CN110882266B - Timosaponin composition and application thereof in treating viral myocarditis - Google Patents

Abstract

The invention discloses a timosaponin composition, which contains 22-34 wt% of timosaponin AIII, 18-30 wt% of timosaponin BII and 10-17 wt% of timosaponin N.

Description

Timosaponin composition and application thereof in treating viral myocarditis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a timosaponin composition and application thereof in treating viral myocarditis.

Background

Myocarditis is an inflammatory reaction of myocardial cells, endocardium, blood vessels, and epicardium caused by various pathogenic microorganisms such as viruses, bacteria, protozoa, rickettsiae, and the like, or other physicochemical factors during or after infection. The clinical most common is Viral Myocarditis (VMC), local or diffuse myocardial inflammatory disease caused by viral infection, which is better than that in young and old years, and has a gradually rising trend in recent years.

Viral myocarditis can cause cardiac dysfunction or general symptoms with different degrees, clinical manifestations are mild and severe, some patients have few clinical symptoms, the prognosis is mostly good, but few patients can suffer from cardiogenic shock, even sudden death and prolonged unhealed patients, and chronic myocarditis or cardiomyopathy can be formed. Almost all viruses may cause myocarditis, and more than 20 viruses are currently known to cause myocarditis, such as echoviruses, influenza viruses, and most commonly coxsackievirus B. Since viral myocarditis seriously harms human health, particularly the health of teenagers, has attracted extensive attention from the medical community and numerous scholars, and although the pathogenesis of viral myocarditis is not completely elucidated, cardiac damage caused by viral myocarditis is proved to be of great significance in reducing the occurrence of dilated cardiomyopathy.

Viral myocarditis is not treated specifically or is not treated definitely by effective medicines, and comprehensive therapies including antiviral, myocardial protection, immunoregulation and the like are mostly adopted. Western medicines have a certain treatment effect on viral myocarditis, but often combine severe toxic and side effects of pelvic inhibition, immune injury and the like. At present, in the aspect of traditional Chinese medicine, scholars give traditional Chinese medicine decoction data after differentiation and typing stages of viral myocarditis patients, and experimental research and clinical observation prove that a single traditional Chinese medicine and a compound empirical formula have certain effect on treating viral myocarditis. For example, the study shows that ginseng and astragalus root have curative effect on myocarditis, the active ingredients of ginseng can reduce myocardial oxygen consumption, improve anoxic zones, increase myocardial blood and oxygen supply, and have protective effect on myocardial cells, and astragalus root can improve myocardial function, play a role of cardiac glycoside, and change myocardial contractility.

Anemarrhena asphodeloides is also called rhizome of Anemarrhena asphodeloides of Liliaceae, and is sweet and bitter in taste and cold in nature, entering lung, stomach and kidney meridians. Has effects in clearing away heat, purging pathogenic fire, nourishing yin, moistening dryness, quenching thirst, and relieving restlessness. Rhizoma anemarrhenae is rich in various components such as saponins, flavonoids, lignin and the like, and has good pharmacological activity, wherein saponins and aglycones thereof are the main components with wide pharmacological activity, and account for about 6% of rhizome content. Changrun Guo et al found that timosaponin BII has an anti-apoptotic effect by inhibiting endothelial cells in high sugar-induced oxidative stress and has potential for preventing cardiovascular complications of diabetes; korean soldiers and the like find that the rhizoma anemarrhenae total saponin can reduce the content of serum total cholesterol, triglyceride and low-density lipoprotein and obviously reduce the area of arterial plaque; the prunus salicina and the like find that timosaponin AIII has obvious antithrombotic effect in vivo and in vitro, but does not influence the in vivo blood coagulation time, and the fact that the timosaponin AIII only influences the aggregation, adhesion and activation of platelets in the process of inhibiting blood coagulation and does not influence various blood coagulation factors and blood cell factors in blood is suggested; kim et al believe that timosaponin B has the effect of blocking the generation of cell inflammatory factor NF-kB, and can realize anti-inflammatory action by inhibiting the pathway of p38 Mitogen Activated Protein Kinase (MAPK); the timosaponin is extracted from Liuzhuo and can inhibit the release of macrophage inflammatory factors TNF-alpha and NO caused by lipopolysaccharide; ying-guang Sun indicates that neomangiferin, mangiferin, timosaponin BII and AIII, etc. are the main active components with EMP anti-inflammatory and anti-asthma effects; UJ Youn et al found that timosaponin AIII has antiviral activity, however, there is no report on timosaponin being used for treating viral myocarditis, and there is no research on pharmacological action of timosaponin on viral myocarditis.

Disclosure of Invention

The invention aims to solve the technical problems, researches the protection of the effective extraction part of rhizoma anemarrhenae on myocardial cells and the treatment effect on viral myocarditis, and provides a new medicine source for the traditional Chinese medicine treatment of viral myocarditis.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a timosaponin composition, which comprises 22-34 wt% of timosaponin AIII, 18-30 wt% of timosaponin BII and 10-17 wt% of timosaponin N.

The timosaponin AIII has a molecular formula of C ₃₉ H ₆₄ O ₁₃ The structural formula is as follows:

。

the timosaponin BII has a molecular formula of C ₄₅ H ₇₆ O ₁₉ The structural formula is as follows:

。

the timosaponin N has a molecular formula of C ₄₅ H ₇₆ O ₃₀ The structural formula is as follows:

。

as a preferred embodiment of the invention, the timosaponin AIII is 26-30 wt%, the timosaponin BII is 22-26 wt%, and the timosaponin N is 12-15 wt%.

In a preferred embodiment of the present invention, the timosaponin aiii is 28 wt%, the timosaponin bii is 24 wt%, and the timosaponin N is 13 wt%.

