Dipsacus root saponin compounds and preparation method thereof and application in preparation of drugs for preventing and treating fatty liver

Abstract

The invention discloses a dipsaponin compound and a preparation method thereof and an application in the preparation of drugs for the prevention and treatment of fatty liver. The preparation method comprises: using dipsaponin medicinal material slices as raw materials, adding an alcohol aqueous solution, heating and refluxing extraction 1-4 times, extracting 1-6h each time, and collecting the extract; the extract is concentrated under reduced pressure to obtain a discontinuous crude extract; after the discontinuous crude extract is dissolved in water, it is loaded into a macroporous adsorption resin, and after washing, impurity removal and elution in sequence, the dipsaponin compound is obtained after the eluent is recovered. The dipsaponin compound provided by the present invention can effectively regulate liver function abnormalities and dyslipidemia caused by fatty liver, inhibit liver tissue oxidative stress response, inhibit lipid deposition in liver cells and block liver cell apoptosis, and has a liver cell repair and protective effect on the pathological changes of fatty liver tissue, high safety, and potential application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a dipsaponin compound and a preparation method thereof, and an application thereof in the preparation of drugs for preventing and treating fatty liver. Background Art

[0002] Dipsacus asper is the dried root of Dipsacus asperoides CYCheng et TMAi (or Dipsacus asper Wall), a plant of the Dipsacus family. It is listed as a top-grade product in the Shennong's Herbal Classic. It has the effects of nourishing the liver and kidney, strengthening tendons and bones, healing fractures, promoting blood circulation, stopping metrorrhagia, and calming the fetus. It is an important medicine for nourishing the liver and kidney and treating bone injuries. It is clinically used to treat low back and knee pain, weak feet, bruises, and fractures caused by liver and kidney deficiency. Dipsacus asper has complex and diverse chemical components, including saponins, iridoids, alkaloids, volatile oils, etc. Modern pharmacological studies have shown that Dipsacus asper extract has pharmacological activities such as inhibiting spontaneous uterine contraction, improving immunity, anti-aging, and antibacterial and anti-inflammatory.

[0003] Fatty liver disease is characterized by diffuse fatty changes in liver cells. It refers to excessive fat accumulation in liver cells that affects the normal function of the liver. It is referred to as fatty liver. Fatty liver is a heterogeneous disease caused by the interaction of genetic susceptibility factors, environmental factors and metabolic stress. It mainly includes alcoholic fatty liver, non-alcoholic fatty liver and special types of fatty liver.

[0004] More than a quarter of adults worldwide have fatty liver disease, and there is no significant difference in the prevalence between Eastern and Western countries. With the increasing number of obese people and people with three highs, the prevalence of fatty liver disease is on the rise, and the age of onset is getting younger, and even children are increasingly suffering from fatty liver disease. In addition, the prevalence of fatty liver disease is as high as more than 50% in patients with obesity, metabolic syndrome, type 2 diabetes, and long-term excessive drinking.

[0005] The liver is the main site of fat metabolism in the human body. Long-term high-fat, high-fructose, high-calorie dietary structure, sedentary lifestyle, and increased alcohol consumption will increase the liver's fat intake and enhance esterification. When fat is overloaded and liver fat metabolism disorders occur, unoxidized fat will be deposited in liver cells. Excessive accumulation will lead to diffuse fat infiltration and diffuse fatty changes in liver cells, i.e. fatty liver. As the disease progresses, it can manifest as simple fatty liver, fatty hepatitis, liver fibrosis and cirrhosis, and may even develop into liver cancer.

[0006] In addition to directly causing decompensated cirrhosis, hepatocellular carcinoma and other lesions, fatty liver can also induce the progression of other chronic liver diseases and participate in the onset of type 2 diabetes and atherosclerosis. At the same time, malignant tumors associated with metabolic syndrome, atherosclerotic cardiovascular and cerebrovascular diseases, and cirrhosis are important factors affecting the quality of life and life expectancy of patients with fatty liver. In addition, a large number of clinical studies have shown that most lipid-lowering drugs are hepatotoxic, mainly because lipid-lowering drugs have a "fat-driving" effect, which "drives" lipids in the blood to the liver, making the fat accumulation in the liver more difficult to handle, making fatty liver more serious, and also causing intrahepatic bile stasis, causing jaundice or drug-induced liver injury, and even leading to cirrhosis and liver failure. The group of patients with fatty liver is huge, and there is currently no specific and effective drug for the treatment of fatty liver on the market in the world, especially for non-alcoholic fatty liver. Only liver-protecting drugs can be used, and long-term medication is required to control the progression of the disease, and it cannot be cured.

[0007] It can be predicted that the harm of fatty liver to human health will continue to increase in the future, and it will become a major epidemic disease for the prevention and treatment of liver diseases worldwide. Therefore, researching and developing new drugs for the treatment and prevention of fatty liver is a new challenge in the field of contemporary medicine, which has important innovative significance and clinical value. The development of new drugs in the field of fatty liver is also a large market with great development potential.

[0008] So far, there has been no report on the preparation of Dipsacus asper saponin compounds and their application in the prevention and treatment of fatty liver diseases.

[0009] In view of this, the present invention is proposed. Summary of the invention

[0010] In order to solve the defects existing in the above-mentioned technology, the present invention provides a dipsaponin compound and a preparation method thereof and application thereof in the preparation of drugs for preventing and treating fatty liver.

[0011] To achieve the above object, the technical solution of the present invention is as follows:

[0012] A method for preparing a dipsaponin compound, comprising:

[0013] S1. Take the medicinal pieces of Dipsacus as raw materials, add alcohol aqueous solution, heat and reflux to extract 1-4 times, each extraction is 1-6h, and the extract is collected;

[0014] S2, concentrating the extract under reduced pressure to obtain a discontinuous crude extract;

[0015] S3. After dissolving the crude extract of Dipsacus asper in water, the extract is loaded onto a macroporous adsorption resin, and washed, impurity-removed and eluted in sequence, and the eluent is recovered to obtain Dipsacus asper saponin compounds.

[0016] In the above technical solution, in step S1, the alcohol aqueous solution is a 65-78v% ethanol aqueous solution.

[0017] Preferably, in the above technical solution, the added mass of the alcohol aqueous solution is 4-7.5 times that of the Dipsacus asper medicinal material slices, heated to reflux 2-3 times, each extraction is 2-4h, and the extracts are collected and combined.

[0018] In the above technical solution, in step S3, the macroporous adsorption resin is AB-8 macroporous adsorption resin.

[0019] Furthermore, in the above technical solution, step S3 is specifically,

[0020] After adding 8-14 times the mass of water to the intermittent crude extract to dissolve it, load it into a resin column filled with AB-8 macroporous adsorption resin with a mass that is 2-3 times the mass of the intermittent crude extract, wash it with 3 times the column volume of water and 2 times the column volume of 30v% ethanol aqueous solution to remove impurities, finally elute it with 3 times the column volume of 70v% ethanol aqueous solution and collect the eluate, recover the ethanol under reduced pressure, and then concentrate and evaporate to dryness.

