CN116425821A

CN116425821A - Dipsacus asperoides saponin compound, preparation method thereof and application thereof in preparing medicine for preventing and treating fatty liver

Info

Publication number: CN116425821A
Application number: CN202211150396.9A
Authority: CN
Inventors: 刘丽宏; 蓝苑元; 李超
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-09-21
Filing date: 2022-09-21
Publication date: 2023-07-14

Abstract

The invention discloses a teasel root saponin compound, a preparation method thereof and application thereof in preparing medicines for preventing and treating fatty liver, wherein the preparation method comprises the following steps: taking radix dipsaci medicinal material decoction pieces as raw materials, adding an alcohol aqueous solution, heating and reflux-extracting for 1-4 times, extracting for 1-6 hours each time, and collecting an extracting solution; concentrating the extractive solution under reduced pressure to obtain intermittent crude extract; and (3) adding water into the intermittent crude extract for dissolution, loading the intermittent crude extract into macroporous adsorption resin, and recovering eluent after washing, impurity removal and elution in sequence to obtain the dipsacus saponin compound. The teasel root saponin compound provided by the invention can effectively regulate liver dysfunction and dyslipidemia caused by fatty liver, inhibit liver tissue oxidative stress reaction, inhibit lipid deposition in liver cells and block liver cell apoptosis, has the effects of repairing and protecting liver cells for histopathological lesions of fatty liver, has high safety and has potential application value.

Description

Dipsacus asperoides saponin compound, preparation method thereof and application thereof in preparing medicine for preventing and treating fatty liver

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a teasel root saponin compound, a preparation method thereof and application thereof in preparing medicines for preventing and treating fatty liver.

Background

Radix Dipsaci is dry root of Dipsacaceae plant Dipsacus asperoides Dipsacus asperoides C.Y. Cheng et T.M.ai (or Dipsacus asper Wall), which is listed as the upper product in Shennong Ben Cao Jing, has effects of nourishing liver and kidney, strengthening tendons and bones, continuing to fracture, promoting blood circulation, stopping metrorrhagia, preventing miscarriage, etc., is an essential drug for nourishing liver and kidney and treating bone injury, and is clinically used for treating soreness of waist and knees, weakness of feet, traumatic injury, fracture, etc. caused by liver and kidney deficiency. The teasel root has complex and diverse chemical components including saponins, iridoids, alkaloids, volatile oils and the like, and modern pharmacological researches show that the teasel root extract has pharmacological activities of inhibiting spontaneous uterine contraction activity, improving immunity, resisting aging, resisting bacteria, diminishing inflammation and the like.

Fatty liver disease changes diffuse fat of liver cells into pathological characteristics, and refers to fatty liver for short, which is affected by excessive fat accumulation in liver cells. Fatty liver is a heterogeneous disease, caused by the interaction of genetic susceptibility factors, environmental factors and metabolic stress, and mainly comprises alcoholic fatty liver, non-alcoholic fatty liver and special type fatty liver.

More than one quarter of adults worldwide have fatty liver, and the prevalence of eastern and western countries is not significantly different; with the increasing population of obesity and three highs, the prevalence of fatty liver shows an increasing trend, and the onset age tends to be younger, even the fatty liver of children is increasing. In addition, fatty liver has a prevalence of up to 50% or more in obese, metabolic syndrome, type 2 diabetes and long-term overdrinking patients.

Liver is the main place of human fat metabolism, the long-term high fat, high fructose, high calorie dietary structure, many sitting and little moving life style and the increase of alcohol consumption all can make the liver ingest fat more, and the esterification is strengthened, and when fat overload appears and liver fat metabolism is disturbed, unoxidized fat can deposit in the hepatocyte, and excessive accumulation just takes place diffuse fat infiltration, and hepatocyte diffuse fat transformation, i.e. fatty liver. With the development of the disease, the liver cancer may be caused by simple fatty liver, steatohepatitis, liver fibrosis and cirrhosis, and may even be caused by liver cancer.

Fatty liver can directly cause pathological changes such as decompensated liver cirrhosis, hepatocellular carcinoma and the like, can induce the progress of other chronic liver diseases, and is involved in the pathogenesis of type 2 diabetes and atherosclerosis. Meanwhile, malignant tumors, atherosclerosis cardiovascular and cerebrovascular diseases and liver cirrhosis related to metabolic syndrome are also important factors affecting the life quality and life expectancy of fatty liver patients. In addition, a great deal of clinical researches show that most of lipid-lowering drugs have liver toxicity, and mainly because the lipid-lowering drugs have the effect of 'expelling lipid', lipid in blood is expelled to the liver, so that fat accumulation exists in the liver originally, a great deal of gushed lipid is more difficult to treat, the fatty liver is more serious, intrahepatic cholestasis can be caused, jaundice or drug-induced liver injury is caused, and even cirrhosis and liver failure are caused. The fatty liver patients are huge, and no specific curative effect medicine for treating fatty liver is currently marketed globally, especially the non-alcoholic fatty liver, and the liver protection medicine can only be used, so that the disease progress needs to be controlled by taking medicine for a long time, and the cure cannot be achieved.

It is predicted that future fatty liver will continue to increase in human health and will become a major epidemic disease for global liver disease control. Therefore, the research and development of new drugs for treating and preventing fatty liver are new challenges in the modern medicine field, have important innovative significance and clinical value, and the development of new drugs in the fatty liver field is also a large market with great development potential.

So far, no report on the preparation of dipsacus root saponins compounds and the application of dipsacus root saponins compounds in preventing and treating fatty liver diseases is seen.

In view of this, the present invention has been made.

Disclosure of Invention

In order to solve the defects in the technology, the invention provides a teasel root saponin compound, a preparation method thereof and application thereof in preparing medicines for preventing and treating fatty liver.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

a preparation method of dipsacus root saponins compound comprises:

s1, taking radix dipsaci medicinal material decoction pieces as raw materials, adding an alcohol aqueous solution, heating and refluxing for extraction for 1-4 times, extracting for 1-6 hours each time, and collecting an extracting solution;

s2, concentrating the extracting solution under reduced pressure to obtain intermittent crude extract;

s3, adding water into the intermittent crude extract to dissolve, loading the intermittent crude extract into macroporous adsorption resin, and recovering eluent to obtain the dipsacus saponin compound after washing, impurity removal and elution in sequence.

In the above technical scheme, in step S1, the alcohol aqueous solution is 65-78v% ethanol aqueous solution.

Preferably, in the above technical scheme, the adding mass of the alcohol aqueous solution is 4-7.5 times of that of the teasel root medicinal material decoction pieces, heating and refluxing for 2-3 times, extracting for 2-4 hours each time, collecting the extracting solution, and combining.

