CN113398186A

CN113398186A - Viola yedoensis makino extract and preparation method and application thereof

Info

Publication number: CN113398186A
Application number: CN202110957291.3A
Authority: CN
Inventors: 陈文静; 程艳; 董小康; 魏然; 宋慧慧; 王怡斐; 李汶
Original assignee: Affiliated Hospital of Shandong University of Traditional Chinese Medicine
Current assignee: Affiliated Hospital of Shandong University of Traditional Chinese Medicine
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-09-17
Anticipated expiration: 2041-08-20
Also published as: CN113398186B

Abstract

The invention provides a Chinese violet extract and a preparation method and application thereof, belonging to the technical field of biological medicines. The invention uses traditional water decoction to process the Chinese violet medicinal material, combines an enzymolysis method to dissociate aglycone in an extracting solution, and then uses ethanol to enrich polar and weak polar compounds in the extracting solution, thereby laying a good material foundation for preparation. Pharmacological tests show that the viola yedoensis makino extract obtained by the method can improve the cardiac function and myocardial tissue inflammation state of mice with viral myocarditis, and the extract has a good myocardial protection effect on the viral myocarditis, so the viola yedoensis makino extract has a good value in practical application.

Description

Viola yedoensis makino extract and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a Chinese violet extract and a preparation method and application thereof.

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.

The herba Violae is Violae herba Violae of ViolaceaeViolaye doensis Makino's dried whole herb has wide distribution in China, and the medicinal material resource is abundant. Viola Yedoensis Makino is bitter and pungent in flavor and cold in nature, and has the effects of clearing heat and removing toxicity, cooling blood and relieving swelling, and is also indicated for surgical diseases in compendium in essential ingredient, but ancient people always have the effects of clearing heat and removing dampness by treating jaundice and pharyngitis. The theory that the big prescription can not be abandoned easily is that the medicine is used for treating sore pyogenic infections, carbuncle, cellulitis, back, erysipelas, snake venom bite and the like. Modern pharmacological research shows that the Chinese violet has the functions of antibiosis,Anti-inflammatory, immunoregulatory, antioxidant, anti-tumor, etc., and also broad-spectrum antiviral (trexue, etc., research progress of chemical components and antiviral action of herba Violae, food and medicine, 3 rd stage 2020).

Herba Violae contains flavonoids, coumarins, steroids, cyclic peptide compounds, amides, organic acids, terpenes, lignans, etc.; recent studies have shown that coumarins are the main antibacterial component; in addition, researches on other components also show that different components in the Chinese violet have different pharmacological effects.

One of the main purposes of the modernization of traditional Chinese medicine is to convert the traditional Chinese medicine 'coarse, large and black' into delicate, efficient and portable dosage form by combining the modern technology. Therefore, extracting effective components or parts and removing impurities to the maximum extent are one of the subjects of the universal research of modernization of traditional Chinese medicine. In recent years, the refining research of the Chinese violet medicinal material is frequently seen, but the ethanol reflux is taken as the first step of extraction due to the limitation of the physicochemical properties of chemical components in the Chinese violet, and the purpose of the ethanol reflux is to extract and obtain flavone and coumarin components in the medicinal material. For example, chinese patents CN105770207A, CN10341749A, CN102526357A, etc. and the scientific literature available at present adopt the above methods mostly because the chemical components are easily dissolved in ethanol.

The viola philippica is a whole herb medicinal material, leaves of the viola philippica account for 80-95% of the whole herb, the chlorophyll content in leaves is huge, most of chlorophyll can be separated out into a solvent after ethanol backflow, and how to remove the chlorophyll needs to be considered in subsequent concentration and filtration steps. Meanwhile, after ethanol extraction, there is basically a step of recovering ethanol for concentration, in which a part of water-insoluble substances is precipitated, but the extraction rate is impaired. Therefore, the technology involved in the above patent requires a great cost if it is converted to a large production, and the extraction rate is not necessarily much higher.

