CN113521132B

CN113521132B - Preparation method of koozhao extract

Info

Publication number: CN113521132B
Application number: CN202010295787.4A
Authority: CN
Inventors: 胡琳; 任杨帆; 李清清; 胡群; 尹开云; 方震东
Original assignee: Kunming Xianghao Technology Co ltd
Current assignee: Kunming Xianghao Technology Co ltd
Priority date: 2020-04-15
Filing date: 2020-04-15
Publication date: 2023-02-10
Anticipated expiration: 2040-04-15
Also published as: CN113521132A

Abstract

The application discloses a preparation method of a quzhazha extract, wherein the quzhazhigan extract contains at least one of quzhazhigan or methyl polydatin. The Qu Zha extract rich in Quzhazhigan or methyl polydatin is found to have application value in the aspect of preventing and treating the non-alcoholic fatty liver disease for the first time.

Description

Preparation method of koozhao extract

Technical Field

The application relates to a preparation method of a koozhiza extract, belonging to the field of medicines.

Background

In normal human livers, lipids account for approximately 5% of the wet weight of the liver. When the accumulation of lipid in liver cell exceeds 5% of liver wet weight, or reaches more than 1/3 area histologically, it is non-alcoholic fatty liver disease (NAFLD).

NAFLD is a clinical syndrome disease characterized primarily by diffuse hepatocellular bullous fat, and has no history of excessive alcohol consumption and other definitive liver damaging factors. The NAFLD disease spectrum includes non-alcoholic simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and cirrhosis (cirrhosis). The incidence of NAFLD has increased year by year in recent years and has become a significant public health problem worldwide in the 21 st century. The prevalence of NAFLD is about 25% worldwide, which has become the first chronic liver disease in our country, and has increased to 58% and 74% in overweight people and patients with type II diabetes.

One longitudinal clinical data analysis showed that approximately 1/3 of NAFL patients progressed to NASH, and once NASH progressed, patients were significantly at increased risk for cirrhosis, hepatocellular carcinoma, and liver failure, and NASH presence also increased non-hepatic adverse consequences (such as cardiovascular and malignant diseases). Most NASH patients do not have obvious clinical symptoms for decades, but some patients develop a rapid progression. The NASH clinical study web fibrosis classification data shows that NASH diagnosed patients will progress to a fibrosis stage on average every 7 years. Therefore, the positive prevention and treatment of the fatty liver has important significance for preventing the progress of chronic liver diseases and improving prognosis.

The pathogenesis of NAFLD is closely related to insulin resistance, obesity, type II diabetes and hyperlipidemia, and due to a complex pathogenesis, a specific medicine aiming at NAFLD is not available in the market. At present, some corresponding therapeutic drugs including insulin sensitizer, lipid-lowering, anti-oxidative stress and liver-protecting drugs, etc. also fail to completely reverse the development of NAFLD.

The common drugs for clinically treating NAFLD are statins lipid-lowering drugs, which can increase the burden of the liver. The lack of drugs for the treatment of NAFLD has prompted researchers to study and develop novel drugs.

Disclosure of Invention

The present application provides a process for the preparation of a koji extract for solving the above technical problems.

The application provides a preparation method of a Quzhazhiza extract, wherein the Quzhazhigan extract contains at least one of Quzhazhigan or MePolygoni Cuspidatin.

Preferably, the preventing and/or treating NAFLD comprises: delay the development of NAFLD caused by high fat diet; or improving the activity of the liver antioxidant enzyme of the NAFLD rat, improving the antioxidant capacity of the liver cells, inhibiting oxidative stress and lipid peroxidation, and reducing the accumulation of lipid in the liver cells; or has obvious down-regulation effect on the liver inflammation level of the NAFLD, and can improve the liver inflammation environment so as to play the role of preventing and treating the NAFLD.

Preferably, the NAFLD comprises: simple non-alcoholic fatty liver disease (NAFL).

Preferably, the NAFLD comprises: nonalcoholic steatohepatitis (NASH).

Preferably, the NAFLD comprises: fatty liver fibrosis.

Need to explain: the application provides the Quzhu extract used in the application, and in order to prepare the required medicament, required auxiliary materials and auxiliary agents can be added according to the conventional requirements of the preparation form of the prepared medicament. The added auxiliary materials and auxiliary agents do not play a role in treatment. Meanwhile, after each quzhang extract is mixed with other treatment components according to requirements, the medicine is prepared, and the quzhang extract plays a role in preventing and/or treating NAFLD in the medicine.