[0021] In a preferred embodiment of the present invention, the method for preparing the dipsaponin compound further comprises:

[0022] The dipsaponin compounds prepared in step S3 are purified by a normal phase silica gel column, and the eluate whose mobile phase is chloroform-methanol in a volume ratio of 7:3 is collected, and the organic solvent is recovered under reduced pressure and then concentrated and evaporated to dryness to obtain a purified dipsaponin compound.

[0023] Another aspect of the present invention also provides dipsaponin compounds prepared by the above preparation method.

[0024] Specifically, in the above technical solution, the molecular structure formula of the dipsaponin compound is:

[0025]

[0026] in:

[0027] In Dipsacus saponin D, R1=Ara, R2=OH, R3=Glc(1→6)Glc;

[0028] 3-O-(4-O-acetyl)-α-L-arabinopyranosyl hederagenin-28-O-β-D-glucopyranose-(16)-β-D-glucopyranoside R1=(4-O-acetyl)Ara, R2=OH, R3=Glc(1→6)Glc;

[0029] In 3-O-α-L-arabinopyranosyloleanolic acid-28-O-β-D-glucopyranose-(1→6)-β-D-glucopyranoside, R1=Ara, R2=H, R3=Glc(1→6)Glc;

[0030] In Dipsacus saponin M, R1=Ara(1→2)rha, R2=OH, R3=Glc(1→6)Glc;

[0031] In Dipsacus saponin C, R1=Xyl(1→4)glc(1→4)glc(1→3)rha(1→4)rha(1→2)ara, R2=OH, R3=H;

[0032] In Dipsacus saponin B, R1=Glc(1→4)rha(1→6)glc(1→3)rha(1→2)ara, R2=OH, R3=H;

[0033] In Akebia saponin PA, R1=Ara, R2=OH, and R3=H.

[0034] In detail, the dipsaponin compounds are akebiasaponin D (3-O-α-L-arabinopyranosyl hederagenine-28-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranoside), chuanxiongsaponin M (3-O-α-L-arabinopyranose-(1→2)-α-L-rhamnose hederagenine 28-O-β-D-glucopyranose-(1→6)-β-D-glucopyranoside), chuanxiongsaponin C (hederagenine-3-O-α-L-arabinopyranose-(1→2)-α-L-rhamnose-(1→4)-α-L-rhamnose-(1→3)-β-D-glucopyranose-(1→4)-β-D-glucopyranose-(1→4)-β-D-pyranose The invention discloses a mixture of (3-O-α-L-arabinopyranosyl oleanolic acid-28-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl) and (3-O-(4-O-acetyl)-α-L-arabinopyranosyl hederagenin-28-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl)) esters of the present invention.

[0035] In another aspect, the present invention also provides the use of the above-mentioned dipsaponin compounds in the preparation of drugs for preventing and treating fatty liver.

[0036] Specifically, the applications include regulating abnormal liver function and abnormal blood lipids and blood sugar caused by fatty liver, inhibiting oxidative stress response of liver tissue, inhibiting lipid deposition in liver cells and blocking liver cell apoptosis, and repairing and protecting liver cells.

[0037] In addition, the present invention also provides a pharmaceutical composition for preventing and treating fatty liver diseases containing the above-mentioned dipsaponin compounds or prepared from the above-mentioned dipsaponin compounds.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) The present invention provides a simple and easy method for extracting Dipsacus asper saponin compounds from Dipsacus asper decoction pieces;

[0040] (2) The dipsaponin compounds provided by the present invention can effectively regulate abnormal liver function and dyslipidemia caused by fatty liver, inhibit the oxidative stress response of liver tissue, inhibit lipid deposition in liver cells and block liver cell apoptosis, and have liver cell repair and protection effects on histopathological lesions such as diffuse fatty degeneration of liver cells, accompanied by varying degrees of inflammatory cell infiltration and hepatocyte fragmentary necrosis, and apoptotic body formation in fatty liver. That is, they have significant pharmacological effects on the treatment and prevention of fatty liver and related diseases, and have extremely low toxicity, high safety, and potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the HPLC fingerprint of the compound prepared in Example 1 of the present invention;

[0042] Figure 2 This is the HPLC fingerprint of the compound prepared in Example 2 of the present invention;

[0043] Figure 3 The anatomical appearance of the liver of each test group in Example 4 of the present invention;

[0044] Figure 4 The liver pathological sections under the microscope of each test group in Example 4 of the present invention;

[0045] Figure 5 The anatomical appearance of the liver of each test group in Example 5 of the present invention;

[0046] Figure 6 The liver pathological sections under a microscope of each test group in Example 5 of the present invention;

[0047] Figure 7 The anatomical appearance of the liver of each test group in Example 6 of the present invention;

[0048] Figure 8 The anatomical appearance of the liver of each test group in Example 7 of the present invention;

[0049] Fig. 9 These are pathological sections of the liver under a microscope of each test group in Example 7 of the present invention. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0051] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0052] In the examples, unless otherwise specified, all the means used are conventional means in the art.

[0053] As used herein, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0054] In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0055] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, biological materials, etc. used in the following examples are all commercially available unless otherwise specified.

[0056] The Dipsacus asper medicinal material slices used in the following examples were purchased from the Chinese medicinal material market in Yulin City, Guangxi Zhuang Autonomous Region, and were identified as the dried roots of Dipsacus asperoides C.Y.Cheng et TMAi (or Dipsacus asper Wall) of the Dipsacus family by the Guangxi Zhuang Autonomous Region Food and Drug Inspection Institute.

[0057] Example 1

[0058] The preparation process of Dipsacus asper saponin compounds is as follows:

[0059] Take 5.0 kg of Radix Dipsaci medicinal pieces, extract them twice with 70v% ethanol aqueous solution under heating reflux, the amount of extraction solvent is 25 kg each time, the extraction time is 2 h each time, combine the two extracts, recover ethanol under reduced pressure, and dry under reduced pressure at 60°C to prepare 2.2 kg of crude extract of Radix Dipsaci.

[0060] The crude extract of Dipsacus asper (2.2 kg) was dissolved in water and diluted to 25 kg, and loaded into a resin column containing 5 kg of AB-8 macroporous adsorption resin (column diameter 10 cm, length 65 cm), washed with 3 times the column volume of water and 2 times the column volume of 30v% ethanol aqueous solution for impurity removal, and finally eluted with 3 times the column volume of 70v% ethanol aqueous solution and the eluate was collected, ethanol was recovered under reduced pressure, concentrated and evaporated to dryness, and 450g of Dipsacus asper saponin compounds were obtained.

