阅读说明：本技术 前列欣胶囊抗炎组分筛选及其在抑制炎症反应中的应用 (Screening of anti-inflammatory components of Qianliexin capsules and application of anti-inflammatory components in inhibition of inflammatory reaction ) 是由 王岱杰 陈龙 崔莉 王晓 孟兆青 于 2021-07-14 设计创作，主要内容包括：本发明提供前列欣胶囊抗炎组分筛选及其在抑制炎症反应中的应用,属于医药技术领域。本发明通过制取获得前列欣胶囊的正丁醇提取物,并研究其对脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7细胞分泌炎症细胞因子的影响及其对NF-κB信号通路中相关蛋白的干预作用,证明前列欣胶囊的正丁醇提取物可通过抑制NF-κB信号通路的激活而减轻LPS诱导的RAW264.7细胞炎症反应,从而表明其可作为治疗炎症相关疾病的药物,因此具有良好的实际应用之价值。(The invention provides screening of an anti-inflammatory component of a Qianliexin capsule and application of the anti-inflammatory component in inhibition of inflammatory reaction, and belongs to the technical field of medicines. According to the invention, the n-butyl alcohol extract of the prostate capsule is obtained, and the influence of the n-butyl alcohol extract on Lipopolysaccharide (LPS) induced mouse macrophage RAW264.7 cell secretion inflammatory cytokine and the intervention effect of the n-butyl alcohol extract on related proteins in an NF-kB signal channel are researched, so that the n-butyl alcohol extract of the prostate capsule can relieve the LPS induced RAW264.7 cell inflammatory reaction by inhibiting the activation of the NF-kB signal channel, thereby showing that the n-butyl alcohol extract can be used as a medicine for treating inflammation related diseases, and therefore, the n-butyl alcohol extract has good practical application value.)

1. The application of the prostate capsule is (a) or (b) as follows:

(a) preparing an inflammation reaction inhibitor;

(b) inhibiting inflammatory response;

the prostate capsule is specifically a n-butyl alcohol extract of the prostate capsule.

2. The use of claim 1, wherein the n-butanol extract of the prostate euphoria capsule is prepared by a method comprising:

taking the Qianliexin capsule powder, heating and refluxing the powder by using ethanol, concentrating the powder under reduced pressure until no alcohol smell exists, extracting the powder by using n-butyl alcohol, and concentrating the extract under reduced pressure until the extract is dried to obtain the Qianliexin capsule powder.

3. The use of claim 2, wherein the ethanol is 50-80% ethanol;

the mass ratio of the Qianliexin capsule powder to the ethanol is 1: 5-15.

4. The use of claim 2, wherein the n-butanol extract is administered in a dose of 10 to 100 μ g/mL.

5. The application of the Qianliexin capsule in preparing medicaments for treating diseases related to inflammatory reaction;

the prostate capsule is specifically a n-butyl alcohol extract of the prostate capsule.

6. The use of claim 5, wherein the disease associated with inflammatory response comprises infectious endocarditis, rheumatoid arthritis, ulcerative colitis.

7. The application of the Qianliexin capsule in the following (1) to (4):

(1) use in inhibiting secretion and/or expression of a related inflammatory factor;

(2) the application in preparing products for inhibiting the secretion and/or expression of related inflammatory factors;

(3) use in inhibiting secretion and/or expression of a protein of interest;

(4) preparing the application of inhibiting the secretion and/or expression of related protein;

wherein the prostate capsule is a n-butanol extract of the prostate capsule.

8. The use of claim 7, wherein the related inflammatory factors comprise NO, IL-6, IL-1 β, TNF- α, and PGE₂；

The related proteins include TLR4, COX-2, p-NF-kappa B p65, p-IKK alpha/beta and iNOS.