In the above technical scheme, in step S3, the macroporous adsorbent resin is AB-8 macroporous adsorbent resin.

Further, in the above technical solution, step S3 is specifically,

adding 8-14 times of water into the intermittent crude extract for dissolution, loading into a resin column filled with AB-8 macroporous adsorption resin with the mass which is 2-3 times of that of the intermittent crude extract, washing with 3 times of water and removing impurities with 2 times of 30v% ethanol water solution, eluting with 3 times of 70v% ethanol water solution, collecting eluent, recovering ethanol under reduced pressure, concentrating, and evaporating to dryness.

In a preferred embodiment of the present invention, the preparation method of the dipsacoside compound further comprises:

refining the dipsacus saponin compound prepared in the step S3 by adopting a normal phase silica gel column, and collecting a mobile phase with a volume ratio of 7:3, recovering the organic solvent under reduced pressure, concentrating and evaporating to dryness to obtain refined product of dipsacus root saponins compound.

The invention also provides the dipsacus root saponin compound prepared by the preparation method.

Specifically, in the above technical scheme, the molecular structural formula of the dipsacus saponin compound is:

wherein:

barrel saponin D (Dipsacus saponin D), R ₁ ＝Ara，R ₂ ＝OH，R ₃ ＝Glc(1→6)Glc；

3-O- (4-O-acetyl) -alpha-L-arabinopyranosyl hederagenin-28-O-beta-D-glucopyranose- (1- & gt 6) -beta-D-glucopyranoside R ₁ ＝(4-O-acetyl)Ara，R ₂ ＝OH，R ₃ ＝Glc(1→6)Glc；

3-O-alpha-L-arabinopyranosyl oleanolic acid-28-O-beta-D-glucopyranose- (1- & gt 6) -beta-D-glucopyranoside, R ₁ ＝Ara，R ₂ ＝H，R ₃ ＝Glc(1→6)Glc；

Radix Dipsaci saponin M (Dipsacus saponin M), R ₁ ＝Ara(1→2)rha，R ₂ ＝OH，R ₃ ＝Glc(1→6)Glc；

Radix Dipsaci saponin C (Dipsacus saponin C), R ₁ ＝Xyl(1→4)glc(1→4)glc(1→3)rha(1→4)rha(1→2)ara，R ₂ ＝OH，R ₃ ＝H；

Radix Dipsaci saponin B (Dipsacus saponin B), R ₁ ＝Glc(1→4)rha(1→6)glc(1→3)rha(1→2)ara，R ₂ ＝OH，R ₃ ＝H；

Radix Dipsaci saponin PA (Akebia saponin PA), R ₁ ＝Ara，R ₂ ＝OH，R ₃ ＝H。

Specifically, the teasel root saponin compound contains akebia saponin D (3-O-alpha-L-arabinopyranosyl hederagenin-28-O-beta-D-glucopyranosyl (1-6) -beta-D-glucopyranoside), teasel saponin M (3-O-alpha-L-arabinopyranose- (1-2) -alpha-L-rhamnopyranoside 28-O-beta-D-glucopyranose- (1-6) -beta-D-glucopyranoside), teasel saponin C (hederagenin-3-O-alpha-L-arabinopyranose- (1-2) -alpha-L-rhamnopyranose- (1-4) -alpha-L-rhamnopyranose- (1-3) -beta-D-glucopyranose- (1-4) -beta-D-xylopyranoside), A mixture of hederagenin B (hederagenin-3-O-alpha-L-arabinopyranosyl- (1- > 2) -alpha-L-rhamnopyranose- (1- > 3) -beta-D-glucopyranose- [ (1- > 6) -alpha-L-rhamnopyranose ] (1- > 4) -beta-D-glucopyranoside), akebia saponin PA (hederagenin-3-O-alpha-L-arabinopyranosyl) and (3-O-alpha-L-arabinopyranosyl oleanolic acid-28-O-beta-D-glucopyranosyl (1- > 6) -beta-D-glucopyranoside) and (3-O- (4-O-acetyl) -alpha-L-arabinopyranosyl hederagenin-28-O-beta-D-glucopyranosyl (1- > 6) -beta-D-glucopyranoside).

The invention also provides an application of the teasel root saponin compound in preparing medicines for preventing and treating fatty liver.

Specifically, the application comprises the steps of regulating liver function abnormality and dyslipidemia caused by fatty liver, inhibiting oxidative stress reaction of liver tissues, inhibiting lipid deposition in liver cells, blocking apoptosis of liver cells, repairing and protecting liver cells.

In addition, the invention also provides a pharmaceutical composition, a health product or a food for preventing and treating fatty liver diseases, which contains the teasel root saponin compounds or is prepared from the teasel root saponin compounds.

Compared with the prior art, the invention has the following advantages:

(1) The invention provides a simple and easy method for extracting dipsacus saponin compounds from dipsacus medicinal decoction pieces;

(2) The dipsacus root saponin compound provided by the invention can effectively regulate liver dysfunction and dyslipidemia caused by fatty liver, inhibit liver tissue oxidative stress reaction, inhibit lipid deposition in liver cells, block hepatic cell apoptosis, and has effects of repairing and protecting liver cells due to histopathological changes such as inflammatory cell infiltration and hepatic cell debris necrosis, apoptotic body formation and the like, namely has remarkable pharmacological effect on treating and preventing fatty liver and related diseases, and has the advantages of extremely low toxicity, high safety and potential application value.

Drawings

FIG. 1 is a HPLC fingerprint of the compound prepared in example 1 of the present invention;

FIG. 2 is a HPLC fingerprint of the compound prepared in example 2 of the present invention;

FIG. 3 is an anatomic exterior view of the liver for each test group in example 4 of the present invention;

FIG. 4 shows microscopic pathological sections of liver in each of the test groups of example 4 according to the present invention;

FIG. 5 is an anatomic exterior view of the liver for each of the test groups of example 5 of the present invention;

FIG. 6 shows microscopic pathological sections of liver in each test group of example 5 according to the present invention;

FIG. 7 is an anatomic exterior view of the liver for each test group in example 6 of the present invention;

FIG. 8 is an anatomic exterior view of the liver for each of the test groups of example 7 of the present invention;

FIG. 9 shows microscopic pathological sections of the liver in each test group of example 7 according to the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent.

It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

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

The terms "comprising," "including," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of 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.

In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biological materials, etc. used in the examples described below are commercially available unless otherwise specified.

The teasel root medicinal material decoction pieces used in the following examples are purchased from the traditional Chinese medicine market of Yulin, guangxi Zhuang nationality, and are verified and identified as dry roots of Sichuan teasel root Dipsacus asperoides C.Y.Cheng et T.M.Ai (or Dipsacus asper Wall) which is a Sichuan intermittent plant through the inspection of food and medicine of Guangxi Zhuang nationality.

