Application of Perilla frutescens extract in the preparation of drugs for preventing and treating liver and intestinal damage

Abstract

The present invention discloses the use of Perilla frutescens or its extract in the preparation of drugs for preventing and treating liver and intestinal damage, wherein the liver and intestinal damage is caused by depression. The Perilla frutescens or its extract of the present invention is used to prepare drugs for preventing and treating liver and intestinal damage caused by depression. By inducing a stress model of mice with irregular long-term stimulation, it is confirmed that Perilla frutescens or its extract can effectively reduce the levels of AST and ALT in the plasma of the depressed animal model, significantly improve liver and intestinal function, improve liver and intestinal tissue damage, and regulate the expression of oxidative stress and immune-related factors in liver and intestinal tissues. The Perilla frutescens or its extract of the present invention provides new options and ideas for the preparation of drugs for preventing and treating liver and intestinal damage caused by depression.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of oriental perilla or an extract thereof in preparing medicines for preventing and treating liver and intestinal damage. Background Art

[0002] Depression is a type of mental illness characterized by mood disorders. It is caused by external environment and psychological quality, and is accompanied by clinical symptoms such as loss of interest, decreased energy, slow movements, loss of appetite or weight, etc. Depression begins in the liver. If the emotions are not smooth, the liver is depressed and the spleen is attacked. The spleen is weak and the appetite is poor, thus causing the disease and leading to liver and intestinal damage.

[0003] Elsholtizia bodinieri Vaniot is a herbaceous plant of the genus Elsholtizia in the family Lamiaceae, and is usually grown in Yunnan Province, Sichuan Province, Gansu Province and other places. Elsholtizia bodinieri is a traditional folk medicine in my country. The whole plant is used as medicine, and it is spicy and flat in nature. It has the functions of dispersing and relieving exterior symptoms, clearing away heat and dampness, regulating qi and nourishing the body. It is used for exogenous wind-cold, cold and fever, headache and body pain, sore throat, toothache caused by deficiency fire, indigestion, acute conjunctivitis, urinary retention and hepatitis. In recent years, domestic scholars have isolated a variety of flavonoids, triterpenes and phenolic components from the roots, stems and leaves of Elsholtizia bodinieri, and confirmed that it has certain antioxidant, lipid-lowering, anti-fatigue, antiviral and antibacterial activities. However, there is no report on the study of Elsholtizia bodinieri improving liver and intestinal damage caused by depression in mice. Summary of the invention

[0004] The main purpose of the present invention is to provide an application of Perilla frutescens or its extract in the preparation of drugs for preventing and treating liver and intestinal damage, aiming to use Perilla frutescens or its extract in the preparation of new drugs for preventing and treating liver and intestinal damage caused by depression, aiming to reduce the plasma AST and ALT content of model mice, improve liver and intestinal tissue damage caused by depression, regulate the expression of oxidative stress and immune-related factors (NQO1, NRF2, IDO1) in liver and intestinal tissues, and improve liver and intestinal function.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] Application of Perilla frutescens or its extract in preparing medicine for preventing and treating liver and intestinal damage, wherein the liver and intestinal damage is caused by depression.

[0007] The medicine prepared from the extract of Perilla frutescens for preventing and treating liver and intestinal damage caused by depression comprises at least one of (1) to (4):

[0008] (1) Reduce the levels of AST and ALT in the plasma of depression model mice and improve liver function;

[0009] (2) Alleviate pathological damage in liver and intestinal tissues of depression model mice;

[0010] (3) Regulate the expression of oxidative stress and immune-related factors (NQO1, NRF2, IDO1) in the liver and intestinal tissues of depression model mice;

[0011] (4) Reduce the risk of liver and intestinal damage caused by depression.

[0012] The present invention has conducted a series of studies on the use of Perilla frutescens or its extract in the preparation of drugs for preventing and treating liver and intestinal damage caused by depression, and confirmed that Perilla frutescens extract can significantly reduce the content of AST and ALT in plasma; improve liver and intestinal tissue damage in depression model mice; and regulate the expression of oxidative stress and immune-related factors in liver and intestinal tissues of depressed mice. In general, it effectively improves the liver and intestinal damage of depression model mice, and has a significant effect on relieving depression. The Perilla frutescens or its extract provided by the present invention has a significant effect on preventing and treating liver and intestinal damage caused by depression; and the Perilla frutescens plant is widely distributed, highly safe, low-priced, and the raw materials are easily available; Perilla frutescens or its extract has a good development and application prospect in the preparation of drugs for preventing and treating liver and intestinal damage caused by depression.

[0013] Preferably, the shiso extract is an extract obtained by extracting shiso with methanol at a mass concentration of 80%.