9. A product characterized in that the active ingredient of the product comprises a Qianliexin capsule;

wherein the prostate capsule is a n-butanol extract of the prostate capsule;

preferably, the product is a pharmaceutical or experimental model tool formulation;

preferably, when the product is a medicament, it also contains at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

10. A method of screening a prostate euphoria capsule anti-inflammatory composition, the method comprising: applying the prostate capsule extracts obtained by adopting different solvent extractions to a cell inflammation model, and detecting the inflammatory reaction of the cell inflammation model;

preferably, the solvent comprises petroleum ether, ethyl acetate and n-butanol, and is further preferably n-butanol;

preferably, the model of cellular inflammation comprises a lipopolysaccharide-induced RAW264.7 model of cellular inflammation.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to screening of an anti-inflammatory component of a Qianliexin capsule and application of the anti-inflammatory component in inhibiting inflammatory reaction.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

In recent years, the incidence of chronic prostatitis in men has been increasing with the increase of pressure in all aspects, and the clinical manifestations are discomfort and pain caused by chronic pelvic inflammation, which may be accompanied by urination abnormality and sexual dysfunction of different degrees, and the quality of life is seriously affected, so that the treatment of chronic prostatitis and the recovery of male fertility are one of the research hotspots of clinical scholars. The Qianliexin capsule is a first-part carrier variety of Chinese pharmacopoeia 2020 edition, consists of 14 Chinese medicinal materials of peach kernel (fried), myrrh (fried), salvia miltiorrhiza, red paeony root, safflower, herba lycopi, cowherb seed (fried), spina gleditsiae, herba patriniae, dandelion, szechwan chinaberry fruit, radix angelicae, pyrrosia lingua and medlar, and has the effects of promoting blood circulation to remove blood stasis, clearing heat and promoting diuresis. It is mainly used for treating stranguria caused by blood stasis accumulation and downward flow of damp-heat, with symptoms of urgent micturition, odynuria, urination difficulty, dribbling, chronic prostatitis, and prostatic hyperplasia. At present, some clinical studies show that the Qianliexin capsules have obvious improvement on clinical symptoms when used for treating damp-heat downward flow type chronic prostatitis patients.

The inflammatory response is a defensive response produced by the host under the stimulation of both internal and external factors. Excessive and chronic inflammatory reactions cause damage to various tissues of the body, thereby participating in the occurrence and development of various diseases, including infectious endocarditis, rheumatoid arthritis, ulcerative colitis and the like. However, the inventors found that the effective anti-inflammatory active site and effective ingredients of the Qianliexin capsule are not currently studied.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides the screening of the anti-inflammatory component of the Qianliexin capsule and the application of the anti-inflammatory component in inhibiting inflammatory reaction. The RAW264.7 cell inflammation model induced by lipopolysaccharide is established to prove that the n-butyl alcohol part in the prostate capsule has the strongest anti-inflammatory effect, and the RAW264.7 cell inflammation reaction induced by LPS can be relieved by inhibiting the activation of an NF-kB signal channel, so that the invention is completed

Specifically, the invention relates to the following technical scheme:

in a first aspect of the invention, there is provided the use of the prostate capsule as (a) or (b) below:

(a) preparing an inflammation reaction inhibitor;

(b) inhibiting inflammatory reaction.

According to the invention, the strongest anti-inflammatory component of the Qianliexin capsule is specifically n-butyl alcohol extract of the Qianliexin capsule.

In a second aspect of the invention, the application of the Qianliexin capsule in preparing a medicament for treating diseases related to inflammatory reaction is provided.

Wherein the prostate capsule is a n-butanol extract of the prostate capsule;

the inflammatory response-related diseases comprise all diseases or symptoms related to inflammatory response, including but not limited to infectious endocarditis, rheumatoid arthritis, ulcerative colitis and the like.

In a third aspect of the present invention, there is provided a use of the prostate capsule in the following (1) to (4):

(1) use in inhibiting secretion and/or expression of a related inflammatory factor;

(2) the application in preparing products for inhibiting the secretion and/or expression of related inflammatory factors;

(3) use in inhibiting secretion and/or expression of a protein of interest;

(4) preparing the application of inhibiting the secretion and/or expression of related protein.

Wherein the prostate capsule is a n-butanol extract of the prostate capsule;

the related inflammatory factorIncluding but not limited to NO, IL-6, IL-1 beta, TNF-alpha and PGE₂；

Such related proteins include, but are not limited to, TLR4, COX-2, p-NF-. kappa. B p65, p-IKK. alpha./beta. and iNOS.

In a fourth aspect of the invention, a product is provided, wherein the active ingredient of the product comprises the prostate capsule.

Wherein the prostate capsule is a n-butanol extract of the prostate capsule.

The product may be a pharmaceutical or experimental model tool formulation.

When the product is a medicament, it may also contain at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

In a fifth aspect of the present invention, there is provided a method for screening an anti-inflammatory component of a prostate capsule, the method comprising: the prostate capsule extracts obtained by adopting different solvent extractions are applied to a cell inflammation model, and the inflammatory response of the cell inflammation model is detected.

