the Japanese pig tooth flower of the same genus plant has strong bacteriostatic effect, strong antioxidant effect and high development value. The method has important significance for researching the application of the extract of the Grifola frondosa in Xinjiang in the food and medicine industries, is expected to be developed into natural preservatives and medicines, and also has great development potential. The Xinjiang pig tooth (Erythronium sibiricum) is used as a tonic in Xinjiang nationality, and is a plant with long history of edible and medicinal use in Kazakh nationality.

Disclosure of Invention

The invention solves the technical problem of providing the application of the plant (Erythronium) of the genus Convolvulus in preparing the medicine for preventing and/or treating the liver diseases.

Extract obtained by extracting and separating bulb or aboveground part of Xinjiang pig tooth (Erythronium sibiricum), and application of the extract and the pharmaceutical composition of the extract of the genus plant in preparing medicines for treating liver diseases, especially liver protection, and treating nonalcoholic fatty liver and liver fibrosis. The invention provides an extract of a plant (Erythronium) of the genus Erythronium, namely, Xinjiang, and finds that the extract has obvious activity of resisting ConA induced acute immunological liver injury. In order to solve the technical problem, the invention provides the following technical scheme:

the first aspect of the technical scheme of the invention provides the extraction and separation process of domestic extraction of the porcine reproductive and respiratory syndrome plant (Erythronium), namely the extraction and separation process of the Sinkiang porcine reproductive and respiratory syndrome (Erythronium) extract:

extracting pulverized bulb part of Erythronium plant (Erythronium) Sinkiang Erythronium Sibiricum with water, and concentrating the extractive solution to obtain fluid extract; adding 95% ethanol into the obtained fluid extract until the ethanol content is 70%, standing, filtering, vacuum drying, and pulverizing to obtain extract ZYH-W of Conyza genus plant (Erythronium) Sinkiang Conyza sativa (Erythronium sibiricum). In a second aspect of the present invention, there is provided a pharmaceutical composition, comprising an extract of the plant belonging to the genus Adenophora (Erythronium) Sinkiang Adenophora (Erythronium sibiricum) and a pharmaceutically acceptable excipient. The third aspect of the technical scheme of the invention provides application of the extract of the plant (Erythronium) in preparing the medicines for protecting the liver and treating the non-alcoholic fatty liver and the hepatic fibrosis. The invention provides an extract of a plant (Erythronium) in the genus of Cona, and finds that the extract has remarkable activity of resisting ConA induced acute immunological liver injury.

It is still another object of the present invention to provide a pharmaceutical composition comprising an extract of a plant of the genus Adenophora (Erythronium), and a carrier commonly used in the pharmaceutical field.

The invention also aims to provide application of the plant (Erythronium) and/or the extract of the plant (Erythronium) in preparing a medicament for treating non-alcoholic fatty liver disease and liver fibrosis, or a composition containing the plant (Erythronium) and/or the extract of the plant (Erythronium).

The invention also aims to provide application of the plant (Erythronium) and/or the extract of the plant (Erythronium) in preparing a liver protection medicament, or application of a composition containing the plant (Erythronium) and/or the extract of the plant (Erythronium) in preparing a liver protection medicament.

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the raw medicinal materials of the plant (Erythronium) of the genus Convolvulus are dried and appropriately crushed to increase the contact area between the medicinal materials and a solvent and improve the efficiency.

The extraction solvent of the raw medicinal materials is water or a mixture of water and alcohols. Preferred alcohols include methanol, ethanol, isopropanol, butanol, and the like. Mixtures of water and alcohols, for example, containing 40% to 80% by volume of the alcohol compound. The solvent amount is 4-14 times of the weight of the raw material medicine during extraction. The extraction can be under static or dynamic conditions, preferably under dynamic conditions. In order to improve the efficiency of extraction, ultrasonic waves or the like may be used. The temperature of extraction is in the range from room temperature (e.g. 20 ℃) to the reflux temperature of the solvent, preferably at reflux temperature. The extraction can be carried out continuously or intermittently, and the intermittent extraction can be repeated for 1-4 times.

After the above steps are finished, combining the filtrates, heating and concentrating the filtrate under normal pressure or reduced pressure to a volume 1-5 times of the weight of the medicinal materials under a dynamic state, and cooling. The extraction solvent is alcohol compound such as methanol, ethanol, isopropanol, butanol, etc., or their mixture; ethanol is preferred. Standing for precipitation, filtering or centrifuging to remove insoluble substances, and washing the insoluble substances with water, generally 1-3 times. The filtrates were combined and further concentrated to a paste.