Example 1

The preparation of the dipsacus root saponins compound comprises the following specific procedures:

taking 5.0kg of radix dipsaci medicinal material decoction pieces, heating and reflux-extracting twice with 70v% ethanol water solution, wherein the extracting solvent is 25kg each time, the extracting time is 2h each time, combining the two extracting solutions, recovering ethanol under reduced pressure, and drying under reduced pressure at 60 ℃ to obtain 2.2kg of radix dipsaci crude extract.

Dissolving and diluting crude radix Dipsaci extract (2.2 kg) with water to 25kg, loading into resin column (column diameter 10cm, length 65 cm) containing 5kg AB-8 macroporous adsorbent resin, sequentially washing with 3 times of column volume water and 2 times of column volume 30v% ethanol water solution to remove impurities, eluting with 3 times of column volume 70v% ethanol water solution, collecting eluate, recovering ethanol under reduced pressure, concentrating, and evaporating to dry to obtain 450g radix Dipsaci saponin compounds.

The content of the dipsacoside compound prepared by HPLC and ELSD through standard curve method measurement and analysis shows that the content of the compound 1 is 65.78%, the content of the compound 2 is 3.35%, the content of the compound 3 is 1.99%, the content of the compound 4 is 8.79%, the content of the compound 5 is 0.20%, the content of the compound 6 is 7.22%, and the content of the compound 7 is 10.87%; HPLC finger print of the obtained dipsacus saponin compounds is shown in figure 1.

Example 2

Refining of dipsacus root saponins compounds, and the specific flow is as follows:

the dipsacoside compound prepared in the example 1 is separated by a normal phase silica gel column (phi 120cm multiplied by 9 cm), and the mobile phase is collected to have a volume ratio of 7:3, recovering the organic solvent under reduced pressure, concentrating and evaporating to dryness to obtain refined product of dipsacus root saponins compound; HPLC finger print of refined product of dipsacus root saponins compound is shown in figure 2.

The composition and mass content ratio of refined product of dipsacus saponin compound were analyzed by HPLC and ELSD through standard curve method, and the content detection results are shown in Table 1 below.

TABLE 1 content detection results of refined product of Dipsacus asperosaponin type Compound

Example 3

The refined product of the dipsacus root saponins compound is separated and purified, and the specific flow is as follows:

(1) The purified product of the dipsacoside compound prepared in example 2 was subjected to component separation and purification using a reverse phase silica gel column (Φ60 cm. Times.5 cm), and the mobile phase was collected as methanol-water 3:7, recovering methanol under pressure, evaporating to dryness to obtain 15.74g of white powder A (compound 6, content 98.22%); the mobile phase was collected as methanol-water 2:8, recovering methanol under pressure, evaporating to dryness to obtain 156.93g of white powder B (compound 1, content 98.45%); the mobile phase was collected as methanol-water 1:9, recovering methanol under pressure, evaporating to dryness to obtain 31.32g of white powder C (compound 7, content 98.05%);

(2) Eluting the residue after separation and purification by using Sephadex LH-20 gel column chromatography (purchased from Beijing Huideyi technology Co., ltd.) and a methanol system, and collecting one fraction every 10 mL; reversed phase high performance liquid chromatography (Agilent 1200) analysis combined fractions a, b, c, d, e and f. Wherein:

separating the fraction f by reverse phase high performance liquid chromatography (Agilent 1200), sequentially collecting eluting components corresponding to absorption peaks at 210nm wavelength, and recovering sample peak with retention time of 27.0min under reduced pressure to obtain 1.9mg white powder D (Akebia saponin PA, compound 5); separating fraction B by reverse phase high performance liquid chromatography (Shimadzu LC-20 AT), sequentially collecting corresponding eluting components when absorption peak appears AT 210nm wavelength, respectively retaining sample peaks with retention time of 19.3min and 19.7min, and recovering under reduced pressure to obtain 1.2mg white powder E (radix Dipsaci saponin C, compound 3) and 3.9mg white powder F (radix Dipsaci saponin B, compound 4); the fraction c and the fraction d were subjected to combined gradient elution by using ODS (5 μm) medium-pressure column chromatography (purchased from Beijing Huideyi technologies Co., ltd.) and the sample eluate having a retention time in the range of 37 to 41min was concentrated and then separated by reversed-phase high performance liquid chromatography, and the corresponding eluate fraction was collected in sequence at a 210nm wavelength as an absorption peak, and the sample peak having a retention time of 5.8min was recovered under reduced pressure to obtain 5.8mg of white powder G (Dipsacus saponin M, compound 2).

The chemical composition analysis and structure identification were as follows:

(1) Appearance: all amorphous white powders;

(2) Solubility: are all easy to dissolve in methanol, ethanol, slightly soluble in low-polarity organic solvents such as acetone, chloroform and the like;

(3) Ultraviolet spectrum: the ultraviolet spectrum of the methanol solution of the compounds 1-6 has a maximum absorption peak at 210 nm;

(4) Optical rotation value:

compound 1, [ α ]25d+20 (c 0.025, meoh);

compound 2, [ α ]25d+8 (c 0.025, meoh);

compound 3, [ α ]25d+8 (c 0.025, meoh);

compound 4, [ α ]25d+16 (c 0.025, meoh);

compound 5, [ α ]25d+87.99 (c 0.025, meoh);

compound 6, [ alpha ]25d+9 (c 0.1, meoh).

(5) High resolution mass spectrometry:

HRESIMS mass spectrum of (3-O-alpha-L-arabinopyranosyl hederagenin 28-O-beta-D-glucopyranose- (1- & gt 6) -beta-D-glucopyranoside and compound 1) shows that the compound is [ M+HCOO ]]The peak is m/z 973.5019 and provides the most probable molecular formula C ₄₇ H ₇₆ O ₁₈ ；

(3-O-alpha-L-arabinopyranose- (1- & gt 2) -alpha-L-rhamnopyranose hederagenin 28-O-beta-D-glucopyranose- (1- & gt 6) -beta-D-glucopyranose ester glycoside; compound 2) HRESIMS mass spectrum, shows its [ M-H ]]The peak is m/z 1073.5599 and provides the most probable molecular formula C ₅₃ H ₈₆ O ₂₂ 。

