Application of schisandra chinensis extract in improvement of chronic alcoholic intestinal injury

Abstract

Pharmacological experiment results show that in a chronic alcoholic intestinal injury model, total lignans of schisandra chinensis can reduce histological injury of intestinal tracts of mice, relieve oxidative injury of small intestines, improve tight connection of the intestinal tracts through a RhoA / ROCK1-MLCK-MLC pathway, improve the intestinal tracts and improve the intestinal tracts, so that the total lignans of schisandra chinensis can be used for preventing and treating chronic alcoholic intestinal injury. The composition can improve intestinal wall villus integrity, reduce intestinal permeability and regulate intestinal flora disorder, so that alcoholic intestinal injury is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a new use of total lignans from Schisandra chinensis Background Art

[0002] In recent years, with the improvement of living standards, the per capita annual consumption of alcohol in China has increased significantly, and the incidence of alcohol-related organ damage diseases has also shown an increasing trend. Long-term exposure to excessive alcohol can directly cause disorders of intestinal motility function and mucosal damage. Common poisoning manifestations include acute or chronic diarrhea, dehydration, poor nutrient absorption. In severe cases, digestive tract ulcers or bleeding may occur, and the risk of intestinal cancer increases. In addition, after long-term alcohol exposure leads to intestinal damage, it will also induce diseases such as endotoxemia and alcoholic liver disease. Therefore, it is of great significance to study drugs for preventing and treating intestinal damage

[0003] Schisandra chinensis is a medicinal plant of the genus Schisandra in the Magnoliaceae family, including Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils., which are widely distributed in China. Schisandra chinensis tastes sour and sweet, and is warm in nature, and belongs to the lung, heart, and kidney meridians, with the effects of astringing and astringing, replenishing qi and promoting fluid production, and tonifying the kidney and tranquilizing the mind. Total lignans of Schisandra chinensis are natural compounds extracted from Schisandra chinensis, and their properties are white crystal powders. Schisandra chinensis has many biological effects, such as liver protection, blood sugar lowering, anti-inflammatory, and improving the cardiovascular and cerebrovascular systems and other pharmacological effects. Previously, studies have shown that Schisandra chinensis lignans can significantly reduce liver lesions, reduce inflammatory responses, restore liver function and blood lipid levels, and enhance the activity of antioxidant enzymes. There are also studies showing that Schisandra chinensis lignans can prevent and treat intestinal radiation damage and relieve inflammatory bowel disease

[0004] From the above description of the prior art, it can be seen that current studies on the treatment of alcoholic liver disease with Schisandra chinensis only focus on the single target organ liver, and do not involve other organ damages related to alcoholic liver injury, especially the study of alcoholic intestinal injury closely related to it. Many traditional Chinese medicine prescriptions for treating alcoholic intestinal injury involve Schisandra chinensis, but they do not emphasize the therapeutic effect of a single traditional Chinese medicine Schisandra chinensis on alcoholic intestinal injury, and the mechanism of action is not clear. Secondly, the research on the treatment of intestinal diseases with Schisandra chinensis mainly focuses on immune-related inflammatory models, and the pathophysiological mechanisms of enteritis simulated by these models are completely different from those of alcoholic intestinal injury. It can be seen that there is currently no research on the prevention and treatment of alcoholic intestinal injury with total lignans of Schisandra chinensis Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a use of Schisandra chinensis extract in improving chronic alcoholic intestinal injury

[0006] The inventors found that a single Schisandra chinensis extract can regulate the expression of proteins related to the RhoA (Ras homolog gene family member) / ROCK1 (Rho-associated coiled-coil containing protein kinase 1) / MLCK (myosin light chain kinase) pathway downstream of oxidative stress, repair tight junctions, improve the intestinal barrier structure, and at the same time regulate the intestinal flora, thereby alleviating or improving intestinal damage induced by chronic alcohol intake.

[0007] Furthermore, the Schisandra chinensis extract of the present invention refers to the ethanol extract of Schisandra chinensis.

[0008] Furthermore, the extraction method of the ethanol extract of Schisandra chinensis of the present invention includes the following steps:

[0009] (1) Soak Schisandra chinensis in edible alcohol, then heat under reflux for extraction, and filter to obtain the ethanol extract of Schisandra chinensis.

[0010] (2) Concentrate under reduced pressure and freeze-dry to obtain the ethanol extract of Schisandra chinensis.

[0011] (3) Dissolve the ethanol extract of Schisandra chinensis obtained in step (2) with an alcohol solution, purify it by macroporous resin, elute successively with water and ethanol of different concentrations, collect the eluate therein, concentrate under reduced pressure and freeze-dry to obtain the total lignans of Schisandra chinensis.

[0012] In the above method, Schisandra chinensis in step (1) refers to the fruit part of Schisandra chinensis.

