CN111000852B

CN111000852B - Application of withanolide extract in physalis angulata in preparation of drugs for preventing or treating non-alcoholic fatty liver diseases

Info

Publication number: CN111000852B
Application number: CN202010009086.XA
Authority: CN
Inventors: 罗建光; 张�浩; 陈馨霖; 项德娟; 张美卉
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2022-03-11
Anticipated expiration: 2040-01-06
Also published as: CN111000852A

Abstract

The invention belongs to the field of medicines and health-care products, and particularly relates to application of a withanolide extract in physalis angulata in preparation of a medicine or health-care product for preventing and treating non-alcoholic fatty liver disease. In the MCD diet-induced NASH model, the withanolide extract can inhibit liver steatosis, reduce ALT and/or AST in plasma and TG and/or TC in liver tissues, and has certain prevention and treatment effects on the occurrence and development of NASH. In an animal model of liver injury caused by a chemical inducer, the withanolide extract has good liver injury protection effect and anti-hepatic fibrosis activity. The withanolide extract has the advantages of outstanding effect and high safety, and is expected to be developed into an effective medicament for treating and preventing non-alcoholic fatty liver disease, protecting liver injury and resisting hepatic fibrosis.

Description

Application of withanolide extract in physalis angulata in preparation of drugs for preventing or treating non-alcoholic fatty liver diseases

The technical field is as follows:

the invention belongs to the field of medicines, and relates to application of a withanolide extract in physalis angulata in preparation of a medicine or health-care product for preventing and treating non-alcoholic fatty liver disease.

Background art:

non-alcoholic fatty liver disease (NAFLD) is a metabolic stress liver injury that is closely related to Insulin Resistance (IR) and genetic predisposition. The number of patients with NAFLD and the influence range exceed those of viral hepatitis, and the NAFLD is one of the most common liver diseases affecting teenagers and adults in China. The disease spectrum of NAFLD includes nonalcoholic simple fatty liver, nonalcoholic steatohepatitis (NASH), liver fibrosis (liver fibrosis), cirrhosis (liver cirrhosis), and liver cancer (HCC). NASH is the progressive stage of NAFLD, and about 10-20% of NASH patients can progress to liver fibrosis, cirrhosis and even liver cancer. NASH is mainly characterized by inflammation and fibrosis, which may be accompanied by cellular injury, inflammatory cell infiltration and hepatocellular ballooning, eventually leading to cirrhosis and hepatocellular carcinoma. With the increasing incidence of NASH, the incidence of NASH-induced liver cancer (NASH-HCC) has increased quietly, and many patients develop liver cancer before cirrhosis. To date, no drug has been approved for the treatment of NASH.

NAFLD is a complex, systemic metabolic disease, the main pathological mechanism of which is insulin resistance and its secondary disturbance of sugar and lipid metabolism. In view of the complex and multifactorial pathological characteristics of NAFLD, the traditional Chinese medicine with the characteristics of multi-path pharmacological action has great advantages in the treatment. Some Chinese patent medicine shell lipid capsules, Dangfei liganning, liver-strengthening capsules, stagnation-removing liver-softening granules and Sanqi fatty liver pills in China have been used for NAFLD clinical treatment, but the medicines are mostly derived from traditional Chinese medicine compound, and have the problems of unclear material basis, and difficult preparation quality controllability and stability.

Physalis angulata (Physalis angulata L.) is a plant of Physalis of Solanaceae, and has functions of clearing heat, promoting urination and removing toxic substance. It can be used for treating common cold, cough due to lung heat, swelling and pain of throat, gingival swelling, jaundice due to damp-heat pathogen, dysentery, edema, pyretic stranguria, pemphigus, and furuncle. It is used clinically to treat jaundice, chronic tracheitis and bacillary dysentery. The withanolide compound is the main effective active ingredient of the plant.