The extract can also be directly refined and concentrated by exchange column and membrane technology, and then prepared into extract or dry powder. Useful exchange columns include: macroporous resin, ion exchange resin, active carbon, sephadex and the like; macroporous resins and activated carbon are preferred.

The extract can be freeze-dried into dry powder, or concentrated liquid can be directly spray-dried into dry powder for various preparation molding.

The invention also relates to pharmaceutical compositions containing as active ingredient an extract according to the invention and conventional pharmaceutical excipients or adjuvants. Typically, the pharmaceutical composition of the invention contains 0.1-95% by weight of the extract of the invention.

The invention also provides a pharmaceutical composition comprising a pharmaceutically effective amount of the extract obtained by the method of the invention as an active ingredient and a pharmaceutically acceptable carrier.

Pharmaceutical compositions of the extracts of the present invention may be prepared according to methods well known in the art. For this purpose, the extract according to the invention can, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, in a suitable administration form or dosage form for use as a human or veterinary medicine.

The extract of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be intestinal or parenteral, such as oral, nasal, oral mucosa, skin, peritoneum or rectum administration, etc., preferably oral administration.

The route of administration of the extract of the invention or the pharmaceutical composition containing it likewise includes administration by injection. Injections include intravenous, intramuscular, subcutaneous, intradermal, and the like.

The administration dosage form can be liquid dosage form or solid dosage form. For example, the liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.

The extract can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various particle delivery systems.

In order to prepare the unit dosage form into tablets, various carriers well known in the art can be widely used. Examples of the carrier are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate and the like; wetting agents and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, etc.; lubricants, for example, talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layered and multi-layered tablets.

For example, to form the dosage unit into a pill, a wide variety of carriers known in the art are used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binders, such as acacia, tragacanth, gelatin, ethanol, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone and the like; disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium dodecylsulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glyceryl tristearate, cacao butter, hydrogenated oil and the like; absorption promoters such as quaternary ammonium salts, stearates, boric acid, liquid paraffin, polyethylene glycol, and the like. The tablets may be further formulated as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layered and multi-layered tablets.

For example, to form the dosage unit into a pill, a wide variety of carriers known in the art are used. Examples of the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc and the like; binding agent, such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, etc. Disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecylsulfate, methylcellulose, etc. For example, for the encapsulation of administration units, the active ingredient extract is mixed with the various carriers mentioned above and the mixture thus obtained is placed in hard gelatin capsules or soft gelatin capsules. The effective component of the extract can also be prepared into microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules or prepared into injection.

For example, the extract of the present invention can be formulated into injectable preparations, such as solutions, suspensions, emulsions, lyophilized powders, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, for the preparation of isotonic injection shooting liquid, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and in addition, a conventional cosolvent, a buffer, a PH adjuster, and the like may be added. These adjuvants are conventional in the art. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.

For administration purposes, to enhance the therapeutic effect, the medicaments or pharmaceutical compositions of the invention may be administered by any known method of administration.

The administration dose of the extract pharmaceutical composition of the present invention depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, character and individual response of the patient or animal, the administration route, administration frequency, therapeutic purpose, and thus the therapeutic dose of the present invention can be widely varied. Generally, the dosage of the pharmaceutical ingredients of the present invention used is well known to those skilled in the art. The prophylactic or therapeutic objectives of the invention can be accomplished by appropriate adjustment of the actual amount of drug contained in the final formulation of the extract composition of the invention to achieve the desired therapeutically effective amount. The amount of the extract of the present invention to be used is 0.001-100g crude drug/kg body weight, preferably 0.01-50g crude drug/kg body weight, and most preferably 0.05-25g crude drug/kg body weight. The above doses may be administered in single dosage form or in divided doses, e.g., two, three or four doses, subject to the clinical experience of the administering physician and including dosage regimens involving the use of other therapeutic means. The total dose required for each treatment can be divided into multiple doses or administered as a single dose. The extract or composition of the present invention can be administered alone or in combination with other therapeutic or symptomatic drugs and adjusted in dosage.