In the traditional theory, most of the medicinal materials are decocted, ground or juiced, and the like, so that the medicinal materials can also exert better medicinal effects. According to records in Qianjin prescriptions for emergency, herba Violae is used alone for treating furuncle and pyogenic infections, the method is to take herba Violae after pounding into juice, and most of the other prescriptions are decocted with water.

As a whole herb medicine, the dosage of the philippine violet herb in the prescription is often larger, such as honeysuckle detoxification decoction, Wushen decoction and the like. Therefore, a method for treating the Chinese violet medicinal material is still needed at present, which can not only accord with the traditional Chinese medicine theory, but also better realize the refining purpose.

Most of the flavone and steroid compounds in plants exist in the form of glycosides, so that the use of enzymes to dissociate aglycones in the extraction process and then the extraction with polar solvents is proposed, so that effective components can be extracted more fully, but the use of enzymes as assistance in the extraction process has more rigorous requirements on extraction conditions (including temperature, pH value, time and the like), and how the influence of the conditions on the final extract is not evaluated by people.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a Chinese violet extract and a preparation method and application thereof. The invention uses traditional water decoction to process the Chinese violet medicinal material, combines an enzymolysis method to dissociate aglycone in an extracting solution, and then uses ethanol to enrich polar and weak polar compounds in the extracting solution. In addition, animal experiments show that the Chinese violet extract prepared by the method has the effect of improving the heart function of mice with viral myocarditis, so that the Chinese violet extract has good practical application value.

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

in one aspect of the invention, the preparation method of the Chinese violet extract is provided, and the preparation method comprises the steps of decocting, carrying out enzymolysis and extracting the Chinese violet;

wherein in the enzymolysis step, the enzyme is any one or more of cellulase, neutral protease, papain and beta-glucosidase.

In a second aspect of the present invention, the extract of Chinese violet prepared by the above preparation method is provided. Experiments prove that the Chinese violet extract has the function of improving the heart function of mice with viral myocarditis.

Therefore, the third aspect of the invention provides the application of the Chinese violet extract in preparing medicines for treating inflammation-related diseases.

Wherein the inflammation-related disease comprises sepsis, pneumonia, hepatitis, rheumatoid arthritis, systemic lupus erythematosus, asthma, viral myocarditis, acute respiratory distress syndrome, pancreatitis, sepsis, ulcerative colitis, bronchitis, and infectious endocarditis, preferably viral myocarditis.

In a fourth aspect of the invention, a pharmaceutical composition is provided, and the active ingredients of the pharmaceutical composition comprise the Chinese violet herb extract.

Further, the pharmaceutical composition also comprises at least one pharmaceutical active ingredient or pharmaceutical carrier.

In a fifth aspect of the present invention, there is provided a method of treating an inflammation-related disorder, the method comprising: administering to the subject an effective dose of the above extract of Viola yedoensis Makino or the above pharmaceutical composition.

The inflammation-related diseases include sepsis, pneumonia, hepatitis, rheumatoid arthritis, systemic lupus erythematosus, asthma, viral myocarditis, acute respiratory distress syndrome, pancreatitis, sepsis, ulcerative colitis, bronchitis, and infectious endocarditis, preferably viral myocarditis.

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

the preparation method of the viola yedoensis makino extract fully considers the characteristics of traditional water decoction medicinal material treatment, simultaneously combines modern theory, utilizes enzyme to free aglycone, further refines the extract, and lays a foundation for better exerting the pharmacological action of the next preparation. The Chinese violet extract obtained by the preparation method can regulate the serum inflammatory factor level of mice with viral myocarditis, reduce the expression of myocardial tissue IGF-1, play a role in myocardial protection, and can be used as a candidate drug for antiviral myocarditis to prepare clinically acceptable drug formulations, so the Chinese violet extract 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 a microscopic observation result of myocardial pathological tissue of a mouse model group in Experimental example 1 of the present invention.

FIG. 2 is a microscopic observation result of the myocardial pathological tissue of the control mice in Experimental example 1 of the present invention.