In recent years, the laboratory utilizes the constructed non-alcoholic fatty liver disease resistant drug cell screening model based on the cholesterol 7-hydroxylase CYP7A1 promoter drug screening target to study the regulation and control of CYP7A1 activity by the withanolide extract, and finds that the extract has different degrees of inhibition effects on the CYP7A1 promoter, which indicates that the extract has potential non-alcoholic fatty liver disease resistant activity. The laboratory deeply studies the non-alcoholic fatty liver disease resisting effect of the withanolide extract, and further verifies the NASH model of MCD diet induction and the chemical inducer CCl from the in vivo animal model on the basis of examining the safety of the extract₄The protective effect of the liver injury caused by the liver injury. The experimental study on the NASH resistant function and the liver injury protection function of the withanolide extract has no reports at home and abroad so far.

The invention content is as follows:

the invention provides a preparation method of withanolide part in physalis angulata and application of withanolide part in drugs or health care products for preventing and treating non-alcoholic fatty liver disease, protecting liver injury and resisting hepatic fibrosis.

The withanolide extract in physalis angulata is applied to the preparation of medicines or health products for preventing or treating non-alcoholic fatty liver diseases.

The application is characterized in that the withanolide extract in the physalis angulata contains any two or a mixture of more than two of Phylysin B, Phylysin D, Phylysin F, Phylysin I and Phylysin H.

The application is characterized in that the weight portions are as follows: the withanolide extract in the physalis angulata is a mixture of 0.1-100 parts of Phylysin B, 0.1-100 parts of Phylysin D, 0.1-100 parts of Phylysin F, 0.1-100 parts of Phylysin I and 0.1-100 parts of Phylysin H.

The application is characterized in that the withanolide extract in the physalis angulata is a pharmaceutical composition consisting of the withanolide extract in the physalis angulata and pharmaceutically acceptable auxiliary materials.

The application is characterized in that the withanolide extract in the physalis angulata is prepared into oral dosage forms and non-oral dosage forms; the oral dosage form is tablet, capsule, powder or granule.

The preparation method of the withanolide extract from physalis angulata of the invention comprises the following steps:

pulverizing dried Physalis angulata, extracting with 95% ethanol under reflux for three times, each for 3 hr. Filtering, combining the filtrates, and concentrating under reduced pressure to dryness at 50 deg.C. And (3) loading the obtained extract according to the ratio of 2:1 (the weight of D101 macroporous resin filler: the weight of concentrate), sequentially carrying out chromatography and purification on the extract by using 20%, 80% and 100% ethanol concentration gradients through D101 macroporous resin, collecting 80% ethanol eluent, and concentrating under reduced pressure to dry to obtain the withanolide extract in the physalis angulata. The structure of the main component is determined by HPLC-DAD through comparison with a Phylysin compound standard substance, and the content of the main Phylysin compound is determined.

The cholesterol 7-hydroxylase CYP7A1 promoter is used as a drug screening target, CYP7A1 luciferase plasmid pGL4.17-CYP7A1 and Renailla plasmid are co-transfected into human normal liver cell LO2, and a dual-luciferase report system is utilized to find that the withanolide extract has inhibition effects on the CYP7A1 promoter in different degrees. In order to further evaluate the efficacy of the compound in treating the non-alcoholic fatty liver disease in vivo, the invention firstly adopts a dose of 600mg/kg to carry out toxicity evaluation on the withanolide extract, the weight of a mouse does not obviously reduce after 14 days of administration, the withanolide extract has no hepatotoxicity and no toxic sign or death, the safety of the medicine is higher, the basis is provided for the pharmacodynamics evaluation in vivo of the compound, and the foundation is laid for the compound to be developed into the medicine or health-care products. The invention relates to an in vivo pharmacodynamic experiment, which firstly evaluates the in vivo NASH resistant function of withanolide extract by an MCD diet-induced NASH model. The liver histopathological section staining result shows that compared with a model group, the fat accumulation and inflammatory cell infiltration conditions of mouse liver cells of the withanolide extract administration group are obviously improved; ALT and AST activities in plasma and TC and TG contents in liver were significantly lower than those in the model group. The results show that the withanolide extract can prevent MCD diet-induced liver steatosis and oxidative stress injury, and has certain prevention and treatment effects on the occurrence and development of NASH. Meanwhile, the invention also utilizes a chemical inducer CCL4 to induce a liver injury model to evaluate the protection effect of the withanolide extract on liver injury. The result of the liver histopathological section staining shows that the withanolide extract can relieve the liver injury induced by CCL4, improve the liver function and the pathological change of the liver of mice in a model group, reduce the degree of the liver fibrosis and delay the occurrence and the development of the liver fibrosis.

