Application of pepper essential oil diethyl ether extract in preparation of medicine for treating hepatic fibrosis

Abstract

The invention discloses an application of a pepper essential oil diethyl ether extract in preparation of a medicine for treating hepatic fibrosis, and finds that the pepper essential oil diethyl ether extract has an improvement effect on different hepatic fibrosis animal models and is good in safety. Therefore, the invention provides the application of the pepper essential oil diethyl ether extract in preparation of the medicine for treating hepatic fibrosis.

Description

Technical Field

[0001] The invention relates to the field of medicine and pharmacy, and in particular to application of an ether extract of Zanthoxylum bungeanum essential oil in preparing a medicine for treating liver tissue fibrosis. Background Art

[0002] Liver fibrosis is a very complex chronic liver disease. It is a common pathological process for various chronic liver diseases such as viral hepatitis, non-alcoholic fatty liver disease, alcoholic liver disease, autoimmune liver disease and cholestatic liver disease. It is the only way for various chronic liver diseases to progress to cirrhosis, liver failure or liver cancer. It is accompanied by abnormal changes in the liver microenvironment, manifested as the accumulation of a large number of inflammatory mediators, liver cell damage, repair, regeneration, and a large number of continuous activation of resting hepatic stellate cells. Domestic and foreign studies have shown that liver fibrosis can be reversed, and effective treatment of liver fibrosis can prevent the development of cirrhosis and even liver cancer. There is an urgent need to develop a safe and efficient new drug.

[0003] Zanthoxylum bungeanum essential oil is a natural essential oil extracted from the fruit of Zanthoxylum bungeanum tree, which has rich health and medicinal value. At present, there are reports on the antibacterial, anti-inflammatory, analgesic, antioxidant and anti-cancer effects of Zanthoxylum bungeanum essential oil. However, there are no reports on the use of Zanthoxylum bungeanum essential oil ether extract as a drug for the treatment of liver fibrosis. Summary of the invention

[0004] The present invention is based on the discovery that the Zanthoxylum bungeanum essential oil ether extract has the effect of treating liver tissue fibrosis, thereby inventing an application of the Zanthoxylum bungeanum essential oil ether extract in preparing a medicine for treating liver tissue fibrosis.

[0005] The technical solution of the present invention is to use an ether extract of Zanthoxylum bungeanum essential oil in preparing a medicine for treating liver tissue fibrosis.

[0006] Preferably, the Zanthoxylum bungeanum essential oil ether extract is prepared according to the following method: add 8 times amount of anhydrous ether to Zanthoxylum bungeanum essential oil, heat at 40°C for 30-60min, filter to obtain ether extract, recover ether under reduced pressure to obtain Zanthoxylum bungeanum essential oil ether extract.

[0007] Preferably, the Zanthoxylum bungeanum essential oil ether extract is a Zanthoxylum bungeanum essential oil ether extract emulsion, which is prepared from the following components in percentage by mass: 0.1-5% Zanthoxylum bungeanum essential oil ether extract, 0.1-10% oil phase, 0.1-6.0% surfactant, 0.1-0.5% co-surfactant, and the balance is water.

[0008] The Zanthoxylum bungeanum essential oil ether extract of the present invention has an improvement effect on various animal models of liver tissue fibrosis and has good safety, and can be used for preparing a medicine for treating liver tissue fibrosis. DETAILED DESCRIPTION

[0009] 1. Prepare the Zanthoxylum bungeanum essential oil ether extract emulsion according to the following steps: Step 1. Take 30g of Zanthoxylum bungeanum essential oil, add 240ml of anhydrous ether, and heat at 40°C for 30min to dissolve the Zanthoxylum bungeanum essential oil; filter with a 0.45 microporous filter membrane to obtain an ether extract, and recover the ether under a reduced pressure of 67kpa to obtain an ether extract of Zanthoxylum bungeanum essential oil; Step 2. Take 1g of Zanthoxylum essential oil ether extract and add it to 5g of soybean oil, heat and stir at 50°C until dissolved to prepare an oil phase; Step 3. Add 2.4 g soybean lecithin and 0.15 g glycerol to 90 ml ultrapure water, stir and dissolve to prepare an aqueous phase; Step 4. After mixing the oil phase and the water phase, ultrasonicate them in an ice water bath at 120 W for 4 min (pulse 3 s, pause 2 s) to obtain a primary emulsion; homogenize the primary emulsion 8 times at 6000 psi, and add ultrapure water to 100 ml to obtain a finished emulsion.

[0010] The Zanthoxylum bungeanum essential oil used was a product of Guangzhou Wuweiyuan Biotechnology Co., Ltd., and the analytically pure anhydrous ether was a product of Sinopharm Chemical Reagent Co., Ltd.