The extract contains only one of Quzhazhigan and methyl polydatin, and can be used for the above purpose.

Specifically, the Quzhazhigan extract can be Quzhazhigan or methyl polydatin.

Preferably, the Quzhazhigan extract at least comprises Quzhazhigan or methyl polydatin.

Preferably, the Quzhazhigan extract is Quzhazhigan.

Preferably, the extract of kozak is methyl polydatin.

Preferably, the quzhazhigan extract at least comprises 50-99 wt.% of quzhazhigan and 1-25 wt.% of polydatin.

Preferably, the quzhazhigan extract at least comprises 50-80 wt.% of quzhazhigan and 20-50 wt.% of methylpiperazine.

Preferably, the extract at least comprises 25wt.% to 40wt.% of quzhazhigan, 5wt.% to 10wt.% of polydatin, 10wt.% to 25wt.% of methyl polydatin, 1wt.% to 5wt.% of resveratrol, 5wt.% to 10wt.% of piceatannol and 5wt.% to 10wt.% of methyl resveratrol.

Specifically, the chemical formula of the Quzhazhigan is (E) -1- (3,5-dihydroxyphenyl) -2- (3-hydroxy-4-O-D-glucopyranosyl) ethylene or 3,5,3',4' -tetrahydroxystilbene-3 ' -O-beta-glucoside, and the structural formula is shown as formula I:

specifically, the polydatin has a chemical formula of 4',5-dihydroxy-stilbene-3-O-glucoside (4', 5-dihydroxy-stilbene-3-O-glucoside, polydatin, piceid), and a structural formula of II:

specifically, the chemical formula of the giant knotweed rhizome glycoside is 5-hydroxy-4 '-methoxystilbene-3-O-glucoside (4' -methoxy-5-hydroxy-stilbene-3-O-glucoside, deoxyhalopontin), and the structural formula is shown as formula III:

specifically, the structural formula of resveratrol is shown as formula IV:

specifically, the structural formula of piceatannol is shown as formula V:

specifically, the structural formula of the methyl resveratrol is shown as a formula VI:

the application discovers for the first time that the Quzhazhigan extract containing at least one of Quzhazhigan, polydatin or methyl polydatin can significantly reduce serum Total Cholesterol (TC), triglyceride (TG), alanine Aminotransferase (ALT), glutamic-oxalacetic transaminase (AST) and low-density lipoprotein (LDL) indexes of NAFLD rats caused by high-fat diet, significantly increase serum high-density lipoprotein (HDL) indexes, and prompt that the Quzhazhigan extract has an obvious treatment and protection effect on experimental NAFLD rats and is beneficial to generation and development of fatty liver of the rats induced by high-fat diet. It is suggested that the medicine has corresponding effect on human body and can play a role in preventing and treating simple non-alcoholic fatty liver disease (NAFL).

The application also finds that the quzhao extract can obviously reduce the Malondialdehyde (MDA) content of liver tissues of NAFLD rats, and increase the activity of superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC), and shows that the quzhao extract can improve the activity of the liver antioxidant of the NAFLD rats, increase the antioxidant capacity of hepatocytes, inhibit oxidative stress and lipid peroxidation, and reduce the accumulation of lipid in the hepatocytes, so that the development of NAFLD can be effectively prevented and treated. It is suggested that the medicine has corresponding effect on human body and can play a role in preventing and treating simple non-alcoholic fatty liver disease (NAFL) and fatty liver fibrosis thereof.

The application further discovers that the quzhang extract can reduce the expression level of matrix metalloproteinase 9 (MMP-9), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and human monocyte chemotactic protein-1 (MCP-1) which are factors related to the inflammation of the rat NAFLD caused by high-fat diet, and the quzhang extract has a remarkable down-regulation effect on the liver inflammation level of the rat NAFLD and can improve the liver inflammation environment so as to play a role in preventing and treating the NAFLD. It is suggested that the medicine has corresponding effect on human body and can prevent and treat nonalcoholic steatohepatitis (NASH).

Pharmacodynamic experiment results show that the Quzhang extract can obviously reduce the content of Malondialdehyde (MDA) in liver tissues and increase the activity of superoxide dismutase (SOD) and the total antioxidant capacity (T-AOC).