Wherein, the solvent includes but is not limited to petroleum ether, ethyl acetate, n-butanol, and preferably n-butanol.

The beneficial technical effects of one or more technical schemes are as follows:

according to the technical scheme, the n-butyl alcohol extract of the prostate capsule is obtained by preparation, the influence of the n-butyl alcohol extract on Lipopolysaccharide (LPS) induced mouse macrophage RAW264.7 cell secretion inflammatory cytokine and the intervention effect of the n-butyl alcohol extract on related proteins in an NF-kB signal channel are researched, and the n-butyl alcohol extract of the prostate capsule is proved to relieve the RAW264.7 cell inflammatory reaction induced by LPS by inhibiting the activation of the NF-kB signal channel, so that the n-butyl alcohol extract can be used as a medicine for treating inflammation related diseases, and has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is an HPLC chromatogram of a control and a sample according to an embodiment of the present invention; a is a control group, B is a sample, wherein, 1, gallic acid, 2, caffeic acid, 3, p-coumaric acid, 4, luteolin-7-O-glucoside, 5, salvianolic acid B, 6, salvianolic acid A, 7, byak-angelicin, 8, isoanisic lactone, 9, oxypeucedanin, 10, isoimperatorin and 11, cryptotanshinone.

FIG. 2 is a graph showing the inhibition of nitric oxide by different extracts of Qianliexin Capsule in the present invention.

FIG. 3 shows the effect of n-butanol fraction of Qianliexin capsules on the viability of RAW264.7 cells in an example of the present invention.

FIG. 4 shows the inhibitory effect of the n-butanol fraction of the Qianliexin capsule on nitric oxide in the example of the present invention.

FIG. 5 shows the effect of n-butanol fraction of Qianliexin Capsule on inflammatory factors secreted by RAW264.7 cells induced by LPS in the example of the present invention.

FIG. 6 shows the effect of n-butanol fraction of Qianliexin Capsule on the expression of iNOS, COX-2, TLR4, p-NF-kappa Bp65, and p-IKK α/β proteins in examples of the present invention.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.

In one embodiment of the present invention, the prostate capsule is provided as the following (a) or (b):

(a) preparing an inflammation reaction inhibitor;

(b) inhibiting inflammatory reaction.

According to the invention, the Qianliexin capsule is specifically an n-butanol extract of Qianliexin capsule.

The preparation method of the n-butanol extract of the prostate capsule comprises the following steps:

taking the Qianliexin capsule powder, heating and refluxing the powder by using ethanol, concentrating the powder under reduced pressure until no alcohol smell exists, extracting the powder by using (equal volume of) n-butyl alcohol, and concentrating the extract under reduced pressure until the extract is dried to obtain the Qianliexin capsule powder.

Wherein the ethanol is 50-80% ethanol, such as 50%, 60%, 70% and 80%;

the mass ratio of the Qianliexin capsule powder to the ethanol is 1: 5-15, such as 1:5, 1:8, 1:10, 1:12 and 1: 15. The n-butanol extract of the Qianliexin capsule prepared by the process contains more than ten kinds of medicinal active ingredients such as gallic acid, caffeic acid, cryptotanshinone and the like through detection, and has a good inhibition effect on inflammatory reaction. It should be noted that the n-butanol extract of the prostate capsule of the present invention can be used as an experimental model tool preparation to inhibit (in vitro) inflammatory reaction of cells, thereby providing convenience for basic research (such as signal pathway, action target) related to inflammation, etc.

Specifically, the n-butanol extract of the Qianliexin capsule has an inhibition effect on the Lipopolysaccharide (LPS) induced macrophage RAW264.7 cell inflammation of a mouse, and is represented as follows: the n-butanol extract of the Qianliexin capsule reduces the secretion and expression level of related inflammatory factors in cell supernatant and related proteins in cells; and the degree of reduction is dose-dependent.

Wherein the dosage of the n-butanol extract is controlled to be 10-100 mug/mL, including but not limited to 12.5,25 and 50 mug/mL.

Such related inflammatory factors include, but are not limited to, NO, IL-6, IL-1 β, TNF- α, and PGE₂；

The related protein is NF-kB signal channel related protein, including but not limited to TLR4, COX-2, p-NF-k B p65, p-IKK alpha/beta and iNOS.

