New indication application of traditional Chinese medicine preparation containing folium mori

Abstract

The invention relates to new indications of a traditional Chinese medicine preparation containing mulberry leaves. The traditional Chinese medicine preparation is prepared from the following traditional Chinese medicinal materials in parts by weight: 12 parts of mulberry leaves, 12 parts of lotus leaves, 12 parts of hawthorn leaves, 15 parts of salvia miltiorrhiza and 15 parts of red peony roots. The invention aims to provide a new clinical application of the medicine for treating metabolic related fatty liver diseases, non-alcoholic fatty liver diseases, hyperlipidemia, cerebral infarction, coronary heart disease, angina pectoris and the like, so that the clinical application indication range of the medicine is expanded, and more medication choices are provided for clinical patients.

Description

Technical Field

[0001] The present invention relates to a new indication use of a traditional Chinese medicine preparation containing mulberry leaves, belonging to the field of new indication uses in traditional Chinese medicine clinical practice. Background Art

[0002] Metabolic diseases are a group of diseases caused by metabolic disorders, including hyperlipidemia, obesity, diabetes, hyperuricemia, and non-alcoholic fatty liver disease (NAFLD), etc. Due to their wide impact, they have become a global health burden. It is estimated that by 2030, the number of patients with obesity, diabetes, and NAFLD globally will reach approximately 156 million, 57.8 million, and 10.1 million respectively. In addition, diseases caused by high blood lipid and high blood sugar, including atherosclerosis, diabetic complications, etc., are highly harmful, seriously affecting the quality of life of patients and even endangering their lives.

[0003] The traditional Chinese medicine preparation referred to herein is Sanyepian, whose formula is derived from the clinical experience formula of Academician Zhang Boli and is composed of five herbs, namely mulberry leaf, lotus leaf, hawthorn leaf, salvia miltiorrhiza, and red peony root, and has the functions of ascending lucidity and descending turbidity, removing stasis and relieving distension. It is used for diabetes mellitus caused by phlegm-turbidity stasis obstruction, with symptoms such as heaviness and discomfort of the head and body, fatigue, fullness and distension in the epigastric region, dry mouth, bitter taste in the mouth, unsmooth bowel movements, dull purple tongue, or spotted tongue, greasy tongue coating, and taut and slippery pulse. In long-term clinical applications, this formula has the effects of improving the clinical symptoms of type 2 diabetes patients, reducing fasting and OGTT 2h blood glucose, and regulating blood lipids, and is applicable to patients with type 2 diabetes with glucose and lipid metabolism disorders.

[0004] The applicant conducted a systematic search and collation of the prior art literature related to this traditional Chinese medicine composition. CN117064936A discloses a pharmaceutical composition comprising: 2 - 30 parts of mulberry leaves, 2 - 30 parts of lotus leaves, 2 - 30 parts of hawthorn leaves, 2 - 30 parts of salvia miltiorrhiza, and 2 - 30 parts of red peony roots. This pharmaceutical composition has an effect in treating diarrhea caused by intestinal flora imbalance, gastrointestinal dysfunction, and dyspepsia. Patent CN100534461A records a pharmaceutical composition for treating diabetes and impaired glucose tolerance, which is made from the following medicinal materials in the following weight ratios: 2 - 40 parts of mulberry leaves, 2 - 40 parts of lotus leaves, 2 - 40 parts of hawthorn leaves, 2 - 40 parts of salvia miltiorrhiza, and 2 - 40 parts of red peony roots. Pharmacological experiments show that this composition has definite curative effects in reducing fasting blood glucose, improving impaired glucose tolerance, reducing serum triglyceride (TG), and insulin (INS), etc. CN102451253A discloses a prescription of a pharmaceutical composition: 12 parts of mulberry leaves, 12 parts of lotus leaves, 12 parts of hawthorn leaves, 15 parts of salvia miltiorrhiza, and 15 parts of red peony roots, and its new use in the preparation of drugs for treating viral colds. CN103830374A discloses a pharmaceutical composition made from the following medicinal materials in the following weight ratios: 6 - 25 parts of mulberry leaves, 6 - 25 parts of lotus leaves, 6 - 25 parts of hawthorn leaves, 6 - 25 parts of salvia miltiorrhiza, and 6 - 25 parts of red peony roots. The application of this pharmaceutical composition in the preparation of drugs for treating, inhibiting, or preventing hyperuricemia.