In recent years, the oxidative stress mechanism of fatty liver and the prevention and treatment effect of antioxidant in fatty liver pathological process are attracting more and more attention. Fatty liver is a pathological change caused by the disturbance of lipid metabolism of liver due to various causes, and the accumulation of a large amount of lipid in liver, and the occurrence of steatosis when the lipid is accumulated in liver tissue for a long time will block the transmission of electron current in respiratory chain, induce pro-oxidant increase and anti-oxidant decrease of oxygen stress, thereby promoting the significant increase of intracellular Reactive Oxygen Species (ROS), and react with intracellular lipid, protein and DNA, resulting in the damage and death of liver cells.

ROS generated after the enhancement of oxygen stress oxidize the biofilm to form a series of lipid free radicals and degradation products such as Malondialdehyde (MDA) and 4-hydroxynonenoic acid (HNE). These substances can further cross-link intracellular proteins to form Mallory corpuscles, and induce immune responses, chemotaxis neutrophils, and cause inflammatory cell infiltration. The lipid peroxide not only increases endogenous ROS and toxicity, but also can inhibit oxidation and the like, and increase the sensitivity to exogenous peroxide toxicity.

The blood MDA level of experimental NAFLD (nafLD) hyperlipidemic rats is obviously increased, the T-AOC level and the SOD activity are obviously reduced, and the condition that the oxidation and antioxidation balance of the body is damaged under the state of non-alcoholic fatty liver is suggested, so that the excessive accumulation of ROS in the body is caused.

The application finds that the Quzhu extract can improve the activity of liver antioxidase of a rat NAFLD, increase the antioxidant capacity of liver cells, and inhibit oxidative stress and lipid peroxidation, so that the accumulation of lipid in the liver cells is effectively reduced, and the purpose of effectively preventing and treating NAFLD is achieved.

In addition, the liver can generate inflammatory factors due to long-term high fat diet, the generation and development of NAFLD are closely independent of inflammatory reaction, and various inflammatory factors play a role in the NAFLD to aggravate the degree of liver lesion. TNF-alpha is an important inflammatory factor mediating liver injury and plays a cytotoxic role in liver disease leading to liver injury. The liver kupffer cell can produce IL-1 beta to aggravate liver inflammation and steatosis, and the IL-1 beta promotes the generation of nitric oxide and apoptosis in islet cells to cause the selective damage of the islet cells, thereby further inducing insulin resistance. Inflammatory cells, represented by neutrophils, produce MMP-9, which is one of the markers of chronic inflammatory changes in NAFLD liver tissue, and can degrade liver basement membrane and destroy basement membrane integrity.

MMP-9 overexpression can destroy the normal matrix environment of Hepatic Stellate Cells (HSC), so that the Hepatic Stellate Cells (HSC) are activated, and the activated HSC expresses a large amount of inflammatory mediators and fibrosis cytokines and stimulates the mass synthesis of extracellular matrix (ECM), thereby promoting the occurrence and development of hepatic fibrosis. The overexpression of MMP-9, IL-1 beta and TNF-alpha forms a mutual vicious circle, and the pathological degree of the liver is continuously increased.

MCP-1 is a chemokine, a potent chemoattractant secreted by macrophages, endothelial cells, hepatic stellate cells and vascular smooth muscle cells, and is associated with fat accumulation in hepatocytes in NAFLD in response to inflammatory stimuli of IL-1 β and TNF- α.

The animal experiment results prove that the quzhao extract can obviously reduce the expression levels of the factors MMP-9, TNF-alpha, IL-1 beta and MCP-1 related to the inflammation of the NAFLD rat caused by high-fat diet, and the quzhao extract has an obvious down-regulation effect on the liver inflammation level of the NAFLD rat and can obviously improve the liver inflammation environment so as to block the process of the NAFLD and play a role in preventing and treating diseases.

The extract of Quzhao in the above application can be prepared from Qu Zha rhizome by conventional extraction and purification method, or by mixing the extracts containing the above components at the above ratio. The ingredients of the Quzhazha extract can also be prepared from other plant materials such as edible radix et rhizoma Rhei (Rheum rhaponticum L.) and rhizoma Polygoni Cuspidati (Polygonum cuspidatum Sieb. Et Zucc.) or obtained by chemical synthesis. The medicinal material, namely the roots and stems of the quzhang are used as raw materials for extraction.