In another embodiment of the invention, the application of the Qianliexin capsule in preparing a medicament for treating inflammation reaction related diseases is provided.

Wherein the prostate capsule is a n-butanol extract of the prostate capsule;

the preparation method of the n-butanol extract of the prostate capsule comprises the following steps:

taking the Qianliexin capsule powder, heating and refluxing the powder by using ethanol, concentrating the powder under reduced pressure until no alcohol smell exists, extracting the powder by using (equal volume of) n-butyl alcohol, and concentrating the extract under reduced pressure until the extract is dried to obtain the Qianliexin capsule powder.

Wherein the ethanol is 50-80% ethanol, such as 50%, 60%, 70% and 80%;

the mass ratio of the Qianliexin capsule powder to the ethanol is 1: 5-15, such as 1:5, 1:8, 1:10, 1:12 and 1: 15. The n-butanol extract of the Qianliexin capsule prepared by the process contains more than ten kinds of medicinal active ingredients such as gallic acid, caffeic acid, cryptotanshinone and the like through detection, and has a good inhibition effect on inflammatory reaction.

The inflammatory response-related diseases comprise all diseases or symptoms related to inflammatory response, including but not limited to infectious endocarditis, rheumatoid arthritis, ulcerative colitis and the like.

In another embodiment of the present invention, there is provided a use of the prostate capsule in the following (1) to (4):

(1) use in inhibiting secretion and/or expression of a related inflammatory factor;

(2) the application in preparing products for inhibiting the secretion and/or expression of related inflammatory factors;

(3) use in inhibiting secretion and/or expression of a protein of interest;

(4) preparing the application of inhibiting the secretion and/or expression of related protein.

Wherein the prostate capsule is a n-butanol extract of the prostate capsule;

the preparation method of the n-butanol extract of the prostate capsule comprises the following steps:

taking the Qianliexin capsule powder, heating and refluxing the powder by using ethanol, concentrating the powder under reduced pressure until no alcohol smell exists, extracting the powder by using (equal volume of) n-butyl alcohol, and concentrating the extract under reduced pressure until the extract is dried to obtain the Qianliexin capsule powder.

Wherein the ethanol is 50-80% ethanol, such as 50%, 60%, 70% and 80%.

The mass ratio of the Qianliexin capsule powder to the ethanol is 1: 5-15, such as 1:5, 1:8, 1:10, 1:12 and 1: 15. The n-butanol extract of the Qianliexin capsule prepared by the process contains more than ten kinds of medicinal active ingredients such as gallic acid, caffeic acid, p-coumaric acid, luteolin-7-O-glucoside, salvianolic acid B, salvianolic acid A, byak-angelicin, isoanisic lactone, oxypeucedanin, isoimperatorin, cryptotanshinone and the like through detection, and has good inhibitory effect on inflammatory response.

Such related inflammatory factors include, but are not limited to, NO, IL-6, IL-1 β, TNF- α, and PGE₂；

The related protein is NF-kB signal channel related protein, including but not limited to TLR4, COX-2, p-NF-k B p65, p-IKK alpha/beta and iNOS.

In a fourth aspect of the invention, a product is provided, wherein the active ingredient of the product comprises the prostate capsule.

Wherein the prostate capsule is a n-butanol extract of the prostate capsule.

The preparation method of the n-butanol extract of the prostate capsule comprises the following steps:

taking the Qianliexin capsule powder, heating and refluxing the powder by using ethanol, concentrating the powder under reduced pressure until no alcohol smell exists, extracting the powder by using (equal volume of) n-butyl alcohol, and concentrating the extract under reduced pressure until the extract is dried to obtain the Qianliexin capsule powder.

Wherein the ethanol is 50-80% ethanol, such as 50%, 60%, 70% and 80%;

the mass ratio of the Qianliexin capsule powder to the ethanol is 1: 5-15, such as 1:5, 1:8, 1:10, 1:12 and 1: 15.

The product may be a pharmaceutical or experimental model tool formulation.

When the product is a medicament, it may also contain at least one other pharmaceutically active ingredient and/or at least one other non-pharmaceutically active ingredient.

In another embodiment of the present invention, the pharmaceutically inactive ingredient may be a pharmaceutically commonly used carrier, excipient, diluent, or the like. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Such pharmaceutically inactive ingredients, which may include carriers, excipients and diluents, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the medicament of the present invention may be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In still another embodiment of the present invention, the subject to which the pharmaceutical composition is administered may be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, and the like.