[0014] Specifically, the extraction method of the methanol extract of Perilla frutescens is as follows: grind the dried whole herb of Perilla frutescens into powder to obtain Perilla frutescens powder; extract the Perilla frutescens powder with 80% methanol for 1 hour, then put it into a round-bottom flask (generally to 1 / 2 to 2 / 3 of the volume of the round-bottom flask), connect a condenser, pass cooling water, heat and reflux in a water bath for 5 hours, filter out the extract, add 80% methanol to the residue and reflux 3 times, combine the filtrate, recover the methanol to obtain a concentrated extract, and finally perform vacuum freeze-drying to obtain a powdered Perilla frutescens alcohol extract; store the powdered Perilla frutescens extract at a low temperature of 4-5°C for use.

[0015] Preferably, the medicament comprises a Perilla frutescens extract.

[0016] Specifically, the dosage of the Perilla frutescens extract is 200-600 mg / day / kg. The present invention has no special requirements on the dosage form, auxiliary materials and preparation method of the drug, and those known to those skilled in the art can be used.

[0017] The new drug for preventing and / or treating liver and intestinal damage caused by depression of the present invention is a drug for preventing and treating liver and intestinal damage caused by depression, which uses the extract of Perilla frutescens as an active ingredient, and can also be added with one or more excipients acceptable to pharmaceutical preparations, or compounded with other active ingredients to play a synergistic role in improving liver and intestinal damage caused by depression. The drug is an oral preparation, which is a granule, pill, tablet, capsule, or powder.

[0018] The drug is taken into the gastrointestinal tract of a mammal, which includes but is not limited to humans.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention develops a new use of the perilla extract, which is used to prepare drugs for liver and intestinal damage caused by depression, and has the effects of soothing the liver and relieving depression, and treating intestinal damage of the liver hyperactivity and spleen deficiency syndrome.

[0021] 2. The present invention has verified through experiments that the extract of Perilla frutescens can effectively reduce the levels of AST and ALT in plasma; improve the liver and intestinal tissue damage of depression model mice; and regulate the expression of oxidative stress and immune-related factors in the liver and intestinal tissues of depressed mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the main component analysis results of the methanol extract of Perilla frutescens of the present invention;

[0023] Figure 2 The bar graph is a graph showing that the basidiomyces frutescens extract of the present invention reduces AST and ALT in mouse plasma;

[0024] Figure 3 The extract of the present invention significantly regulates the expression of oxidative stress and immune-related factors in the liver and intestinal tissues of mice;

[0025] Figure 4 This is a HE staining image of liver injury tissue sections of mice with depression model improved by the Perilla frutescens extract of the present invention;

[0026] Figure 5 This is a HE staining image of the intestinal injury tissue section of the depression model mouse improved by the Perilla frutescens extract of the present invention. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0028] The terms "first", "second" and "third" in this application are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined as "first", "second" and "third" can explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. All directional indications in the embodiments of the present application (such as up, down, left, right, front, back...) are only used to explain the relative position relationship, movement, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication also changes accordingly. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not limited to the listed steps or units, but optionally also includes steps or units that are not listed, or optionally also includes other steps or units inherent to these processes, methods, products or devices.

[0029] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0030] The C57BL / 6J mice used in the following examples of the present invention were purchased from Beijing Sibeifu Experimental Animal Co., Ltd. Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0031] Example 1 Preparation of Perilla frutescens Extract

[0032] (1) Extraction of methanol extract of Perilla frutescens:

[0033] The dried whole herb of Perilla frutescens was ground into powder with a high-speed grinder, and passed through an 80-mesh sieve to obtain Perilla frutescens powder. The Perilla frutescens powder was extracted with 80% methanol water with ultrasound assistance for 1 hour, and then put into a round-bottom flask (generally to 1 / 2 to 2 / 3 of the volume of the round-bottom flask), connected to a condenser, passed cooling water, heated and refluxed in a water bath for 5 hours, and the extract was filtered out. The residue was then added with a new solvent and refluxed for 3 times, the filtrate was combined, and the methanol was recovered to obtain a concentrated extract, and finally vacuum freeze-dried. The obtained sample was ground into powder with a mortar to obtain Perilla frutescens alcohol extract, which was stored at 4°C for subsequent use.