FIG. 3 is a microscopic observation result of the myocardial pathological tissue of the mice in the treatment group in Experimental example 1 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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 invention 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 in accordance with 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.

In a typical embodiment of the invention, the preparation method of the Chinese violet extract is provided, and the preparation method comprises the steps of decocting, carrying out enzymolysis and extracting the Chinese violet;

The specific method of the decoction step comprises the following steps: soaking herba violae in water, decocting for 2-3 times, mixing decoctions, and filtering to obtain a decoction;

the amount of water added in each decoction is controlled to be 1g of herba violae medicinal material per 4-6 g of water.

The enzymolysis step specifically comprises: concentrating the decoction under reduced pressure, adding enzyme, and performing enzymolysis to obtain enzymolysis solution;

the final density of the decoction after decompression concentration is controlled to be 0.60-1.10 (80 ℃).

The amount of the added enzyme is controlled to be 1g of enzyme, the amount of the added enzyme is 80-160 g, such as 80g, 100g, 120g, 140g and 160g, the enzymolysis treatment temperature is 40-65 ℃, such as 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and 65 ℃, the enzymolysis treatment time is controlled to be 0.5-2.5 h, such as 0.5h, 1h, 1.5h, 2h and 2.5h, the enzymolysis pH is controlled to be acidic, further controlled to be 3.0-6.5, such as pH is 3.0, 4.0, 5.0, 6.0 and 6.5, and the enzyme is beta-glucosidase. By optimizing enzymolysis conditions, the enzymolysis is more sufficient, and the active ingredients (such as aglycone and the like) of the Chinese violet medicament can be fully released.

In the extraction step, an organic solvent is selected as an extractant, ethanol is further preferably selected, and after the ethanol is added, the concentration of the ethanol in the solution is controlled to be 40-80%, such as 40%, 50%, 55%, 60%, 70%, 80%, and preferably 55%, so as to obtain an ethanol extract. The polar and weak polar compounds in the ethanol are effectively enriched by using the ethanol.

In another embodiment of the present invention, the preparation method further includes a step of concentrating and drying the prepared ethanol extract, and specifically includes: recovering ethanol from the ethanol extract to obtain extract, and drying the extract to obtain the Chinese violet extract.

In another embodiment of the present invention, the viola yedoensis makino extract prepared by the above preparation method is provided. Experiments prove that the Chinese violet extract has the function of improving the heart function of mice with viral myocarditis.

Therefore, in another embodiment of the present invention, the use of the above extract of Chinese violet herb for preparing a medicament for treating inflammation-related diseases is provided.

Wherein the inflammation-related disease comprises sepsis, pneumonia, hepatitis, rheumatoid arthritis, systemic lupus erythematosus, asthma, viral myocarditis, acute respiratory distress syndrome, pancreatitis, sepsis, ulcerative colitis, bronchitis, and infectious endocarditis, preferably viral myocarditis. Experiments prove that the Chinese violet extract prepared by the invention can improve the cardiac function and myocardial tissue inflammation state of mice with viral myocarditis, and the extract has a better myocardial protection effect on the viral myocarditis.

In another embodiment of the invention, the active ingredient of the pharmaceutical composition comprises the Chinese violet herb extract.

In yet another embodiment of the present invention, the pharmaceutical composition further comprises at least one pharmaceutically active ingredient or a pharmaceutical carrier. Wherein the pharmaceutically active ingredient may be a dandelion extract.

The pharmaceutical composition containing the Chinese violet extract can be administrated in a unit dosage form. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage forms can be true solutions, colloids, microparticles, emulsions, and suspensions. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of from 1% by weight to 98% by weight, typically about 80% by weight. For convenience, preservatives, buffers and the like may be dissolved directly in the carrier.

Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.

The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

In yet another embodiment of the present invention, there is provided a method of treating a disease associated with inflammation, the method comprising: administering to the subject an effective dose of the above extract of Viola yedoensis Makino or the above pharmaceutical composition.