[0013] The applicable alcohol concentration for soaking can be 70-95 degrees, such as 70 degrees, 80 degrees, 90 degrees, 95 degrees, etc.

[0014] The applicable soaking time can be 12-48 hours, such as 12h, 24h, 48h, etc.

[0015] The appropriate solid-liquid ratio can be 1 kg: 6-10 L. For example, it can be 1 kg: 6 L, 1 kg: 7 L, 1 kg: 8 L, 1 kg: 10 L, etc. The appropriate reflux extraction time can be 1-4 hours, such as 1h, 2h, 3h, 4h, etc. The appropriate elution alcohol concentration can be 50-95%, such as 50%, 70%, 95%, etc.

[0016] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0017] 1) The extraction process of the Schisandra chinensis extract is simple, and the components are simple and safe, which is conducive to the industrial application of Schisandra chinensis.

[0018] 2) The Schisandra chinensis extract of the present invention has a significant effect in improving chronic alcoholic intestinal damage, specifically including:

[0019] (1) Alleviating the indicators related to intestinal histological damage, including the ratio of small intestine organ weight to body weight and the ratio of small intestine length to body weight.

[0020] (2) Reduce the oxidative stress response in the intestine, including total antioxidant capacity (T-AOC), reactive oxygen species (ROS), nitric oxide induction and enzyme (iNOS), Ras homolog gene family member A (RhoA), Rho kinase 1 (ROCK1), myosin light chain kinase (MLCK), and myosin light chain (MLC).

[0021] (3) Restore the content of tight junction proteins (Claudin1, Occludin, ZO-1), reduce intestinal permeability, and improve the villus damage under H&E pathological examination.

[0022] (4) Improve the intestinal flora, including the levels of Lactobacillus, Bifidobacterium, Enterococcus, Enterobacter, and Clostridium perfringens. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Pathological pictures of HE staining of mice in each group in Example 1 (×200). DETAILED DESCRIPTION OF THE INVENTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings and tables in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] EXAMPLE

[0026] This example provides a method for extracting Schisandra chinensis extract, which specifically includes: washing 5 kg of Schisandra chinensis fruits, soaking them in 8 times the amount of 95-degree edible alcohol overnight, heating and refluxing with high-concentration edible alcohol 3 times, each time for 2 h, filtering the extract, concentrating under reduced pressure, and drying to obtain Schisandra chinensis extract. Then, it is dissolved in 30% edible alcohol for standby, purified by passing through AB-8 macroporous resin at room temperature, the elution speed is 2.5 BV / h, the elution is carried out with 95% edible alcohol, the eluate is collected, concentrated under reduced pressure, and dried to obtain total lignans of Schisandra chinensis.

[0027] EXPERIMENTAL EXAMPLE

[0028] In this experimental example, the Schisandra chinensis extract obtained in the example was used for the experiment on alleviating intestinal injury induced by long-term alcohol consumption. In the following statements, "NC" represents the "normal group", "MC" represents the "model group", "PC" represents the "silymarin group", "L-SAE" represents the "low-dose ethanol extract group of Schisandra chinensis", "H-SAE" represents the "high-dose ethanol extract group of Schisandra chinensis", "L-STL" represents the "low-dose total lignans group of Schisandra chinensis", and "H-STL" represents the "high-dose total lignans group of Schisandra chinensis".

[0029] 1. Test materials and methods

[0030] Male SPF-grade C57BL / 6J mice at 6 weeks of age were randomly divided into 7 groups of 12 mice each after 5 days of adaptive feeding, namely the normal group (NC), the alcohol consumption group (MC), the silymarin group (PC), the low-dose ethanol extract group of Schisandra chinensis (L-SAE, 0.9 g / kg), the high-dose ethanol extract group of Schisandra chinensis (H-SAE, 1.8 g / kg), the low-dose total lignans group of Schisandra chinensis (L-STL, 200 mg / kg), and the high-dose total lignans group of Schisandra chinensis (H-STL, 400 mg / kg). The feeding temperature of all mice was 24.0 ± 2.0 °C, the environmental relative humidity was 40 - 60%, and the light cycle was 12 h.

[0031] 2. Establishment of the chronic alcoholic intestinal injury model and administration

[0032] Model establishment process: A 42-degree edible alcohol was used to establish the model. Mice in each alcohol consumption group were administered according to the gradient dose increase method. The initial dose was set at 3.6 g / kg, and it was increased by 0.7 g / kg every 5 days until it reached 6.4 g / kg, and this dose was maintained until the end of the experiment. Mice in the normal group were gavaged with 10 ml / kg of pure water until the end of the experiment.