Because the product is directly prepared by a common water extraction or alcohol extraction method of timosaponin, wherein the content of timosaponin BII is generally higher than that of timosaponin AIII, and the content of timosaponin N is often lower than 5%, the preparation method of the timosaponin composition can be used for respectively extracting and purifying timosaponin BII, timosaponin AIII and timosaponin N in timosaponin, then detecting the purity of the timosaponin BII, and mixing the timosaponin composition within the content range according to the weight ratio with or without adding auxiliary materials. However, preferably, the invention provides a preferred preparation method of the timosaponin composition, which can be used for solving the solubility difference of timosaponin bii, timosaponin aiii and timosaponin N in different solvents and the characteristic of converting timosaponin biii into timosaponin aiii under a certain environment, and comprises the following steps:

(1) extracting rhizoma anemarrhenae total saponins: taking a rhizoma anemarrhenae medicinal material, carrying out reflux extraction for 2 times, the first time for 2 hours and the second time for 1.5 hours by using 70% ethanol in an amount which is 6-10 times that of the rhizoma anemarrhenae medicinal material, combining ethanol extract, and concentrating under reduced pressure to obtain concentrated solution with the relative density of 1.25-1.35;

(2) extracting timosaponin BII: taking a half volume of the concentrated solution obtained in the step (1), loading HP20 adsorption resin into a column, filtering the concentrated solution, then sampling, washing with water to remove impurities, then eluting with 3BV of 50% ethanol, collecting the eluent to recover ethanol, then sampling the obtained liquid with an SP825 macroporous resin column, eluting with 4BV of 50% ethanol, collecting the eluent to recover ethanol, concentrating to obtain a concentrated solution with the relative density of 1.25-1.35, and performing spray drying to obtain timosaponin BII powder;

(3) preparing timosaponin AIII: taking 60-65 wt% of timosaponin BII powder obtained in the step (2), adding anhydrous acetic acid-acetic acid buffer solution with pH of 4.8, uniformly stirring, heating to 53 ℃, adding beta-glucosidase, reacting for 2h, extracting a product for 3 times by using ethyl acetate, combining ethyl acetate extract liquor, and recovering a solvent to obtain timosaponin AIII powder;

(4) extracting timosaponin N: taking the rest concentrated solution obtained in the step (1), packing HPD100 macroporous resin into a column, filtering the concentrated solution, sampling the filtrate, eluting with 6BV of water to remove impurities, eluting with 6BV of 40% ethanol to remove impurities, eluting with 6BV of 60% ethanol to remove lower column, recovering ethanol from the eluent, dissolving the ethanol in water, introducing into an ODS column, eluting with 40% acetone, collecting the eluent, recovering acetone, concentrating and drying to obtain timosaponin N powder;

(5) preparation of the saponin composition: mixing the timosaponin BII powder obtained in the step (2), the timosaponin AIII powder obtained in the step (3), the timosaponin N powder obtained in the step (4) and a filler according to a ratio of 1.70-1.75: 1.90-1.95: 1:2 to obtain the timosaponin composition.

The preparation method can obtain the timosaponin composition with timosaponin AIII by weight percent of 22-34%, timosaponin BII by weight percent of 18-30% and timosaponin N by weight percent of 10-17%. The method saves medicinal material resources, has reasonable step design, and simplifies operation steps.

The timosaponin composition is used for preparing a medicament for treating viral myocarditis. The medicament for treating viral myocarditis comprises a medicinal excipient or carrier and a timosaponin composition with a therapeutically effective amount, and the preparation form of the medicament for treating viral myocarditis is a solid preparation, such as tablets, capsules, pills and the like.

As a preferred embodiment of the invention, the therapeutically effective amount is 5-20 mg/kg-d based on the total saponin content.

In summary, compared with the prior art, the invention has the following beneficial effects: the applicant of the invention discovers that the effect of treating viral myocarditis by independently using timosaponin AIII and timosaponin BII is very weak, and in vitro and rat in vivo CVB3 virus infection tests show that timosaponin AIII has a certain antiviral effect and timosaponin BII has a certain anti-inflammatory effect, but the protective effect of both the timosaponin AIII and the timosaponin BII on myocardial cells is weak when the timosaponin AIII and the timosaponin BII are used independently, while in the commonly extracted timosaponin composition, the content of timosaponin BII is higher than that of timosaponin AIII, the protective and therapeutic effect of the timosaponin composition on viral myocarditis is not obvious, but surprisingly, under the condition of containing higher contents of timosaponin AII and timosaponin BII, more than 10% of timosaponin N can play a good synergistic effect, so as to greatly improve the pharmaceutical activity of timosaponin AIII and timosaponin protecting cardiac muscle, the three can obviously inhibit the proliferation of viruses under a certain content ratio, and play a role in protecting myocardial cells and tissues, and clinical researches show that the timosaponin composition can improve the treatment effective rate of the viral myocarditis by combining with conventional treatment means.

Detailed Description

The present invention will be described in further detail with reference to specific examples. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.