[0011] The prepared Zanthoxylum bungeanum essential oil ether extract emulsion is milky white, without obvious wall hanging phenomenon, no obvious stratification and other unstable phenomena, indicating that the prepared emulsion meets the appearance quality requirements of the emulsion. The emulsions were placed at 4°C for 1 day, 7 days, and 30 days, respectively, and were marked as A, B, and C, and placed together to observe their appearance morphology, and no obvious stratification phenomenon was observed, indicating that the emulsion has good stability.

[0012] 2. Experimental study on the therapeutic effect of Zanthoxylum bungeanum essential oil ether extract emulsion on carbon tetrachloride-induced liver fibrosis model Experimental rats: 60 SPF Wistar male rats, eight weeks old, were purchased from Beijing Weitonglihua Experimental Animal Company, license number: SCXK (Beijing): 2019-25, weighing 200-220g, with an average weight of 214.27±6.07g.

[0013] Experimental method: The rats were divided into 4 groups by random number table method, namely normal group, model group, positive control group and embodiment group of the present invention (hereinafter referred to as embodiment group), with 15 rats in each group. Except for the normal group, the other groups were used to prepare liver fibrosis models, and the first dose of 3ml / kg of peanut oil containing 40% CCl4 was injected into the rat abdominal cavity, and then continuously injected for 8 weeks, with 2ml / kg injected every 3 days. From the second day of the first injection of rats in each group, according to the dose conversion between humans and animals, the positive control group was gavaged with 0.11mg / kg of colchicine suspension, the embodiment group was gavaged with 2.2g / kg of Zanthoxylum essential oil ether extract emulsion, and the model group and the normal group were gavaged with equal doses of normal saline, once a day, for 8 consecutive weeks.

[0014] Observation indicators: 1. Observe the pathological morphology of liver tissue and the degree of liver fibrosis in rats. After the last administration, the rats were anesthetized by intraperitoneal injection of 0.8 ml / 100 g of 2.4% chloral hydrate, and then killed by cervical dislocation. The liver tissue was selected, washed with buffer, fixed with 10% formaldehyde, dehydrated with alcohol gradient, embedded in paraffin and sliced ​​with a thickness of 4 μm. The liver tissue was stained with hematoxylin-eosin (HE) and the pathological morphology of liver tissue in each group of rats was observed under a light microscope. Grading of liver fibrosis: no liver fibrosis was seen in the liver tissue (grade 0); perisinusoidal fibrosis, fibrosis in the portal area and surrounding areas, or fibrous scars in the lobules, but no effect on the integrity of the lobules (grade I); fibrous septa were seen, but most of the lobular structures were still retained (grade II); there were many fibrous septa, disordered lobular structures, but no cirrhosis (grade III); diffuse fibrosis, extensive damage to the liver parenchyma, early cirrhosis and pseudolobules (grade IV).

[0015] 2. Collect 6 ml of rat abdominal aorta blood, and detect serum liver function indicators including albumin (ALB), alanine aminotransferase (ALT), globulin (GLB), serum total protein (TP) and aspartate aminotransferase (AST) by ELISA method. At the same time, relevant cytokines including VEGF, TGF-β1, TNF-α and CTGF content were detected.

[0016] Experimental results: 1. Pathological morphology of liver tissue and liver fibrosis grade of rats in each group The liver lobule structure of rats in the normal group did not change, the hepatocytes were regular cord-like structures, the central vein wall was clear, and there was no inflammatory cell infiltration and collagen fiber hyperplasia; the liver lobule structure in the liver tissue of rats in the model group was severely damaged, the hepatic cords were arranged in disorder, there was significant inflammatory cell infiltration and fatty degeneration, and liver cell necrosis, swelling and atrophy increased; the liver cell arrangement of rats in the positive control group and the embodiment group tended to be normal, the inflammatory cell infiltration decreased, and the conditions of liver cell necrosis, swelling and atrophy were significantly improved. The liver fibrosis grade of rats in the model group was significantly higher than that in the normal group, and the liver fibrosis grade of rats in the positive control group and the embodiment group was significantly lower than that in the model group, and the difference was statistically significant (P < 0.05), as shown in Table 1.

[0017] Table 1 Comparison of liver fibrosis grades in rats of each group (n=15)

[0018] 2. Serum VEGF, CTGF, TN-α and TGF-β levels in rats of each group Compared with the normal group, the levels of serum VEGF, CTGF, TNF-α and TGF-β1 in the model group were increased; compared with the model group, the levels of serum VEGF, CTGF, TNF-α and TGF-β1 in the positive control group and the example group were decreased, and the differences were statistically significant (P<0.05), as shown in Table 2 for details.

[0019] Table 2 Comparison of serum VEGF, CTGF, TNF-α and TGF-β1 levels in rats of each group (unit: pg / ml, n=15, ±s)

[0020] Note: Compared with the normal group, a :P<0. 05; Compared with the model group, b :P<0.05 3. Liver function indicators of rats in each group Compared with the normal group, the serum GLB, ALB and TP levels of the rats in the model group were decreased, and the ALT and AST levels were increased; compared with the model group, the serum GLB, ALB and TP levels of the rats in the positive control group and the embodiment group were increased, and the ALT and AST levels were decreased, and the differences were statistically significant (P < 0.05), as shown in Table 3 for details.