(hederagenin-3-O-alpha-L-arabinopyranose- (1- & gt 2) -alpha-L-rhamnopyranose- (1- & gt 4) -alpha-L-rhamnopyranose- (1- & gt 3) -beta-D-glucopyranose- (1- & gt 4) -beta-D-xylopyranose glycoside, and Compound 3) HRESIMS mass spectrum, and its [ M-H ] is shown ]The peak is m/z 1351.6541 and provides the most probable molecular formula C ₆₄ H ₁₀₄ O ₃₀ 。

(hederagenin-3-O-alpha-L-arabinopyranose- (1- > 2) -alpha-L-rhamnopyranose- (1- > 3) -beta-D-glucopyranose- [ (1- > 6) -alpha-L-rhamnopyranose](1→4) - β -D-glucopyranoside; HRESIMS mass spectrum of Compound 4), showing its [ M-H ]]The peak is m/z1219.6117 and provides the most probable molecular formula C ₅₉ H ₉₆ O ₂₆ 。

(hederagenin-3-O-alpha-L-arabinopyranoside; compound 5) HRESIMS mass spectrum, showing that it is [ M+Na ]]The +peak is m/z 627.3897 and provides the most probable molecular formula C ₃₅ H ₅₆ O ₈ 。

The compound 3-O-alpha-L-arabinopyranosyl oleanolic acid 28-O-beta-D-glucopyranose- (1- & gt 6) -beta-D-glucopyranoside; HRESIMS mass spectrum of Compound 6), showing its [ M+Na ]]The +peak is m/z 935.5050 and provides the most probable molecular formula C ₄₇ H ₇₆ O ₁₇ 。

(6) Nuclear magnetic resonance spectroscopy:

NMR measurements on the above-mentioned Compounds 1 to 6 according to Compounds 1 to 6 ¹ H-NMR ¹³ C-NMR, nuclear magnetic resonance Spectroscopy of Compounds 1-6 was studied and examined ¹³ The C-NMR signals were assigned as shown in tables 2 and 3.

TABLE 2 Compounds 1-6 ¹³ Assignment of peaks in the C-NMR spectrum

TABLE 3 glycosides of Compounds 1-6 ¹³ Assignment of peaks in the C-NMR spectrum

The final determined structure is as follows:

akebia 1 saponin D (Dipsacus saponin D) R ₁ ＝Ara，R ₂ ＝OH，R ₃ ＝Glc(1→6)Glc

2 Dipsacus asperoides saponin M (Dipsacus saponin M) R ₁ ＝Ara(1→2)rha，R ₂ ＝OH，R ₃ ＝Glc(1→6)Glc

3 Dipsacus asperoides saponin C (Dipsacus saponin C) R ₁ ＝Xyl(1→4)glc(1→4)glc(1→3)rha(1→4)rha(1→2)ara，R ₂ ＝OH，R ₃ ＝H

4 Dipsacus asperoides saponin B (Dipsacus saponin B) R ₁ ＝Glc(1→4)[rha(1→6)]glc(1→3)rha(1→2)ara，R ₂ ＝OH，R ₃ ＝H

5 Akebia saponin PA (Akebia saponin PA) R ₁ ＝Ara，R ₂ ＝OH，R ₃ ＝H

6 3-O-alpha-L-arabinopyranosyl oleanolic acid 28-O-beta-D-glucopyranose- (1.fwdarw.6) -beta-D-glucopyranoside R ₁ ＝Ara，R ₂ ＝H，R ₃ ＝Glc(1→6)Glc

7 3-O- (4-O-acetyl) -alpha-L-arabinopyranosylHederagenin-28-O-beta-D-glucopyranosyl (1- & gt 6) -beta-D-glucopyranoside R ₁ ＝(4-O-acetyl)Ara，R ₂ ＝OH，R ₃ ＝Glc(1→6)Glc

EXAMPLE 4 prevention and treatment of non-alcoholic fatty liver disease with secoisolariciresinol diglucoside

70 Kunming mice (provided by Experimental animal science department of Beijing university medical department) for healthy adult experiments (SPF grade, male and female halves, weight 20+ -2 g) were randomly divided into seven groups (10 each, male and female halves) with a test period of 45d, and the method specifically comprises:

normal control group: feeding common feed, and lavaging to administer physiological saline;

model group: feeding high-fat feed, and performing gastric lavage to administer physiological saline;

positive control group: feeding high-fat feed, and performing gastric administration to obtain fenofibrate (produced by Li Bofu Ni pharmaceutical company of France) with a concentration of 0.15 g/kg.d;

low dose dipsacoside group: feeding high-fat feed, and filling 0.25 g/kg.d of dipsacus root saponins compound;

High dose dipsacoside group: feeding high-fat feed, and filling 0.50 g/kg.d of dipsacus root saponins compound;

low dose dipsacus saponin compound refined product group: feeding high-fat feed, and pouring 0.25 g/kg.d of refined dipsacus root saponins compound;

high dose dipsacus saponin compound refined product group: feeding high-fat feed, and pouring 0.50 g/kg.d of refined dipsacus root saponins compound;

the stomach was irrigated once daily at 0.2mL/20g (volume/body weight).

After the experiment is finished, the patient is fasted for 12 hours after the last 1 time of gastric lavage, is free to drink water, is sacrificed after blood is taken from eyeballs, and is subjected to low-temperature preservation at-20 ℃ to separate plasma, and various biochemical indexes are measured; cervical spining, killing, laparotomy, rapidly separating body fat (abdominal omentum and fat around kidney after abdomen), taking livers, weighing respectively, cutting out a large liver leaf, placing in 4% formalin for fixation, HE staining, and observing pathological changes.

SPSS 11.5 software was used for significance testing of data statistics and differences.

Experimental results are expressed as mean ± standard deviation, and a single factor analysis of variance is used to perform a comparison of the mean values between the groups, and a statistical treatment is performed by an inter-group t-test, with P <0.05 being a significant level, as follows.

TABLE 4 influence on liver function index in non-alcoholic fatty liver mice

And (3) injection: in comparison with the normal group, ^# P<0.05， ^## P<0.01; in comparison with the set of models, ^* P<0.05， ^** P<0.01。

the data in the table show that compared with the normal control group, the transaminase AST, ALT, MDA, FFA, liver index and fat index of the blood of the mice in the model group are obviously increased (P < 0.01), SOD is obviously reduced (P < 0.01), and the non-alcoholic fatty liver disease and liver function abnormality are reflected.

Compared with the model group, the positive control group mice AST, ALT, MDA, SOD, FFA and other indexes have no obvious difference, and the positive drug fenofibrate (hypolipidemic drug) has no effect on liver function abnormality caused by non-alcoholic fatty liver caused by obesity.

The transaminase AST, ALT, MDA, FFA and liver index of the mice blood of the low-dose teasel saponin compound group, the high-dose teasel saponin compound group, the low-dose teasel saponin compound refined product group and the high-dose teasel saponin compound refined product group are obviously reduced (P <0.01 or P < 0.05), and the SOD is obviously increased (P <0.01 or P < 0.05).