Preparation of timosaponin composition

Example 1

(1) Taking a rhizoma anemarrhenae medicinal material, performing reflux extraction for 3 times by using 70% ethanol in an amount which is 10 times that of the rhizoma anemarrhenae medicinal material, respectively extracting for 2h, 1.5h and 1h, combining extracting solutions, concentrating until the relative density is 1.15, after extracting n-butanol, filtering part of the n-butanol, concentrating into an extract, applying to D101 macroporous resin, eluting by using 50% ethanol with 4BV, collecting eluent, performing reversed-phase ODS column chromatography, concentrating and drying a product, crushing to obtain timosaponin AIII powder, and determining that the purity of the timosaponin AIII is 95.4%;

(2) taking a rhizoma anemarrhenae medicinal material, carrying out reflux extraction for 3 times by 10 times of 70% ethanol, respectively extracting for 2h, 1.5h and 1h, merging extracting solutions, filtering, concentrating into an extract, adding HPD100 macroporous resin, sequentially eluting with water and 20% ethanol to remove impurities, eluting with 5BV of 50% ethanol, collecting eluent, concentrating into an extract, adding HPD400 macroporous resin, eluting with 4BV of 45% ethanol, collecting eluent, concentrating, and drying to obtain timosaponin BII with the purity of 90.7%;

(3) taking a rhizoma anemarrhenae medicinal material, carrying out reflux extraction for 3 times by 10 times of 70% ethanol, respectively extracting for 2h, 1.5h and 1h, merging extracting solutions, filtering, concentrating the extracting solution into an extract, packing ADS-17 resin into a column, filtering the concentrated solution, loading the filtrate into a sample, eluting by 6BV water to remove impurities, eluting by 6BV 60% ethanol into the lower column, recovering ethanol from the eluent, dissolving in water, introducing into an ODS column, eluting by 40% methanol, collecting the eluent, recovering methanol, concentrating and drying to obtain timosaponin N powder, and detecting that the purity of the timosaponin N powder is 92.4%;

(4) and uniformly mixing 31g of timosaponin AIII powder, 25g of timosaponin BII powder, 14g of timosaponin N powder and 30g of filler lactose to obtain the timosaponin composition, wherein the timosaponin AIII content is 29.6%, the timosaponin BII content is 22.7% and the timosaponin N content is 12.9%.

Example 2

(1) Extracting rhizoma anemarrhenae total saponins: taking a rhizoma anemarrhenae medicinal material, carrying out reflux extraction for 2 times, the first time for 2 hours and the second time for 1.5 hours by using 70% ethanol in an amount which is 8 times that of the rhizoma anemarrhenae medicinal material, combining ethanol extract, and concentrating under reduced pressure to obtain concentrated solution with the relative density of 1.28;

(2) extracting timosaponin BII: loading the concentrated solution obtained in the step (1) with the volume of 1/2 into a column by using HP20 adsorption resin, filtering the concentrated solution, loading the sample, washing with water to remove impurities, eluting with 50% ethanol at 3BV, collecting the eluent to recover ethanol, loading the obtained liquid onto an SP825 macroporous resin column, eluting with 50% ethanol at 4BV, collecting the eluent to recover ethanol, concentrating to obtain the concentrated solution with the relative density of 1.32, and performing spray drying to obtain timosaponin BII powder with the purity of 94.3% by detection;

(3) preparing timosaponin AIII: taking 65% by weight of timosaponin BII powder obtained in the step (2), adding an anhydrous acetic acid-acetic acid buffer solution with the pH value of 4.8, uniformly stirring, heating to 53 ℃, adding beta-glucosidase, reacting for 2 hours, extracting a product for 3 times by using ethyl acetate, combining ethyl acetate extract liquor, recovering a solvent to obtain timosaponin AIII powder, and determining that the purity of timosaponin AIII is 96.6%;

(4) extracting timosaponin N: taking the rest concentrated solution obtained in the step (1), packing HPD100 macroporous resin into a column, filtering the concentrated solution, sampling the filtrate, eluting with 6BV of water to remove impurities, eluting with 6BV of 40% ethanol to remove impurities, eluting with 6BV of 60% ethanol to remove lower column, recovering ethanol from the eluent, dissolving the ethanol in water, introducing into an ODS column, eluting with 40% acetone, collecting the eluent, recovering acetone, concentrating and drying to obtain timosaponin N powder, and detecting the purity of the timosaponin N powder to be 92.3%;

(5) preparation of the saponin composition: and (3) uniformly mixing 26g of timosaponin BII powder obtained in the step (2), 29g of timosaponin AIII powder obtained in the step (3), 15g of timosaponin N powder obtained in the step (4) and 30g of filler lactose to obtain the timosaponin composition, wherein the content of timosaponin AII, timosaponin BII and timosaponin N is 28.0%, 24.5% and 13.8% respectively.

Example 3

The timosaponin prepared in example 1 was mixed uniformly according to 28g of timosaponin aiii powder, 28g of timosaponin bii powder, 16g of timosaponin N powder and 28g of filler lactose to obtain a timosaponin composition, wherein the timosaponin aiii content was 26.7%, the timosaponin bii content was 25.4%, and the timosaponin N content was 14.7%.

Example 4

The timosaponin prepared in example 1 was mixed uniformly according to 24g of timosaponin aiii powder, 33g of timosaponin bii powder, 11g of timosaponin N powder and 32g of filler lactose to obtain a timosaponin composition, wherein the timosaponin aiii content was 22.9%, the timosaponin bii content was 29.9%, and the timosaponin N content was 10.1%.

Example 5

The timosaponin prepared in example 2 was mixed uniformly according to 35g of timosaponin aiii powder, 28g of timosaponin bii powder, 18g of timosaponin N powder and 19g of filler lactose to obtain a timosaponin composition, wherein the timosaponin aiii content was 33.8%, the timosaponin bii content was 18.8%, and the timosaponin N content was 16.6%.

Comparative example 1

The timosaponin prepared in example 1 was mixed uniformly according to 13g of timosaponin aiii powder, 39g of timosaponin aiii powder, 1.5g of timosaponin N powder and 46.5g of filler lactose to obtain a timosaponin composition, wherein the timosaponin aiii content was 12.4%, the timosaponin aiii content was 35.3%, and the timosaponin N content was 1.39%.

Comparative example 2

The timosaponin prepared in example 2 was mixed uniformly according to 19g of timosaponin aiii powder, 27g of timosaponin bii powder, 13g of timosaponin N powder and 41g of filler lactose to obtain a timosaponin composition, wherein the timosaponin aiii content was 18.3%, the timosaponin bii content was 25.4%, and the timosaponin N content was 11.9%.