[0061] The prepared Dipsacus saponins were analyzed by HPLC and ELSD with the standard curve method. The results showed that the content of compound 1 was 65.78%, the content of compound 2 was 3.35%, the content of compound 3 was 1.99%, the content of compound 4 was 8.79%, the content of compound 5 was 0.20%, the content of compound 6 was 7.22%, and the content of compound 7 was 10.87%. The HPLC fingerprint of the prepared Dipsacus saponins is as follows: Figure 1 shown.

[0062] Example 2

[0063] The specific process of refining Dipsacus asper saponin compounds is as follows:

[0064] The dipsaponin compounds prepared in Example 1 were separated using a normal phase silica gel column (Φ120 cm×9 cm), and the eluent whose mobile phase was chloroform-methanol in a volume ratio of 7:3 was collected, and the organic solvent was recovered under reduced pressure and then concentrated and evaporated to dryness to obtain a refined dipsaponin compound; the HPLC fingerprint of the refined dipsaponin compound obtained was as follows: Figure 2 shown.

[0065] The components and mass content ratio of the refined products of Dipsacus asper saponins were determined and analyzed by HPLC and ELSD using the standard curve method. The content detection results are shown in Table 1 below.

[0066] Table 1 Content test results of refined products of Dipsacus asper saponins

[0067]

[0068] Example 3

[0069] The specific process of separation and purification of the refined products of Dipsacus asper saponin compounds is as follows:

[0070] (1) The refined product of the dipsaponin compound prepared in Example 2 was separated and purified by using a reverse phase silica gel column (Φ60 cm×5 cm), and the eluent whose mobile phase was methanol-water 3:7 was collected, methanol was recovered under pressure, and evaporated to dryness to obtain 15.74 g of white powder A (compound 6, content 98.22%); the eluent whose mobile phase was methanol-water 2:8 was collected, methanol was recovered under pressure, and evaporated to dryness to obtain 156.93 g of white powder B (compound 1, content 98.45%); the eluent whose mobile phase was methanol-water 1:9 was collected, methanol was recovered under pressure, and evaporated to dryness to obtain 31.32 g of white powder C (compound 7, content 98.05%);

[0071] (2) The residue after separation and purification by reverse phase silica gel column was eluted by Sephadex LH-20 gel column chromatography (purchased from Beijing Huideyi Technology Co., Ltd.) and methanol system, and one fraction was collected for every 10 mL; reverse phase high performance liquid chromatography (Agilent 1200) was used to analyze and combine to obtain fractions a, b, c, d, e and f. Among them:

[0072] The fraction f was separated by reverse phase high performance liquid chromatography (Agilent 1200), and the elution components corresponding to the absorption peak at a wavelength of 210 nm were collected in sequence, and the sample peak with a retention time of 27.0 min was recovered under reduced pressure to obtain 1.9 mg of white powder D (Akebia saponin PA, compound 5); the fraction b was separated by reverse phase high performance liquid chromatography (Shimadzu LC-20AT), and the elution components corresponding to the absorption peak at a wavelength of 210 nm were collected in sequence, and the sample peaks with retention times of 19.3 min and 19.7 min were recovered under reduced pressure to obtain 1.2 mg of white powder E (Dipsaconin C, compound The fractions c and d were combined for gradient elution using ODS (5 μm) medium-pressure column chromatography (purchased from Beijing Huideyi Technology Co., Ltd.), and the sample eluate with a retention time of 37-41 min was concentrated and separated by reverse-phase high performance liquid chromatography, and the elution components corresponding to the absorption peak at a wavelength of 210 nm were collected in sequence, and the sample peak with a retention time of 5.8 min was recovered under reduced pressure to obtain 5.8 mg of white powder G (Dipsacoside M, compound 2).

[0073] The chemical composition analysis and structure identification are as follows:

[0074] (1) Appearance: All are amorphous white powder;

[0075] (2) Solubility: They are easily soluble in methanol and ethanol, and slightly soluble in low-polarity organic solvents such as acetone and chloroform;

[0076] (3) UV spectrum: The UV spectrum of the methanol solution of compound 1-6 has a maximum absorption peak at 210 nm;

[0077] (4)Optical rotation value:

[0078] Compound 1, [α]25D+20 (c 0.025, MeOH);

[0079] Compound 2, [α]25D+8 (c 0.025, MeOH);

[0080] Compound 3, [α]25D+8 (c 0.025, MeOH);

[0081] Compound 4, [α]25D+16 (c 0.025, MeOH);

[0082] Compound 5, [α]25D+87.99 (c 0.025, MeOH);

[0083] Compound 6, [α]25D+9 (c 0.1, MeOH).

[0084] (5) High-resolution mass spectrometry:

[0085] The HRESIMS mass spectrum of (3-O-α-L-pyranosyl arabinosyl hederagenin 28-O-β-D-pyranose glucopyranose-(1→6)-β-D-pyranose glucopyranoside; compound 1) showed that its [M+HCOO-]-peak was m / z 973.5019, and the most likely molecular formula was C 47 H 76 O 18 ;

[0086] (3-O-α-L-arabinopyranose-(1→2)-α-L-rhamnopyranose hederagenin 28-O-β-D-glucopyranose-(1→6)-β-D-glucopyranose ester; compound 2) HRESIMS mass spectrum, showing its [MH]-peak at m / z 1073.5599, and providing the most likely molecular formula as C 53 H 86 O 22 .

[0087] (Hederasapogenin-3-O-α-L-arabinopyranose-(1→2)-α-L-rhamnose-(1→4)-α-L-rhamnose-(1→3)-β-D-glucopyranose-(1→4)-β-D-glucopyranose-(1→4)-β-D-xylopyranose ester; Compound 3) HRESIMS mass spectrum, showing its [MH]-peak at m / z 1351.6541, and providing the most likely molecular formula as C 64 H 104 O 30 .

[0088] (Hederasapogenin-3-O-α-L-arabinopyranose-(1→2)-α-L-rhamnose-(1→3)-β-D-glucopyranose-[(1→6)-α-L-rhamnose](1→4)-β-D-glucopyranose ester; compound 4) HRESIMS mass spectrum, showing its [MH]-peak at m / z 1219.6117, and providing the most likely molecular formula as C 59 H 96 O 26 .

[0089] (Hederasapogenin-3-O-α-L-arabinopyranosyl ester glycoside; Compound 5) HRESIMS mass spectrum, showing its [M+Na]+ peak at m / z 627.3897, and providing the most likely molecular formula as C 35 H 56 O8.

[0090] The HRESIMS mass spectrum of compound 3-O-α-L-arabinopyranose oleanolic acid 28-O-β-D-pyranose glucopyranose-(1→6)-β-D-pyranose glucopyranose ester; compound 6) showed that its [M+Na]+ peak was m / z 935.5050, and the most likely molecular formula was C 47 H 76 O 17 .

[0091] (6) Nuclear Magnetic Resonance Spectroscopy:

[0092] The NMR test of the above compounds 1-6 showed that 1 H-NMR and 13 C-NMR, the NMR spectra of compounds 1-6 were studied and 13 The C-NMR signals were assigned and are shown in Tables 2 and 3.