[0034] (2) UPLC-MS / MS principal component analysis of ethanol extract of Perilla frutescens:

[0035] UPLC-MS / MS was used to conduct a qualitative analysis of the main components of the alcohol extract of Perilla frutescens. An Agilent SB-C18 column (1.8 μm, 2.1 mm × 100 mm) was used; mobile phase A was ultrapure water (containing 0.1% formic acid), and mobile phase B was acetonitrile (containing 0.1% formic acid); elution program: 0 min, 5% B; 9 min, 95% B; 10 min, 95% B; 11 min, 5% B. Column temperature 40 ° C; injection volume 4 μL, flow rate 0.35 mL / min, analysis results are shown in Figure 1 shown.

[0036] Example 2 Evaluation of the activity of Perilla frutescens extract in preventing and treating liver damage caused by depression, liver depression, spleen invading liver, spleen deficiency and poor appetite, and intestinal damage of liver hyperactivity and spleen deficiency syndrome.

[0037] The Perilla frutescens extract used in this example was prepared in Example 1.

[0038] (1) Experimental animal grouping:

[0039] The C57BL / 6J mice purchased above were randomly divided into the following groups: blank group (C), model group (M), high-dose group of ethanol extract of Perilla frutescens (H), low-dose group of ethanol extract of Perilla frutescens (L), and positive drug paroxetine group (Y). The high-dose group of ethanol extract of Perilla frutescens (H) was gavaged with 600 mg / kg per day, the low-dose group of ethanol extract of Perilla frutescens (L) was gavaged with 200 mg / kg per day, and the positive drug paroxetine group (Y) was gavaged with 10 mg / kg per day. Each group of mice, 10 in each group, was gavaged for 7 consecutive days.

[0040] (2) Establishment of animal models of depression

[0041] Male 8-week-old C57BL / 6J mice were selected. The model group (M) mice received three different stress stimuli every day, and the same stimuli were not given continuously, so the animals could not predict the stimulation mode and adapt. The stress stimulation methods were as follows: (A) living in a reversed day and night life for 48 hours per week; (B) living in a humid cage for 48 hours per week; (C) living in a 45-degree tilted cage for 48 hours per week; (D) fasting for 48 hours per week; (E) restraining but not compressing for 5 hours in a well-ventilated environment (50ml centrifuge tube). The depression animal model was established by giving irregular stimulation for three consecutive weeks. The mice were fasted but not watered for 12 hours before being killed, and then killed by cervical dislocation. Fresh blood and liver, intestinal tissue and other samples were quickly collected after cervical dislocation.

[0042] (3) Perilla frutescens significantly reduced the expression of AST and ALT in the plasma of depression model mice

[0043] Biochemical reaction kits were used to detect the expression of AST (aspartate aminotransferase) and ALT (alanine aminotransferase), the markers of liver tissue damage in model mice.

[0044] AST, ALT biochemical reaction kit detection:

[0045] (a) Sample pretreatment: Serum (plasma) and other liquid samples to be tested: direct sampling and testing.

[0046] (b) The reagent kit is kept at a constant temperature of 37°C.

[0047] (c) Sample detection: set up control wells and assay wells, add 20 μL of matrix solution to each well, add 5 μL of sample to the assay wells, and leave it untreated in the control wells. Then place the wells in a constant temperature environment at 37 degrees for 30 minutes, add 20 μL of 2,4-dinitrophenylhydrazine solution, and add 5 μL of sample to the control wells. Place the wells in a constant temperature environment at 37 degrees again for 20 minutes, and finally add 200 μL of 0.4 mol / L sodium hydroxide solution. Gently shake the wells horizontally to mix, and place them at room temperature for 15 minutes. Use a microplate reader to measure the OD value of each well at a wavelength of 510 nm. Use the absolute OD value (OD value of the assay well minus OD value of the control well) to check the standard curve and obtain the corresponding AST / GPT activity unit.

[0048] The results are as follows Figure 2 As shown in the table, *P<0.05, **P<0.01, ***P<0.001 are statistically significant. Figure 2 It can be seen that the extract of Perilla frutescens can significantly reduce the expression of ASL and ALT in the plasma of depression model mice, indicating that the active ingredients of Perilla frutescens can significantly improve liver damage in depression model mice.

[0049] (4) Perilla frutescens significantly regulates the expression of oxidative stress and immune-related factors in the liver and intestinal tissues of depression model mice

[0050] Western blotting was used to detect the expression of oxidative stress and immune-related factors NQO1, NRF2, and IDO1 in the liver and intestinal tissues of model mice. Figure 3 As shown, *P<0.05, **P<0.01, ***P<0.001 were statistically significant.

[0051] Depend on Figure 3 It can be seen that the extract of Perilla frutescens can significantly increase the expression of NQO1 and NRF2 in the liver tissue of depression model mice, significantly reduce the expression of IDO1, and significantly reduce the expression of IDO1 in the intestinal tissue of depression model mice. This shows that the active ingredients of Perilla frutescens can significantly regulate the expression of related oxidative stress and immune-related factors in the liver and intestinal tissues of depression model mice.