Comparative example 3

The timosaponin prepared in example 2 was uniformly mixed according to 47g of timosaponin AIII powder, 15g of timosaponin BII powder, 15g of timosaponin N powder and 23g of filler lactose to obtain a timosaponin composition, wherein the timosaponin AIII content was 45.4%, the timosaponin BII content was 14.1% and the timosaponin N content was 13.8%.

Comparative example 4

The timosaponin prepared in example 2 was mixed uniformly according to 25g of timosaponin AIII powder, 40g of timosaponin BII powder, 21g of timosaponin N powder and 14g of filler lactose to obtain a timosaponin composition, wherein the timosaponin AIII content was 24.1%, the timosaponin BII content was 37.7% and the timosaponin N content was 19.4%.

Comparative example 5

The timosaponin prepared in example 2 was uniformly mixed according to 39g of timosaponin AIII powder, 16g of timosaponin BII powder, 27g of timosaponin N powder and 18g of filler lactose to obtain a timosaponin composition, wherein the timosaponin AIII content was 37.6%, the timosaponin BII content was 15.1% and the timosaponin N content was 24.9%.

Secondly, the research on the protective effect of timosaponin on CVB3 infected rat myocardial cells (in vitro experiment)

Test grouping and test sample

Taking 20 SD rats born at 1-3d, taking out hearts of the SD rats, culturing whole heart cardiac muscle cells, culturing and freezing by using commercially available Hela cells, and performing in-vitro anti-CVB 3 virus test, wherein 17 groups are provided in the test, namely a normal cell group, a virus control group, a blank adjuvant group, a positive control group and an A-M group, so as to observe the in-vitro protection effect of different timosaponins and timosaponin compositions on the rat cardiac muscle cells, and the test sample conditions of each test group are shown in Table 1.

TABLE 1 test sample Condition for each test group

Test group	Cell processing	Added medicine/adjuvant ingredients
			Group of Normal cells	Adding culture medium without virus and medicine	——
Virus control group	Adding virus	——
			Blank auxiliary material group	Adding virus, adding lactose	No drug was added, but 0.5ml of 0.05g/ml lactose solution was added
Positive control group	Adding virus, adding alpha-interferon	280u/ul alpha-interferon 0.5ml
			Group A	Adding virus, adding timosaponin AIII	Timosaponin AIII prepared in example 1, the timosaponin AIII has a purity of 95.4%, and 0.5ml of a solution having a total saponin concentration of about 0.05g/ml is prepared
Group B	Adding virus, adding timosaponin BII	Timosaponin BII prepared in example 2, the timosaponin BII had a purity of 94.3%, and was prepared as a solution with a total saponin concentration of about 0.05g/ml, which was added in an amount of 0.5ml
			Group C	Adding virus, adding timosaponin N	Timosaponin N prepared in example 1, the timosaponin N has a purity of 92.4%, and is prepared by adding 0.5ml of a solution having a total saponin concentration of about 0.05g/ml
Group D	Adding virus, adding timosaponin composition	The timosaponin composition prepared in example 1 contains timosaponin AIII, timosaponin BII, timosaponin N, and timosaponin AIII 29.6% 12.9%, and lactose in balance, and adding 0.5ml of solution with total saponin concentration of about 0.05g/ml
			Group E	Adding virus and timosaponin	The timosaponin composition prepared in example 2 contains timosaponin AIII, timosaponin BII and timosaponin N, wherein timosaponin AIII and timosaponin BII are 28.0% and 24.5%, respectively Is 13.8 percent, is prepared into a solution with the total saponin concentration of about 0.05g/ml, and is added with 0.5ml
Group F	Adding virus, adding timosaponin composition	The timosaponin composition prepared in example 3 contains timosaponin AIII (26.7%), timosaponin BII (25.4%) and timosaponin N (N) Is 14.7%, and 0.5ml of solution with total saponin concentration of about 0.05g/ml is prepared
			Group G	Adding virus, adding timosaponin composition	The timosaponin composition prepared in example 4 contains timosaponin AIII 22.9%, timosaponin BII 29.9%, and timosaponin N 10.1% of total saponin is prepared by adding 0.5ml of total saponin solution with concentration of about 0.05g/ml
Group H	Adding virus, adding timosaponin composition	The timosaponin composition prepared in example 5 contains 33.8% of timosaponin AIII, 18.8% of timosaponin BII and 18.8% of timosaponin N 16.6% of total saponin is prepared by adding 0.5ml of total saponin solution with concentration of about 0.05g/ml
			Group I	Adding virus, adding timosaponin composition	The timosaponin composition prepared in comparative example 1 contains timosaponin AIII 12.4%, timosaponin BII 35.3%, and timosaponin N 1.39%, preparing a solution with total saponin concentration of about 0.05g/ml, adding 0.5ml
J group	Adding virus, adding timosaponin composition	The timosaponin composition prepared in comparative example 2 contains timosaponin AIII (18.3%), timosaponin BII (25.4%) and timosaponin N (N) 11.9% of the total saponin content was prepared as a solution with a total saponin concentration of about 0.05g/ml, 0.5ml was added
			Group K	Adding virus, adding timosaponin composition	The timosaponin composition prepared in comparative example 3 contains timosaponin AIII 45.4%, timosaponin BII 14.1%, and Anemarrhena soapGlycoside N content 13.8%, preparing a solution with total saponin concentration of about 0.05g/ml, adding 0.5ml
Group L	Adding virus, adding timosaponin composition	The timosaponin composition prepared in comparative example 4 contains timosaponin AIII 24.1%, timosaponin BII 37.7%, and timosaponin N 19.4% of the total saponin is prepared by adding 0.5ml of the solution with the total saponin concentration of about 0.05g/ml
			M groups	Adding virus, adding timosaponin composition	The timosaponin composition prepared in comparative example 5 contains timosaponin AIII 37.6%, timosaponin BII 15.1%, and timosaponin N Is 24.9%, and 0.5ml of solution with total saponin concentration of about 0.05g/ml is prepared

In the above test groups, interferon-alpha and lactose were both commercially available, and timosaponin or a composition thereof used in groups A to M was prepared. In the above examples and comparative examples, the saponin composition was prepared by mixing the prepared saponin with lactose as an excipient, but in practice, it may be prepared by mixing with any other commonly used inert excipient such as starch, cellulose, mannitol, etc., as long as the excipient does not exert pharmacological effects on cardiac muscle cells.

