CN100528179C - Application of chrysanthemum indicum extract in the preparation of medicine for preventing and curing alcoholic liver disease - Google Patents

Abstract

The present invention relates to a novel use of wild chrysanthemum ethanol extract, that is an application in the preparation of the drugs for the prevention and treatment of alcoholic liver disease. The present invention explores a new medical use of the wild chrysanthemum ethanol extract and also develops a new application field. The wild chrysanthemum ethanol extract can not only cure the symptoms, but can also cure the disease permanently, which is safe, non-toxic, strong in pharmacological effects and indicates the good medicinal prospect; the invention has rich sources of the raw materials, low price and mature preparation process, which can be made into various preparations for oral administration and is easy to use.

Description

Application of wild chrysanthemum ethanol extract in the preparation of drugs for preventing and treating alcoholic liver disease

technical field

The invention relates to the application of the ethanol extract of the wild chrysanthemum, in particular to the application of the ethanol extract of the wild chrysanthemum in the preparation of medicines for preventing and treating alcoholic liver disease.

Background technique

Wild chrysanthemum is the flower head of Chrysanthemum indicum L., a plant of Compositae. It is picked when the flowers first bloom in autumn and winter, dried or steamed and used as medicine. It is one of the commonly used Chinese herbal medicines in most areas of my country. Widely distributed in Northeast my country, North China, Northwest China, East China, Southwest China and other places, in Anhui Province, it is mainly distributed in eastern Anhui, southern Anhui and other places. The wild resources are relatively rich, and they are mostly born on rocky hillsides, grasslands, fields, roadsides, etc.

Flos Chrysanthemum nature and flavor are bitter, acrid, be slightly cold, return liver, heart meridian, have the effect of dispelling wind-heat, reducing swelling and detoxifying, anti-infection, anti-virus, among the people cure furuncle carbuncle, conjunctival congestion and swelling pain, headache and dizziness. Clinically, wild chrysanthemum is widely used to prevent acute and chronic infectious diseases such as colds and meningitis, and is also used to treat carbuncles, boils, hypertension, hyperlipidemia, and tumors. Most of the domestic research is limited to the extraction and identification of the chemical components of the wild chrysanthemum, and the pharmacodynamics research also focuses on the antibacterial and antiviral activities.

Chemical composition of wild chrysanthemum:

There are many chemical components in wild chrysanthemum, such as flavonoids, terpenes, volatile oil, palmitic acid, polysaccharides, β-carotene, protein, amino acid, purine, choline, tannin, vitamins, chlorophyll and other components.

Flavonoids are important medicinal ingredients in wild chrysanthemum. As early as 1942, people isolated the flavonoid luteolin glucoside from wild chrysanthemum. Funded by the National Fund for Major Basic Research Preliminary Research, the School of Pharmacy of our school extracted and separated the wild chrysanthemum, and used the xylene-induced mouse ear swelling model to conduct an orthogonal design on several effective parts and dosages. The anti-inflammatory active site and dosage were screened out through repeated experiments. After identification by the Department of Natural Medicine and Chemistry of the College of Pharmacy, it was determined that the anti-inflammatory effective part of the wild chrysanthemum is the ethanol extract of the wild chrysanthemum, in which the total flavonoid content is as high as 60%, so it is also called the total flavonoids of Chrysanthemum indicum (TFC) . According to thin-layer chromatography and high-performance liquid chromatography, TFC mainly contains flavonoids such as luteolin, mongoside, and luteolin-7-glucoside.

Alcoholic liver disease and its drug treatment

Long-term excessive drinking, through ethanol itself and its derivative acetaldehyde, can cause fatty degeneration, necrosis and regeneration of liver cells repeatedly, leading to alcoholic liver disease (Alcoholic liver disease, ALD), including alcoholic fatty liver (Alcoholic fatty liver ), alcoholic hepatitis (Alcoholic hepatitis), liver fibrosis (Alcoholic fibrosis) and liver cirrhosis (Alcoholic cirrhosis) and other manifestations. Its histological diagnosis was divided into four types: alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis and alcoholic liver cirrhosis. In European and American countries, alcoholic liver disease is one of the main causes of death among young and middle-aged people. It is estimated that about 15.3 million people in the United States were alcoholics in 1993, and there were more than 2 million people with alcoholic liver disease. Every year, 26,000 people died of liver cirrhosis. At least 40% or 90% of the patients had a history of alcoholism. In my country, with the improvement of living conditions, the number of alcoholics tends to increase. Although there is no accurate statistics on the incidence of alcoholic liver disease, it is not uncommon. Because domestic liver disease is mainly caused by hepatitis virus, the number of carriers of hepatitis virus is more, which may cover up the liver disease that is actually caused by alcohol. Therefore, a correct understanding of alcohol-induced liver damage, timely diagnosis and prevention are of great significance.