Quzhao, a Tibetan medicine material, the plant source is Rheum lhasaense A.J.Li et P.K.Hsiao, belonging to Rheum genus plant of Polygonaceae, produced in eastern Tibet, sichuan and the like, is successfully introduced and domesticated in Shanshan plant gardens of Shanghai Shangri. The Quzhu herb is selected from wild or planted Rheum palmatum rhizome.

The research on the chemical components and the pharmacological activity of the Lhasa rhubarb finds that the Lhasa rhubarb contains high-content stilbene compounds which mainly comprise Quzhazhigan and methyl polydatin. Unlike other rhubarb plants, quzha contains almost no anthraquinone chemical components with potential toxic and side effects.

In another aspect of the present application, there is provided a medicament for preventing and/or treating NAFLD comprising the extract Qu Zha as described above;

preferably, the pharmaceutical dosage form is a fixed oral dosage form or an injectable dosage form.

Is prepared by the preparation method of a fixed oral preparation or an injection preparation in the prior art.

The beneficial effects that this application can produce include:

1) The application provided by the application discovers for the first time that the Qu Zha extract rich in Quzhazhigan and methyl polydatin has application value in the aspect of preventing and treating non-alcoholic fatty liver. Pharmacodynamic experiment results show that the Quzha kouzi extract can remarkably reduce serum Total Cholesterol (TC), triglyceride (TG), alanine Aminotransferase (ALT), glutamic-oxalacetic transaminase (AST) and low-density lipoprotein (LDL) indexes of NAFLD rats caused by high-fat diet, remarkably increase serum high-density lipoprotein (HDL) indexes, prompt that the Quzha kouzi extract has a remarkable treatment and protection effect on non-alcoholic fatty liver induced by high-fat diet, and is beneficial to delaying or preventing NAFLD induced by high-fat diet, especially occurrence of simple non-alcoholic fatty liver.

(2) The application provided by the application is rich in the stilbene components such as Quzhazhigan, methyl polydatin and the like, and the components are clear and controllable. Because the used plant-based source contains rich stilbene substances and the extract hardly contains anthraquinone impurities, the extract rich in the stilbene components such as quzhazhigan, methyl polydatin and the like can be obtained only by a conventional extraction and purification method, and the extract raw material for preparing the medicament can be obtained without complicated purification and refining.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

Examples

Unless otherwise specified, the raw materials, solvents and auxiliaries in the examples of the present application were purchased commercially and were not treated.

The instrumentation used was tested:

the multifunctional microplate reader is a cassette SpectraMax Paradigm multifunctional microplate reader produced by Molecular Devices; the full-automatic chemiluminescence apparatus is a UniCel DxI 800 immunoassay system produced by Beckmann Coulter. The refrigerated centrifuge is a high-speed refrigerated centrifuge 3k15 type produced by Sigma in Germany; the full-automatic biochemical analyzer is a UniCel DxC 800 Synchron full-automatic biochemical analyzer produced by Beckmann Coulter.

Example 1 preparation of Quzhazha extract samples 1-12

a. Washing artificially-planted or wild koozhiza plants with water to remove soil, hanging and air-drying in a ventilation place, separating underground rhizomes and overground stems and leaves, and respectively crushing the underground rhizomes and the overground stems and leaves into coarse particles by a crusher;

b. cutting underground rhizome and overground stem and leaf, soaking in 75% ethanol for 24 hr, percolating with 75% ethanol at percolation rate of 20ml/min, and mixing extractive solutions;

performing high performance liquid chromatography (a chromatographic column using octadecylsilane chemically bonded silica as a filler, methanol-6% acetic acid (19) as a mobile phase, 319nm detection wavelength and high performance liquid chromatography operation according to appendix V of 2010 version of Chinese pharmacopoeia) on the obtained extracting solution, determining the content of each substance in the extracting solution, and entering step c when the content of each substance meets the requirement;

c. and c, adding water into the extract of the quzha in the step b for precipitation, collecting the precipitate, and directly drying and refining to obtain the quzha extract samples 1-11.

The contents of the substances in the quzao extract samples 1-11 are shown in table 1, and the percentages of the substances in the table represent the mass ratio of the substances to the total mass of the quzao extract.