(II) test method

1. Culture of cells

1.1 Whole myocardial cell culture

Culturing 20 SD rats born for 1-3 days, sterilizing chest with 75% alcohol, taking out heart, placing into 4 deg.C precooled D-HANK 'S buffer solution, removing blood, placing into culture dish, adding D-HANK' S buffer solution, cutting heart into 1mm pieces ³ Tissue pieces of size, then the tissue pieces were placed in a container containing 15ml of 0.1% trypsin (in D-HANK'S buffer solution preparation), digesting in water bath at 37 ℃ for 20 minutes, standing, sucking supernatant, adding 15ml of 0.1% trypsin (prepared by D-HANK' S buffer solution) for digesting for 15 minutes, repeatedly digesting for 7 times, collecting supernatant, adding 20% FBS-containing MEM culture solution to terminate digestion, culturing for 1 hour adherent thereto, removing fibroblasts, collecting cardiomyocyte suspension, staining and counting placenta blue, adjusting cell density to 5 × 10 ⁵ And/ml, inoculating a 24-well cell culture plate with 1ml per well, inoculating a 96-well cell culture plate with 100ul per well, culturing the culture plate inoculated with the cardiac muscle cells in an incubator with 5% carbon dioxide at 37 ℃ for 24 hours, and continuously culturing for 24 hours after liquid is changed.

1.2 Hela cell culture method

(1) Cell passage: pouring out the culture solution in the Hela cell culture bottle, adding 0.1% trypsin, after the cells are rounded, blowing and sucking by using a dropper to separate from the adherent wall, transferring the cells to a centrifuge tube, centrifuging at 800rpm for 5 minutes, removing the supernatant, adding 20% FBS DMEM culture solution, blowing and sucking uniformly, and then inoculating the cells to the culture bottle.

(2) Freezing and storing cells: taking cells with good adherence and good growth, pouring out original culture solution, adding 0.1% trypsin, after the cells are rounded, blowing and sucking by using a dropper to separate the cells from the adherence, transferring the cells to a centrifuge tube, centrifuging at 1000rpm for 3 minutes, removing supernatant, and storing in a refrigerator at-80 ℃.

(3) Cell recovery: taking out the frozen tube, putting into 37 ℃ warm water, shaking to dissolve, transferring to a centrifuge tube, adding 10ml of culture solution, mixing uniformly, centrifuging at 1000rpm for 5 minutes, removing supernatant, adding 20ml of culture solution, mixing uniformly, and transferring to a culture bottle.

2. Timosaponin cytotoxicity assay

Dissolving timosaponin of groups A-M in 1% DMSO to obtain in vitro experimental solution, centrifuging the solution to obtain supernatant, filtering with microporous membrane, sterilizing, diluting with 20% FBS MEM to 11 concentrations, adding into 96 wells in which single layer of cardiomyocytes are cultured, culturing at 37 deg.C in 5% carbon dioxide incubator for 5 days, and measuring maximum nontoxic concentration (TD) by Cytopathic (CPE) method ₀ ). The results are TD of timosaponin of each test group ₀ 43.5-87.3 mg/mL.

3. CVB3 Virus virulence assay

The virus was serially diluted 10-fold in serum-free medium, 10 each ^-1 -10 ^-8 Diluting, inoculating to 96-well plate with cultured monolayer cardiomyocyte, culturing at 37 deg.C in 5% carbon dioxide incubator for 1 hr, discarding virus solution, washing with Hank's solution for three times, adding cell culture solution, culturing in incubator, periodically replacing cell culture solution, observing CPE, and determining that cytopathic effect reaches above "++" as positive and CPE no longer appears at highest dilution. Calculation of TCID by Reed-Muench method ₅₀ (ii) a Distance ratio (= 50% lesion rate-50%)/(> 50% lesion rate- < 50% lesion rate); LgTCID ₅₀ = log of viral dilution > 50% + distance ratio x log of dilution factor 100TCID selection ₅₀ As the inoculation concentration for the real experiment. The TCID of the Coxsackie B3 virus used in this experiment was calculated ₅₀ A virus concentration of 3.51, i.e.a virus concentration which is capable of causing CPE in 50% of the cells, of 10 ^3.51 /ml。

4. Study on protective effect of timosaponin on myocardial cells

According to the settings shown in Table 1, 0.5mL of 100TCID virus diluent was added to cardiomyocytes that had been regularly pulsed after 48h of culture ₅₀ After 1h, adding 0.5ml of different drug solutions into the group A-M for continuous culture, wherein the drug solutions are solutions with the total saponin concentration of 0.05g/ml, which are obtained by diluting the saponin substances of the group A-M with MEM (20% FBS), only adding culture solution into normal cell groups, only adding virus into virus control groups, adding 0.05g/ml lactose solution prepared by adding virus and MEM culture solution containing 20% FBS into blank auxiliary material groups, 0.5ml of virus and 280u/ul alpha-interferon into positive control groups, respectively measuring the pulsation rate of each cell in the 2 nd and 5d under an inverted microscope, collecting the cells and the culture solution of each group in the 2 nd and 5d, repeatedly freezing and thawing to release virus particles, centrifuging to remove supernatant, inoculating the diluted virus solution to the cultured Hela cell 96 of a pore plate, and measuring TCID of each group ₅₀ The data is analyzed by SPSS13.0 medical statistic software, the measured data is represented by mean plus or minus standard deviation, single-factor variance analysis and pairwise comparison are adoptedThe results are shown in tables 2 and 3, compared to the LSD analysis method.