The treatment of alcoholic liver disease is mainly aimed at: (1) reducing the severity of alcoholic liver disease; (2) preventing or reversing liver fibrosis; (3) improving the existing secondary malnutrition; (4) improving the severity of alcoholic liver cirrhosis treat. In recent years, drugs for the treatment of alcoholic liver disease include:

1. During glucocorticoid-induced alcoholic liver disease, there is an inflammatory reaction in the liver, swelling and necrosis of liver cells, and collagen production and deposition; there is evidence that immune factors are involved in the initiation and development of alcoholic liver disease. Glucocorticoids can inhibit the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, thereby inhibiting the pro-inflammatory effects of leukotrienes and prostaglandins, and can still promote albumin synthesis and prevent type I collagen production. Therefore, it was proposed that it can be used to treat alcoholic liver disease, but the results of many current studies are inconsistent. Many factors including gender, severity of liver disease, renal function, nutritional status and even geography can contribute to different outcomes. It is generally believed that hormones have no obvious effect on mild and moderate cases, and only severe cases can benefit from hormones. As for whether hormones have an impact on the long-term survival rate of patients, or whether long-term hormone therapy can prevent the development of cirrhosis, there is still a lack of research.

2. Insulin and glucagon Intravenous infusion of insulin and glucagon has a certain effect on alcoholic liver disease, but blood sugar should be detected during treatment to prevent fatal hypoglycemia.

3. Propylthiouracil The curative effect of propylthiouracil treatment is mainly seen in severe cases, and those with a relatively small amount of alcohol.

4. Antioxidants Reduced glutathione, taurine, carotene, vitamin E, r-evening primrose, selenium organic compounds, etc., may reduce oxygen stress damage and liver fibrosis induced by lipid peroxidation, It can relieve the toxicity of exogenous toxic substances.

5. Polyunsaturated lecithin is a stabilizer of liver sinusoidal endothelium and liver cell membrane, which can reduce lipid peroxidation, but it should be used with caution for those who cannot quit drinking because it is rich in unsaturated fatty acids.

6. Lipid-lowering drugs have objections and need to be explained. Many lipid-lowering drugs may tend to concentrate blood lipids in the liver for metabolism, but instead promote lipid accumulation and damage liver function.

7. Traditional Chinese medicine that inhibits liver fibrosis has a long history of using traditional Chinese medicine for promoting blood circulation and removing blood stasis to treat chronic liver diseases in my country, such as peach kernel, salvia miltiorrhiza, angelica, tetrandrine, fleece-flower root, hawthorn, turmeric, medlar, chuanxiong, Alisma, yellow Cen, sealwort, rhubarb, etc. can improve liver microcirculation, prevent liver cell degeneration and necrosis, reduce collagen fiber production or enhance collagenase activity, etc., which are helpful for the treatment of alcoholic hepatitis and liver fibrosis.

Contents of the invention

The purpose of the present invention is to provide a new application of the ethanol extract of the wild chrysanthemum, that is, a new application in pharmacy.

In fact, the present invention relates to the application of the ethanol extract of wild chrysanthemum in the preparation of drugs for preventing and treating alcoholic liver disease.

Physical properties of ethanol extract of wild chrysanthemum flower: dark brown powder, easy to absorb moisture, poor in water solubility, easily soluble in lipophilic organic solvents, and has a special fragrance.

The content of total flavonoids in the ethanol extract of Wild Chrysanthemum is 50-65% (HPLC method; colorimetric method). There are also saponins (15-20%, colorimetric method), caffeic acid (1-2%, HPLC) and chlorogenic acid (1-3%, HPLC). The ethanol extract of wild chrysanthemum is further separated to obtain luteolin, luteolin and mongoside. As determined by HPLC, the contents of the three flavonoids are luteolin (10-15%), luteolin (2-4%), and mongoside (8-12%), respectively.