TABLE 1 percentage content of active ingredients in each Quzhang extract sample

Example 2: preparing oral dispersible tablet of Quzhazha extract 1-5

(1) Prescription

Raw materials	Dosage of
		QuzhaExtract of plant	200.00g
Hydroxypropyl-beta-cyclodextrin	175.00g
		Sodium carboxymethyl starch	60.00Lg
Pregelatinized starch	40.00g
		Magnesium stearate	25.00g
Is made into	1000 tablets

(2) Method for producing

Preparing a inclusion compound of the quzha extract: dissolving the prescription amount of Quzhazha extract in 2.5L50% ethanol, grinding, adding the prescription amount of hydroxypropyl-beta-cyclodextrin, filtering the obtained solution with microporous membrane until the solution is clear, and separating the clathrate from the mixture. Adding hydroxypropyl-beta-cyclodextrin, sodium carboxymethyl starch and pregelatinized starch according to the formula amount by an equivalent gradual addition method, uniformly mixing, adding magnesium stearate, uniformly mixing, and tabletting to obtain the oral dispersible tablets 1-5 of the quzha extract.

The correspondence between dispersible tablets 1-5 and the Quzha extract used in each dispersible tablet is shown in the following table:

dispersible tablet numbering	Quzha extract sample number
		1	1
2	2
		3	3
4	4
		5	5

EXAMPLE 3 preparation of Quzhu extract injection 1-5

(1) Prescription

(2) Method for producing

Weighing 2.50L of absolute ethyl alcohol, adding 1.50L of propylene glycol and phosphate buffer solution, mixing well, adding 100.00g of the quzhazhigan extract obtained in examples 1 and 2, performing ultrasonic dissolution, and fixing the volume of the buffer solution to scale. Filtering with 0.45 μm microporous membrane, and coarse filtering. The secondary filtrate is filtered through a 0.22 mu m microporous filter membrane and is filtered through a 1 ten thousand molecular weight cutoff polyether sulfone membrane to remove pyrogen. Filling nitrogen into the brown bottle, and encapsulating to obtain 1-5 parts of injection.

The correspondence between the injections 1-5 and the Quzha extracts used in the injections is shown in the following table:

injection numbering	Quzha extract sample number
		1	1
2	2
		3	3
4	4
		5	5

EXAMPLE 4 improving Effect of Quzha extract on high fat diet-induced non-alcoholic fatty liver disease (NAFLD) in SD rats

1. Experimental methods

1.1 clean SD rat is fed with common feed for 1 week, and the experiment is started, with room temperature of 22 + -2 deg.C and humidity of 55 + -15%. Randomly selecting 10 rats as a normal group, feeding the rats by using a common feed, taking 60 rats as a modeling group, feeding the rats by using a high-fat feed (5% of lard, 10% of sucrose, 3% of egg yolk, 5% of cholesterol and 59% of a basal feed) for 4 weeks, and then dividing the rats into a model group and Qu Zha extract samples 1-5 groups, wherein each group comprises 10 rats.

The normal group and the model group are perfused with normal saline, the administration group is perfused with 40mg/kg of normal saline for 8 weeks, and mouse serum and liver are taken after 12 weeks of experiments. Fasting is carried out for 12 hours before material taking, water is not forbidden, chloral hydrate is used for anesthesia on day 2, eyeballs are removed, blood is taken, livers are quickly dissected and weighed, and normal saline at 4 ℃ is used for washing for standby.

1.2 serum physiological and biochemical index detection: the contents of serum Total Cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C) and high density lipoprotein (HDL-C) were measured with a blood lipid biochemical kit (Shanghai Jingjing diagnostics technologies Co., ltd.).

Mouse whole blood 3000 r.min ^-1 Centrifuging for 10min, collecting serum, operating according to the kit instructions, and measuring absorbance values of TC and TG with an enzyme-labeling instrument at 510nm wavelength; the absorbance of LDL and HDL was measured at 546nm, and the concentration of each marker in serum was calculated. The levels of serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) were measured by ELISA, which was performed according to the instructions of the kit, and the absorbance at 510nm was measured by an enzyme reader to calculate the concentration of each index in serum.