TABLE 2 Effect of timosaponin on the beating rate of CVB 3-infected cardiomyocytes (n = 10)

Test group	Dosage (0.5 ml)	2d beat rate/min	5d beat rate/min
				Group of normal cells	/	134.2±4.254	113.8±2.117
Virus control group	/	76.45±1.547	0
				Blank auxiliary material group	0.05g/ml	78.63±2.345	0
Positive control group	250u/ul	97.45±1.325	96.29±2.481* ＃
				Group A	0.05g/ml	72.41±3.547	56.72±3.257*
Group B	0.05g/ml	80.87±1.545	0
				Group C	0.05g/ml	71.54±1.842	0
Group D (example 1)	0.05g/ml	97.54±2.455	86.44±1.942* ＃
				Group E (example 2)	0.05g/ml	96.37±2.314	95.67±1.487* ＃
Group F (example 3)	0.05g/ml	94.16±3.697	88.87±2.541* ＃
				Group G (example 4)	0.05g/ml	89.24±4.632	78.45±1.974* ＃
Group H (example 5)	0.05g/ml	85.14±3.422	80.33±2.317* ＃
				Group I (comparative example 1)	0.05g/ml	68.66±1.954	32.15±1.652*
Group J (comparative example 2)	0.05g/ml	62.18±1.366	36.84±3.478*
				Group K (comparative example 3)	0.05g/ml	74.54±2.145	60.24±2.487*
Group L (comparative example 4)	0.05g/ml	65.87±3.122	42.21±2.698*
				Group M (comparative example 5)	0.05g/ml	78.45±2.647	39.15±1.354*

Compared to the virus control group,: p < 0.05, D-H group vs. A, #: p is less than 0.05.

TABLE 3 TCID on cardiomyocytes following timosaponin intervention ₅₀ Influence (n = 10)

Test group	Dosage (0.5 ml)	2d TCID 50	5d TCID 50
				Group of Normal cells	/	0	0
Virus control group	/	4.15±0.06	4.98±0.13
				Blank auxiliary material group	0.05g/ml	4.35±0.27	5.00±0.34
Positive control group	250u/ul	0*	0*
				Group A	0.05g/ml	2.87±0.24*	3.14±0.18*
Group B	0.05g/ml	4.27±0.14	5.48±0.23
				Group C	0.05g/ml	5.07±0.21	5.59±0.18
Group D (example 1)	0.05g/ml	1.45±0.05* ＃	1.34±0.09* ＃
				Group E (example 2)	0.05g/ml	1.27±0.09* ＃	1.05±0.12* ＃
Group F (example 3)	0.05g/ml	1.48±0.16* ＃	1.27±0.11* ＃
				Group G (example 4)	0.05g/ml	1.98±0.21* ＃	2.01±0.13* ＃
Group H (example 5)	0.05g/ml	1.87±0.17* ＃	2.14±0.06* ＃
				Group I (comparative example 1)	0.05g/ml	3.64±0.13	4.02±0.14
Group J (comparative example 2)	0.05g/ml	3.97±0.16	4.14±0.25
				Group K (comparative example 3)	0.05g/ml	4.05±0.12	4.34±0.31
Group L (comparative example 4)	0.05g/ml	3.55±0.54*	3.78±0.17*
				Group M (comparative example 5)	0.05g/ml	4.95±0.34	5.12±0.25

Compared to the virus control group,: p < 0.05, D-H group vs. A, #: p is less than 0.05.

The results show that cell clustering and rounding can be seen after 2 days of the virus control group, the fluctuation rate of the myocardial cells is reduced, 5 days of the cells die, timosaponin AIII, timosaponin BII and timosaponin N are used for processing alone and timosaponin composition of I-M groups is used for processing, the results of timosaponin BII and timosaponin N and the results of the virus control group are not obviously different, the cells die after 5 days, but timosaponin AIII has a certain protection effect, while the timosaponin composition prepared in examples 1-5 has the same protection effect as alpha-interferon, and the myocardial cells fluctuate obviously after 5 days, and the effect is obviously different compared with the effect of using timosaponin AIII alone; when cells are infected with CVB 32D, the interferon alpha group can reduce the virus titer to 0, the A group, the K group and the L group can reduce the virus titer but have not very obvious effect, the D-H group can reduce the virus titer obviously, and at 5D, only the A group, the D-H group and the L group can reduce the virus titer but have more obvious effect on the D-H group.

Thirdly, research on therapeutic effect of timosaponin on myocarditis of Balb/C mice infected with CVB3

(I) pretreatment

Virus titer was determined by plaque formation assay and Hela cells (8X 10) ⁵ One/well) were inoculated in a six-well plate, the cell fusion rate reached 90% after 24h incubation at 37 ℃, washed 2 times with PBS, infected with each group of CVB3 virus diluted in a gradient in volume of 200ul, infected at 37 ℃ for 1h, washed with PBS to remove the virus supernatant, added with 2ml of agar-MEM medium (2 × MEM: 1.5% agar =1: 1). After 72h, fixation was performed for 30min with Carlo's fixative, followed by 1% crystal violet staining, virus titer was calculated using the number of plaques, and then the virus titer was diluted 10-fold to 8 titers, each 10 ⁰ 、10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 Diluting the virus with DMEM, inoculating 0.3ml of CVB3 virus to the abdominal cavity of a mouse, and calculating LD of the virus by applying statistical results of a Reed-Muench method ₅₀ Is 2.7X 10 ⁹ PFU. And performing acute toxicity experiments on the medicaments in the groups A, E, F and L to determine the safe administration dose.