[0093] Table 2 Compounds 1-6 13 C-NMR spectrum peaks

[0094]

[0095]

[0096] Table 3 Glycosides of Compounds 1-6 13 C-NMR spectrum peaks

[0097]

[0098]

[0099] The final structure is as follows:

[0100]

[0101] Example 4: The preventive and therapeutic effects of saponin compounds on non-alcoholic fatty liver disease

[0102] 70 healthy adult Kunming mice (SPF grade, half male and half female, weighing 20±2g) were randomly divided into seven groups (10 mice in each group, half male and half female) for 45 days.

[0103] Normal control group: fed with normal feed and given normal saline by gavage;

[0104] Model group: fed with high-fat diet and given normal saline by gavage;

[0105] Positive control group: fed with high-fat diet and given fenofibrate (produced by French Libofani Pharmaceutical Company) 0.15 g / kg·d by gavage;

[0106] Low-dose Dipsacus saponins group: fed with high-fat diet, gavage with 0.25 g / kg·d of Dipsacus saponins;

[0107] High-dose Dipsacus saponins group: fed with high-fat diet, gavage with 0.50 g / kg·d of Dipsacus saponins;

[0108] The low-dose group of refined Dipsacus saponin compounds was fed with a high-fat diet and intragastrically administered with 0.25 g / kg·d of refined Dipsacus saponin compounds;

[0109] The high-dose group of refined Dipsacus saponin compounds was fed with a high-fat diet and intragastrically administered with 0.50 g / kg·d of refined Dipsacus saponin compounds;

[0110] The mice were gavaged once a day at 0.2 mL / 20 g (volume / body weight).

[0111] After the experiment, the mice were fasted for 12 hours after the last oral gavage, with free access to water. Blood was collected from the eyeballs and the mice were killed, and the plasma was separated and stored at -20°C for the measurement of various biochemical indices. The mice were killed by dislocating the neck, and the laparotomy was performed to quickly separate the body fat (the greater omentum in the abdomen and the fat around the kidneys in the posterior abdomen). The liver was taken out and weighed separately. A larger liver lobe was cut off and fixed in 4% formalin. The liver was stained with HE to observe the pathological changes.

[0112] SPSS11.5 software was used for data statistics and significance test of differences.

[0113] The experimental results are expressed as mean ± standard deviation. One-way analysis of variance was used to compare the means among multiple groups, and the inter-group t-test was used for statistical analysis. P < 0.05 was considered significant. The results are as follows.

[0114] Table 4 Effects on liver function indexes in mice with non-alcoholic fatty liver disease

[0115]

[0116] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0117] The data in the above table show that compared with the normal control group, the blood transaminase AST, ALT, MDA, FFA, liver index and fat index of the mice in the model group were significantly increased (P<0.01), and SOD was significantly decreased (P<0.01), reflecting non-alcoholic fatty liver disease and abnormal liver function.

[0118] Compared with the model group, there were no significant differences in the AST, ALT, MDA, SOD, FFA and other indicators of mice in the positive control group, indicating that the positive drug fenofibrate (lipid-lowering drug) had no effect on the abnormal liver function caused by obesity-induced non-alcoholic fatty liver.

[0119] The blood transaminase AST, ALT, MDA, FFA, liver index and fat index of mice in the low-dose of Dipsacus asper saponin compound group, high-dose of Dipsacus asper saponin compound group, low-dose of Dipsacus asper saponin compound refined product group and high-dose of Dipsacus asper saponin compound refined product group were significantly decreased (P<0.01, or P<0.05), and SOD was significantly increased (P<0.01, or P<0.05).

[0120] Comprehensive analysis of the above results shows that Dipsacus asper saponin compounds and refined Dipsacus asper saponin compounds have obvious protective and restorative effects on abnormal liver function caused by obesity-induced non-alcoholic fatty liver disease.

[0121] Table 5 Effects on blood lipid indexes in mice with non-alcoholic fatty liver disease

[0122]

[0123] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0124] The data in the above table show that compared with the normal control group, the leptin, CHOL and LDL in the blood of the mice in the model group were significantly increased (P<0.01, or P<0.05), and the HDL was significantly decreased (P<0.01). The mice developed non-alcoholic fatty liver disease, hyperlipidemia, and symptoms of liver lipid oxidation damage.

[0125] Compared with the model group, CHOL and TG in the positive control group were significantly decreased (P<0.01, or P<0.05), and there were no differences in other indicators.

[0126] The leptin, CHOL and LDL levels of mice in the low-dose Dipsacus saponin compound group, high-dose Dipsacus saponin compound group, low-dose Dipsacus saponin compound refined product group and high-dose Dipsacus saponin compound refined product group were significantly decreased (P<0.01, or P<0.05), and the HDL levels were significantly increased (P<0.01).

[0127] Comprehensive analysis of the above results shows that Dipsacus asper saponin compounds and refined Dipsacus asper saponin compounds have obvious repairing effects on hyperlipidemia and lipid peroxidation damage caused by non-alcoholic fatty liver disease.

[0128] The anatomical appearance of the liver Figure 3 As shown, the normal control group ( Figure 3-1 ) mice had dark red livers, smooth surfaces, sharp edges, and soft textures; the model group ( Figure 3-2 ) and positive control group ( Figure 3-3 ) had enlarged livers, full of fat, whitish in color, blunted edges, brittle and greasy in cross section, and some had severe fatty liver inflammation such as jaundice; low-dose Dipsacus saponins group ( Figure 3-4 ) and low-dose Dipsacus asper saponins group ( Figure 3-5 The liver color and size of the mice in the control group were close to those in the control group, and were significantly improved compared with the model group.

[0129] Figure 4 The results of liver pathological sections under a microscope show that the model group ( Figure 4-2 ) mice had typical pathological features of non-alcoholic fatty liver disease, mainly manifested by a large number of fat particles in the liver cells, round and tense fat vacuoles of varying sizes in the cytoplasm of the liver cells, mainly located in the peripheral area of ​​the liver lobules, squeezing the nucleus to one side, and most of the liver cells were enlarged. The hepatic sinusoids were occluded due to the compression of the enlarged liver cells; the positive control group ( Figure 4-3 ) Compared with the model group, the fat particles in the mouse liver cells did not decrease significantly; while the low-dose Dipsacus saponins group ( Figure 4-4 ) and low-dose Dipsacus asper saponins group ( Figure 4-5The liver tissue morphology of the mice was similar to that of normal mice. The fat vacuoles in the hepatocytes were significantly reduced, and some mice no longer had fat vacuoles. The hepatic sinusoids reappeared, and the volume of hepatocytes almost returned to normal.

[0130] Example 5 Comparison of the preventive and therapeutic effects of refined saponin compounds and monomeric active ingredients on non-alcoholic fatty liver disease

[0131] 120 healthy adult Kunming mice (SPF grade, half male and half female, weighing 20±2g) were randomly divided into 6 groups (20 mice in each group, half male and half female) for 45 days.