The withanolide extract is safe and effective when being taken orally or not when being used for non-alcoholic steatohepatitis, liver injury protection and hepatic fibrosis prevention and treatment. The oral administration can be made into any conventional dosage forms, such as tablet, capsule, powder, granule, etc.; it can be made into injection for non-oral administration.

The withanolide extract dosage can be adjusted according to factors such as administration mode, disease degree, patient age, existence of previous medical history and the like.

When the invention is used for preparing the medicine or health care product for preventing and treating the non-alcoholic fatty liver disease, protecting the liver injury and resisting the hepatic fibrosis, the auxiliary materials and the preparation method can adopt any pharmaceutically acceptable form.

Drawings

FIG. 1 is a liquid phase diagram of withanolide extract from Physalis angulata;

FIG. 2-inhibitory effect of withanolide extract on cholesterol 7-hydroxylase CYP7A1 promoter activity; wherein: p <0.05, p <0.01 compared to control (zero dosing concentration).

Figure 3-effect of withanolide extract on toxicity of mouse organs;

FIG. 4-HE staining of MCD diet-induced NASH model mouse liver tissue sections by withanolide extract;

wherein: a is the HE staining result of the liver tissue section of the mouse in the control group (feeding model control MCS diet); b is the HE staining result of the liver tissue section of the mice in the NASH model group (fed with MCD diet); C-E is the HE staining result of the liver tissue section of mice of the withanolide extract (7.5, 15, 30mg/kg) administration group.

FIG. 5-results of Withania somnifera lactone extract staining MCD diet-induced liver tissue sections of NASH model mice with oil Red O; wherein: a is the oil red O staining result of the liver tissue section of the mouse in the control group (feeding the model and controlling the MCS diet); b is the oil red O staining result of the liver tissue section of the mice in the NASH model group (fed with MCD diet); C-E is the result of oil red O staining of liver tissue sections of mice of a drug administration group with withanolide extract (7.5, 15, 30 mg/kg). FIG. 6-Withanolide extract vs CCl₄The pathological changes affecting the resulting liver injury; wherein: a is control group (without CCl)₄) HE staining results of mouse liver tissue sections; b is CCl₄The HE staining result of the liver tissue section of the mouse in the model group; C-E is the HE staining result of the liver tissue section of mice of the withanolide extract (2.5, 5, 10mg/kg) administration group.

FIG. 7-Effect of withanolide extract on the degree of liver fibrosis in CCl4 model mice; wherein: a is control group (without CCl)₄) The result of Sirius red staining of mouse liver tissue sections; b is CCl₄The result of Sirius red staining of a liver tissue section of a mouse in a model group; C-E is sleepResults of Sirius red staining of liver tissue sections of mice in the administration group of the solanolide extract (2.5, 5, 10 mg/kg).

FIG. 8-Withanolide extract vs CCl₄Influence of glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) levels in serum of a model mouse, wherein A is detection of ALT activity level, and B is detection of AST activity level; wherein: p<0.05,**p<0.01，***p<0.001 was compared to the model groups, respectively.

Detailed Description

The invention is described in detail below with reference to the drawings and examples, which are intended to be illustrative of the invention and not limiting.

Example 1 extraction preparation and qualitative analysis of withanolide from Physalis angulata

1. Extraction and preparation of withanolide from physalis angulata

Pulverizing 5kg dried Physalis angulata, extracting with 20L 95% ethanol under reflux for three times, each for 3 hr, filtering, mixing filtrates, and concentrating under reduced pressure at 50 deg.C to dry to obtain 440g extract. Loading the extract at a ratio of 2:1 (weight of D101 macroporous resin filler: weight of concentrate), purifying by D101 macroporous resin chromatography sequentially with 20%, 80% and 100% ethanol concentration gradient, eluting 8 column volumes each, collecting 80% ethanol eluate, and concentrating under reduced pressure at 50 deg.C to dry to obtain withanolide extract (225g) from Physalis angulata.