(II) test method

150 pure male Balb/c mice with 4 weeks age are adopted and randomly divided into 15 groups of 10 mice each, a blank control group, a virus control group and a positive control group are arranged in the test, 3 dosage groups of drug height, middle and low of A group, E group, F group and L group are respectively arranged in the test, the virus control group is that each mouse is injected with 0.3ml of medicine with the titer of 9 multiplied by 10 in the abdominal cavity ⁹ PFU/ml CVB3 virus solution without drug administration, and after 0.5 hour, gavage 0.3ml physiological saline daily, blank control group is normal control mice without virus inoculation, gavage 0.3ml physiological saline daily, and positive control group is intraperitoneal injection alpha interferon 10 daily after intraperitoneal injection of equal amount of virus solution for 0.5 hour ⁴ IU/g body weight, once/d, 7d continuously, the same amount of virus liquid as the virus control group is injected into the abdominal cavity of the other groups, and the tested drugs are perfused into the stomach, wherein the concentration of the perfusate in the groups A, E, F and L (prepared by water) is 2.4mg/ml in the low dose group, 4.8mg/ml in the medium dose group, 9.6mg/ml in the high dose group, and the test period is 2 weeks. Counting the death conditions of each group of mice in the experimental period at 7d and 14d respectively, taking out the hearts of the rats, washing the hearts with PBS, dividing the hearts into two parts along the long axis of the left ventricle, fixing one half with 10% neutral formaldehyde, performing HE staining, observing the pathological change conditions of myocardial tissues, integrating the pathological change conditions of the hearts, calculating the pathological change free value to be 0, and calculating the pathological change area<25 percent of 1 minute, 25 to 50 percent of 2 minutes, 51 to 75 percent of 3 minutes,>4 points are calculated according to 75 percent; the other half of myocardium is used for detecting virus titer by repeatedly freezing and dissolving myocardium tissue for 3 times, and diluting with Hank's solution as serial 1 for 10 times ^-1 To 10 ^-8 Diluting, inoculating to micro cell culture plate of Hela cell monolayer, adsorbing at 37 deg.C for 1 hr, removing virus solution, washing with Hank's solution, culturing in 10% MEM culture medium containing calf serum for 5 days, and calculating myocardial virus titer TCID by Reed-Mueneh method ₅₀ . The results are shown in tables 4 and 5.

TABLE 4 pathological integral of myocardial tissue of mice in each test group

Test group	Integral of pathology (7 d)	Pathological integral (14 d)
			Blank control group	0	0
Virus control group	3.54±0.25	3.87±0.18
			Positive control group	0.95±0.14*	0.73±0.18*
Group A high dose	2.09±0.16*	1.88±0.14*
			Group A Medium dose	2.22±0.05*	2.07±0.21*
Group A Low dose	3.22±0.14	3.09±0.17
			Group E (example 2) high dose	1.02±0.18* ＃	0.89±0.15* ＃
Dose in group E (example 2)	1.22±0.01* ＃	1.10±0.13* ＃
			Group E (example 2) Low doses	1.18±0.05* ＃	1.04±0.12* ＃
Group F (example 3) high dose	1.43±0.16* ＃	1.22±0.08* ＃
			Dose in group F (example 3)	1.54±0.15* ＃	1.34±0.15* ＃
Group F (example 3) Low dose	1.64±0.11* ＃	1.47±0.17* ＃
			High dose in group L (comparative example 4)	2.82±0.11	3.24±0.12
Dosage in group L (comparative example 4)	3.02±0.19	3.48±0.20
			Low dose for group L (comparative example 4)	3.27±0.18	3.35±0.13

Compared to the virus control group,: p < 0.05, and E and F groups compared to the same dose in group A, #: p is less than 0.05.

TABLE 5 Virus Titre (TCID) of myocardial tissue in mice of each test group ₅₀ ) Results

Test group	7d	14d
			Blank control group	0	0
Virus control group	2.57±0.18	3.48±0.02
			Positive control group	1.02±0.51*	0.98±0.43*
Group A high dose	2.04±0.09	2.16±0.05*
			Dose in group A	2.18±0.04	2.26±0.18*
Group A Low dose	2.22±0.15	2.33±0.16
			Group E (example 2) high dose	1.03±0.14* ＃	1.14±0.07* ＃
Dose in group E (example 2)	1.28±0.07* ＃	1.38±0.09* ＃
			Group E (example 2) Low doses	1.32±0.18* ＃	1.42±0.21* ＃
Group F (example 3) high dose	1.19±0.15* ＃	1.25±0.33* ＃
			Dose in group F (example 3)	1.28±0.07* ＃	1.34±0.07* ＃
Group F (example 3) Low dose	1.33±0.12* ＃	1.40±0.05* ＃
			High dose in group L (comparative example 4)	2.02±0.16	2.26±0.19*
Dosage in group L (comparative example 4)	2.58±0.18	2.88±0.24
			Low dose for group L (comparative example 4)	2.86±0.19	3.14±0.13

Compared to the virus control group,: p < 0.05, and E and F groups compared to the same dose in group A, #: p is less than 0.05.

The results show that the timosaponin composition of the high, medium and low dose groups of the group E and the group F can obviously reduce the virus Titer (TCID) ₅₀ ) The group A and the group L adopt independent timosaponin AIII and saponin compositions with other proportions to have weaker protective effect on rat myocardial cells, while the group E and the group F adopt the composition of timosaponin AIII, timosaponin BII and timosaponin N which are matched in a certain proportion to obviously improve the protective capability of the saponin composition on the myocardial cells under three dosages, namely high, medium and low dosages, which shows that the combined use of the three components has unexpected synergistic effect and plays an unexpected role in viral myocardial damage mediated by CVB 3.