[0132] Normal control group: fed with normal feed and given normal saline by gavage;

[0133] Model group: fed with high-fat diet and given normal saline by gavage;

[0134] The group of refined Dipsacus saponin compounds was fed with high-fat diet and intragastrically administered with 0.50 g / kg·d of refined Dipsacus saponin compounds;

[0135] Compound 1 group: fed with high-fat diet and intragastrically administered with compound 1 at a dose of 0.50 g / kg·d;

[0136] Compound 2 group: fed with high-fat diet and intragastric administration of compound 2 at a dose of 0.50 g / kg·d;

[0137] Compound 3 group: fed with high-fat diet and intragastric administration of compound 3 at a dose of 0.50 g / kg·d;

[0138] The mice were gavaged once a day at 0.2 mL / 20 g (volume / body weight).

[0139] After the experiment, the mice were fasted for 12 hours after the last oral gavage, with free access to water. Blood was collected from the eyeballs and the mice were killed, and the plasma was separated and stored at -20°C for the measurement of various biochemical indices. The mice were killed by dislocating the neck, and the laparotomy was performed to quickly separate the body fat (the greater omentum in the abdomen and the fat around the kidneys in the posterior abdomen). The liver was taken out and weighed separately. A larger liver lobe was cut off and fixed in 4% formalin. The liver was stained with HE to observe the pathological changes.

[0140] SPSS11.5 software was used for data statistics and significance test of differences. The experimental results were expressed as mean ± standard deviation, and one-way analysis of variance was used for comparison of means among multiple groups, and t-test between groups was used for statistical processing, with P < 0.05 as the significant level. The results are as follows.

[0141] Table 6 Effects on liver function indexes of female mice with non-alcoholic fatty liver disease

[0142]

[0143] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0144] Table 7 Effects on liver function indices of male mice with non-alcoholic fatty liver disease

[0145]

[0146] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0147] The data in the above table show that compared with the normal control group, the blood transaminase AST, ALT, MDA, FFA, liver index and fat index of the mice in the model group were significantly increased (P<0.01, or P<0.05), SOD was significantly decreased (P<0.01, or P<0.05), and the mice developed non-alcoholic fatty liver disease; compared with the model group, the AST, ALT, MDA, FFA, liver index and fat index of the mice in the refined group of Dipsacus saponins were significantly decreased (P<0.01), and SOD was significantly increased (P<0.01, or P<0.05); while the blood transaminase AST, ALT and FFA of the mice in the compound 1 group, compound 2 group and compound 3 group were significantly decreased (P<0.01, or P<0.05), and there were no significant changes in the MDA, SOD, liver index and fat index.

[0148] It can be seen that the refined products of Dipsacus asper saponin compounds have a significant therapeutic effect on obesity-induced non-alcoholic fatty liver disease, while compound group 1, compound group 2 and compound group 3 only show a certain trend of action. That is to say, the effective combination of compounds 1-7 in a certain mass ratio has a more significant pharmacological activity, and its effect is significantly better than that of the active ingredients of Dipsacus asper saponin compounds 1, compound 2 and compound 3.

[0149] Table 8 Effects on blood lipid indexes in female mice with non-alcoholic fatty liver disease

[0150]

[0151] Note: Compared with the normal group, # P<0.05, ##P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0152] Table 9 Effects on blood lipid indexes in male mice with non-alcoholic fatty liver disease

[0153]

[0154] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0155] The above data show that compared with the normal control group, the CHOL and LDL of the mice in the model group were significantly increased (P<0.01, or P<0.05), TG and HDL were significantly decreased (P<0.01, P<0.05), and the blood glucose of male mice was significantly increased (P<0.01). The mice developed non-alcoholic fatty liver disease, hyperlipidemia, hyperglycemia, liver lipid oxidation damage and other symptoms; compared with the model group, the CHOL and LDL of the mice in the refined group of Dipsacus saponins were significantly decreased (P<0.01, or P<0.05), HDL was significantly increased (P<0.05), and the blood glucose of male mice was significantly decreased (P<0.01); the CHOL, TG, HDL, LDL and blood glucose of the mice in the compound 1 group, compound 2 group and compound 3 group did not change significantly. It can be seen that the refined group of Dipsacus saponins, compound 1, compound 2 and compound 3 have a significant repair effect on hyperlipidemia, hyperglycemia and lipid peroxidation damage caused by non-alcoholic fatty liver.

[0156] The anatomical appearance of the liver Figure 5 As shown, the normal control group ( Figure 5-1 ) mice had dark red livers, smooth surfaces, sharp edges, and soft textures; compared with the normal control group ( Figure 5-1 ) compared with the model group ( Figure 5-2 ) The body fat index and liver index of mice were significantly increased, the liver was enlarged, full of fat, yellowish in color, with obvious fat particles, blunt edges, brittle texture, greasy cross-section, and some livers showed severe fatty hepatitis such as jaundice; the group of refined dipsacon saponin compounds ( Figure 5-3 ) mice liver color and size were close to the normal group, and were significantly improved compared with the model group; Compound 1 group ( Figure 5-4 )、Compound 2 Group ( Figure 5-5 ) and compound group 3 ( Figure 5-6 ) mice still had a certain degree of liver enlargement, with a yellowish color, which was different from the model group ( Figure 5-2) showed some improvement compared with the normal control group ( Figure 5-1 ) compared with the control group, there are still certain manifestations of fatty liver.

[0157] Figure 6 The liver pathological sections under the microscope show that the model group ( Figure 6-2 ) mice showed typical pathological features of non-alcoholic fatty liver disease, mainly manifested by a large number of fat vacuoles in the peripheral hepatocytes of the hepatic lobules, hepatocyte enlargement, even hepatocyte rupture and diffuse fatty hepatitis, and the hepatic sinusoids were blocked due to compression by hepatocyte enlargement; the refined group of Dipsacus saponin compounds ( Figure 6-3 ) mice liver tissue morphology was similar to that of normal mice, fat vacuoles in hepatocytes were significantly reduced, some no longer had fat vacuoles, hepatic sinusoids reappeared, and the volume of hepatocytes returned to normal; Compound 1 group ( Figure 6-4 )、Compound 2 Group ( Figure 6-5 ) and compound group 3 ( Figure 6-6 A small amount of fat vacuoles were still visible in the liver cells of the ) mice, and certain fatty liver symptoms still existed, but compared with the model group, there was a clear decreasing trend.