Advantageous technical effects

The invention adopts a ConA-induced acute immunological liver injury model, and proves that the extract of the Xinjiang odontophyllum (Erythronium sibiricum) has obvious liver protection activity, and the AST reducing function is equivalent to that of the bicyclol and even slightly superior to that of the bicyclol; the compound has obvious inhibition effect on human liver cell lipid accumulation caused by OA, and the activity is superior to that of fenofibrate; also shows significant inhibition of TGF-beta 1 induced hepatic stellate cell activation. Therefore, the extract of the plant of the genus Adenophora (Erythronium) of the present invention can be used for the preparation of a medicament for the prevention and/or treatment of liver diseases, including the treatment of non-alcoholic fatty liver disease and liver fibrosis.

Drawings

FIG. 1 is a photograph showing representative pathological conditions of liver tissues of mice in each group (H.E.; X200)

FIG. 2 Effect of test substances on lipid accumulation in human hepatocytes by OA. (n-6-9)

Detailed Description

Extraction experiments

Example 1 extract of Adenophora hyoscyami (Erythronium sibiricum)

1000g of coarse powder of a dried bulb part of Xinjiang pig tooth flower (Erythronium sibiricum), 10000 ml of distilled water is used for hot reflux, three times (10000 ml/time) of extraction are carried out, 1.5-2.0 hours of extraction are carried out each time, 4000ml of aqueous extract is obtained after decompression and concentration of extracting solution, ethanol is added until the alcohol content reaches 70 percent (volume ratio), precipitation and filtration are carried out, and 155.0g of faint yellow powdery solid (ZYH-W) is obtained after vacuum drying of concentrated filtrate.

Pharmacological experiments

Experimental example 1. extract of Adenophora saxifraga (Erythronium sibiricum) ZYH-W carbon tetrachloride (CCL)₄)

Activity test for induced acute toxic liver injury

Experimental method

Model building and drug delivery

The male ICR mice are randomly grouped after adapting to the environment, 9 mice in each group are respectively a blank control group and a CCL₄Model group, positive control drug bicyclol (200 mg. kg-1) group, ZYH-W200 mg. kg-1 and 600 mg. kg-1 dose group. The animals in each group of ZYH-W were gavaged 1 time a day for 5 days, the positive control drug, bicyclol, was gavaged 1 time in the afternoon, the morning and afternoon, respectively, and the animals in the blank control group and CCL4 model group were given the same amount of solvent. All groups were injected with 0.15% CCL in the abdominal cavity 2h after the last administration₄Peanut oil solution was used 1 time. The dosage was 10 ml/kg-1. The mice were fasted for 16h without water deprivation and then treated.

Determination of biochemical index

ICR mouse eyeball blood is taken, and after the blood is kept still for 2 hours at room temperature, the blood is centrifuged (3500rpm, 15min) to prepare serum. According to the instruction of the detection kit, the ALT, AST and LDH contents in the serum are detected by a full-automatic biochemical analyzer.

Histopathological observation of liver

Taking a liver large leaf specimen at the same part, fixing by 4% paraformaldehyde, carrying out conventional dehydration and transparency, paraffin embedding and slicing, carrying out H.E. staining, and carrying out optical microscopy on pathological states of the liver, photographing and analyzing.

Data analysis

Data are expressed as mean ± standard deviation. Data between groups were significantly different by t-test with P < 0.05.

Results of the experiment

Animal body weight and general State

After the animals in the test object group are continuously administrated for 5 days, the state of the mice is good, the weight is normally increased, the phenomenon of obviously reducing the weight of the animals is avoided, and the weight of the animals in each group has no obvious difference compared with a blank control group and a model group. There was no animal death. Suggesting that the mice have good tolerance to the test substances under the current dosing scheme.

Influence on biochemical indexes of liver function

The results are shown in tables 1-3. The ccl4 can cause significant liver tissue damage, which is shown in that the serum ALT, AST and LDH levels are all significantly increased compared with the blank control group, the serum ALT level is an internationally recognized liver damage biomarker, and the level is positively correlated with the liver damage degree. ZYH-W200 mg/kg, 600mg/kg all have obvious improvement effect on toxic injury in liver tissue caused by CCL 4. ZYH-W600 mg/kg had slightly better effect on reducing serum AST and LDH levels than the positive control drug bicyclol.

Table 1 effect of test substances on the serum ALT content in mice with acute toxic liver injury induced by CCL4 (n-9)

^###P is less than 0.001, compared with a blank control group;^*P＜0.05，^**P＜0.01,^***p is less than 0.001, compared with the model group.