[0052] (5) Perilla frutescens significantly alleviates liver and intestinal tissue damage caused by depression in mice

[0053] Since depression can cause pathological damage to liver and intestinal tissues, mice in the blank group (Control), model group (Model), high-dose EBEH group (EBEH), low-dose EBEL group (EBEL), and positive group (PAROXETINE) were taken out by aseptic technique, and the liver and intestinal tissues were washed with sterile pre-cooled PBS and fixed to make paraffin sections.

[0054] Preparation of paraffin sections:

[0055] (a) Sampling: Fresh colon tissue was fixed with 10% formalin solution for more than 24 hours. The colon tissue was taken out of the fixative and the target part of the tissue was trimmed with a scalpel in a fume hood. The trimmed tissue and the corresponding label were placed in a dehydration box;

[0056] (b) Dehydration and wax dipping: Place the dehydration box in a dehydrator and dehydrate with graded alcohols in sequence. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, paraffin I melted at 65℃ for 1 hour, paraffin II melted at 65℃ for 1 hour, paraffin III melted at 65℃ for 1 hour;

[0057] (c) Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, take the tissue out of the dehydration box and place it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. Cool in a -20℃ freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block;

[0058] (d) Sectioning: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 4 μm. Float the slices on 40°C warm water on a slide spreader to flatten the tissue. Pick up the tissue on a glass slide and bake the slices in a 60°C oven. After the wax is dried and melted, take them out and store them at room temperature for later use.

[0059] HE staining method is as follows:

[0060] (a) Dewaxing of paraffin sections: sequentially place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min, and then wash with tap water;

[0061] (b) Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 min, wash with tap water, differentiate with differentiation solution, wash with tap water, blue with bluing solution, and rinse with running water;

[0062] (c) Eosin staining: The sections were dehydrated in 85% and 95% graded alcohol for 5 min each, and then stained in eosin solution for 5 min.

[0063] (d) Dehydration and sealing: the sections were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, and xylene II for 5 min to make them transparent, and then sealed with neutral gum;

[0064] (e) Microscope examination, image acquisition and analysis.

[0065] The results of the section of Perilla frutescens extract improving the pathological damage of liver tissue caused by depression in mice are as follows Figure 4 As shown. Figure 4 It can be seen that the extract of Perilla frutescens can significantly reduce the pathological damage of liver cells, inflammatory cell infiltration and blood cell exudation in liver tissue, indicating that the extract of Perilla frutescens can significantly improve the pathological damage of liver tissue in depressed mice.

[0066] The results of the section of Perilla frutescens extract improving the pathological damage of intestinal tissue caused by depression in mice are as follows Figure 5 As shown. Figure 5 It can be seen that the extract of Perilla frutescens can significantly reduce the edema of the intestinal muscle layer tissue, significantly reduce the infiltration of inflammatory cells in the tissue, and maintain the integrity of the intestinal epithelial cells. This shows that the extract of Perilla frutescens can significantly improve the pathological damage of intestinal tissue in depressed mice.

[0067] In summary, the extract of Perilla frutescens can significantly reduce the AST and ALT content in the plasma of model mice; improve the liver and intestinal tissue damage of depression model mice; and regulate the expression of oxidative stress and immune-related factors in the liver and intestinal tissues of depressed mice. The extract of Perilla frutescens provided by the present invention has a significant effect on preventing and treating liver and liver hyperactivity and spleen deficiency syndrome-induced intestinal damage caused by depression.

[0068] The specific implementation methods of the invention are described in detail above, but they are only examples, and the invention is not limited to the specific implementation methods described above. For those skilled in the art, any equivalent modification or substitution of the invention is also within the scope of the invention, therefore, the equalization, modification, improvement, etc. made without departing from the spirit and principle of the invention should be included in the scope of the invention.

Claims

1. Use of a Perilla frutescens extract in the preparation of a drug for preventing and treating liver and intestinal damage caused by depression; The extraction method of the perilla extract is as follows: grinding the dried perilla whole plant into powder to obtain perilla powder; extracting the perilla powder with 80% methanol for 1 hour, then putting it into a round-bottom flask, heating and refluxing it in a water bath for 5 hours, filtering out the extract, adding 80% methanol to the filter residue and refluxing it for 3 times, combining the filtrate, recovering the methanol to obtain a concentrated extract; vacuum freeze-drying the concentrated extract to obtain a powdered perilla alcohol extract; and storing the powdered perilla extract at a low temperature of 4-5°C for use.

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