Comprehensive analysis of the results shows that the dipsacus saponin compounds and refined dipsacus saponin compounds have obvious effects of protecting and repairing liver function abnormality caused by non-alcoholic fatty liver caused by obesity.

TABLE 5 influence on lipid index in non-alcoholic fatty liver mice

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Note that: in comparison with the normal group, ^# P<0.05， ^## P<0.01; in comparison with the set of models, ^* P<0.05， ^** P<0.01。

the above table data shows that the leptin, CHOL and LDL in the blood of mice in the model group were significantly increased (P <0.01, or P < 0.05), HDL was significantly decreased (P < 0.01), and the mice developed nonalcoholic fatty liver lesions, hyperlipidemia, and liver lipid oxidative damage symptoms compared to the normal control group.

Compared with the model group, the positive control group mice CHOL and TG were significantly reduced (P <0.01, or P < 0.05), and other indexes were not different.

Leptin, CHOL, LDL are significantly reduced (P <0.01, or P < 0.05), HDL is significantly increased (P < 0.01) in mice of low-dose dipsacoside group, high-dose dipsacoside group, low-dose dipsacoside refined group, and high-dose dipsacoside refined group.

Comprehensive analysis of the results shows that the dipsacus saponin compounds and refined dipsacus saponin compounds have obvious repairing effect on hyperlipidemia and lipid peroxidation damage caused by non-alcoholic fatty liver.

The anatomical appearance of the liver is shown in figure 3, and the liver of the normal control group (figure 3-1) mice has dark red color, smooth surface, sharp edge and soft texture; the liver of the model group (figure 3-2) and the positive control group (figure 3-3) is enlarged, full of fat, white color, blunted edge, crisp and fragile, greasy section, and jaundice of some livers; the color and the size of the mouse livers of the low-dose dipsacoside compound group (figures 3-4) and the low-dose dipsacoside compound refined product group (figures 3-5) are similar to those of the control group, and compared with the model group, the liver color and the liver size of the mice of the low-dose dipsacoside compound group and the low-dose dipsacoside compound refined product group are obviously improved.

FIG. 4 shows microscopic pathological sections of liver, showing that the liver of mice in model group (FIG. 4-2) has been characterized by typical nonalcoholic fatty liver disease, mainly characterized by massive fat globules in the liver cells, and fat vacuoles of unequal size, round shape and tension in the cytoplasm of the liver cells, mainly located in peripheral areas of the liver lobules, squeezing the nucleus to one side, most of the liver cells being enlarged, and the liver sinuses being occluded by hepatomegaly compression; the positive control group (fig. 4-3) showed no significant decrease in fat particles in the hepatocytes of mice compared to the model group; the liver tissue morphology of the mice of the low dose dipsacoside compound group (figures 4-4) and the low dose dipsacoside compound refined product group (figures 4-5) is similar to that of normal mice, fat vacuoles in liver cells are remarkably reduced, and some fat vacuoles are not generated, liver sinuses reappear, and the liver cell volume is almost recovered to be normal.

Example 5 comparison of the effects of refined secoisolariciresinol diglucoside Compounds with the monomer active ingredient on the prevention and treatment of non-alcoholic fatty liver disease

120 Kunming mice (supplied by Experimental animal science department of Beijing university medical department) for healthy adult experiments (SPF grade, male and female halves, weight 20+ -2 g) were randomly divided into 6 groups (20 each, male and female halves) with a test period of 45d, and the method specifically comprises:

refined product group of dipsacus saponins compounds: feeding high-fat feed, and pouring 0.50 g/kg.d of refined dipsacus root saponins compound;

compound 1 group: feeding high-fat feed, and filling stomach compound 1 with the dosage of 0.50 g/kg.d;

group 2: feeding high-fat feed, and filling stomach compound 2 with the dosage of 0.50 g/kg.d;

group 3: feeding high-fat feed, and filling stomach compound 3 with the dosage of 0.50 g/kg.d;

the stomach was irrigated once daily at 0.2mL/20g (volume/body weight).

SPSS 11.5 software was used for significance testing of data statistics and differences. Experimental results are expressed as mean ± standard deviation, and a single factor analysis of variance is used to perform a comparison of the mean values between the groups, and a statistical treatment is performed by an inter-group t-test, with P <0.05 being a significant level, as follows.

TABLE 6 influence on liver function index in female mice with non-alcoholic fatty liver disease

TABLE 7 influence (x.+ -.s) of liver function index of male mice with non-alcoholic fatty liver disease

the data in the 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 are obviously increased (P <0.01 or P < 0.05), SOD is obviously reduced (P <0.01 or P < 0.05), and the mice have nonalcoholic fatty liver disease; compared with the model group, the teasel root saponin compound refined product group mice AST, ALT, MDA, FFA, liver index, fat index and other indexes are obviously reduced (P < 0.01), and SOD is obviously increased (P <0.01, or P < 0.05); the blood transaminases AST, ALT and FFA of mice in the group 1, the group 2 and the group 3 are obviously reduced (P <0.01 or P < 0.05), and the indexes such as MDA, SOD, liver index and fat index are not obviously changed.

Therefore, the refined product of the dipsacus saponin compound has remarkable treatment effect on the non-alcoholic fatty liver caused by obesity, and the group 1, the group 2 and the group 3 only show a certain trend of effect, namely, the effective combination of the compounds 1-7 according to a certain mass ratio has more remarkable pharmacological activity, and the effect is obviously better than that of the dipsacus saponin active ingredients of the

compounds

1, 2 and 3.

TABLE 8 influence on blood lipid index of non-alcoholic fatty liver female mice

table 9 influence on lipid index of non-alcoholic fatty liver Male mice

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the data show that compared with the normal control group, the CHOL and LDL of the model group are obviously increased (P <0.01 or P < 0.05), TG and HDL are obviously reduced (P <0.01 and P < 0.05), blood sugar of the male mice is obviously increased (P < 0.01), and the mice have symptoms of non-alcoholic fatty liver disease, hyperlipidemia, hyperglycemia, liver lipid oxidative damage and the like; compared with the model group, the dipsacus saponin compound refined product group mice have obviously reduced CHOL and LDL (P <0.01 or P < 0.05), obviously increased HDL (P < 0.05), and obviously reduced blood sugar (P < 0.01) of the male mice; none of the CHOL, TG, HDL, LDL and blood glucose in the compound 1, compound 2 and compound 3 mice were significantly altered. Therefore, the refined dipsacus root saponins compound, the compound 1, the compound 2 and the compound 3 have obvious repairing effect on hyperlipidemia, hyperglycemia and lipid peroxidation damage caused by the non-alcoholic fatty liver.