2. Qualitative analysis of withanolide extract from Physalis angulata

The reference substances Physialin B, Physialin D, Physialin F, Physialin I and Physialin H were prepared by the present study, with a purity > 95%, and the specific structures are as follows:

(1) chromatographic conditions are as follows: ZORBAX SB-C18 column (4.6X 250mm,5 μm), column temperature 25 deg.C, flow rate 1.0mL/min, wavelength scanning range 200 and 400nm, detection wavelength 230nm, sample injection 10 μ L, mobile phase acetonitrile (A) and deionized water (B), gradient elution program: 0-30min, 20-70% A.

(2) Preparing a test sample:

20.2314mg of dried Physalis angulata lactone extract is put into a 10ml volumetric flask, diluted to the mark with acetonitrile and shaken well to obtain a test solution.

(3) Preparation of standards

Accurately weighing each control, adding acetonitrile to prepare control solutions respectively containing physiolin B0.1862 mg/ml, physiolin D0.1338 mg/ml, physiolin F0.0872 mg/ml, physiolin I0.0856 mg/ml and physiolin H0.0899 mg/ml.

(4) And (4) analyzing results:

by comparing the ultraviolet absorption of withanolides compounds, it can be judged that 80% ethanol eluate extracted in the research institute is mainly withanolides compounds. In addition, the withanolide extract from the physalis angulata mainly comprises five physical withanolide compounds of Phylysin B, Phylysin D, Phylysin F, Phylysin I and Phylysin H (figure 1) by comparison with the standard, wherein Phylysin B is the most main component. In addition, the contents (weight percentage) of each compound were determined to be respectively 16.50% for physiolin B, 10.95% for physiolin D, 7.85% for physiolin F, 8.08% for physiolin I and 8.20% for physiolin H.

Withanolide extracts described in the following examples were prepared for example 1.

Example 2 Dual-luciferase reporter System assay for detecting the inhibition of Cholesterol 7-hydroxylase CYP7A1 promoter Activity by Withania Somnifera lactone extract

Human normal hepatocytes L02 were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum at 37 ℃ under 5% CO 2. After the confluence of cells was about 95%, 2X 10 cells per well⁴Individual cells were plated in 96-well white plates. And after the confluence degree of the cells is about 90 percent, changing the cells into a DMEM medium (Gibco) without fetal bovine serum for serum-free culture for 24 hours, co-transfecting the pGL4.17-CYP7A1 and the renalla plasmid into L02 cells by using Lipo2000, adding the withanolide extract after 6 hours of transfection, setting at least 3 multiple wells in each group of experiments, and continuing to culture for 24 hours. Following the procedure described in the Dual-GloTM Luciferase Assay System, the medium was aspirated and rinsed once with PBS. Adding 20 μ L/well of 1 XPLB, lysing the cells (within 15 min), adding 50 μ L/well of luciferase substrateDetection was carried out by adding 50. mu.L/well of the termination reaction solution. The results showed that the activity of the CYP7a1 promoter was inhibited with withanolide extract (fig. 2).

Example 3C57 mouse toxicity test

8C 57BL/6 mice were randomly divided into a control group and a withanolide extract administration group, and each group had 4 mice. The withanolide extract administration group was gavaged with 600mg/kg (control group was given 0.5% CMC-Na), and the body weight, physical condition (e.g., whether the breath was rapid, whether the skin had bleeding spots, etc.), and whether there was death were recorded daily for 14 days. Mice were sacrificed after 14 days. The results show (see figure 3), the weight of the mice does not obviously reduce after 14 days of administration, pathological staining of organs is observed, and toxic signs and death do not occur, which indicates that the medicine safety is higher.