The subject refers to an animal, preferably a mammal, most preferably a bovine, who has been the object of treatment, observation or experiment. By "therapeutically effective amount" is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medical response in a tissue system, animal, or other subject sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated. It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment may be determined by methods known in the art.

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. The reagents and materials involved in the preparation method in the embodiment are all medicinal reagents or analytically pure or chemically pure reagents, and the reagents and materials involved in the pharmacological test are all chemically pure or biochemical reagents, which can be obtained in the market. The experimental procedures involved in pharmacological testing are well known to those skilled in the art.

Example 1 determination of the content of Flavonoids and coumarins in Viola yedoensis Makino extract

The purpose of this example is to measure the content of flavonoids and coumarins in the extract of Viola yedoensis Makino by HPLC. The specific method comprises the following steps:

(1) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler for the chromatographic column; the sample amount is 10 mul, the column temperature is 30 ℃, and the detection wavelength is 290 nm; the mobile phase A is acetonitrile; the mobile phase B is 14mmol/L ammonium acetate aqueous solution; gradient elution (0-10 min, A10-25%, 10-20min, A25-60%).

(2) Control solution: accurately weighing aesculin, aesculetin, quercetin, luteolin and naringenin respectively, placing into a volumetric flask, adding methanol to obtain mixed solutions with concentrations of 100 μ g/ml, 50 μ g/ml and 25 μ g/ml, and making into mixed reference solution.

(3) Test solution: precisely weighing 5mg of herba Violae extract powder, placing in a volumetric flask, adding methanol, ultrasonically dissolving, diluting to scale, shaking, filtering, and collecting the filtrate as sample solution.

(4) Content determination: respectively and precisely sucking the reference solution and the test solution, injecting into a liquid chromatograph, and measuring to obtain the content of flavonoids and coumarins in the herba Violae extract, wherein the flavonoids comprise quercetin, luteolin and naringenin, and the coumarins comprise aesculin and aesculetin.

Example 2 preparation of viola yedoensis makino extract:

(1) decocting: soaking herba Violae in water for 30min, decocting for 3 times (5 times of the amount of the medicinal materials per time, 1 hr, and 0.5 hr), mixing decoctions, and filtering to obtain water decoction;

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 0.8 (80 ℃), and beta-glucosidase is added for enzymolysis, and the enzymolysis conditions are as follows: each 1g of enzyme corresponds to 140g of medicinal materials; the enzymolysis temperature is 50 ℃, the enzymolysis time is 1 hour, the enzymolysis pH value is 4.5, and the enzymolysis liquid is obtained by stirring in due time;

(3) extraction: adding ethanol into the enzymolysis solution to make the ethanol concentration of the solution reach 55%, stirring thoroughly, standing overnight, and filtering to obtain ethanol extract;

(4) concentrating and drying: recovering ethanol from the ethanol extract to obtain extract, and drying the extract to obtain the Chinese violet extract.

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 15.6% of aesculin, 18.5% of aesculetin, 7.6% of quercetin, 9.7% of luteolin and 4.2% of naringenin.

Example 3 preparation of viola yedoensis makino extract:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 0.6 (80 ℃), and cellulase is added for enzymolysis, and the enzymolysis conditions are as follows: 160g of medicinal materials are added for every 1g of enzyme; the enzymolysis temperature is 65 ℃, the enzymolysis time is 0.5 hour, the enzymolysis pH value is 4, and the enzymolysis liquid is obtained by stirring in due time during the enzymolysis process;

(3) extraction: adding ethanol into the enzymolysis solution to make the ethanol concentration of the solution reach 40%, stirring thoroughly, standing overnight, and filtering to obtain ethanol extract;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: aesculin 13.9%, aesculetin 16.7%, quercetin 3.3%, luteolin 5.1%, and naringenin 2.6%.