The anatomical appearance of the liver is shown in figure 5, and the liver of the normal control group (figure 5-1) mice has dark red color, smooth surface, sharp edge and soft texture; compared with the normal control group (figure 5-1), the body fat index and liver index of the mice in the model group (figure 5-2) are obviously increased, the liver is enlarged and full of fat, the color is yellow, obvious fat particles can be seen, the edge becomes blunt, the quality is fragile and fragile, the section is greasy, and the jaundice and other serious steatohepatitis appear in some livers; the color and the size of the liver of the mice in the refined product group (shown in figures 5-3) of the dipsacus saponins compound are close to those of the liver of the mice in the normal group, and compared with the model group, the dipsacus saponins compound in the liver of the mice is obviously improved; mice from group 1 (fig. 5-4), group 2 (fig. 5-5) and group 3 (fig. 5-6) still showed a degree of hepatomegaly, a yellow color, an improvement over the model group (fig. 5-2), and a fatty liver performance that was still present in comparison to the normal control group (fig. 5-1).

FIG. 6 is a microscopic pathological section of the liver, showing that the liver of the mice in the model group (FIG. 6-2) is characterized by typical nonalcoholic fatty liver disease, mainly characterized by massive fatty vacuoles filled in peripheral liver cells of liver lobules, hepatomegaly, even with rupture of liver cells and diffuse steatohepatitis, and the liver sinuses are occluded due to compression of hepatomegaly; the liver tissue morphology of the mice of the refined product group of dipsacus saponins compounds (figures 6-3) is similar to that of normal mice, fat vacuoles in liver cells are remarkably reduced, fat vacuoles are not generated, liver sinuses reappear, and the liver cell volume is restored to be approximate to normal; small amounts of fatty vacuoles were still visible in hepatocytes of mice in compound 1 (fig. 6-4), compound 2 (fig. 6-5) and compound 3 (fig. 6-6), and some fatty liver performance was still present, but there was a clear trend of reduction compared to the model group.

Example 6 refined secoisolariciresinol diglucoside and monomer active ingredient pair CCl ₄ Comparison of protective action against acute liver injury in mice

60 healthy adult mice (SPF grade, male, weight 20+ -2 g) were randomly divided into 5 groups (12 animals per group) and fed 1d for free diet; the method specifically comprises the following steps:

Normal control group: lavage to administer physiological saline;

model group: lavage to administer physiological saline;

refined product group of dipsacus saponins compounds: the refined product of the gastrolavage teasel root saponins compound is 0.50 g/kg.d;

group 4: gastric lavage compound 4 at a dose of 0.50 g/kg.d;

group 5 compounds: gastric lavage compound 5 at a dose of 0.50 g/kg.d;

each group of mice was dosed, given by gavage 1 time a day, 0.2mL/20g each time, for 7 consecutive days. Except for the normal control group, the remaining mice were intraperitoneally injected with 10mL/kg of 0.2% CCl 2h after the last dose ₄ Taking eyeball blood after 18 hours and separating serum, preserving at low temperature of-20 ℃ and measuring various biochemical indexes; mice were sacrificed by cervical vertebra removal after blood collection, livers were rapidly dissected and extracted, rinsed with normal saline, dried with filter paper, weighed, right lobes of livers were taken out and placed in 4% formaldehyde fixative, HE stained, pathological changes observed, and the remaining livers were stored in a-20deg.C refrigerator.

Table 10 vs CCl ₄ Protective effect on acute liver injury of mice

the data show that compared with the normal control group, ALT and AST in the serum of the mice in the model group are obviously increased, MDA content in the liver is obviously increased, SOD and GSH content is obviously reduced, liver weight and liver index are obviously increased, and the liver is obviously damaged, thus indicating CCl ₄ Successful molding; compared with a model group, the liver weight and liver index of a mice in a refined product group of the dipsacus saponin compound are obviously reduced, ALT and AST in serum are obviously reduced, MDA content in the liver of the mice is obviously reduced, and the content of SOD and GSH is obviously increased; ALT and AST in the serum of the mice in the compound 4 group and the compound 5 group are obviously reduced, and the contents of MDA, SOD and GSH in the liver are not obviously changed.

The anatomical appearance of liver is shown in figure 7, the liver of the normal control group (figure 7-1) has reddish brown appearance, soft and elastic texture, clear structure of liver lobule, regular cell arrangement, uniform size, clear demarcation, cell nucleus in the center of cell, and round and clear; model group (fig. 7-2) mice liver has yellow macroscopic color, increased oiliness, increased envelope tension, roughly in the form of granule, small She Bianxing necrosis of liver visible under light microscope, and pathological changes mainly including sheet necrosis, cytoplasmic aggregation, water sample denaturation and fat denaturation; the refined product group (shown in figures 7-3) of dipsacus saponins compounds has the advantages that the fat vacuoles in the liver cells of mice are obviously reduced, the degeneration of the liver cells is light, the liver sinuses are visible, and the liver tissue morphology is similar to that of normal mice; the arrangement of liver hepatocytes in mice of compound 4 (FIGS. 7-4) and compound 5 (FIGS. 7-5) was still disturbed to some extent, and the cytoplasmic loose sink region and liver lobules remained scattered in punctate or focal necrosis, with still a greater amount of inflammatory cell infiltration.

In conclusion, the refined product of the dipsacus root saponins compound can obviously improve CCl ₄ The mechanism of the liver peroxidation injury of mice is probably to enhance the activity of cell antioxidant enzyme by resisting the injury of lipid peroxide to liver cells and improve the antioxidant capacity of the liver cells, thereby maintaining the integrity of liver cell plasma membranes and ensuring that the liver cells exert normal defense and compensation functions; and compound 4 and compound 5 pair CCl ₄ The liver peroxidation damage of mice has no obvious effect.

EXAMPLE 7 control action on fatty liver in ovariectomized rats

50 healthy adult laboratory Kunming rats (SPF grade, female, weight 200+ -2 g) were randomly divided into 5 groups (10 per group); the method specifically comprises the following steps:

normal control group: lavage to administer physiological saline;

model group: lavage to administer physiological saline;

group 6: gastric lavage compound 6 at a dose of 0.50 g/kg.d;

group 7: the dosage of the gastric lavage compound 7 is 0.50 g/kg.d.