[0021] Table 3 Comparison of liver function indicators in rats of each group (n= 15, ±s)

[0022] Note: Compared with the normal group, a:P<0. 05; Compared with the model group, b :P<0.05 The research results showed that the arrangement of liver cells in the rats in the embodiment group tended to be normal, the infiltration of inflammatory cells decreased, and the conditions of liver cell necrosis, swelling and atrophy were significantly improved. The liver fibrosis grade of the rats in the embodiment group was significantly lower than that in the model group, and the difference was statistically significant. The serum liver function index test showed that the liver function of the rats in the embodiment group could be restored, indicating that the Zanthoxylum essential oil ether extract emulsion of the embodiment of the present invention can significantly improve liver fibrosis induced by carbon tetrachloride.

[0023] 3. Experiment on the therapeutic effect of Zanthoxylum bungeanum essential oil ether extract emulsion on rats with immune liver fibrosis Experimental rats: 60 SPF Wistar male rats, eight weeks old, were purchased from Beijing Weitonglihua Experimental Animal Company, license number: SCXK (Beijing): 2019-25, weighing 200-220g, with an average weight of 210.46±5.33g.

[0024] Experimental methods: The rats were divided into 4 groups by random number table method, namely normal group, model group, positive control group and embodiment group of the present invention (hereinafter referred to as embodiment group), with 15 rats in each group. Except for the normal group, rats in other groups were intraperitoneally injected with 0.5 mL of sterile porcine serum to establish rat immune liver fibrosis model, and the normal group was intraperitoneally injected with 0.5 mL of normal saline twice a week. At the same time as modeling, according to the dose conversion between humans and animals, the positive control group was gavaged with 0.11 mg / kg of colchicine suspension, the embodiment group was gavaged with 2.2 g / kg of Zanthoxylum essential oil ether extract emulsion, and the model group and the normal group were gavaged with the same dose of normal saline, once a day, for 8 consecutive weeks. At the beginning of the 8th week, two rats in the model group were randomly killed and HE staining was performed, which was consistent with the pathological histological changes of liver fibrosis level 3-4, indicating that the modeling was successful.

[0025] Experimental results: After paraffin embedding, liver tissue was cut into 4 μm thick sections. After dewaxing, hydration and antigen retrieval, the primary antibodies wnt1 (1:200), B-catenin (1:100), and DKK1 (1:100) were incubated at 4 °C overnight. The secondary antibodies were incubated at 37 °C for 30 min, and the sections were developed by DAB, counterstained, blued, and sealed with neutral gum. Four high-power fields (×400) were randomly selected from each section for observation and positioning under a microscope. The cells with brown-yellow staining in the cytoplasm were wnt1 and β-catenin positive cells, and the cells with brown-yellow or brown staining in the nucleus were DKK1 positive cells. The average optical density (OD) value was calculated using ImageJ. The experimental results are shown in Table 4.

[0026] Table 4 Protein expression levels of wnt1, B-catenin and DKK1 in rats of each group (n=15, ±s)

[0027] Note: Compared with the normal group, a :P<0. 05; Compared with the model group, b :P<0.05 The experimental results showed that compared with the model group, the expression levels of wnt1 and β-catenin proteins in the example group were reduced, and the expression level of DKK1 protein was increased, and was better than that in the positive control group, indicating that the ether extract emulsion of Zanthoxylum essential oil in the example of the present invention can improve immune liver fibrosis in rats.

Claims

1. Use of an ether extract of Zanthoxylum bungeanum essential oil in the preparation of a drug for treating liver fibrosis.

2. The use of the Zanthoxylum bungeanum essential oil ether extract according to claim 1 in the preparation of a medicament for treating liver fibrosis, characterized in that The Zanthoxylum bungeanum essential oil ether extract is prepared according to the following method: adding 8 times amount of anhydrous ether to Zanthoxylum bungeanum essential oil, heating at 40° C. for 30-60 min, filtering to obtain ether extract, and recovering ether under reduced pressure to obtain Zanthoxylum bungeanum essential oil ether extract.

3. Use of the Zanthoxylum bungeanum essential oil ether extract according to claim 1 or 2 in the preparation of a medicament for treating liver fibrosis, characterized in that: The Zanthoxylum bungeanum essential oil ether extract is a Zanthoxylum bungeanum essential oil ether extract emulsion, which is prepared from the following components in percentage by mass: 0.1-5% of Zanthoxylum bungeanum essential oil ether extract, 0.1-10% of oil phase, 0.1-6.0% of surfactant, 0.1-0.5% of co-surfactant, and the balance is water.

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