Chrysanthemum chrysanthemum ethanol extract (TFC, 250, 500mg·kg ^-1 ) can significantly reduce the elevated levels of TG, AST, ALT in the serum of mice with acute alcoholic liver disease and the elevated MDA content in liver homogenate (compared with model group Compared with the model group, p<0.05), the activities of SOD, GSH, and GSH-Px in the liver homogenate were significantly improved (compared with the model group, p<0.05), and the excessively high levels of TNF-α and IL-1β caused by alcoholic liver disease were significantly inhibited (compared with the model group, p<0.05); at the same time, the results of histopathological examination indicated that TFC (250, 500 mg·kg ^-1 ) could significantly improve the degree of hepatic steatosis and reduce the inflammatory response of liver tissue.

In order to better understand the essence of the present invention, the following pharmacological tests and results of the ethanol extract of Chrysanthemum chrysanthemum will be used to illustrate its new use in the field of pharmacy.

The prevention and treatment effect test of wild chrysanthemum ethanol extract on acute alcoholic liver disease is as follows:

(1) Materials

Animals Kunming mice, male, weighing 22±2g; provided by the Experimental Animal Center of Anhui Medical University (certificate number: Wanyishidongzhun No. 02).

Main drugs and reagents Ethanol extract of wild chrysanthemum (TFC, self-made), Kaixilai (Henan Xinyi Pharmaceutical Co., Ltd., batch number: 060716), absolute ethanol (Shanghai Chemical Reagent Co., Ltd., batch number: 200610206), ALT , AST assay kit (Nanjing Jiancheng Bioengineering Institute, batch number: 20070118, 20070115), TG assay kit (Zhejiang Dongou Bioengineering Co., Ltd., product number: AO-10017), SOD, MDA, Coomassie brilliant blue protein assay Kit (Nanjing Jiancheng Institute of Bioengineering, batch number: 20070123, 20070122, 20061010), GSH, GSH-Px assay kit (Nanjing Jiancheng Institute of Bioengineering First Branch, batch number: 20070123, 20070122), TNF-α, IL-1β radioimmunoassay kit (Beijing North Institute of Biotechnology, batch number: 070120, 070120).

Main instruments BP211D electronic balance (manufactured by Saftarius, Germany), DL-5M low-speed refrigerated centrifuge (product of Changsha Xiangyi Centrifuge Instrument Co., Ltd.), TGL-16H high-speed centrifuge (product of Zhuhai Heima Medical Instrument Co., Ltd.), 722S spectrophotometer (product of Shanghai Precision Scientific Instrument Co.), GSY-8 electric heating constant temperature water bath (product of Yicheng Company of Beijing Medical Equipment Factory), Multiskan MK3 microplate reader (product of Leiber Company of the Netherlands), SW-CJ-IF ultra-clean workbench (Products of Jiangsu Sujing Group Suzhou Antai Air Technology Co., Ltd.), etc.

(2) Method

Model preparation, grouping and specimen collection

，体重22±2g，随机分成6组，每组10只。设正常对照组(Normal)、模型组(Model)、TFC低剂量组(125mg·kg^-1)、中剂量组(250mg·kg^-1)、高剂量组(500mg·kg^-1)和凯西莱(TP)阳性对照组(20mg·kg^-1)。Kunming mice fed normally for one week,

, weighing 22±2g, were randomly divided into 6 groups, 10 in each group. Normal control group (Normal), model group (Model), TFC low dose group (125mg·kg ^-1 ), middle dose group (250mg·kg ^-1 ), high dose group (500mg·kg ^-1 ) and Casey Lay (TP) positive control group (20 mg·kg ^-1 ).

During the experiment, intragastric (ig) administration (20ml·kg ^-1 ) was administered according to body weight, once a day for 7 consecutive days, and the intragastric drug was made into a suspension of 5 g·L ^-1 carboxymethylcellulose sodium , weigh the body weight before gavage every day to determine the amount of gavage, and the model group and the normal control group are given the corresponding volume of vehicle. 6 hours before the last administration, start fasting (not forbidden to drink), 1 hour after the last administration, except the normal control group mice ig corresponding volume of normal saline, the mice in other groups were ig 50% alcohol 6g·kg ^-1 once, After continuing to fast for 6 hours, blood was collected from the eyes of the mice, serum was separated, and relevant indicators were tested. At the same time, the animals were killed by necking, the liver was taken out and weighed, and a small piece of liver tissue at a distance of 0.5 cm from the edge of the left lobe of the liver was taken for histopathological examination, and liver homogenate was routinely prepared.