Double sided Ovariectomy (OVX) after anesthesia by intraperitoneal injection of 45mg/kg of 3% sodium pentobarbital, the rat ovarian tissue was surgically excised and the capsule was intact; after the skin, muscle and peritoneum were incised by the sham operation in the normal control group, only small pieces of adipose tissue were excised, but ovaries were not removed; the normal feed is fed for 3 months after operation. After ovariectomy for 3 months, rats were given gavage for 3 months and body weight was measured weekly to adjust the dose.

After 6 months, the experiment was ended and blood, fat and liver specimens were taken for testing after the rats were anesthetized with uratam. Separating serum from liquid blood, preserving at-20deg.C, and measuring various biochemical indexes; removing cervical vertebra after blood taking, rapidly dissecting and picking liver, rinsing with normal saline, drying filter paper, weighing, placing right leaf of liver in 4% formaldehyde fixing solution, HE staining, observing pathological changes, and storing the rest liver in a refrigerator at-20deg.C.

TABLE 11 influence on liver function index of ovariectomized rat fatty liver

the data show that the model group rats have significantly higher blood transaminase AST, ALT, FFA and liver index (P <0.01, or P < 0.05) than the normal control group rats with non-alcoholic fatty liver disease; compared with the model group, the indexes of the dipsacus root saponin compound refined product group rat AST, ALT, FFA, liver index and the like are obviously reduced (P is less than 0.01 or P is less than 0.05), and the dipsacus root saponin compound refined product has good treatment effect on the ovarian-removed rat fatty liver; while the blood transaminases AST and ALT of rats in the group 6 and the group 7 are obviously reduced (P < 0.05), and other indexes are not obviously changed. Therefore, compared with the compound 6-7, the refined product of the dipsacoside compound has more obvious treatment effect on fatty liver caused by estrogen secretion disorder caused by ovariectomized rats.

Table 12 influence on lipid index of ovariectomized rat fatty liver

the data show that compared with a normal control group, the blood sugar, the CHOL and the LDL of rats in a model group are obviously increased (P < 0.01), the TG and the HDL are obviously reduced (P <0.01, P < 0.05), and the rats have abnormal indexes such as hyperlipidemia, hyperglycemia, oxidative damage of liver lipid and the like; compared with the model group, the blood sugar, CHOL and LDL of rats in the refined group of dipsacus saponin compounds are obviously reduced (P <0.01, P < 0.05), and HDL is obviously increased (P < 0.01); while compound 6, compound 7 rats had no significant differences in blood glucose, CHOL and TG, HDL was significantly increased (P < 0.05) and LDL was significantly decreased (P < 0.05). Therefore, the refined product of the dipsacus root saponins has obvious repairing effect on hyperlipidemia and hyperglycemia caused by fatty liver caused by estrogen secretion disorder caused by ovariectomized rats and lipid peroxidation damage; and the compound 6 and the compound 7 only have a certain repairing effect on liver lipid peroxidation damage, and have no effect on hyperlipidemia and hyperglycemia caused by fatty liver.

The anatomical appearance of the liver is shown in figure 8, and the liver of the rat in the normal control group (figure 8-1) has dark red color, smooth surface, sharp edge and soft texture; the liver of the rat in the model group (figure 8-2) is enlarged, full of fat, white, dull edge, crisp and fragile, greasy section, and jaundice of some liver; the color and the size of the rat liver of the refined product group (figure 8-3) of the dipsacus saponin compound are close to those of the normal group, and the refined product group is obviously improved compared with the model group (figure 8-2); the liver of rats in the group 6 (FIGS. 8-4) and the group 7 (FIGS. 8-5) was slightly improved, but the manifestation of steatohepatitis with hepatomegaly still existed.

FIG. 9 shows microscopic pathological sections of liver, showing that the liver of the rats in the model group (FIG. 9-2) has been characterized by typical nonalcoholic fatty liver disease, which is mainly characterized by the filling of liver cells with a large amount of fat particles, the appearance of fat vacuoles of unequal size, round shape and tension in the cytoplasm of liver cells, which are mainly located in the peripheral areas of liver lobules, squeezing nuclei to one side, most of liver cells being enlarged, and liver sinuses being occluded by hepatomegaly compression; the liver tissue morphology of the rat in the refined product group of dipsacus saponins (figures 9-3) tends to be similar to that of a normal mouse, fat vacuoles in liver cells are remarkably reduced, fat vacuoles are not generated, liver sinuses reappear, and the liver cell volume is almost recovered to be normal; while the fat particles in the liver cells of the rats in the group 6 (FIGS. 9-4) and the group 7 (FIGS. 9-5) tended to be reduced, but there was still a phenomenon of steatohepatitis.

EXAMPLE 8 Effect of inhibiting adipocyte growth and proliferation

Inoculating 3T3-L1 cells of preadipocytes of mice with 96-well culture plate, culturing until the 3 rd day, changing cell culture solution, grouping and adding dipsacoside compound, refined dipsacoside compound and compound 1-7 with certain concentration to make final concentration of the medicine reach 0, 37.5, 75, 150, 300 and 600 μg.mL respectively ^-1 Each treatment set was repeated 6 times at 0. Mu. Mol.mL ^-1 For control, MTT assay was performed at the 48h time point of sample action. The absorbance of each group was measured at 490nm with an enzyme-linked detector, and the cell viability of each experimental group was calculated. The results are shown in the following table.

TABLE 13 influence on preadipocyte viability

The results of the above table show that the concentration of dipsacus saponin compound, dipsacus saponin compound refined product and compound 1-7 is 37.5-150. Mu.g.mL compared with 0. Mu. Mol.mL-1 ^-1 Within this range, the survival rate of the mouse preadipocytes 3T3-L1 cells was slightly reduced, within which the cell survival rate was not greatly changed, and the state of the visible cells was good under observation under a microscope; while when the concentration of the drug is highThe degree is 300-600 mug.mL ^-1 When the cell growth inhibitor is used, the survival rate of the mouse preadipocyte 3T3-L1 cells is obviously reduced, namely, the cell growth inhibitor has an inhibiting effect on the growth of the mouse preadipocyte 3T3-L1 cells.