[0005] From the technical content of the above patent documents, it can be seen that the clinical diseases of this drug mainly focus on: diarrhea caused by intestinal flora imbalance, gastrointestinal dysfunction, dyspepsia, reducing fasting blood glucose, reducing serum triglyceride (TG), hyperuricemia, etc. During the process of carrying out clinical relevant research on the traditional Chinese medicine preparation of the present invention, it was surprisingly and unexpectedly found that it can also treat new indications of other metabolic diseases, further expanding and extending the indication range of this traditional Chinese medicine preparation. Summary of the Invention

[0006] The purpose of the present invention is to provide a new indication use of a traditional Chinese medicine preparation containing mulberry leaves, specifically involving providing a new clinical use for treating diseases such as metabolic - related fatty liver disease, non - alcoholic fatty liver, hyperlipidemia, cerebral infarction, and coronary heart disease angina, so as to expand the clinical application indication range of this drug and provide more medication options for clinical patients.

[0007] The technical solution of this invention patent application is as follows:

[0008] The application of a traditional Chinese medicine preparation containing mulberry leaves in the preparation of drugs for treating metabolic - related fatty liver.

[0009] Preferably, the application of the traditional Chinese medicine preparation in the preparation of drugs for treating non - alcoholic fatty liver.

[0010] Preferably, the application of the traditional Chinese medicine preparation in the preparation of drugs for treating hyperlipidemia.

[0011] As a preference of the present invention, the application of the traditional Chinese medicine preparation in the preparation of drugs for treating atherosclerosis and cardio-cerebrovascular diseases.

[0012] As a further preference of the present invention, the application of the traditional Chinese medicine preparation in the preparation of drugs for treating cerebral infarction and angina pectoris of coronary heart disease.

[0013] The traditional Chinese medicine preparation of the present invention is made from the following Chinese medicinal materials by weight: 12 parts of mulberry leaves, 12 parts of lotus leaves, 12 parts of hawthorn leaves, 15 parts of salvia miltiorrhiza, and 15 parts of red peony roots.

[0014] The preparation method of the traditional Chinese medicine preparation of the present invention comprises the following steps:

[0015] (1) Take the mulberry leaves and lotus leaves in the above weights and reflux extract them together. The extraction solvent is 50-70% ethanol and water. The extract is concentrated and dried into a dry extract for standby.

[0016] (2) Reflux extract the hawthorn leaves. The extraction solvent is 50-70% ethanol, and the eluent is 50-70% ethanol. Concentrate and dry the above ethanol eluate into a dry extract for standby.

[0017] (3) Take salvia miltiorrhiza and percolate and extract it with 50% ethanol. Use a macroporous resin column washed with acid water with a pH of 4, and the eluent is 70% ethanol. Concentrate and dry the above ethanol eluate into a dry extract for standby.

[0018] (4) Then take red peony roots and extract them with water. First, precipitate with 80% ethanol, filter, concentrate the filtrate to remove ethanol, and then adsorb with macroporous resin. The eluent is 70% ethanol. Concentrate and dry the above ethanol eluate into a dry extract for standby. The dry extract of the drug of the present invention is obtained from the above steps (1)-(4). Take 200 g of its dry extract powder, add 300 g of dextrin, 2 g of protein sugar, and an appropriate amount of 70% ethanol to granulate, and dry at 60 °C to obtain the traditional Chinese medicine granule of the present invention.

[0019] The dosage form of the traditional Chinese medicine preparation of the present invention can also be made into different oral preparations such as capsules, tablets, dripping pills, oral liquids, dispersible tablets, etc. by adding corresponding pharmaceutical excipients.

[0020] The beneficial effects of the traditional Chinese medicine preparation of the present invention:

[0021] (1) The traditional Chinese medicine preparation (three leaves) of the present invention can significantly improve the activities of ALT (glutamic-pyruvic transaminase) and AST (aspartate aminotransferase) in the liver serum of rabbits. Compared with the model group, it has increased (P < 0.05). These data indicate that the traditional Chinese medicine preparation of the present invention has a certain therapeutic effect on metabolism-related fatty liver and non-alcoholic fatty liver.

[0022] In the three dosages of the traditional Chinese medicine preparation of the present invention, the hydropic degeneration and fatty degeneration of rabbit liver tissues are alleviated, and the swelling of aortic intimal epithelial cells is alleviated. These experimental data also indicate that the traditional Chinese medicine preparation of the present invention can reduce hyperlipidemia in rats and has a certain therapeutic effect on non-alcoholic fatty liver treatment.