Example 4 preparation of viola yedoensis makino extract:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 1.1 (80 ℃), neutral protease is added for enzymolysis, and the enzymolysis conditions are as follows: each 1g of enzyme corresponds to 80g of medicinal materials; the enzymolysis temperature is 40 ℃, the enzymolysis time is 2.5 hours, the enzymolysis pH value is 6, and the enzymolysis liquid is obtained by stirring in due time during the enzymolysis process;

(3) extraction: adding ethanol into the enzymolysis solution to make the ethanol concentration of the solution reach 80%, stirring thoroughly, standing overnight, and filtering to obtain ethanol extract;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 14.3 percent of aesculin, 16.4 percent of aesculetin, 4.1 percent of quercetin, 5.9 percent of luteolin and 3.3 percent of naringenin.

Example 5 preparation of extract of Viola yedoensis Makino:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 0.7 (80 ℃), papain and beta-glucosidase (1: 2) are added for enzymolysis, and the enzymolysis conditions are as follows: every 1g of enzyme corresponds to 100g of medicinal materials; the enzymolysis temperature is 45 ℃, the enzymolysis time is 2.0 hours, the enzymolysis pH value is 5, and the enzymolysis liquid is obtained by stirring in due time during the enzymolysis process;

(3) extraction: adding ethanol into the enzymolysis solution to make the ethanol concentration of the solution reach 60%, stirring thoroughly, standing overnight, and filtering to obtain ethanol extract;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 14.6% of aesculin, 16.9% of aesculetin, 4.5% of quercetin, 5.3% of luteolin and 3.0% of naringenin.

Example 6 preparation of viola yedoensis makino extract:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 1.08 (80 ℃), neutral protease and beta-glucosidase (1: 3) are added for enzymolysis, and the enzymolysis conditions are as follows: each 1g of enzyme corresponds to 110g of medicinal materials; the enzymolysis temperature is 60 ℃, the enzymolysis time is 1.0 hour, the enzymolysis pH value is 6.5, and the enzymolysis solution is obtained by stirring in due time during the enzymolysis process;

(3) extraction: adding ethanol into the enzymolysis solution to make the ethanol concentration of the solution reach 65%, stirring thoroughly, standing overnight, and filtering to obtain ethanol extract;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 14.2 percent of aesculin, 17.5 percent of aesculetin, 5.6 percent of quercetin, 8.3 percent of luteolin and 3.1 percent of naringenin.

Example 7 preparation of viola yedoensis makino extract:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 1.02 (80 ℃), and beta-glucosidase is added for enzymolysis, and the enzymolysis conditions are as follows: each 1g of enzyme corresponds to 120g of medicinal materials; the enzymolysis temperature is 50 ℃, the enzymolysis time is 1 hour, the enzymolysis pH value is 4.0, and the enzymolysis solution is obtained by stirring in due time during the enzymolysis process;

(3) extraction: adding ethanol into the enzymolysis solution to make the ethanol concentration of the solution reach 50%, stirring thoroughly, standing overnight, and filtering to obtain ethanol extract;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 14.9% of aesculin, 17.3% of aesculetin, 6.8% of quercetin, 9.2% of luteolin and 3.9% of naringenin.

Example 8 preparation of viola yedoensis makino extract:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 0.94 (80 ℃), and beta-glucosidase, cellulase and neutral protease (4: 1: 1) are added for enzymolysis, and the enzymolysis conditions are as follows: 150g of medicinal materials are added per 1g of enzyme; the enzymolysis temperature is 55 ℃, the enzymolysis time is 1.5 hours, the enzymolysis pH value is 5.5, and the enzymolysis solution is obtained by stirring in due time during the enzymolysis process;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 15.1% of aesculin, 17.7% of aesculetin, 5.4% of quercetin, 7.9% of luteolin and 3.6% of naringenin.

Example 9 preparation of viola yedoensis makino extract:

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 0.90 (80 ℃), and beta-glucosidase, cellulose and papain (4: 1: 2) are added for enzymolysis, and the enzymolysis conditions are as follows: each 1g of enzyme corresponds to 110g of medicinal materials; the enzymolysis temperature is 50 ℃, the enzymolysis time is 1.0 hour, the enzymolysis pH value is 5.0, and the enzymolysis solution is obtained by stirring in due time during the enzymolysis process;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: 14.6 percent of aesculin, 17.3 percent of aesculetin, 4.7 percent of quercetin, 6.2 percent of luteolin and 2.8 percent of naringenin.