Example 4 MCD-induced NASH model

The 25 male C57BL/6 mice were randomly and evenly divided into a normal control group, a model gavage low dose group, a medium dose group and a high dose group. The normal control group mice were fed with methionine choline normal content feed (MCS), and the model group, model gavage withanolide extract group were fed with methionine and choline deficient feed (MCD). During the molding, the stomach is filled, and the volume of the filled stomach is calculated according to 10 mL/kg. The administration dosage of the model gavage Withania somnifera lactone extract low-dosage group is 7.5mg/kg, the administration dosage of the model gavage Withania somnifera lactone extract medium-dosage group is 15mg/kg, the administration dosage of the model gavage Withania somnifera lactone extract high-dosage group is 30mg/kg, and the administration dosages of the normal group control group and the model group are 0.5% CMC-Na. Mice are raised for four weeks, blood is taken to kill after fasting without water supply for 12 hours, liver tissues are removed, cleaned, wiped dry and packaged. Putting the liver of a mouse with proper size into a 4% paraformaldehyde solution for histopathological detection of the liver; the rest liver is used as biochemical index detection group, and is placed in liquid nitrogen and stored at-80 deg.C. Standing at room temperature for 1h, centrifuging at 4000rpm at 4 deg.C for 10min, collecting upper layer serum, and packaging at-80 deg.C. HE staining results showed that the withanolide extract administered group had improved liver damage, significantly reduced liver fat accumulation, reduced necrotic cells and inflammatory cells, and more significant prophylactic effects in the high dose group compared to the medium and low dose groups, as compared to the NASH model group (fig. 4). The withanolide extract has liver injury protecting effect.

Example 5 effects of withanolide extract on hepatic steatosis, lipid deposition in NASH model mice.

The fat accumulation of the liver is judged by using a liver tissue section staining method, and fig. 5 shows an oil red O staining result, wherein the serious fat accumulation in a model group can be clearly observed, and the fat accumulation condition in the liver of a mouse in an administration group shows the reduction of dose dependence.

Example 6 effect of withanolide extract on serum liver function in NASH model mice.

Feeding mice for four weeks, fasting without water prohibition for 12h, taking blood for killing, standing the blood at normal temperature for 1h, centrifuging at 4000rpm and 4 ℃ for 10min, taking upper serum for subpackaging, and detecting the activity values of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the plasma of the mice by using the kit. As shown in Table 1, the activity value of ALT in the model group fed with MCD feed for four weeks reaches 183.10IU/L, while the activity value of enzyme in the normal control group is 8.52IU/L, and the increase of the enzyme activity value of the model group is obvious relative to that of the normal group (P < 0.001); after the Withania somnifera lactone extract is 7.5mg/kg and 15mg/kg of the administered mice, the activity value of ALT in the liver is reduced relative to that of the model group; after the mice in the administration group are perfused with the withanolide extract for 30mg/kg for four weeks, the activity value of ALT in the liver is obviously reduced relative to that in the model group (P < 0.05). The result shows that the withanolide extract has certain treatment effect on the elevation of ALT activity value in serum caused by liver injury of mice and has obvious dose dependence. When MCD feed is fed for four weeks, the vitality value of AST in the model group reaches 82.10IU/L, and is obviously increased (P is less than 0.001) relative to the vitality value of 20.50IU/L of a normal control group; after the mice in the administration group are subjected to gavage with the withanolide extract for 7.5mg/kg, 15mg/kg and 30mg/kg, the activity value of AST in the liver is obviously reduced relative to that of the model group (P < 0.05). The result shows that the withanolide extract has a certain prevention effect on the increase of AST activity value in serum caused by liver injury of mice.

TABLE 1 Effect of withanolide extract on serum ALT/AST levels in NASH model mice

	MCS	MCD	7.5mg/kg	15mg/kg	30mg/kg
						Plasma ALT(IU/L)	8.52±0.81	183.10±24.95^###	156.70±16.95^###	126.50±14.34^###	93.69±23.51^#*
Plasma AST(IU/L)	20.50±2.31	82.10±9.93^###	57.50±3.54^###*	49.05±5.93^##*	45.76±6.16^##*

#p<0.05,##p<0.01,###p<0.001vs MCS.*p<0.05,**p<0.01,***p<0.001vs MCD

Example 7 effect of withanolide extract on the content of TC, TG in the liver of NASH model mice.