[0023] ⑵、As can be seen from the pharmacological experimental results, in the experiments of TC, TG, LDL-C, HDL-C, and TC-HDL / HDL in the sera of experimental rabbits, in the three dosage groups of the traditional Chinese medicine preparation of the present invention, the serum TC cholesterol of rabbits can be significantly reduced compared with the model group (P < 0.01). The traditional Chinese medicine preparation (three leaves) of the present invention can reduce the hypercholesterolemia rabbit model induced by high-fat diet, reduce serum TC, LDL-C, and TC-HDL / HDL; reduce the liver index, and the contents of TC (total cholesterol) and TG (triglyceride) in the liver tissues of hypercholesterolemia rabbits; the above pharmacological experimental results further confirm the efficacy of the traditional Chinese medicine preparation of the present invention in effectively treating hyperlipidemia.

[0024] ⑶、Compared with the model group, the traditional Chinese medicine preparation (three leaves) of the present invention can reduce the whole blood viscosity of rats (P < 0.05). Compared with the model group, the plasma viscosity is significantly increased (P < 0.01), the plasma viscosity of the rats in the traditional Chinese medicine preparation group of the present invention is significantly reduced (P < 0.01), and the content of plasma fibrinogen in the rats in the traditional Chinese medicine preparation group of the present invention is significantly reduced compared with the model group (P < 0.01). The traditional Chinese medicine preparation (three leaves) of the present invention can reduce the whole blood viscosity at high, medium, and low shear rates of rats with acute blood stasis model, reduce plasma viscosity, fibrinogen content, erythrocyte sedimentation rate, and hematocrit, and improve blood rheology.

[0025] The above pharmacological experimental results further indicate that the traditional Chinese medicine preparation of the present invention can effectively treat atherosclerosis and cardiovascular and cerebrovascular diseases.

[0026] ⑷、The pharmacological experimental results show that the traditional Chinese medicine preparation (three leaves) of the present invention can reduce the whole blood viscosity at high, medium, and low shear rates of rats with acute blood stasis model, reduce plasma viscosity, fibrinogen content, erythrocyte sedimentation rate, and hematocrit, and improve blood flow, which also suggests that the traditional Chinese medicine preparation of the present invention can also be used in the treatment of atherosclerosis, cardiovascular and cerebrovascular diseases, cerebral infarction, and coronary heart disease angina pectoris.

[0027] To more fully understand the implementation of the present invention, the present invention will be further described below through typical examples. The following and through pharmacodynamics are used to prove the clinically beneficial therapeutic effects of the traditional Chinese medicine composition of the present invention. Detailed implementation manners

[0028] To better understand the present invention, several experimental examples are listed below to illustrate its new uses in the field of traditional Chinese medicine pharmacy. The following experiments are intended to illustrate the present invention rather than limit the present invention.

[0029] Example 1 Pharmacological and Pharmacodynamic Tests of Traditional Chinese Medicine Preparation (Three-Leaf Tablets) for Hyperlipidemia and Metabolic-Associated Fatty Liver 1 Test Materials

[0030] 1.1 Drugs and Preparation

[0031] 1.1.1 Test drug: Three-Leaf Tablets, brown dry extract powder, provided by the Department of Pharmacy, Tianjin University of Traditional Chinese Medicine. The extraction rate is 8.8 g dry extract powder / 100 g crude drug (the crude drug content per gram of dry extract powder is 11.36 g), batch number: 2020806. Before the test, it was prepared into a liquid medicine of 0.64 g, 0.32 g, 0.16 g crude drug / ml with 0.5% CMC, and administered to rabbits by gavage at a dose of 3.2 g, 1.6 g, 0.8 g crude drug / kg, and the administration volume was 5 ml / kg.

[0032] 1.1.2 Positive control drug

[0033] Xuezhikang Capsules: Commercially available, produced by Beijing Beida Weixin Biotechnology Co., Ltd., batch number: 20210810. Before the test, it was prepared into a liquid medicine of 0.026 g / ml with 0.5% CMC, and administered to rabbits by gavage at a dose of 0.13 g / kg, and the administration volume was 5 ml / kg.

[0034] 1.2 Composition of High-Fat Feed

[0035] The feed ingredients include: 10% lard, 1% cholesterol, and 89% basal feed mixed. Assisted by Tianjin Wuqing Keda Breeding Center.

[0036] 1.3 Test Reagents

[0037] Cholesterol, batch number: 070802, provided by Tianjin Yingbo Biochemical Reagent Co., Ltd. Cholesterol (TC) kit, batch number: 20200471; triglyceride (TG) kit, batch number: 20201731; low-density lipoprotein cholesterol (LDL-C) kit, batch number: 20210121; high-density lipoprotein cholesterol (HDL-C) kit, batch number: 20200121. Alanine aminotransferase (ALT) kit, batch number: 20210591. All the above kits were provided by Zhong Sheng Bei Kong Biotechnology Co., Ltd., and the fibrinogen (FIB) kit, batch number: BH-25102, was provided by Shanghai Beihai Biotechnology Engineering Co., Ltd.