Table 2 effect of test substances on serum AST content in mice with acute toxic liver injury induced by CCL4 (n-9)

TABLE 3 Effect of the test Agents on CCL4 induced serum LDH content in acute toxic liver injury mice (n-9)

Influence on pathological damage of liver tissue

The results are shown in FIG. 1 and Table 4. Fig. 1 is a photograph of representative liver pathology for each group of animals, and table 4 shows the histopathology score statistics. The result shows that the liver cells of the blank control group animals are normal in shape, polygonal, free from degeneration and necrosis, large and round in cell nucleus, clear in liver lobule structure, complete in shape, orderly in liver cell cord arrangement, normal in the shapes of the central hepatic vein and the region of the junction, smooth in the inner wall of the blood vessel, and free from inflammatory cell infiltration. The liver tissue of the model group animal has obvious damage, a great amount of liver cells are degenerated and necrotized, large-area inflammatory cell infiltration exists, and the central vein of the liver has obvious expansion. Each ZYH-W dosage group has obvious improvement effect on liver histopathology, which is shown in that the degeneration and necrosis degree of liver cells are reduced, inflammatory cells are reduced, and the accumulation area of inflammation/necrosis lesion is obviously reduced.

TABLE 4 ZYH-W vs. CCl₄Induced acute toxic liver injury mouse liver histopathological histology score

^###P is less than 0.001, compared with a blank control group;^*P＜0.05，^**P＜0.01,^***p < 0.001, compared to model group.

Experimental example 2 Activity test of extract ZYH-W of Adianthus communis (Erythronium sibiricum) against acute immunological liver injury induced by Clerodendranthin A (ConA)

Experimental methods

Model building and drug delivery

ICR male mice, after acclimatization, were randomly divided into 5 groups, blank control group, model group, ZYH-W250 mg/kg, ZYH-W500 mg/kg, and positive control drug bicyclol 200mg/kg, each group containing 10-12 animals. The test substance groups were administered 5 times a day, 1 time a day, and 5 times a total of 5 times by gavage administration started 5 days before the model building. The bicyclol is administered by gavage 1 time each in the afternoon, the morning and afternoon of the day before molding. The animals of the blank control group and the model group were given the same amount of solvent. 2h after the last administration, mice in each group were injected with 20mg/kg ConA 1 times into the tail vein except for the blank control group, which was injected with the same amount of physiological saline into the tail vein. After the mice were fasted for 16h without water deprivation, the mice were sacrificed and blood was taken to prepare serum.

Determination of serum biochemical indicators

The method comprises the following steps of taking eyeballs of mice and blood, standing blood samples for 1-1.5 hours, centrifuging at 4000rpm for 10min, separating serum, and detecting the contents of ALT, AST and LDH in the serum by a full-automatic biochemical analyzer.

Data analysis

Data are mean. + -. standard deviation of data

And (4) showing. Comparison between groups by t-test, with p<0.05 indicated a significant difference.

Results of the experiment

Biochemical level of serum

Serum ALT, AST and LDH levels were analyzed by a full-automatic biochemical analyzer, and the results are shown in tables 5-7. The phytohemagglutinin ConA 20mg/kg administered into tail vein for 1 time can cause significant liver tissue damage, and serum ALT, AST and LDH levels are all significantly increased, and have statistical difference compared with blank control group. ZYH-W250 mg/kg and 500mg/kg are administrated for 5 days in advance, and the drug has obvious protective effect on mice immune liver injury caused by ConA, wherein the activity of 250mg/kg is better than 500 mg/kg. The activity of ZYH-W on serum AST was comparable to that of bicyclol. The positive control drug bicyclol is administrated for three times, and also shows a remarkable protective effect on liver injury caused by ConA.

TABLE 5 influence of the test substances on the serum ALT content of the mice with ConA-induced acute immunological liver injury

TABLE 6 influence of test substances on the AST content in the serum of mice with ConA-induced acute immunological liver injury

^###P is less than 0.001, compared with a blank control group;^*p is less than 0.05, compared with the model group.

TABLE 7 Effect of test substances on ConA-induced acute immunological liver injury mouse serum LDH content

^##P is less than 0.01, compared with a blank control group;^*P＜0.05，^**p is less than 0.01, compared with the model group.