Determination of serum biochemical indicators Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TG) were determined according to the kit specifications.

Determination of liver biochemical indexes and cytokines Take the liver from the same part and accurately weigh 0.5g, make 10% liver homogenate in ice water, centrifuge at 3000rpm at 4°C for 10min, extract the supernatant, and measure superoxide according to the kit method Dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), glutathione peroxidase (GSH-Px) content, and tumor necrosis factor-α in the homogenate were determined by radioimmunoassay (TNF-α) and interleukin-1β (IL-1β) levels.

Histopathological examination of the liver The liver tissue was fixed with 10% neutral formalin, and the paraffin-embedded sections were subjected to conventional HE staining. The steatosis and inflammatory activity of the liver tissue were observed under a light microscope. The diagnostic criteria of liver histology refer to the diagnostic criteria of alcoholic liver disease formulated by the Fatty Liver and Alcoholic Liver Disease Group of the Hepatology Branch of the Chinese Medical Association.

(3) Results

1. Effect of ethanol extract of wild chrysanthemum on serum AST, ALT, TG levels in mice with acute alcoholic liver disease

It can be seen from Table 1 that the ethanol extract of wild chrysanthemum (TFC, 250, 500 mg·kg ^-1 ) can significantly reduce the levels of ALT, AST, and TG in the serum of mice with acute alcoholic liver disease, indicating that it has the ability to stabilize liver cell membranes and mitochondrial membranes, improve Lipid metabolism in the liver, reducing the absorption of lipids by the liver, and reducing the cytotoxic effect of free fatty acids on the liver.

Table 1 The effect of the ethanol extract of the wild chrysanthemum on the serum ALT, AST, and TG of alcohol-induced acute liver injury mice (x ± s, n = 10)

^## P＜0.01, compared with the normal group; ^** P＜0.01, ^* P＜0.05, compared with the model group

2. The effect of ethanol extract of wild chrysanthemum on the content of SOD, MDA, GSH and GSH-Px in liver homogenate of mice with alcoholic liver disease

It can be seen from Tables 2 and 3 that the ethanol extract of chrysanthemum chrysanthemum (TFC, 250, 500 mg·kg ^-1 ) can significantly reduce the content of MDA in the liver homogenate of mice with acute alcoholic liver disease, and increase the SOD, GSH and GSH- The activity of _PX shows that the ethanol extract of wild chrysanthemum may have the effect of inhibiting the formation of peroxidation substances and promoting the production of antioxidant substances, and can prevent the development of alcoholic fatty liver by increasing the activity of SOD, GSH and GSH-Px in liver tissue. and outcome.

Table 2 The effect of the ethanol extract of the wild chrysanthemum on the SOD and MDA of the liver tissue of mice with acute liver injury caused by alcoholism (x ± s, n = 10)

^## P＜0.01, compared with the normal group; ^** P＜0.01, ^* P＜0.05, compared with the model group

Table 3 The effect of ethanol extract of Chrysanthemum chrysanthemum on liver GSH and GSH-Px of mice with acute liver injury caused by alcoholism (x ± s, n = 10)

^## P＜0.01, compared with the normal group; ^** P＜0.01, ^* P＜0.05, compared with the model group

3. Effect of ethanol extract of wild chrysanthemum on the content of TNF-α and IL-1β in liver homogenate of mice with acute alcoholic liver disease

It can be seen from Table 4 that compared with the normal group, the contents of TNF-α and IL-1β in the liver tissue of all mice given alcohol (50%, 6 mg·kg ^-1 ) were significantly increased, indicating that under the stimulation of alcohol, the mice The liver tissue of the mice was damaged to a certain extent, resulting in the massive secretion of TNF-α and IL-1β. However, after giving the ethanol extract of Chrysanthemum chrysanthemum (TFC, 250, 500 mg·kg ^-1 ), the contents of TNF-α and IL-1β in the liver tissue were significantly reduced, suggesting that its regulatory effect on cytokines may be its therapeutic effect on alcoholic liver disease. important mechanism.