1.3 detecting physiological and biochemical indexes of liver: adding normal saline into liver tissue at 4 deg.C to make into 100 g.L ^-1 Centrifuging the liver homogenate at 3500r/min at 4 deg.C for 10min, collecting supernatant, and storing at-70 deg.C for detecting physiological and biochemical indexes of liver. The total antioxidant capacity (T-AOC) is measured by a colorimetric method, malondialdehyde (MDA) is measured by a TBA method, and superoxide dismutase (SOD) is measured by a hydroxylamine method, which are operated according to the operation method of the kit specification.

The results are expressed as protein per gram of liver tissue. The expression levels of matrix metalloproteinase 9 (MMP-9), tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and human monocyte chemotactic protein-1 (MCP-1) are determined by enzyme-linked immunosorbent assay, and operation and calculation are carried out according to the instructions of rat MMP-9, rat TNF-alpha and rat MCP-1ELISA kit (Heifeier biology).

1.4 data processing: data to

The normal distribution data is tested by t, and the bias distribution data is tested by rank sum.

2. The experimental results are as follows:

2.1 Effect on NAFLD rat serum Biochemical index

The results for the quzao extract samples 6-11 are similar to those listed in table 2, and the experimental results for the representative samples 1-5 are shown in table 2.

TABLE 2 Effect of Quzhang extracts on the biochemical indices of NAFLD rat serum: (

n＝10，TC，TC，LDL,HDL:mmol/L；AST,ALT:U/L)

Note: in comparison with the normal group, ^Δ p is less than 0.05; in comparison with the set of models, ^* P＜0.05， ^** P＜0.01

the experimental results in table 2 show that compared with the normal group, the serum TC, TG, LDL, ALT and AST contents of the model group mice are obviously increased, the HDL content is obviously reduced (P is less than 0.05), and the model building is successful. Compared with the model group, the serum TG, TC and LDL contents of mice of each administration group are obviously reduced (less than 0.01, P is less than 0.05), the HDL content is obviously increased (P is less than 0.05), and the AST and ALT contents or activities are obviously reduced (P is less than 0.01, P is less than 0.05).

The results show that after the high fat feeding action, the blood fat TC, TG and LDL levels are obviously increased, the liver function indexes AST and ALT levels are obviously increased, and the liver cells are damaged to a certain extent. 1-5 groups of the Quzhazha extracts can obviously reduce the content of TC, TG, LDL, AST and ALT in serum of NAFLD rats, increase the content of HDL and prompt that liver cell damage of each group of the Quzhazha extracts is protected and relieved. The effect of the Quzhazha extract samples 6-11 was similar to that of Qu Zha extract samples 1-5.

2.2 Effect on the expression level of TNF-alpha, IL-1 beta, MCP-1, MMP-9 proteins in NAFLD rat serum

The effect of the quzha extract samples 6-11 is similar to the results listed in table 3, and the experimental results of the effect of the typical quzha extract samples 1-5 on the expression level of TNF-alpha, IL-1 beta, MCP-1, mmp-9 protein in liver tissue of NAFLD rat are shown in table 3.

TABLE 3 influence of the extract of each Quzhu on the expression level of TNF-alpha, IL-1 beta, MCP-1, MMP-9 protein in NAFLD rat serum

Group of	TNF-α	IL-1β	MCP-1	MMP-9
					Is normal and normal	1.49±0.21	1.33±0.13	0.78±0.22	0.98±0.10
Model (model)	2.53±0.24 ^Δ	1.87±0.16 ^Δ	1.13±0.17 ^Δ	1.32±0.09 ^Δ
					Quzha extract 1	2.11±0.31 ^*	1.35±0.12 ^*	0.88±0.19 ^*	1.07±0.12 ^*
Quzhao extract 2	2.18±0.20 ^*	1.43±0.15 ^*	0.83±0.21 ^*	1.01±0.09 ^*
					Quzha extract 3	1.82±0.19 ^*	1.36±0.11 ^*	0.78±0.11 ^*	0.96±0.10 ^*
Quzha extract 4	1.76±0.22 ^*	1.31±0.11 ^**	0.67±0.15 ^**	0.88±0.09 ^**
					Quzha extract 5	1.98±0.18 ^*	1.39±0.17 ^*	0.73±0.13 ^*	0.99±0.11 ^*

Note: as compared with the normal group, the test results, ^Δ p is less than 0.05; in comparison with the set of models, ^* P＜0.05， ^** P＜0.01

the experimental results in Table 3 show that the expression levels of NF-alpha, IL-1 beta, MCP-1 and MMP-9 in the model group are remarkably increased compared with those in the normal group. Compared with a model group, each quzha extract can obviously reduce the expression level of the protein (P is less than 0.05, P is less than 0.01), and the suggestion that each quzha extract has down-regulation effect on the liver inflammation level of NAFLD rats. The effect of the Quzhazha extract samples 6-11 was similar to that of Qu Zha extract samples 1-5.