[0033] Administration process: The doses of each Schisandra chinensis extract and the positive drug were kept consistent. The residual ethanol content and total lignans content of each extract needed to be detected in advance, and they were adjusted with pure water and high-proof edible alcohol to a 42-degree alcoholic solution containing the extract for the above model establishment and administration process.

[0034] 3. Sample collection

[0035] After the last gavage, the mice were fasted but allowed to drink water for 12 h. Mice in the normal group were gavaged with an equal volume of distilled water. After gavaging with FITC-Dextran, blood was collected from the orbital cavity, centrifuged to obtain serum for biochemical detection, and the small intestine of the mice was quickly taken out and specimens were retained for relevant detection.

[0036] 4. Test operations

[0037] Calculation of body weight, organ index, and small intestine weight / length ratio: Record the final body weight. After dissecting the mice, weigh the small intestine and measure its length. Calculate the small intestine organ ratio according to 100 × small intestine weight (g) / mouse body weight (g), and calculate the small intestine weight / length ratio according to intestinal segment weight (g) / intestinal segment length (m).

[0038] Histopathology: Wash the small intestine and soak it in tissue fixative, and observe the tissue morphology by H&E staining method.

[0039] Determination of oxidative stress response: Use the kits produced by Nanjing Jiancheng Bioengineering Institute and Jiangsu Enzyme Immunoassay Industry Co., Ltd. to measure the biochemical indexes of the small intestine of mice, including T-AOC, ROS, iNOS, RhoA, ROCK1, MLCK, and MLC, for analyzing the changes in the oxidative stress level and mechanism pathway of the mouse intestine.

[0040] Determination of tight junction proteins and permeability: Use the Claudin-1, Occludin, and ZO-1 ELISA kits produced by Nanjing Jiancheng Bioengineering Institute to measure the levels of tight junction proteins in the small intestine of mice, and measure the content of serum FITC-Dextran at a wavelength of 435 / 528 nm to reflect intestinal permeability.

[0041] Microbial culture: Use the culture medium produced by Qingdao Haibo Biology to measure the numbers of Lactobacillus, Bifidobacterium, Enterococcus, Enterobacter, and Clostridium perfringens in the cecum of mice, and determine the regulatory effects of each group on the intestinal flora.

[0042] 5. Experimental results

[0043] Data are expressed as mean ± standard deviation (SD). One-way ANOVA analysis was used for comparison among multiple groups by GraphPad Prism 8.0, and independent sample t (LSD’s post hoc test) was used for comparison between two groups. A P < 0.05 indicates significant differences.

[0044] 5.1 Effects of Schisandra chinensis on body weight and organ index of mice

[0045] Analysis of the results of this experimental example showed that compared with the NC group, the body weight of mice in the MC group decreased, and the small intestine index and small intestine weight / length ratio both increased significantly; compared with the MC group, the body weights of the drug (PC) and the Schisandra chinensis extract and total lignans groups (L-SAE, H-SAE, L-STL, H-STL) all increased significantly. As shown in Table 1, the small intestine index and small intestine weight / length ratio both decreased significantly, indicating that total lignans of Schisandra chinensis have the effect of alleviating intestinal tissue damage.

[0046] Table 1 Histological changes of mice in each group (n = 6)

[0047]

[0048] Note: # indicates significant difference compared with the normal group (P<0.05), * indicates significant difference compared with the model group (P<0.05).

[0049] 5.2 Effects of Schisandra chinensis on intestinal pathological changes in mice

[0050] The results of HE staining of the small intestine showed that in the NC group, the cell morphology of the small intestine tissue was normal, the villi were arranged neatly, the intestinal wall was uniform, the cell structure was complete, and the cell gap was dense; in the MC group, the structure was disordered, the villi were arranged irregularly, showing a fractured or atrophied state, and the intestinal wall was thinned; after the intervention of Schisandra chinensis, the cell morphology was significantly changed and the intestinal wall was thickened, indicating that the ethanol extract and total lignans of Schisandra chinensis have a protective effect on the integrity of the intestinal wall and villi, as shown in Table 2 and Figure 1 as shown.

[0051] Table 2 Statistical results of HE staining of mice in each group

[0052]

[0053] Note: # indicates significant difference compared with the normal group (P<0.05), * indicates significant difference compared with the model group (P<0.05).

[0054] 5.3 Effects of Schisandra chinensis on oxidative stress response in mice

[0055] To deeply understand the mechanism of Schisandra chinensis in alleviating alcoholic intestinal injury, the present invention detected the oxidative stress response and changes in specific downstream pathways. The results of oxidative stress showed that compared with the NC group, the T-AOC in the MC group decreased significantly, while the ROS and iNOS increased significantly, and RhoA, ROCK1, and MLCK increased, while MLC decreased; compared with the MC group, the T-AOC of the ethanol extract and total lignans of Schisandra chinensis increased significantly, the levels of ROS and iNOS decreased significantly, RhoA, ROCK1, and MLCK decreased, and MLC increased, indicating that the ethanol extract and total lignans of Schisandra chinensis can inhibit the occurrence of intestinal oxidative stress and exert an intestinal protection effect through the RhoA / ROCK1-MLCK-MLC pathway, as shown in Table 3.