[0038] 1.4 Test Animals

[0039] 80 healthy male white big-ear rabbits, weighing (2.3 ± 0.2) kg, were provided by Xinglong Experimental Animal Breeding Factory in Haidian District, Beijing, with the certificate number: SCXK(Jing) 2021-0006. They were raised in the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine, and the environmental facilities met the secondary standards of the Experimental Animal Environmental Facilities Standard of the Ministry of Health of the People's Republic of China.

[0040] 1.5 Main instruments for the experiment

[0041] Semi-automatic biochemical analyzer (Model 200 of 95 Edition, VWR, Netherlands; Microlab 300, USA); series blood viscosity meter (LG-R80, Beijing Shidi Scientific Instrument Co., Ltd.); centrifuge (LD5-2A, Beijing Medical Centrifuge Factory); bench-top high-speed refrigerated centrifuge (TGLL-18G, Taicang Medical Instrument Factory); vortex mixer (GL-88B, Qilin Medical Instrument Factory, Haimen, Jiangsu); electrothermal constant temperature water bath (GB11241-89, Beijing Medical Equipment Factory); precision electronic balance (JA1003, Shanghai Tianping Instrument Factory); YouSheng ACS-A type electronic weighing scale (Shanghai Shenheng Electronic Co., Ltd.).

[0042] 2 Experimental methods

[0043] 2.1 Model establishment and grouped administration of drugs

[0044] 80 white big-ear rabbits were taken. After being fed with common feed for adaptation, the rabbits were divided into 2 groups according to the random block method of serum TC: 10 rabbits in the blank group and 70 rabbits in the high-fat feed modeling group. The rabbits in the blank group continued to be fed with common feed, while the rabbits in the high-fat feed modeling group were fed with high-fat feed. After 6 weeks, blood was taken from the marginal ear vein of the rabbits to detect the contents of fasting serum TC, TG, LDL, HDL, and GLU. 60 rabbits with successful high-fat modeling were selected and randomly divided into 5 groups according to the random block method of serum TC: model group, positive control drug - Xuezhikang group, Sanyepian 3.2 g crude drug / kg group, Sanyepian 1.6 g crude drug / kg group, Sanyepian 0.8 g crude drug / kg group, with 12 rabbits in each group. The rabbits in the drug administration groups were given intragastric administration according to the grouping principle for 30 days. The blank group and the model group were given intragastric administration of an equal volume of 0.5% CMC. During the drug administration period, the rabbits were still fed with a small amount of high-fat feed to maintain the model.

[0045] 2.2 Specimen preparation

[0046] At the end of the experiment, the rabbits were fasted (water not prohibited) for 10 h. 1 h after the last intragastric administration, the animals were anesthetized by intravenous injection of 30 mg / kg of 3% pentobarbital sodium through the marginal ear vein. Blood was taken from the abdominal aorta, and 4 ml of whole blood was anticoagulated with heparin sodium to detect the whole blood viscosity (230S -1 、115S -1 、46S -1 、23S -1 、11.5S-1 , 5.75S -1 ), after centrifugation, take the whole blood and inject it into a clean test tube and let it stand still at 4°C, 3500 r·min -1 , centrifuge for 10 min, separate the serum, detect fasting GLU and ALT, and aliquot the remaining serum into 1.5 ml cryopreservation tubes and store them in a -80°C refrigerator for further detection of relevant biochemical indicators.

[0047] After blood collection, quickly dissect the rabbit liver, rinse the liver blood with physiological saline, dry it with filter paper and weigh it. Take a small piece of liver tissue from the middle of the anterior lobe and fix it with 10% formalin for further examination; take another 300 mg of liver tissue from the fixed position in the middle of the liver lobe for detecting the contents of TC (cholesterol) and TG (triglyceride) in the liver tissue. Dissect a small section of the rabbit aorta and fix it with 10% formalin for further examination.

[0048] 2.3 Detection indexes and methods

[0049] Observation of animal growth and feeding: Observe the changes in the spirit, activity, food intake and water intake of the animals during the experiment, and record the animal body weight once a week or every two weeks. Serum biochemical detection: Use a semi-automatic biochemical analyzer (purchase the corresponding reagent kit) to detect serum TC, TG, LDL-C, HDL-C, GLU, ALT, and FIB. TC, TG, LDL-C, HDL-C, and GLU are detected by the end point method; ALT is detected by the continuous monitoring method; FIB is detected by the immunoturbidimetry method. Liver index (%) = liver weight / body weight × 100%.