Comparative example 1 preparation of extract of viola yedoensis makino:

this comparative example refers to the technique disclosed in CN 102462701A.

(2) enzymolysis: the decoction is moderately decompressed and concentrated to the density of 1.04 (80 ℃), cellulase is added, and the enzymolysis conditions are as follows: the amount of the added enzyme is 0.8 percent of the weight of the water decoction concentrated solution; the enzymolysis temperature is 50 ℃, the enzymolysis time is 2 hours, the enzymolysis pH value is 4, and the enzymolysis liquid is obtained by stirring in due time during the enzymolysis process;

(5) Content determination: according to the method described in example 1, the contents of flavonoids and coumarins in the extract of Viola yedoensis Makino are determined as follows: aesculin 10.7%, aesculetin 12.5%, quercetin 1.8%, luteolin 2.6%, and naringenin 0.6%.

Experimental example 1

Experiment for influence of herba Violae extract on mice with viral myocarditis

The present example discusses the effects of Viola yedoensis Makino extract on viral myocarditis model mouse mortality, serum creatine kinase isoenzyme (CK-MB), Lactate Dehydrogenase (LDH), aspartate Aminotransferase (AST), inflammatory factors (IL-6, IL-10, TNF-alpha), and cardiomyopathy, to initially evaluate the effect of Viola yedoensis Makino extract on viral myocarditis. The method comprises the following specific steps:

(1) preparing a model: 60 BALC/c mice were selected and randomly divided into model group (20), treatment group (20), and control group (20) according to body weight.

Wherein, each abdominal cavity of the model group and the treatment group of mice is injected with 0.1ml of CNB3 virus solution containing Coxsackie virus B3 (CVB 3) 100TTCID50/0.1ml, and the injection is continuously performed for 3 times to establish a mouse viral myocarditis model.

The control group was injected with an equal amount of physiological saline at the same time.

(2) The preparation of the medicine comprises the following steps: a proper amount of the Chinese violet herb extract obtained in the best embodiment of the embodiment 2 is taken and added with distilled water to be fully stirred into suspension for use when in use.

(3) Administration: after the first injection of the virus solution, the intragastric administration is started for 2 h. Wherein the treatment group is administered with 6g/kg body weight of herba Violae extract; the model group and the control group were simultaneously administered an equal volume of distilled water for intragastric administration. The administration is 1 time daily, and 14 days are continuously administered.

(4) And (4) daily observation: general condition and mortality

(5) And (3) determining the heart function of the mouse: mice were anesthetized by intraperitoneal injection of chloral hydrate, depilated with a topical depilatory, and subjected to transthoracic echocardiography using a small animal ultrasound system. Analyzing the structure and function of the left ventricle by adopting the long axis and short axis sections beside the sternum, displaying the inner diameter (LVEDD) of the left ventricle at the end diastole, the thickness (LVPWs) of the posterior wall of the left ventricle and the thickness (LVAWs) of the anterior wall of the left ventricle under the M mode, and calculating the left ventricle Ejection Fraction (EF) and the left ventricle shortening rate (FS).

(6) Serum myocardial enzyme and inflammatory factor assay: after the last administration, fasting and water deprivation are carried out for 12h, weighing is carried out, the eye artery is taken blood, centrifugation is carried out for 6min at 4000r/min, supernatant fluid is taken, and the CK-MB, AST and LDH activities and the IL-6, IL-10 and TNF-alpha contents in the serum are measured by an ELISA method.

(7) Myocardial histopathological changes: the heart of a mouse is separated after blood is taken, the heart is fixed by 4% paraformaldehyde, dehydrated, embedded by paraffin, sliced conventionally, stained by HE, and the pathological change of the myocardium is observed under a microscope.