2.3 Effect on NAFLD rat liver T-AOC, SOD and MDA

The effect of quzha extract samples 6-11 was similar to the results listed in table 2, and the results of the experiment on the effect of typical quzha extracts 1-5 on liver tissue of NAFLD rats are shown in table 4.

TABLE 4 Effect of extracts of Fiveleaf akebia on NAFLD rat liver tissue T-AOC, SOD and MDA

Group of	T-AOC(U/L)	SOD(U/mg)	MDA(nmol/mg)
				Is normal	2.31±0.13	2.49±0.29	6.19±0.95
Model (model)	0.87±0.09 ^△△	1.97±0.26 ^△	7.07±0.86 ^△
				Quzha extract 1	1.67±0.13 ^**	2.07±0.21 ^*	6.35±1.09 ^*
Quzhao extract 2	1.45±0.08 ^*	2.08±0.20 ^*	6.33±1.05 ^*
				Quzha extract 3	1.86±0.05 ^*	2.16±0.17 ^*	6.46±0.96 ^*
Quzha extract 4	1.97±0.07 ^**	2.21±0.12 ^*	6.36±1.05 ^*
				Quzha extract 5	1.76±0.04 ^*	2.18±0.18 ^*	6.39±1.05 ^*

Note: in comparison with the normal group, ^△ P＜0.05, ^△△ p is less than 0.01; in comparison with the set of models, ^* P＜0.05， ^** P＜0.01

the experimental results in Table 4 show that compared with the normal group, the T-AOC and SOD activity of the modeling group is obviously reduced (P is less than 0.01 and P is less than 0.05), and the MDA content is obviously increased (P is less than 0.05). Compared with the model group, the liver MDA level of each quzhang extract group is obviously reduced, the T-AOC activity and the SOD activity are obviously increased, and the fact that each quzhang extract group can regulate the antioxidant capacity of liver cells is suggested. The effect of the Quzhazha extract samples 6-11 was similar to that of Qu Zha extract samples 1-5.

Reference throughout this specification to "one embodiment," "another embodiment," "an embodiment," "a preferred embodiment," or the like, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment described generally in this application. The appearances of the same phrase in various places in the specification are not necessarily all referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with any embodiment, it is submitted that it is within the scope of the disclosure to effect such feature, structure, or characteristic in connection with other embodiments.

Although the present application has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the scope and spirit of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.

Claims

1. A preparation method of a Quzhang extract is characterized by comprising the following steps:

a. taking artificially planted or wild kozak plants, washing with water to remove soil, hanging and air-drying in a ventilation place, separating underground rhizomes and overground stems and leaves, and respectively crushing the underground rhizomes and the overground stems and leaves into coarse particles by a crusher;

performing high performance liquid chromatography on the obtained extractive solution with chromatographic column using octadecylsilane chemically bonded silica as filler; methanol-6% acetic acid 19:81 is a mobile phase; the detection wavelength is 319nm; performing high performance liquid chromatography operation detection according to appendix V of 2010 edition of Chinese pharmacopoeia, determining the content of each substance in the extracting solution, and entering step c when the content of each substance meets the requirement;

c. c, adding water into the extract of the quzha in the step b for precipitation, collecting the precipitate, and directly drying and refining to obtain the quzha extract;

the extract contains Quzhazhigan 35 wt%, polydatin 8 wt%, polydatin methyl 25 wt%, resveratrol 5 wt%, piceatannol 6 wt%, and resveratrol methyl 10 wt%;

the application of the kozakh extract in preparing a medicine for preventing and/or treating NAFLD; the NAFLD is non-alcoholic fatty liver induced by high fat diet.

2. The method of claim 1, wherein the pharmaceutical dosage form of the Quzhang extract is an oral dosage form or an injection dosage form.