Table 4 The effect of ethanol extract of Chrysanthemum chrysanthemum on liver TNF-α and IL-1β of alcohol-induced acute liver injury mice (x±s, n=10)

^## P＜0.01, ^# P＜0.05, compared with the normal group ^** P＜0.01, ^* P＜0.05, compared with the model group

4. The effect of ethanol extract of wild chrysanthemum on the pathological changes of liver in mice with acute alcoholic liver disease

Histopathological examination: the liver cells in the normal group were normal in shape, the structure of the hepatic lobule was clear, the cords of the liver cells were arranged neatly, the size of the liver cells was relatively consistent, the nucleus was in the middle, the cytoplasm was light red, and a few scattered lipid droplets were occasionally seen in the central vein area , normal hepatic sinusoids, no significant degeneration, necrosis (Figure 1). The mice in the model group had typical fatty liver lesions: the arrangement of liver cell cords was disordered, the liver cells were swollen, and the cells were filled with lipid droplets of different sizes. Cell infiltration, hepatic sinusoidal narrowing, cytoplasm and nuclei were lightly stained, but no liver fibrosis was seen (Figure 2). The wild chrysanthemum ethanol extract (TFC, 125mg kg-1) group had no significant effect on reducing the degree of hepatic steatosis and liver tissue inflammation in acute alcoholic liver disease model mice (Figure 3), and the wild chrysanthemum ethanol extract (TFC, 250mg·kg-1, 500mg·kg-1) groups significantly reduced the degree of hepatic steatosis in model mice, and the inflammatory response of liver tissue was also significantly reduced (Fig. 4, Fig. 5). ·kg ^-1 ) group also significantly reduced the degree of hepatic fatty degeneration of the model mice, and the inflammatory response of the liver tissue was also significantly reduced (Fig. 6).

From the above results, it can be concluded that the beneficial effects of the present invention are:

A, the present invention has excavated new medical application to the wild chrysanthemum ethanol extract, has opened up a new application field.

B. The wild chrysanthemum flower ethanol extract of the present invention can treat both the symptoms and the root cause, is safe and non-toxic, has strong pharmacological effects, and indicates good medicinal prospects.

C. The wild chrysanthemum ethanol extract of the present invention has abundant sources of raw materials, low price, mature preparation technology, can be made into various oral dosage forms, and is easy to use.

D. The wild chrysanthemum ethanol extract of the present invention has a good preventive effect on acute alcoholic liver disease, and there is no harm in taking the medicine for a long time.

Description of drawings

Figure 1 is the morphology (HE×200) of the mouse liver cells in the normal group,

Figure 2 is a typical fatty liver lesion (HE × 200) diagram in mice in the acute alcoholic liver disease model group,

Figure 3 is a picture of the acute alcoholic liver disease model mice + TFC 125mg·kg ^-1 drug group (HE×200),

Figure 4 is a picture of the acute alcoholic liver disease model mice +TFC 250mg·kg ^-1 drug group (HE×200),

Figure 5 is a picture of the acute alcoholic liver disease model mice + TFC 500mg·kg ^-1 drug group (HE×200),

Fig. 6 is a picture of the acute alcoholic liver disease model mice + Kexili drug group (HE × 200).

Detailed ways

Below in conjunction with embodiment, the present invention will be further described.

Example:

Dry wild chrysanthemum (5kg) with 80% ethanol, 50L, reflux extraction twice, each time for 3h, combine the extracts, recover the solvent until there is no alcohol smell (10L), put on AB-8 macroporous resin (5kg), use 25L water Eluted to remove water-soluble impurities, then eluted with 2L of 50% ethanol, combined the ethanol eluents, recovered the solvent, and obtained the ethanol extract of Chrysanthemum chrysanthemum (500±50g). The content of total flavonoids in the ethanol extract of Chrysanthemum chrysanthemum was determined by HPLC method to be 55%; the content of total flavonoids determined by colorimetry was 65%. There are also saponins (20%, colorimetric method), caffeic acid (1.5%, HPLC) and chlorogenic acid (2.5%, HPLC).

The total flavonoids of wild chrysanthemum were further separated to obtain luteolin, luteolin and mongoside. As determined by HPLC, the contents of the three flavonoids were luteolin (15%), luteolin (4%) and mongoside (12%), respectively. The above compounds are the main flavonoids in the ethanol extract of chrysanthemum chrysanthemum.

The above ethanol extracts of Chrysanthemum chrysanthemum were prepared into a suspension with 0.5% CMC-Na before use, and the doses used were expressed as dry powder.

The recommended clinical dose of the ethanol extract of wild chrysanthemum flower: 62.5-125mg/day, orally, in three divided doses.

Claims (1)

1. The application of chrysanthemum indicum extract in pharmacy is characterized in that: the application of chrysanthemum indicum extract in the medicine of preparation control alcoholic liver disease.

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