The 6-hole cell culture plate is inoculated with cells and is divided into a normal cell control group, an induction group, a teasel saponin compound refined product group and a compound 1-7 group. Normal cells were routinely cultured with DMEM/Ham's containing 10% ncs, and after cells of the inducer and sample intervention groups (including teasel saponin compound group, teasel saponin compound purified product group and compound 1-7 group) were cultured until the cells were 100% full, an induction solution i (DMEM, 10% fetal bovine serum, 0.5 mmol.l-1 ibmx,1 μmol.l-1 dexamethasone, 1.7 μmol.l-1 insulin) was added. Adding dipsacoside compounds with different concentrations, refined dipsacoside compounds and compounds 1-7 (37.5, 75, 150, 300 μg mL) into the induction liquid I of the sample intervention group ^-1 ) The induction group was changed from no drug to equal amount of PBS. After 14h incubation, 0.25% trypsin was added with 0.02% EDTA for digestion, centrifugation at 1000rpm for 5min, cells were collected, washed with PBS, centrifugation at 1000rpm for 10min, repeated 3 times, pre-chilled 75% ethanol at 4℃was fixed at 4℃for 48h, washed with PBS to wash off ethanol, 2mL PBS was used to resuspend cells, RNaseA enzyme was incubated at 50. Mu.g.mL-1℃for 30min, after cooling, each sample was added with PI to a final concentration of 1.5 mg.mL-1, and stained with ice bath for 30min in the absence of light, and after filtration with 300 mesh nylon mesh, the cell cycle was detected with flow cytometry. The results are shown in the following table.

TABLE 14 Effect on S-phase cell viability of preadipocytes

The results of the table show that the concentration of the dipsacus saponin compound, the refined dipsacus saponin compound and the compounds 1 to 7 is 37.5 to 600 mu g.mL ^-1 Within the scope, the cells of the S phase of the mouse preadipocyte 3T3-L1 cells are occupiedThe ratio is in a dose-dependent relationship, the cell ratio in the S division phase is reduced along with the increase of the drug concentration, and the state of the visible preadipocytes is good under the observation of a microscope, so that the dipsacoside compound, the refined dipsacoside compound and the compounds 1-7 can effectively inhibit the 3T3-L1 cell growth cycle of the mouse preadipocytes, thereby inhibiting the proliferation of the preadipocytes.

EXAMPLE 9 acute toxicity study

100 healthy adult mice (SPF-grade, male and female halves, weight 20.+ -.2 g) of Kunming species (supplied by Experimental animal sciences department of medical sciences of Beijing university) were randomly divided into 5 groups (male and female halves, 20 each). The dipsacus saponin compound, refined dipsacus saponin compound and compounds 1-3 are respectively prepared into suspension with concentration of 0.5g/mL by distilled water, and the suspension is administrated by one-time gastric lavage according to 0.8mL/20g, and then continuous observation is carried out for 7 days.

The results show that at the dosage, the mice have good mental and activity states and have no poisoning and death phenomena; the dosage of the drug is more than 20 times of the effective dosage. Therefore, the teasel root saponins compound has good safety.

EXAMPLE 10 preparation of Dipsacus asperoides saponins

1. Preparation of Compound 4 injection

Prescription 1 takes 1000mg of compound 4, 20mg of solubilizer F68, 60mg of cosolvent propylene glycol and 30mg of antioxidant sodium sulfite; the anhydrous glucose is adjusted to be isotonic, the pH value of the buffer salt is adjusted to 7, and the redistilled water is added to 150mL.

Prescription 2 takes 1000mg of compound 4, 25mg of solubilizer F68, 2000mg of HP-beta-cyclodextrin and 50mg of antioxidant sodium sulfite. Dissolving the prescription amount HP-beta-cyclodextrin in 40mL of ethanol, adding the compound 4 powder under stirring at 600rpm, and continuously stirring for 2h for later use; adding sodium bisulphite and F68 into 50mL of aqueous solution for dissolution, injecting HP-beta-cyclodextrin ethanol solution of the compound 4 under stirring until the compound 4 is completely dissolved, then pre-freezing at-60 ℃, and vacuum drying to obtain white loose freeze-dried powder.

2. Preparation method of dipsacus root saponins compound and dipsacus root saponins compound refined product

Prescription 1 teasel root saponins compound 10g, hydroxypropyl methyl cellulose 3g, starch 2g, lactose 1g, ethyl cellulose 0.5g and PVP0.5 g. Taking the prescription amount of dipsacus saponin compound, hydroxypropyl methylcellulose, ethylcellulose, starch and lactose, respectively sieving with a 80-mesh sieve, uniformly mixing, adding 3% PVP aqueous solution to prepare a soft material, granulating with a 20-mesh sieve, drying at 60 ℃ for 4h, finishing with a 18-mesh sieve, adding a proper amount of magnesium stearate, fully uniformly mixing, and tabletting with a 6mm flat punch to obtain tablets with the weight of 0.32g.

Prescription 2 teasel root saponin compound refined product 10g, CMC-Na0.5 g, gelatinized starch 0.5g, microcrystalline cellulose 2g,90% ethanol solution as adhesive, and proper amount of magnesium stearate as lubricant. Taking the prescription amount of teasel saponin active ingredient extract B, CMC-Na, gelatinized starch and microcrystalline cellulose, respectively sieving with 80 mesh sieve, mixing uniformly, preparing soft material with 90% ethanol, granulating with 20 mesh sieve, drying at 60deg.C for 4h, grading with 18 mesh sieve, adding appropriate amount of magnesium stearate, mixing uniformly, and tabletting with 6mm flat punch to obtain tablet with weight of 0.32g.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention.

It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A preparation method of dipsacus root saponins compound is characterized in that,

comprising the following steps:

2. The preparation method of the dipsacus saponin compound as defined in claim 1, which is characterized in that,

in the step S1 of the process,

the alcohol aqueous solution is 65-78v% ethanol aqueous solution;

preferably, the adding mass of the alcohol aqueous solution is 4-7.5 times of that of the teasel root medicinal material decoction pieces, the heating reflux is carried out for 2-3 times, each time of extraction is carried out for 2-4 hours, and the extracting solutions are collected and then combined.

3. The preparation method of the dipsacus saponin compound as defined in claim 1, which is characterized in that,

In the step S3, the macroporous adsorption resin is AB-8 macroporous adsorption resin.

4. The preparation method of dipsacus saponin compounds according to claim 3, which is characterized in that,

the step S3 is specifically performed by,

5. The preparation method of dipsacus root saponins compound according to any one of claims 1 to 4, characterized in that,

further comprises:

6. The teasel root saponins compound prepared by the preparation method of any one of claims 1 to 5.

7. The teasel root saponin compound according to claim 6, wherein,

The molecular structural general formula is as follows:

8. the use of a teasel root saponin compound as defined in claim 6 or 7 in the preparation of a medicament for preventing and treating fatty liver.

9. The use according to claim 8, wherein,

the applications include the use of a combination of a plurality of different types of materials,

regulating liver function and blood lipid abnormality caused by fatty liver, inhibiting oxidative stress of liver tissue, inhibiting lipid deposition in liver cells, blocking apoptosis of liver cells, repairing and protecting liver cells.

10. A pharmaceutical composition, health product or food for preventing and treating fatty liver disease comprising the dipsacoside compound of claim 6 or 7 or prepared from the dipsacoside compound of claim 6 or 7.