Example 6

Capsule preparation

260g of timosaponin BII powder, 290g of timosaponin AIII powder, 150g of timosaponin N powder, 180g of filler starch and 70g of microcrystalline cellulose are uniformly mixed to obtain mixed powder, 50g of hydroxypropyl methylcellulose is prepared into 5% aqueous solution, the aqueous solution is added into the mixed powder, the mixed powder is stirred and granulated, the obtained mixture is dried and then filled into a capsule shell by 0.20 g/granule to obtain a timosaponin capsule, and the content of timosaponin AIII, timosaponin BII and timosaponin N is detected to be 28.2%, 24.3% and 13.2%.

Research on clinical treatment effect of timosaponin composition

96 patients with viral myocarditis are selected to be diagnosed in a certain hospital, and are randomly divided into timosaponin groups and control groups of 48 cases respectively, wherein the timosaponin groups comprise 25 men and 23 women, the age is 32-48 years old, the course of disease is 12d-10 months, and the control groups comprise 20 men and 28 women, the age is 30-50 years old, and the course of disease is 9d-12 months. The general data of two groups of patients are compared, and the difference is not statistically significant and has comparability.

The cases are brought into the standard composite viral myocarditis diagnosis standard, allergic or specific physique patients are excluded, patients who are not resistant to the study medicine are combined with serious cardiovascular diseases, liver diseases and other primary diseases, and the like.

The control group adopts conventional treatment including symptomatic treatment and antiviral treatment, and the timosaponin group is added with the timosaponin composition capsule prepared in example 6 on the basis of conventional treatment, 3 times a day, 2 capsules each time, and two groups of patients are treated for 1 month.

The treatment effect judgment standard is as follows: no clinical symptoms, and no abnormality in examination such as electrocardiogram and the like is significant; the clinical symptoms are obviously improved, and the examination such as electrocardiogram and the like is basically recovered to be effective; none of the above indexes is reached or deteriorated to be ineffective, and the effective rate = (effective + effective)/nx100%. The results are shown in Table 6.

TABLE 6 comparison of clinical efficacy of two groups of patients with viral myocarditis

Group of	n (example)	Obvious effect (example)	Effective (example)	Invalid (example)	Effective rate (%)
						Control group	48	15	22	11	77.08
Timosaponin group	48	24	19	5	89.58

Claims (5)

1. The application of the timosaponin composition in preparing the medicament for treating the viral myocarditis is characterized in that the effective components of the medicinal composition are timosaponin AIII, timosaponin BII and timosaponin N, wherein the weight percentage of the timosaponin AIII is 22-34%, the weight percentage of the timosaponin BII is 18-30%, and the weight percentage of the timosaponin N is 10-17%.

2. The use of claim 1, wherein the timosaponin AIII is present in an amount of 26 to 30 wt%, the timosaponin BII is present in an amount of 22 to 26 wt%, and the timosaponin N is present in an amount of 12 to 15 wt%.

3. The use according to claim 1, wherein the timosaponin AIII is 28 wt%, the timosaponin BII is 24 wt%, and the timosaponin N is 13 wt%.

4. The use according to claim 1, wherein the timosaponin composition is prepared by the following steps:

(1) extracting rhizoma anemarrhenae total saponins: taking a rhizoma anemarrhenae medicinal material, carrying out reflux extraction for 2 times, the first time for 2 hours and the second time for 1.5 hours by using 70% ethanol in an amount which is 6-10 times that of the rhizoma anemarrhenae medicinal material, combining ethanol extract, and concentrating under reduced pressure to obtain concentrated solution with the relative density of 1.25-1.35;

(2) extracting timosaponin BII: taking half volume of the concentrated solution obtained in the step (1), packing HP20 adsorption resin into a column, filtering the concentrated solution, then loading the sample, washing with water to remove impurities, then eluting with 50% ethanol of 3BV, collecting the eluent to recover ethanol, then loading the obtained liquid with an SP825 macroporous resin column, eluting with 50% ethanol of 4BV, collecting the eluent to recover ethanol, concentrating to obtain a concentrated solution with the relative density of 1.25-1.35, and spray drying to obtain timosaponin BII powder;

(3) preparing timosaponin AIII: taking 60-65 wt% of timosaponin BII powder obtained in the step (2), adding anhydrous acetic acid-acetic acid buffer solution with pH of 4.8, uniformly stirring, heating to 53 ℃, adding beta-glucosidase, reacting for 2h, extracting a product for 3 times by using ethyl acetate, combining ethyl acetate extract liquor, and recovering a solvent to obtain timosaponin AIII powder;

(4) extracting timosaponin N: taking the rest concentrated solution obtained in the step (1), packing HPD100 macroporous resin into a column, filtering the concentrated solution, sampling the filtrate, eluting with 6BV of water to remove impurities, eluting with 6BV of 40% ethanol to remove impurities, eluting with 6BV of 60% ethanol to remove lower column, recovering ethanol from the eluent, dissolving the ethanol in water, introducing into an ODS column, eluting with 40% acetone, collecting the eluent, recovering acetone, concentrating and drying to obtain timosaponin N powder;

(5) preparation of the saponin composition: mixing the timosaponin BII powder obtained in the step (2), the timosaponin AIII powder obtained in the step (3), the timosaponin N powder obtained in the step (4) and a filler according to a ratio of 1.70-1.75: 1.90-1.95: 1:2 to obtain the timosaponin composition.

5. The use according to any one of claims 1 to 4, wherein the medicament for the treatment of viral myocarditis is in the form of a solid preparation.