[0158] Example 6 Comparison of the protective effects of refined saponin compounds and monomeric active ingredients on CCl4-induced acute liver injury in mice

[0159] Sixty healthy adult Kunming mice (SPF grade, male, weight 20±2g) were randomly divided into 5 groups (12 mice in each group) and fed adaptively for 1 day with free diet. Specifically, they were:

[0160] Normal control group: normal saline was given by gavage;

[0161] Model group: normal saline was given by gavage;

[0162] The group of refined Dipsacus saponin compounds: oral administration of refined Dipsacus saponin compounds 0.50 g / kg·d;

[0163] Compound 4 group: intragastric administration of compound 4, the dose was 0.50 g / kg·d;

[0164] Compound 5 group: intragastric administration of compound 5, the dose was 0.50 g / kg·d;

[0165] According to the dosage, mice in each group were given oral administration once a day, 0.2mL / 20g each time, for 7 consecutive days. Except for the normal control group, the rest of the mice were intraperitoneally injected with 10mL / kg of 0.2% CCl4 2h after the last administration, fasting but not water, and blood was collected by removing the eyeballs 18h later and the serum was separated and stored at -20℃ for various biochemical indicators to be tested; after blood collection, the mice were killed by dislocating the cervical vertebrae, and the liver was quickly dissected and removed, rinsed with physiological saline, dried with filter paper and weighed, the right lobe of the liver was placed in 4% formaldehyde fixative, HE stained, and pathological changes were observed, and the rest of the liver was stored in a -20℃ refrigerator.

[0166] SPSS11.5 software was used for data statistics and significance test of differences. The experimental results were expressed as mean ± standard deviation, and one-way analysis of variance was used for comparison of means among multiple groups, and t-test between groups was used for statistical processing, with P < 0.05 as the significant level. The results are as follows.

[0167] Table 10 Protective effect on acute liver injury induced by CCl4 in mice

[0168]

[0169]

[0170] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0171] The above data show that compared with the normal control group, the ALT and AST in the serum of the mice in the model group were significantly increased, the MDA content in the liver was significantly increased, the SOD and GSH contents were significantly decreased, and the liver weight and liver index were significantly increased, indicating liver damage, indicating that the CCl4 model was successful; compared with the model group, the liver weight and liver index of the mice in the refined group of Dipsacus saponin compounds were significantly decreased, the ALT and AST in the serum were significantly decreased, the MDA content in the liver of the mice was significantly decreased, and the SOD and GSH contents were significantly increased; the ALT and AST in the serum of the mice in the compound 4 group and the compound 5 group were significantly decreased, and the contents of MDA, SOD and GSH in the liver did not change significantly.

[0172] The anatomical appearance of the liver Figure 7 As shown, the normal control group ( Figure 7-1 ) The liver of mice was reddish brown in appearance, soft and elastic in texture. Under the microscope, the liver lobule structure was clear, the cells were neatly arranged, uniform in size, and clearly demarcated. The nucleus was located in the center of the cell, round and clear; the model group ( Figure 7-2) The liver of mice was yellowish, oily, and had increased capsule tension, and was generally granular. Under the microscope, it was observed that the liver lobules were obviously degenerated and necrotic. The lesion type was mainly lamellar necrosis, cytoplasmic aggregation, accompanied by watery degeneration and fatty degeneration. The refined group of Dipsacus saponin compounds ( Figure 7-3 ) mice had significantly fewer fat vacuoles in liver cells, milder liver cell degeneration, visible liver sinusoids, and liver tissue morphology similar to that of normal mice; Compound 4 group ( Figure 7-4 ) and compound group 5 ( Figure 7-5 ) The arrangement of hepatocytes in the liver of mice was still disordered to a certain extent, the cytoplasm was loosened, and there were still scattered punctate or focal necrosis in the portal areas and hepatic lobules, and there were still a large number of inflammatory cells infiltrating.

[0173] In summary, the refined products of Dipsacus asper saponins can significantly improve CCl4-induced liver peroxidation damage in mice. The mechanism may be that it is through counteracting the damage of lipid peroxides to hepatocytes, enhancing the activity of cellular antioxidant enzymes, and improving the antioxidant capacity of hepatocytes, thereby maintaining the integrity of the hepatocyte plasma membrane and enabling hepatocytes to perform normal defense and compensatory functions; while compounds 4 and 5 have no obvious effect on CCl4-induced liver peroxidation damage in mice.

[0174] Example 7 Preventive and therapeutic effects on fatty liver in ovariectomized rats

[0175] Fifty healthy adult Kunming rats (SPF grade, female, weighing 200±2g) were randomly divided into 5 groups (10 rats in each group), including:

[0176] Normal control group: normal saline was given by gavage;

[0177] Model group: normal saline was given by gavage;

[0178] The group of refined Dipsacus saponin compounds: oral administration of refined Dipsacus saponin compounds 0.50 g / kg·d;

[0179] Compound 6 group: intragastric administration of compound 6, the dose was 0.50 g / kg·d;

[0180] Compound 7 group: Compound 7 was administered intragastrically at a dose of 0.50 g / kg·d.

[0181] Bilateral ovariectomy (OVX) was performed after intraperitoneal injection of 45 mg / kg of 3% sodium pentobarbital for anesthesia. The ovarian tissue of the rats was surgically removed with the capsule intact. The normal control group underwent sham surgery to cut the skin, muscle and peritoneum, and only a small piece of fat tissue was removed, but the ovaries were not removed. After surgery, the rats were fed with ordinary feed for 3 months. Three months after ovariectomy, the rats were gavaged with drugs for 3 months, and the body weight was measured weekly to adjust the dosage.

[0182] The experiment was terminated after 6 months. The rats were anesthetized with urethane and blood, fat and liver samples were collected for testing. The serum was separated from the liquid blood and stored at -20℃ for testing of various biochemical indicators. After blood collection, the rats were killed by dislocating the cervical vertebrae, and the liver was quickly dissected and removed. It was rinsed with saline, dried with filter paper and weighed. The right lobe of the liver was placed in 4% formaldehyde fixative and stained with HE to observe pathological changes. The rest of the liver was stored in a -20℃ refrigerator.

[0183] SPSS11.5 software was used for data statistics and significance test of differences. The experimental results were expressed as mean ± standard deviation, and one-way analysis of variance was used for comparison of means among multiple groups, and t-test between groups was used for statistical processing, with P < 0.05 as the significant level. The results are as follows.

[0184] Table 11 Effects on liver function indexes of fatty liver in ovariectomized rats

[0185]

[0186]

[0187] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group, * P<0.05, ** P<0.01.

[0188] The above data show that compared with the normal control group, the blood transaminase AST, ALT, FFA, and liver index of the rats in the model group were significantly increased (P<0.01, or P<0.05), and the rats had non-alcoholic fatty liver disease; compared with the model group, the AST, ALT, FFA, liver index and other indicators of the rats in the group of refined Dipsacus saponins were significantly reduced (P<0.01, or P<0.05), which shows that the refined Dipsacus saponins have a good therapeutic effect on fatty liver in ovariectomized rats; while the blood transaminase AST and ALT of the rats in the compound 6 group and the compound 7 group were significantly reduced (P<0.05), and there was no significant change in other indicators. Therefore, compared with compound 6-7, the refined Dipsacus saponins have a more obvious therapeutic effect on fatty liver caused by estrogen secretion disorders in ovariectomized rats.