[0050] Biochemical detection of liver tissue: Accurately weigh 300 mg of liver tissue from the fixed position in the middle of the liver lobe, add 3 ml of ethanol / acetone (1:1) mixture, homogenize, put it into a glass tube, seal and let it stand still, then centrifuge at 3000 rpm for 10 minutes, take the supernatant to detect TC and TG, and the determination method is the same as that of serum.

[0051] 2.4 Data statistics

[0052] The experimental data are expressed as mean ± standard deviation (-x ± s), and are statistically processed using the Spss11.0 software package. One-way ANOVA is used for comparison among multiple groups of measurement data, t-test is used for comparison between two groups, and Raddit analysis is used for rank data. The significance levels are set at 0.05 and 0.01.

[0053] 3 Experimental results

[0054] 3.1 Serum TC, TG, LDL-C, HDL-C, TC-HDL / HDL

[0055] Before drug treatment, compared with the blank group, the serum cholesterol TC in the model group was significantly increased (P < 0.01), and there was no significant difference in serum TC between the model group and each drug-administered group (P > 0.05). After 30 days of drug treatment, the serum TC of the rabbits in the model group was significantly higher than that in the blank group (P < 0.01). From the above results, it can be seen that the drugs in the three-dose groups of the three-leaf tablets and Xuezhikang group can significantly reduce the serum TC of rabbits compared with the model group (P < 0.01).

[0056] Table 1 Effects of three-leaf tablets on serum cholesterol TC in rabbits with hypercholesterolemia

[0057]

[0058]

[0059] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲▲ P < 0.01

[0060] Before and after drug treatment, compared with the blank group, there was no increase in serum triglyceride TG in the model group (P > 0.05). After drug treatment, the serum TG of the rabbits in the 3.2 g crude drug / kg group of the three-leaf tablets was lower than that in the model group (P < 0.05).

[0061] Table 2 Effects of three-leaf tablets on serum triglyceride TG in rabbits with hypercholesterolemia

[0062]

[0063] Note: Compared with the model group ▲ P < 0.05

[0064] Before drug treatment, compared with the blank group, the serum LDL-C in the model group was significantly increased (P < 0.01), and there was no significant difference in serum LDL-C between the model group and each drug-administered group (P > 0.05). After 30 days of drug treatment, compared with the blank group, the serum LDL-C in the model group was significantly increased (P < 0.01). The three-dose groups of the three-leaf tablets and Xuezhikang group significantly reduced the serum LDL-C compared with the model group (P < 0.01).

[0065] Table 3 Effects of three-leaf tablets on serum LDL-C in rabbits with hypercholesterolemia

[0066]

[0067] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲▲ P < 0.01

[0068] Before drug treatment, due to the increase in serum TC in the model group, the serum HDL-C in the model group of rabbits increased significantly. Compared with the blank group (P < 0.01), there was no significant difference in serum HDL-C between the model group and each drug-administered group (P > 0.05).

[0069] After 30 days of drug treatment, the serum HDL-C in the model group was significantly higher than that in the blank group (P < 0.01). In the groups of 3.2 g crude drug / kg of three-leaf, 1.6 g crude drug / kg of three-leaf, and Xuezhikang group, the serum HDL-C was significantly lower than that in the model group (P < 0.01). The results are shown in Table 4.

[0070] Table 4 Effects of three-leaf on serum HDL-C in rabbits with hypercholesterolemia

[0071]

[0072]

[0073] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲▲ P < 0.01

[0074] Before drug treatment, compared with the blank group, the serum TC-HDL / HDL in the model group increased significantly (P < 0.01). Compared with each drug-administered group, there was no significant difference in serum TC-HDL / HDL in the model group (P > 0.05). After 30 days of drug treatment, compared with the blank group, the serum TC-HDL / HDL in the model group increased significantly (P < 0.01). In the three-dose groups of three-leaf, the serum TC-HDL / HDL was significantly lower than that in the model group (P < 0.01). The results are shown in Table 5.

[0075] Table 5 Effects of three-leaf on TC-HDL / HDL in rabbits with hypercholesterolemia

[0076]

[0077] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲ P < 0.05 ▲▲ P < 0.01

[0078] 4.3 Liver index, TC and TG contents in liver tissue

[0079] Compared with the blank group, the liver index in the model group was significantly increased (P < 0.01). The liver indices of rabbits in the three-dose groups of the three-leaf tablets and the Xuezhikang group were lower than those in the model group (P < 0.01 or P < 0.05). Compared with the blank group, the contents of TC and TG in the liver tissue were significantly increased (P < 0.01). The contents of TC and TG in the liver tissue of rabbits in the 3.2 g crude drug / kg group of the three-leaf tablets and the Xuezhikang group were significantly lower than those in the model group (P < 0.01). The TG in the liver tissue of rabbits in the 1.6 g crude drug / kg group and 0.8 g crude drug / kg group of the three-leaf tablets was lower than that in the model group (P < 0.05).