[0056] Table 3 Oxidative stress levels of mice in each group

[0057]

[0058] Note: # indicates significant difference compared with the normal group (P<0.05), * indicates significant difference compared with the model group (P<0.05).

[0059] 5.4 Effects of Schisandra chinensis on tight junctions and intestinal permeability in mice

[0060] The content of intestinal tight junctions is directly related to the integrity of the intestinal mechanical barrier. Intestinal permeability reflects the integrity of intestinal barrier function. In this invention, the above-mentioned indicators are detected to further verify the intestinal protection mechanism. The test results of intestinal permeability and tight junctions show that compared with the NC group, the permeability in the MC group increases, and the contents of tight junction proteins Claudin1, Occludin, and ZO-1 decrease; compared with the MC group, after the intervention with the ethanol extract of Schisandra chinensis and total lignans, the intestinal permeability decreases, and the contents of Claudin1, Occludin, and ZO-1 tight junction proteins increase, indicating that the ethanol extract of Schisandra chinensis and total lignans can reduce intestinal permeability and restore intestinal structure, having an intestinal protection effect, as shown in Table 4.

[0061] Table 4 Statistical results of intestinal permeability and tight junction proteins in mice of each group

[0062]

[0063]

[0064] Note: # Significantly different from normal, P < 0.05; * Significantly different from the model, P < 0.05.

[0065] 5.5 Effects of Schisandra chinensis on the intestinal flora of mice

[0066] The results of microbial detection show that compared with the NC group, the numbers of Lactobacillus and Bifidobacterium in the MC group decrease, while the numbers of Enterococcus, Enterobacter, and Clostridium perfringens increase. After the intervention with the ethanol extract of Schisandra chinensis and total lignans, the numbers of Lactobacillus and Bifidobacterium increase significantly, and the numbers of Enterococcus and Clostridium perfringens decrease, indicating that the ethanol extract of Schisandra chinensis and total lignans can regulate the intestinal flora, as shown in Table 5.

[0067] Table 5 Statistical data of intestinal microbe culture in mice of each group

[0068]

[0069] Note: # Significantly different from normal, P < 0.05; * Significantly different from the model, P < 0.05.

[0070] In summary, the ethanol extract of Schisandra chinensis and total lignans can significantly relieve the symptoms of chronic alcoholic intestinal injury. Although the invention has disclosed relatively detailed embodiments, it is not intended to limit the invention. Anyone familiar with this technology can make some modifications and decorations without departing from the scope of the invention. The protection scope of the invention shall be defined by the claims.

Claims

1. Use of a Schisandra chinensis extract in the preparation of a health product or drug for improving chronic alcoholic intestinal injury.

2. The use according to claim 1, characterized in that, The Schisandra chinensis extract achieves its effect by regulating the expression mechanism of proteins related to the RhoA (Ras homolog gene family member) / ROCK1 (Rho kinase 1)-MLCK (myosin light chain kinase) pathway, which is downstream of oxidative stress.

3. The use according to claim 1, characterized in that, The Schisandra chinensis extract is an ethanol extract of Schisandra chinensis.

4. The use according to claim 3, characterized in that, The extraction method of the ethanol extract of Schisandra chinensis includes the following steps: (1) Soak Schisandra chinensis in edible alcohol, then heat under reflux for extraction, and filter to obtain the ethanol extract of Schisandra chinensis; (2) Concentrate under reduced pressure and freeze-dry to obtain the ethanol extract of Schisandra chinensis; (3) Dissolve the ethanol extract of Schisandra chinensis obtained in step (2) with an ethanol solution, purify it by macroporous resin, elute successively with water and ethanol solutions of different concentrations, collect the eluate, concentrate under reduced pressure, and freeze-dry to obtain the total lignans of Schisandra chinensis.

5. The use according to claim 4, characterized in that, In step (1), Schisandra chinensis refers to the fruit part of Schisandra chinensis. The alcohol concentration for soaking is 70-95 degrees, the soaking time is 12-48 hours, the material-liquid ratio is 1 kg: 6-10 L, and the reflux extraction time is 1-4 hours.

6. The use according to claim 4, characterized in that In step (3), it is dissolved with a 30% ethanol solution and eluted with a 50-95% ethanol solution.

7. The use according to claim 6, wherein In step (3), it is eluted with a 50%, 70%, and / or 95% ethanol solution.

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