Experimental example 3 Activity test of extract ZYH-W of Adenophora plant (Erythronium purpuricum) in Sinkiang Adenophora on oleic acid-induced acute immunological liver injury

Experimental methods

5.1 Effect of extracts of plants of the genus Adenophora (Erythronium) on human hepatocyte proliferation

Taking HepG2 cells in logarithmic growth phase, and mixing 5X 10 cells⁴Each/ml cell was inoculated into a 96-well plate at 100. mu.l per well, cultured for 24 hours, added with ZYH-W (1. mu.M, 5. mu.M, 10. mu.M, 25. mu.M, 50. mu.M) and fenofibrate at 12.5. mu.M at various concentrations, cultured for 24 hours, removed of the supernatant, added with 100. mu.l MTT (0.5mg/ml, prepared using serum-free DMEM medium), placed in a cell incubator for culture for 4 hours, added with 150. mu.l DMSO per well, and placed in a microplate reader at 570nm for absorbance measurement.

5.2 Effect of extracts of plants of the genus Adenophora (Erythronium) on Oleic Acid (OA) -induced lipid accumulation in human hepatocytes

Taking HepG2 cells in logarithmic growth phase, and mixing 1X 10 cells⁵Each cell/ml was inoculated into a 96-well plate at 100. mu.l/well, cultured for 24 hours, and then different concentrations of ZYH-W (1. mu.M, 5. mu.M, 10. mu.M, 25. mu.M, 50. mu.M), fenofibrate at 12.5. mu.M, and 240. mu.M Oleic Acid (OA) were added under dark conditions, and the culture was continued for 24 hours. The supernatant was discarded, washed with PBS 3 times, and each well was added with 100. mu.l of 4% paraformaldehyde, allowed to stand at room temperature for 40min, and the cells were fixed. Discarding the supernatant, washing with PBS for 3 times, adding 100 μ l of newly prepared oil red dye solution into each hole, standing in dark for 1h, washing with PBS for 3 times, adding 50 μ l of isopropanol into each hole, oscillating with a mixing oscillator for several minutes, and measuring absorbance at 520nm wavelength of an enzyme-labeling instrument. The absorbance values represent the relative lipid content.

Statistical analysis

Data are mean. + -. standard deviation of data

And (4) showing. Comparison between groups was by t-test, with p<0.05 indicated a significant difference.

Results of the experiment

Effect on human hepatocyte proliferation

The results are shown in Table 8, ZYH-W1 mu M, 5 mu M, 10 mu M, 25 mu M, 50 mu M and fenofibrate 12.5 mu M act on human hepatocyte HepG2 cells for 24h, no obvious toxicity is caused to the cells, the cell survival rate is all more than 90%, and the nontoxic concentration is used for research on oleic acid-induced hepatocyte lipid droplet deposition.

Effect on OA-induced lipid accumulation in human hepatocytes

In-vitro lipid-lowering activity of ZYW-H on hepatocyte lipid stacks is evaluated by using an in-vitro hepatocyte lipid stack model induced by oleic acid. The results are shown in Table 9 and FIG. 2, and the absorbance values (OD) are high or low and represent the lipid particle content in the liver cells. The OA acts on HepG2 cells for 24h, and the lipid level in the liver cells is obviously increased compared with that of a blank control group, and has obvious statistical difference. The doses of ZYW-H1 μ M, 5 μ M, 10 μ M, 25 μ M and 50 μ M can obviously reduce the lipid particle accumulation in the liver cells caused by OA, and the statistical difference is compared with that of a model control group. The positive control drug Fenofibrate (Fenofibrate)12.5 μ M also significantly improved the lipid accumulation of HepG2 cells. Under the current experimental scheme, ZYW-H has better activity in reducing liver fat than fenofibrate.

Table 8 effect of test agents on human hepatocyte proliferation. (n-6-9)

Table 9 effect of test substances on lipid accumulation in human hepatocytes by OA. (n-6-9)

Experimental example 4 evaluation of Activity of ZYH-W extract of Adenophora plant (Erythronium purpuricum) in Sinkiang Adenophora on TGF-. beta.activated hepatic stellate cells

Experimental method

4.1 cytotoxicity of test substances against HSC-T6 cells

Rat hepatic stellate cell HSC-T6 cells are inoculated in a 96-hole cell culture plate, cultured in an incubator for 24 hours, subjected to synchronization treatment for 24 hours by changing a serum-free DMEM culture medium, added with a compound to be detected, and simultaneously provided with a solvent DMSO control group (1:1000), and each drug is provided with 4 parallel holes. After the drug continues to act on HSC-T6 cells for 24h, the culture solution is discarded, 100 muL of MTT (0.5mg/ml) solution is added into each well, the culture is continued for 4h, the MTT solution is discarded, 150 muL of DMSO is added into each well, the mixture is shaken by a mixing shaker, and the absorbance value is measured at the wavelength of 570nm of an enzyme labeling instrument.