[0080] Table 6 Effects of the three-leaf tablets on liver index, TC and TG contents in liver tissue of rabbits with hypercholesterolemia

[0081]

[0082] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲ P < 0.05 ▲▲ P < 0.01

[0083] 4.4 Serum ALT activity

[0084] Compared with the blank group, the traditional Chinese medicine preparation (three-leaf tablets) of the present invention could significantly improve the activities of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in the liver serum of rabbits, with an increase (P < 0.05). These data indicated that the traditional Chinese medicine preparation of the present invention had a certain therapeutic effect on metabolism-related fatty liver and non-alcoholic fatty liver. The results are shown in Table 7.

[0085] Table 7 Effects of the three-leaf tablets on liver function indexes of rabbits with hypercholesterolemia

[0086]

[0087] Note: Compared with the model group ▲ P < 0.05

[0088] 4.5 Pathological observation of the liver and aorta

[0089] The liver volume of rabbits in the model group was increased, with a gray-yellow and greasy color. Microscopically, diffuse hydropic degeneration and fatty degeneration occurred in all lobules of the liver tissue; the aortic intima was not smooth. Microscopically, the intimal epithelial cells were swollen, losing continuity, and foam cells phagocytosing lipids appeared under the intima. The intima at the lesion site was thickened and protruded into the lumen, but did not invade the deep layer of the intima, belonging to early lesions.

[0090] In the three-dose groups of the three-leaf tablets, the hydropic degeneration and fatty degeneration of the liver tissue in rabbits were alleviated, and the number of cases of swelling of the aortic intimal epithelial cells decreased, with the lesions alleviated.

[0091] 5. Summary of Experimental Results

[0092] The traditional Chinese medicine preparation (Three-Leaf Tablets) of the present invention can reduce the hypercholesterolemia rabbit model induced by high-fat diet, reduce serum TC, LDL-C, TC-HDL / HDL; reduce the liver index, liver tissue TC (total cholesterol), and TG (triglyceride) content of hypercholesterolemia rabbits; the traditional Chinese medicine preparation of the present invention can increase the activity of serum ALT (alanine aminotransferase). The three doses of the traditional Chinese medicine composition of the present invention can alleviate the hydropic degeneration and fatty degeneration of rabbit liver tissue, and alleviate the swelling of aortic intimal epithelial cells. These experimental data also indicate that the traditional Chinese medicine preparation of the present invention can reduce hyperlipidemia in rats and has a therapeutic effect on non-alcoholic fatty liver.

[0093] Example 2 Pharmacological Experiment on the Therapeutic Effect of the Traditional Chinese Medicine Preparation of the Present Invention on Atherosclerosis

[0094] 2 Test Materials

[0095] 2.1 Drugs and Preparation

[0096] 2.1.1 Test Drugs

[0097] Three-Leaf Tablets, brown dry extract powder, provided by the Pharmacy Department of the Traditional Chinese Medicine Research Center of Tianjin University of Traditional Chinese Medicine. The extract yield is 8.8 g dry extract powder / 100 g crude drug (the crude drug content per gram of dry extract powder is 11.36 g), batch number: 2020806. Before the test, it was prepared into a medicinal solution of 0.6 g, 0.3 g, 0.15 g crude drug / ml with distilled water, and administered to rats by gavage at a dose of 6 g, 3 g, 1.5 g crude drug / kg, and the administration volume was 1 ml / 100 g.

[0098] 2.1.2 Positive Control Drugs

[0099] Compound Danshen Dripping Pills: Commercially available, produced by Tianjin Tasly Pharmaceutical Co., Ltd., batch number: 20201122. Before the test, it was prepared into a medicinal solution of 0.015 g / ml with distilled water, and administered to rats by gavage at a dose of 0.15 g / kg, and the administration volume was 1 ml / 100 g.

[0100] 2.2 Test Reagents

[0101] Adrenaline Hydrochloride Injection, batch number: 202110221, provided by Tianjin KingYork Amino Acid Co., Ltd. Heparin Sodium Injection, batch number: 20200601, provided by Tianjin Biochemical Pharmaceutical Factory. Chloral Hydrate, batch number: 20211215, provided by Tianjin Hebei Haijing Fine Chemical Factory. Fibrinogen (FIB) Kit, batch number: BH-25101, provided by Shanghai Beihai Biotechnology Engineering Co., Ltd.