[0189] Table 12 Effects on blood lipid indexes of fatty liver in ovariectomized rats

[0190]

[0191] Note: Compared with the normal group, # P<0.05, ## P<0.01; compared with the model group,* P<0.05, ** P<0.01.

[0192] The above data show that compared with the normal control group, the blood glucose, CHOL and LDL of the rats in the model group were significantly increased (P<0.01), TG and HDL were significantly decreased (P<0.01, P<0.05), and the rats showed abnormal indicators such as hyperlipidemia, hyperglycemia, and liver lipid oxidation damage; compared with the model group, the blood glucose, CHOL, and LDL of the rats in the group of refined dipsacon saponins were significantly decreased (P<0.01, P<0.05), and HDL was significantly increased (P<0.01); while there were no significant differences in blood glucose, CHOL and TG of the rats in the compound 6 group and compound 7 group, HDL was significantly increased (P<0.05), and LDL was significantly decreased (P<0.05). Therefore, the refined dipsacon saponins have a significant repair effect on hyperlipidemia, hyperglycemia and lipid peroxidation damage caused by fatty liver caused by estrogen secretion disorders in ovariectomized rats; while compounds 6 and 7 only have a certain repair effect on liver lipid peroxidation damage, and have no effect on hyperlipidemia and hyperglycemia caused by fatty liver.

[0193] The anatomical appearance of the liver Figure 8 As shown, the normal control group ( Figure 8-1 ) rats' livers were dark red, smooth, sharp-edged, and soft; the model group ( Figure 8-2 ) rats had enlarged livers, full of fat, whitish color, blunt edges, brittle texture, and greasy cut surface. Some of the livers showed severe fatty liver inflammation such as jaundice. The refined group of Dipsacus saponin compounds ( Figure 8-3 ) rats' liver color and size were close to those of the normal group and compared with the model group ( Figure 8-2 ) were significantly improved; Compound 6 group ( Figure 8-4 )、Compound 7 Group ( Figure 8-5 ) showed slight improvement in the liver, but still showed signs of fatty hepatitis with liver enlargement.

[0194] Fig. 9 The results of liver pathological sections under a microscope show that the model group ( Figure 9-2 ) rats' livers had developed typical pathological features of non-alcoholic fatty liver disease, mainly manifested by a large number of fat particles in the liver cells, the appearance of round, tense fat vacuoles of varying sizes in the liver cell cytoplasm, mainly located in the peripheral area of ​​the liver lobule, squeezing the nucleus to one side, most of the liver cells were swollen, and the liver sinusoids were occluded due to the compression of the swollen liver cells; the refined group of Dipsacus saponin compounds ( Figure 9-3 ) rats' liver tissue morphology was similar to that of normal mice, with a significant reduction in fat vacuoles in hepatocytes, and some no longer having fat vacuoles, sinusoids reappeared, and the volume of hepatocytes almost returned to normal; while compound 6 group ( Figure 9-4 )、Compound 7 Group ( Figure 9-5 ) There was a certain trend of decreasing fat particles in rat liver cells, but fatty liver hepatitis still existed.

[0195] Example 8 Inhibition of adipocyte growth and proliferation

[0196] Mouse preadipocytes 3T3-L1 cells were inoculated in 96-well culture plates and cultured on the third day. The cell culture medium was replaced and the cells were divided into groups and a certain concentration of Dipsacus root saponin compounds, refined Dipsacus root saponin compounds and compounds 1-7 were added to make the final concentrations of the drugs reach 0, 37.5, 75, 150, 300 and 600 μg·mL, respectively. -1 Each treatment group was set up with 6 replicates, with 0 μmol·mL -1 As a control, MTT assay was performed at the 48h time point of sample action. The absorbance of each group was measured at 490nm using an enzyme-linked detector, and the cell survival rate of each experimental group was calculated. The results are shown in the table below.

[0197] Table 13 Effect on the survival rate of preadipocytes

[0198]

[0199] The results in the table above show that compared with 0 μmol·mL-1, the concentrations of Dipsacus saponins, Dipsacus saponins refined products and compounds 1-7 were 37.5-150 μg·mL -1 The survival rate of mouse preadipocyte 3T3-L1 cells was slightly reduced within the concentration range. The cell survival rate did not change much within this concentration range, and the cells were in good condition under the microscope. When the drug concentration was 300-600 μg mL -1 When treated with 24h, the survival rate of mouse preadipocytes 3T3-L1 cells was significantly reduced, which means that it had an inhibitory effect on the growth of mouse preadipocytes 3T3-L1 cells.

[0200] Cells were inoculated into 6-well cell culture plates and divided into normal cell control group, induction group, Dipsacus saponin compound group, Dipsacus saponin compound refined group and Compound 1-7 group. Normal cells were routinely cultured with DMEM / Ham's containing 10% NCS. After the cells of the induction group and sample intervention group (including Dipsacus saponin compound group, Dipsacus saponin compound refined group and Compound 1-7 group) were cultured until 100% of the cells were confluent, induction solution I (DMEM, 10% fetal bovine serum, 0.5mmol·L-1IBMX, 1μmol·L-1 dexamethasone, 1.7μmol·L-1 insulin) was added. Different concentrations of Dipsacus saponin compounds, Dipsacus saponin compound refined group and Compound 1-7 (37.5, 75, 150, 300μg·mL-1 ), the induction group was not treated with drugs but with an equal amount of PBS. After 14 hours of culture, the cells were digested with 0.25% trypsin plus 0.02% EDTA, centrifuged at 1000rpm for 5 minutes, collected, washed with PBS, centrifuged at 1000rpm for 10 minutes, repeated 3 times, fixed with 75% ethanol precooled at 4℃ for 48 hours, washed with PBS to remove the alcohol, resuspended with 2mL PBS, incubated with 50μg·mL-1 RNaseA at 37℃ for 30 minutes, cooled, added PI to each sample to a final concentration of 1.5mg·mL-1, stained in an ice bath away from light for 30 minutes, filtered with a 300-mesh nylon mesh, and detected the cell cycle by flow cytometry. The results are shown in the table below.

[0201] Table 14 Effects on the survival rate of S phase cells of preadipocytes

[0202]

[0203]

[0204] The results in the table above show that the saponin compounds, the refined saponin compounds and compounds 1-7 have a concentration of 37.5-600 μg·mL -1 Within this range, the proportion of cells in the S phase of mouse preadipocytes 3T3-L1 cells was dose-dependent, and the proportion of cells in the S division phase decreased with the increase of drug concentration. Moreover, under the microscope, it can be seen that the preadipocytes are in good condition. It can be seen that the Dipsacus saponin compounds, the refined Dipsacus saponin compounds and compounds 1-7 can effectively inhibit the growth cycle of mouse preadipocytes 3T3-L1 cells, thereby inhibiting the proliferation of preadipocytes.