Cell survival (%) × (mean OD of administered cells/mean OD of solvent control cells) × 100.

4.2 Effect of test Agents on TGF-. beta.1 hepatic stellate cell proliferation

The HSC-T6 cells are inoculated in a 96-well cell culture plate, cultured in an incubator for 24h, subjected to synchronization treatment for 24h by changing a serum-free DMEM culture medium, added with a non-toxic concentration compound to be detected and TGF-beta (the final concentration is 10ng/mL), and simultaneously provided with a solvent control group and a TGF-beta (the final concentration is 10ng/mL) model group. The action of HSC-T6 cells was continued for 24 h. The culture medium was discarded, 100. mu.L of MTT (0.5mg/mL) solution was added to each well, the culture was continued for 4 hours, the MTT solution was discarded, 150. mu.L of DMSO was added to each well, the mixture was shaken by a shaker, and the absorbance at a wavelength of 570nm in a microplate reader was measured.

Statistical analysis

Data are mean. + -. standard deviation

Results of the experiment

Cytotoxic effects on hepatic stellate cells

The results are shown in Table 10, and the ZYH-W1 mu M, 5 mu M, 10 mu M and 25 mu M effects-synchronized HSC-T6 cells are 24h, have no obvious toxicity to the cells, and have the cell survival rate of more than 90 percent. This non-toxic concentration was used for subsequent studies.

Effect on TGF-beta 1 activation of hepatic stellate cell proliferation

The results are shown in Table 11, the TGF-beta 110 ng/mL stimulates the HSC-T6 cells for 24h, the proliferation of the hepatic stellate cells is obviously increased compared with a blank control group (P is less than 0.01), and the hepatic stellate cells are obviously activated. Under the current experimental scheme, ZYH-W5. mu.M, 10. mu.M and 25. mu.M concentrations show significant inhibitory activity on TGF-beta 1-induced hepatic stellate cell activation. Suggesting that the liver-protecting peptide has anti-hepatic fibrosis activity related to hepatic stellate cell pathogenesis.

Table 10 effect of test substances on hepatic stellate cell proliferation. (n is 3)

TABLE 11 Effect of test substances on TGF-. beta.1 hepatic stellate cell proliferation. (n is 5)

Conclusion of the experiment

ZYH-W shows obvious liver protection activity in a ConA and CCL4 induced acute immune liver injury mouse model, and AST reduction effect is equivalent to or even slightly superior to that of bicyclol; the compound has obvious inhibition effect on human liver cell lipid accumulation caused by OA, and the activity is superior to that of fenofibrate; also shows significant inhibition of TGF-beta 1 induced hepatic stellate cell activation. The extract of the Xinjiang pig tooth flower (Erythronium sibiricum) can be used for preparing the medicines for protecting the liver and treating the non-alcoholic fatty liver and the hepatic fibrosis.

Claims

1. The application of the extract of the Adhatoda sugawa (Erythroniumsibiricum) in preparing the medicine for preventing and/or treating the non-alcoholic fatty liver disease or the hepatic fibrosis disease is characterized in that the preparation method of the extract of the Adhatoda sugawa (Erythroniumsibiricum) comprises the following steps:

the bulb of Xinjiang Condrania insignis (Erythroniumsibiricum) is dried, crushed, extracted with water, methanol or ethanol, or a mixture of water and methanol or a mixture of water and ethanol, the extracts are combined, concentrated and purified.

2. Use according to claim 1, characterized in that the drying is oven drying, drying in the shade, air drying, vacuum drying, freeze drying.

3. Use of a pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of non-alcoholic fatty liver or liver fibrosis diseases, wherein the pharmaceutical composition comprises the extract of the flower of Adenophora hyoscyami (Erythroniumsibiricum) of claim 1 as an active ingredient and a pharmaceutically acceptable carrier.