[0102] 2.3 Test Animals

[0103] Sixty Wistar rats, with an equal number of males and females, weighing (260±10) g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., and the certificate number was: SCXK (Jing) 2021-0006. They were housed in the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine, and the environmental facilities met the secondary standards of the Experimental Animal Environmental Facilities Standards of the Ministry of Health of the People's Republic of China.

[0104] 2.4 Main instruments for the experiment

[0105] Series blood viscosity meter (LG-R80, provided by Beijing Shidi Scientific Instrument Co., Ltd.); semi-automatic biochemical analyzer (Model 200 of the 95th edition, VWR, Netherlands); centrifuge (LD5-2A, Beijing Medical Centrifuge Factory); vortex mixer (GL-88B, Qilin Medical Instrument Factory, Haimen, Jiangsu); precision electronic balance (JA1003, Shanghai Tianping Instrument Factory); electronic balance (DT1000, Changshu Weighing Instrument Factory); microscope image capture system (Penguin 600CL, USA).

[0106] 3 Experimental methods

[0107] 3.1 Model establishment, grouping for drug administration and specimen preparation

[0108] After 60 rats were adaptively fed with ordinary feed, they were randomly divided into 6 groups according to body weight by the randomized block method: blank group, model group, positive drug compound Danshen dripping pills control group, Sanyepian 6 g crude drug / kg group (high-dose group), Sanyepian 3 g crude drug / kg group (medium-dose group), Sanyepian 1.5 g crude drug / kg group (low-dose group), with 10 rats in each group.

[0109] (1) Blank group: intragastric administration of 1 ml / 100 g of distilled water; (2) Model group: intragastric administration of 1 ml / 100 g of distilled water; (3) Positive drug: Compound Danshen Dripping Pills control group: intragastric administration of Compound Danshen Dripping Pills liquid medicine, with the daily dosage of 0.15 g / kg; (4) 6 g crude drug / kg group of Sanyepian: the daily dosage is 6 g crude drug / kg; (5) 3 g crude drug / kg group of Sanyepian: the daily dosage is 3 g crude drug / kg; (6) 1.5 g crude drug / kg group of Sanyepian: the daily dosage is 1.5 g crude drug / kg. Each group was intragastrically administered once a day for 14 consecutive days, and the volume of each drug administration was calculated according to 1 ml / 100 g body weight. 1 hour after the last intragastric administration of the drug or distilled water, except for the blank group, the animals in other groups were subcutaneously injected with 0.9 mg / kg of 0.1% hydrochloric acid adrenaline, twice in total, with an interval of 4 hours. Before the second injection, the rats were immersed in ice water for 5 minutes to establish a blood stasis syndrome model, and the blank group was injected with an equal volume of normal saline. After the rats were fasted (water not prohibited) for 18 hours, they were anesthetized by intraperitoneal injection of 280 mg / kg of 3.5% chloral hydrate, and blood was taken from the abdominal aorta, anticoagulated with sodium heparin, and various indicators were detected.

[0110] 3.2 Detection indexes and methods

[0111] Detection of whole blood viscosity and plasma viscosity: The whole blood viscosity was detected by using an LG-R80 series blood viscosity meter. The remaining blood was centrifuged at 3500 r·min -1 , for 10 min to separate plasma, and the plasma viscosity was detected. Fibrinogen content: The plasma fibrinogen content was detected by using a semi-automatic biochemical analyzer (purchasing the corresponding kit), and determined by immunoturbidimetry. Erythrocyte sedimentation rate: Heparinized whole blood was added to an erythrocyte sedimentation rate measuring tube, filled to the scale and then left standing for 1 h, and the reading was taken. Hematocrit: The erythrocyte sedimentation rate measuring tube was centrifuged at 3500 r·min -1 , for 30 min, and the reading was taken.

[0112] 3.3 Statistics

[0113] The experimental data were expressed as mean ± standard deviation , and statistically processed by using the Spss11.0 software package. One-way ANOVA was used for comparison among multiple groups of measurement data, t-test was used for comparison between two groups, and Raddit analysis was used for ranked data. The significance levels were set at 0.05 and 0.01.

[0114] 4 Experimental results

[0115] 4.1 Determination of whole blood viscosity of rats

[0116] Compared with the blank group, the whole blood viscosities at 230I / S, 115I / S, 46I / S, 23I / S, 11.5I / S, and 5.75I / S in the model group were significantly increased (P < 0.01). The whole blood viscosities (230I / S, 115I / S, 46I / S, 23I / S, 11.5I / S, and 5.75I / S) of rats in the three-dose groups of the three-leaf tablets and the compound Danshen group were lower than those in the model group (P < 0.01 or P < 0.05). The results are shown in Tables 8, 9, and 10.