In another embodiment of the present invention, there is provided a method for screening an anti-inflammatory component of a prostate capsule, the method comprising: the prostate capsule extracts obtained by adopting different solvent extractions are applied to a cell inflammation model, and the inflammatory response of the cell inflammation model is detected.

Wherein, the solvent includes but is not limited to petroleum ether, ethyl acetate, n-butanol, and preferably n-butanol. The extract obtained by n-butanol extraction has optimal effect in inhibiting inflammatory reaction.

The cell inflammation model comprises a lipopolysaccharide-induced RAW264.7 cell inflammation model.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

1 instruments and materials

Mouse monocyte macrophage (RAW264.7) cell line was purchased from the institute of basic research of chinese medical academy of sciences.

Qianliexin capsules (available from Shandong Hongji Tang pharmaceutical group Co., Ltd.), gallic acid, caffeic acid, p-coumaric acid, luteolin-7-O-glucoside, salvianolic acid B, salvianolic acid A, byak-angelicin, isoanisidine, oxypeucedanin, isoimperatorin, cryptotanshinone (greater than 99% in content, available from Shanghai-sourced leaf Biotech Co., Ltd.), lipopolysaccharide (Sigma in the United states), DMEM high-sugar medium, and PBS (Wuhan Saervel Biotech Co., Ltd.); fetal bovine serum (Israel Biological Industries, Inc.); BCA protein quantification kit (shanghai bi yuntian biotechnology limited); rabbit anti-TLR 4 monoclonal antibody, rabbit anti-COX-2 monoclonal antibody, rabbit anti-iNOS monoclonal antibody, rabbit anti-p 65 monoclonal antibody, rabbit anti-p-NF-kB p65 monoclonal antibody, rabbit anti-p-IKK alpha/beta monoclonal antibody, and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG-HRP) (Cell Signaling Technology, USA)) (ii) a ECL chemiluminescence kits (beijing solibao technologies ltd); mouse TNF-alpha/IL-6/IL-1 beta ELISA kit (EBscience Biotechnology Co., Ltd., USA); PGE₂ELISA kit (Wuhan Huamei bioengineering Co., Ltd.).

Agilent high performance liquid chromatograph (model 1260, DAD detector, Agilent corporation, usa); HF90 carbon dioxide incubator (shanghai scientific instruments ltd); eclipse Ts2 inverted microscope (Nikon corporation, japan); HFsafe-1200LC biosafety cabinet (shanghai science instruments ltd); spark multifunctional microplate reader (Tecan, Switzerland); PowerPac model 300 electrophoresis apparatus (BIO-RAD, USA); 170-3930 type membrane transfer instrument (BIO-RAD, USA); ChemiScope 6000Exp chemiluminescence imaging system (shanghai volitan scientific instruments ltd).

2 method

2.1 analysis of the N-butanol fraction of Qianliexin Capsule

2.1.1 preparation of test specimens

Weighing 50g of Qianliexin capsule powder, heating and refluxing for 3 times by using 70% ethanol in an amount which is 10 times that of the Qianliexin capsule powder, extracting for 1 hour each time, combining extracting solutions, concentrating under reduced pressure until no alcohol smell exists (about 200mL), and sequentially extracting by using petroleum ether, ethyl acetate and n-butanol with equal volumes respectively. The extract was concentrated to dryness under reduced pressure and weighed to obtain 1.159g of petroleum ether extract, 2.7913g of ethyl acetate extract, 2.5617g of n-butanol extract and 25.98g of water layer extract.

2.1.2 chromatographic conditions

A chromatographic column: YMC Pack ODS-AC₁₈(4.6 mm. times.250 mm, 5 μm), acetonitrile (A) -0.5% aqueous formic acid (B) was selected as the mobile phase, and the flow rate was set to 1.0 mL/min; the sample amount of the sample is 10 mu L; detection wavelength: 300 nm; column temperature: 25 ℃; elution was performed according to the gradient elution procedure of table 1, as shown in the table below.

TABLE 1 gradient elution procedure

2.1.3 preparation of control solutions

The reference solution is prepared by precisely weighing reference substances such as gallic acid, caffeic acid, p-coumaric acid, luteolin-7-O-glucoside, salvianolic acid B, salvianolic acid A, angelica sinensis, isoanisic lactone, oxypeucedanin, isoimperatorin and cryptotanshinone, adding methanol to prepare a standard solution with the concentration of each component of 0.1g/L, and filtering with a 0.22 μm microporous membrane. Elution was performed according to the gradient elution procedure described above.