(8) As a result:

general status, death status of mice:

the control group mice had normal eating, activity and the like during the experiment, hair color and luster, sensitive response and normal weight, and the mice did not die during the experiment;

the mice in the model group have reduced food intake and activity, shrunken hair, lusterless hair, slow response to stimulation, emaciation and 8 deaths during the experiment (mortality rate is 40%);

the treated mice generally showed a significant improvement over the model group, with 2 deaths (mortality 10%) during the experiment.

The differences were statistically significant (P <0.05) when the mortality of 3 groups of mice was compared.

② the mouse heart function test result: see Table 1

Note: p <0.05, compared to control group; p <0.05, compared to model groups

As can be seen from the above table,

the EF and FS of the model group mice are obviously reduced compared with the control group (P is less than 0.05), and the heart function is effectively improved after the model group mice are treated by the viola yedoensis makino extract (P is less than 0.05).

Thirdly, the detection result of the myocardial enzyme level in the serum: see Table 2

Note: p <0.05, compared to control group; p <0.05, compared to model groups

As can be seen from the above table, the serum CK-MB, AST and LDH levels of the model group mice are obviously increased compared with the control group (P < 0.05); compared with the model group, the serum CK-MB, AST and LDH levels of the mice in the treatment group are obviously reduced, and the difference has statistical significance (P < 0.05).

Fourthly, the detection result of the mouse serum inflammatory factor: see Table 3

Note: p <0.05, compared to control group; p <0.05, compared to model groups

As can be seen from the above table, compared with the control group, the serum IL-6 and TNF-alpha levels of the model group mice are obviously increased, and the IL-10 content is obviously reduced (P < 0.05); compared with the model group, the serum IL-6 and TNF-alpha level of the mice in the treatment group is obviously reduced, the IL-10 content is obviously increased, and the difference has statistical significance (P < 0.05).

The histopathological changes of the myocardium are detailed in figures 1-3.

The analysis of the attached figure shows that the myocardial cells of the control group mice are arranged regularly, and the proliferation of interstitial fibers and inflammatory cells is not seen; the mouse model group has the advantages that the mouse model group has the symptoms of myocardial cell enlargement, myocardial fiber arrangement disorder and myocardial interstitial edema, and a large amount of inflammatory cell infiltration and multifocal necrosis are seen; the pathological lesion degree of the mice in the treatment group is improved compared with that of the mice in the model group.

It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A preparation method of a Chinese violet extract is characterized by comprising the steps of decocting, carrying out enzymolysis and extracting the Chinese violet;

wherein in the enzymolysis step, the used enzyme is beta-glucosidase;

the enzymolysis step specifically comprises: concentrating the decoction under reduced pressure, adding enzyme, and performing enzymolysis to obtain enzymolysis solution; the final density of the decoction after reduced pressure concentration is controlled to be 0.60-1.10, 80 ℃; the amount of the added enzyme is controlled to be 80-160 g corresponding to 1g of the enzyme to the Chinese violet medicinal material, the enzymolysis treatment time is controlled to be 0.5-2.5 h, and the enzymolysis pH is controlled to be 3.0-6.5;

in the extraction step, an extracting agent selects an organic solvent;

the organic solvent is ethanol, and after the ethanol is added, the concentration of the ethanol in the solution is controlled to be 40-80%.

2. The method of claim 1, wherein the step of decocting comprises: soaking herba violae in water, decocting for 2-3 times, mixing decoctions, and filtering to obtain a decoction;

3. The method according to claim 1, further comprising the step of concentrating and drying the prepared extract.

4. The extract of Viola yedoensis Makino prepared by the method of any one of claims 1-3.

5. The use of the extract of Viola yedoensis Makino as claimed in claim 4 for the preparation of a medicament for treating viral myocarditis.

6. A pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the extract of Viola yedoensis Makino as claimed in claim 4.

7. The pharmaceutical composition of claim 6, further comprising at least one pharmaceutically active ingredient or a pharmaceutical carrier.