[0117] Table 8 Effects of the three-leaf tablets on the whole blood viscosity of rats with acute blood stasis model

[0118]

[0119] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲▲ P < 0.01

[0120] Table 9 Effects of the three-leaf tablets on the whole blood viscosity of rats with acute blood stasis model

[0121]

[0122] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲ P < 0.05 ▲▲ P < 0.01

[0123] Table 10 Effects of the three-leaf tablets on the whole blood viscosity of rats with acute blood stasis model

[0124]

[0125] Note: Compared with the blank group *P < 0.05 * P < 0.01; compared with the model group ▲ P < 0.05 ▲▲ P < 0.01

[0126] 4.2 Plasma viscosity and fibrinogen content

[0127] Compared with the blank group, the plasma viscosity was significantly increased (P < 0.01). The plasma viscosities of rats in the 6 g crude drug / kg group and 3 g crude drug / kg group of the three-leaf tablets were significantly lower than those in the model group (P < 0.01), and the plasma viscosity of rats in the compound Danshen group was lower than that in the model group (P < 0.05).

[0128] Compared with the blank group, the plasma fibrinogen content in the model group was significantly increased (P < 0.01). The plasma fibrinogen content in the three-dose groups of Sanye Pian was significantly lower than that in the model group (P < 0.01), and the plasma fibrinogen content in the compound Danshen dripping pills group was lower than that in the model group (P < 0.05). The results are shown in Table 11.

[0129] Table 11 Effects of Sanye Pian on Plasma Viscosity and Fibrinogen Content in Rats with Acute Blood Stasis Model

[0130]

[0131] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲ P < 0.05 ▲▲ P < 0.01

[0132] 4.3 Erythrocyte Sedimentation Rate and Hematocrit

[0133] Compared with the blank group, the erythrocyte sedimentation rate and hematocrit in the model group were significantly increased (P < 0.01). The erythrocyte sedimentation rate in the 6 g crude drug / kg group of Sanye Pian and the compound Danshen group was significantly lower than that in the model group (P < 0.01); the hematocrit in the 6 g crude drug / kg group and 3 g crude drug / kg group of Sanye Pian was lower than that in the model group (P < 0.05), and the hematocrit in the compound Danshen group was significantly lower than that in the model group (P < 0.01). The experimental results are shown in Table 12.

[0134] Table 12 Effects of Sanye Pian on Erythrocyte Sedimentation Rate and Hematocrit in Rats with Acute Blood Stasis Model

[0135]

[0136]

[0137] Note: Compared with the blank group ** P < 0.01; compared with the model group ▲ P < 0.05 ▲▲ P < 0.01

[0138] 5. Summary of Experimental Results

[0139] In summary, the traditional Chinese medicine preparation (Sanye Pian) of the present invention can reduce the whole blood viscosity at high, medium, and low shear rates in rats with acute blood stasis model, reduce plasma viscosity, fibrinogen content, erythrocyte sedimentation rate, and hematocrit, and improve blood flow, which also indicates that the traditional Chinese medicine preparation of the present invention can also be used in the treatment of atherosclerosis, cardiovascular and cerebrovascular diseases, cerebral infarction, and coronary heart disease angina.

Claims

1. Application of a traditional Chinese medicine preparation containing mulberry leaves in the preparation of a drug for treating metabolism-related fatty liver.

2. The Chinese medicine preparation according to claim 1, characterized in that: Application of the traditional Chinese medicine preparation in preparing medicine for treating non-alcoholic fatty liver disease.

3. Application of a traditional Chinese medicine preparation containing mulberry leaves in the preparation of drugs for treating hyperlipidemia.

4. The Chinese medicine preparation according to claim 3, characterized in that: The Chinese medicine preparation is used in preparing medicines for treating atherosclerosis and cardiovascular and cerebrovascular diseases.

5. The Chinese medicine preparation according to claim 3, characterized in that: The Chinese medicine preparation is used in preparing medicines for treating cerebral infarction and angina pectoris caused by coronary heart disease.

6. The Chinese medicine preparation according to claim 1, characterized in that: The Chinese medicine preparation is made of Chinese medicinal materials in the following weight ratio and dosage: 12 parts of mulberry leaves, 12 parts of lotus leaves, 12 parts of hawthorn leaves, 15 parts of salvia miltiorrhiza, and 15 parts of red peony root.

7. The Chinese medicine preparation according to claim 1, characterized in that: The dosage form of the traditional Chinese medicine preparation can be: granules, capsules, oral liquids, tablets.

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