Mice are raised for four weeks, blood is taken to kill after fasting without water supply for 12 hours, liver tissues are removed, cleaned, wiped dry and packaged. And taking the liver with proper size, and detecting the content of TC and TG by adopting a kit. As shown in table 2, after the model group was fed with MCD feed for four weeks, the TC content in the liver increased to 0.13mmol/gprot, the TC content in the liver of the normal control group was 0.08mmol/gprot, and the TC content in the model group was significantly increased relative to the normal control group (P < 0.05); after the withanolide extract is infused in the period of administration of the drug group for 7.5mg/kg and 15mg/kg four weeks, the content of TC in the liver of the mice in the drug group is reduced relative to that in the model group, and after the withanolide extract is infused in the mice in the drug group for 30mg/kg four weeks, the content of TC in the liver of the mice in the drug group is obviously reduced relative to that in the model group (P < 0.05). The results show that the withanolide extract can remarkably prevent the accumulation of TC in the liver and has certain dose dependence. After the model group is fed by MCD feed for four weeks, the content of TG in the liver is increased to 0.35mmol/gprot, the content of TG in the liver of the normal control group is 0.13mmol/gprot, and the content of the model group is increased relative to the normal control group (P is less than 0.001); after the mice in the administration group are subjected to intragastric gavage and the withanolide extract is 7.5mg/kg, 15mg/kg and 30mg/kg for four weeks, the content of TG in the liver is obviously reduced relative to that in the model group (P is less than 0.001). The results show that the withanolide extract can obviously prevent the accumulation of TG in the liver and has certain dose dependence.

TABLE 2 Effect of Withania somnifera lactone extract on TG and TC content in the liver of NASH model mice

Example 8CCl4 model of liver injury

C57 male mice (body weight 22-25g) were randomly assigned to control group, CCl₄Inducing group, and Withania Somnifera lactone extract (2.5, 5, 10mg/kg) administration group, each group containing 10 animals. For CCl₄Induction group, Withanolide extract-administered group, was subjected to CCl three times per week for 4 weeks₄The intraperitoneal injection induces a hepatic fibrosis model, and the intraperitoneal injection of olive oil is performed on a control group. The withanolide extract is administered to CCl for the first time₄Injection with CCl₄Administration is carried out after injection. At the last CCl₄Liver tissue was harvested 2 days after induction and HE staining of pathological sections was performed. The results show that the withanolide extract administration group and CCl₄The degree of liver damage was improved compared to the induction group (fig. 6). The withanolide extract has liver injury protecting effect.

Example 9 Withania somnifera lactone extract on CCl₄The effect of the pathological structural changes of the liver of the model mouse.

The Sirius red (Sirius red) staining is specific staining of collagen fibers, paraffin sections are stained with Sirius red, and the fibrosis condition of liver tissues of mice is observed. The results show that the degree of liver tissue fibrosis of the model mouse is obviously increased, and the degree of fibrosis is obviously inhibited after the withanolide extract is administrated (figure 7). The withanolide extract can obviously inhibit the generation of collagen fibers and inhibit the hepatic fibrosis of a model mouse.

Example 10 Withania somnifera lactone extract on CCl₄Induce the effects of serum liver function in mice.

Fasting for 12h before sampling, anesthetizing the mice with 3.5% chloral hydrate, taking blood from abdominal aorta, standing for 1h at room temperature, centrifuging at 3000rpm for 5min, and taking serum for serum biochemical index detection. The results show that the withanolide extract can reduce glutamic-oxalacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT), and the withanolide extract can improve CCl₄Induced liver damage in mice (figure 8).

Claims

1. The application of withanolide extract in Physalis angulata in preparing medicine for preventing or treating non-alcoholic fatty liver disease;

the withanolide extract in the physalis angulata is prepared by the following steps: pulverizing dried Physalis angulata, extracting with 95% ethanol under reflux for three times, each for 3 hr; filtering, mixing filtrates, and concentrating under reduced pressure at 50 deg.C to dry; according to the weight of D101 macroporous resin filler: sampling at the weight ratio of 2:1, purifying by D101 macroporous resin chromatography sequentially with 20%, 80% and 100% ethanol concentration gradient, collecting 80% ethanol eluate, and concentrating under reduced pressure to dry to obtain Physalis angulata lactone extract.

2. The use according to claim 1, wherein the withanolide extract from Physalis angulata comprises

A mixture of physalin B, D, F, I and H.

3. The use according to any one of claims 1-2, wherein the medicament is a combination of withanolide extract from Physalis angulata and pharmaceutically acceptable excipients.

4. The use according to claim 3, wherein the medicament is formulated into oral and non-oral dosage forms; the oral dosage form is tablet, capsule, powder or granule.