2.2 cell culture

Frozen RAW264.7 cells were taken out of liquid nitrogen and immediately placed in a 37 ℃ water bath for thawing. Thawing, centrifuging to remove supernatant, transferring into culture dish, adding 10% FBS high sugar culture medium, and adding 5% CO₂And cultured at 37 ℃. And (3) after the cells grow to 90%, discarding cell supernatant, adding a newly configured high-sugar culture medium containing 10% serum, scraping the cells by using a cell scraper, fully blowing the cells in the dish by using a suction pipe, uniformly mixing, and sucking supernatant culture solution. And moving the culture dish into a new culture dish and continuing culturing.

2.3MTT method for testing cytotoxicity of drugs

When the cell density is over 80 percent of the culture dish, scraping the cells by a cell scraper, blowing uniformly, counting the cells, and counting the cells by 2 x 10 in a 96-well plate⁵Adding 100 mu L of high-sugar culture medium into each hole of a density plate per mL, culturing for 24h, discarding the supernatant, adding 100 mu L of cell culture medium (12.5,25,50,100,200 mu g/mL) of prepared samples with different concentrations respectively, continuing culturing for 24h, adding 10 mu L of MTT (5 mu g/mL) solution, continuing culturing for 4h, discarding the supernatant, adding 100 mu L of DMSO solution into each hole, shaking and mixing uniformly, and determining the OD value at 490 nm.

2.4 determination of the Nitrogen monoxide content

When the cell density is over 80 percent over the culture dish, the cell is scraped by a cell scraper, the cell is blown evenly, the cell is counted, and the number of the cells is 2 multiplied by 10 in a 96-well plate⁵Adding 100 μ L of high sugar culture medium into each well of density plate, culturing for 24 hr, discarding supernatant, adding 100 μ L of cell culture medium (50 μ g/mL) containing petroleum ether extract, ethyl acetate extract and n-butanol extract with different concentrations of lipopolysaccharide, and using caffeic acid (50 μ g/mL) as the active ingredientAnd (3) taking 50 mu L of supernatant after continuously culturing for 24h as a positive control drug, adopting a nitric oxide detection kit for determination, shaking and uniformly mixing, determining an OD value at 540nm, and calculating the nitric oxide inhibition rate.

2.5 measurement of IL-6, IL-1. beta., TNF-. alpha.and PGE by ELISA₂In an amount of

RAW264.7 cells were treated at 5X 10⁴And/well inoculating the cells in 48-well cell culture plates, culturing overnight in an incubator, discarding the supernatant, adding 100 μ L of prepared cell culture media (12.5,25,50 μ g/mL) containing samples with different concentrations and caffeic acid, culturing for 24h, and setting a normal cell control group and a model cell control group. Each concentration was provided with 5 multiple wells. Cell supernatants were harvested after 24h of stimulation and stored at-20 ℃. According to mouse IL-6, IL-1 beta, TNF-alpha and PGE₂The ELISA detection kit of (1).

2.6Western Blot to detect expression of iNOS/COX-2/TLR 4/p-NF-kappa B p65/p-IKK alpha/beta protein

RAW264.7 cells were treated at 5X 10⁴And/well inoculating in 6-well cell culture plate, stimulating for 24h according to 2.5 sample adding step, washing each group of cells for 3 times respectively by using PBS, adding proper amount of lysate into the cells, cracking for half an hour on ice, blowing for many times, transferring the cells into a 1.5mL centrifuge tube, and centrifuging for 15min at 12000r/min at 4 ℃. Extracting supernatant, packaging into new centrifuge tube, adding sample buffer, and denaturing at 100 deg.C for 5 min. And (4) carrying out sample electrophoresis according to the result of protein quantification. After SDS-PAGE electrophoresis, a semidry transfer membrane is used for transferring to a PVDF membrane, after blocking, primary antibody is added for overnight at 4 ℃, TBST is used for washing for 3 times, each time is 10min, and goat-anti-rabbit secondary antibody marked by HRP is added. Preparing chemical luminous liquid, putting the film on an exposure plate, dripping a proper amount of luminous liquid on the film, and exposing and analyzing the picture by using a full-automatic fluorescence and visible light gel imaging analysis system.