[0205] Example 9 Acute toxicity study

[0206] 100 healthy adult Kunming mice (SPF grade, half male and half female, weighing 20±2g) were randomly divided into 5 groups (half male and half female, 20 mice in each group). Dipsacus saponins, refined Dipsacus saponins and compound 1-3 were respectively prepared into a suspension with a concentration of 0.5g / mL with distilled water, and administered by oral gavage at a concentration of 0.8ml / 20g, and then observed for 7 consecutive days.

[0207] The results showed that at this dose, the mice were in good spirits and active state, and no poisoning or death occurred; this dose was equivalent to more than 20 times the effective dose. This shows that the safety of Dipsacus asper saponin compounds is good.

[0208] Example 10 Preparation of Dipsacus asper saponins

[0209] 1. Preparation of Compound 4 Injection

[0210] Prescription 1: Take 1000 mg of compound 4, 20 mg of solubilizer F68, 60 mg of co-solvent propylene glycol, and 30 mg of antioxidant sodium sulfite; adjust isotonicity with anhydrous glucose, adjust the pH value to 7 with buffer salt, and add redistilled water to 150 mL.

[0211] Prescription 2: Take 1000mg of compound 4, 25mg of solubilizer F68, 2000mg of HP-β-cyclodextrin, and 50mg of antioxidant sodium sulfite. Take the prescribed amount of HP-β-cyclodextrin and dissolve it in 40mL of ethanol. Add compound 4 powder under stirring at 600rpm, continue stirring for 2h, and set aside; take sodium bisulfite and F68 and add 50mL of aqueous solution to dissolve, inject the HP-β-cyclodextrin ethanol solution of compound 4 under stirring until compound 4 is completely dissolved, then pre-freeze at -60℃, and vacuum dry to obtain an off-white loose freeze-dried powder.

[0212] 2. Preparation of Dipsacus saponins and refined products of Dipsacus saponins

[0213] Prescription 1: 10g of Dipsacus saponins, 3g of hydroxypropyl methylcellulose, 2g of starch, 1g of lactose, 0.5g of ethyl cellulose and 0.5g of PVP. Take the prescribed amount of Dipsacus saponins, hydroxypropyl methylcellulose, ethyl cellulose, starch and lactose, pass through an 80-mesh sieve respectively, mix evenly, add 3% PVP aqueous solution to make a soft material, granulate with a 20-mesh sieve, dry at 60°C for 4h, granulate with an 18-mesh sieve, add an appropriate amount of magnesium stearate, mix well, and press tablets with a 6mm flat punch. The tablet weight is 0.32g.

[0214] Prescription 2: 10g of refined product of Dipsacus saponin compounds, 0.5g of CMC-Na, 0.5g of gelatinized starch, 2g of microcrystalline cellulose, 90% ethanol solution as a binder, and an appropriate amount of magnesium stearate as a lubricant. Take the prescription amount of Dipsacus saponin active ingredient extract B, CMC-Na, gelatinized starch, and microcrystalline cellulose, pass through an 80-mesh sieve respectively, mix evenly, use 90% ethanol to make a soft material, use a 20-mesh sieve to granulate, dry at 60℃ for 4h, and granulate with an 18-mesh sieve. After adding an appropriate amount of magnesium stearate, mix thoroughly, and use a 6mm flat punch to press the tablets. The tablet weight is 0.32g.

[0215] The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0216] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A mixture comprising a dipsaponin compound, characterized in that: The molecular structure formula of the dipsaponin compound is: The mixture comprises akebiasaponin D, keisaponin M, keisaponin C, keisaponin B, akebiasaponin PA, (3-O-α-L-arabinopyranosyloleanolic acid-28-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranoside) and (3-O-(4-O-acetyl)-α-L-arabinopyranosylhederagenolgenin-28-O-β-D-glucopyranosyl (1→6)-β-D-glucopyranoside); Among them, in Akebia saponin D, R1=Ara, R2=OH, R3=Glc(1→6)Glc; In 3-O-(4-O-acetyl)-α-L-arabinopyranosyl hederagenin-28-O-β-D-glucopyranose-(1→6)-β-D-glucopyranoside, R1=(4-O-acetyl)Ara, R2=OH, R3=Glc(1→6)Glc; 3-O-α-L-arabinopyranosyloleanolic acid-28-O-β-D-glucopyranose -(1→6)-β-D-pyranoglucoside, R1=Ara, R2=H, R3=Glc(1→6)Glc; In Dipsacus asper saponin M, R1=Ara(1→2)rha, R2=OH, R3=Glc(1→6)Glc; In Dipsacus asper saponin C, R1= Xyl(1→4)glc(1→4)glc(1→3)rha(1→4)rha(1→2)ara, R2=OH, R3=H; In Dipsacus asper saponin B, R1=Glc(1→4)rha(1→6)glc(1→3)rha(1→2)ara, R2=OH, R3=H; In Dipsacus asper saponin PA, R1=Ara, R2=OH, R3=H; The preparation method of the mixture comprises: Take 5.0 kg of Radix Dipsaci, heat and reflux extract twice with 70v% ethanol aqueous solution, the amount of extraction solvent is 25 kg each time, the extraction time is 2 hours each time, combine the two extracts, recover ethanol under reduced pressure, and dry under reduced pressure at 60°C to prepare 2.2 kg of Radix Dipsaci crude extract; The crude extract of Radix Dipsaci was dissolved in water and diluted to 25 kg, and loaded into a resin column containing 5 kg of AB-8 macroporous adsorption resin, the diameter of the resin column was 10 cm, and the length was 65 cm, and washed with 3 times the column volume of water and 2 times the column volume of 30v% ethanol aqueous solution to remove impurities, and finally eluted with 3 times the column volume of 70v% ethanol aqueous solution and the eluate was collected, ethanol was recovered under reduced pressure, and concentrated and evaporated to dryness to obtain 450g of Radix Dipsaci saponin compounds; The prepared dipsaponin compounds are separated using a Φ120cm×9cm normal phase silica gel column, and the eluent whose mobile phase is chloroform-methanol in a volume ratio of 7:3 is collected, and the organic solvent is recovered under reduced pressure and then concentrated and evaporated to dryness to obtain the mixture containing dipsaponin compounds.

2. Use of the mixture containing dipsaponin compounds according to claim 1 in the preparation of drugs for preventing and treating fatty liver.

3. Use of the mixture containing dipsaponin compounds according to claim 1 in the preparation of a drug for regulating abnormal liver function and abnormal blood lipids and blood sugar caused by fatty liver, inhibiting oxidative stress response of liver tissue, inhibiting lipid deposition in hepatocytes and blocking hepatocyte apoptosis, and repairing and protecting hepatocytes.

4. A pharmaceutical composition for preventing and treating fatty liver disease, characterized in that: A mixture containing the dipsaponin compounds according to claim 1.

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