3 results

3.1 analysis of the composition of the prostasin-n-butanol fraction (QLXB)

The chromatographic analysis result of the prostate euphoria n-butyl alcohol part shows that the 11 components can be detected in the n-butyl alcohol part, the separation degree is good, and the contents of gallic acid, caffeic acid, cryptotanshinone and the like are high.

3.2 Effect of QLXB on cell viability

Cell viability assays are an important basis for evaluating drug cytotoxicity. The cell survival rate of the blank group is taken as 100 percent as a control, and the cell survival rate of the n-butanol extract part of the prostate capsule under the dosage of 0-100 mu g/mL is respectively detected. As shown in FIG. 3, the cell viability was above 100% at the dose of 0-25. mu.g/mL, however, the cell viability of RAW264.7 decreased gradually with the increase of QLXB concentration, wherein the cell viability was about 90% at the dose of 50. mu.g/mL, and the cell viability was also better. However, at 100 μ g/mL, cell viability decreased significantly, indicating that this concentration can cause cell damage and is cytotoxic. Therefore, the safe dose range of the n-butanol fraction of the prostate capsule can be studied at a dose of 50 μ g/mL.

3.3 Effect of QLXB on the amount of NO produced by RAW264.7 macrophages

RAW264.7 macrophages can produce a large amount of nitric oxide under the induction of lipopolysaccharide, and the nitric oxide can influence the synthesis and release of other inflammatory factors. As shown in fig. 2 and 4, the nitric oxide content was significantly increased (p <0.05) in the LPS group compared to the blank group, and the prostate-euphoric n-butanol fraction had the strongest anti-inflammatory activity. After the n-butanol part of the Qianliexin capsule is added, each group has obvious inhibition effect (p is less than 0.05) on the content of nitric oxide in RAW264.7 macrophage, under the dosage of 50 mu g/mL, the generation inhibition rate of the nitric oxide reaches more than 67.12 percent, the inhibition effect is obvious, and the inhibition effect is more consistent with the inhibition effect of positive medicine caffeic acid.

3.4 Effect of QLXB on inflammatory factors produced by RAW264.7 macrophages

Macrophages secrete a large number of proinflammatory factors (e.g., IL-1 beta, IL-6, IL-12, TNF-alpha, and the like) under stimulation by lipopolysaccharide, wherein TNF-alpha is the earliest and most important inflammatory mediator in the inflammatory response process, IL-1 beta, IL-6, and PGE₂Is involved in the occurrence and development processes of various inflammatory diseases and is a promoter of inflammatory response. As shown in FIG. 5, the inflammatory factors IL-1. beta., IL-6, TNF-. alpha.and PGE in the model group were observed after LPS stimulation₂Display of production amountSignificant increase (p)<0.05), QLXB group to RAW264.7 macrophage inflammatory factors IL-1 beta, IL-6, TNF-alpha and PGE₂Has a remarkable inhibitory effect on secretion (p)<0.05) and exhibit a dose-dependent relationship. The inhibitory effect of QLXB group on inflammatory factors at high concentrations is more consistent with that of caffeic acid (50 μ g/mL), and these results indicate that QLXB can inhibit the production of inflammatory mediators and the progression of inflammatory responses.

3.5 influence of QLXB on expression of iNOS/COX-2/TLR4/p-NF- κ BETA p65/p-IKK α/β protein

In the LPS-induced RAW264.7 cell model, iNOS and COX-2 are key rate-limiting enzymes for the release of various inflammatory mediators; TLR4 in the Toll-like receptor can specifically recognize lipopolysaccharide, and p-NF-kappa BETA 65/p-IKK alpha/beta is a key protein in an NF-kappa BETA pathway. Therefore, the expression of the above proteins plays an important role in inflammation. As shown in FIG. 6, Western blot shows that the expression level of the proteins is significantly increased in cells of the model group by using iNOS/COX-2/TLR 4/p-NF-kappa BETA 65/p-IKK alpha/beta protein as compared with that of the blank group; the QLXB different dose groups can obviously inhibit the expression of the protein and have obvious dose dependence.

In conclusion, the experimental results show that the n-butyl alcohol part of the prostate capsule has a remarkable anti-inflammatory effect, can remarkably inhibit macrophages caused by LPS from releasing proinflammatory factors and inflammatory mediators, has an action mechanism closely related to the inhibition of the activation of an NF-kB channel, and provides an experimental basis for the clinical efficacy of the prostate capsule.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.