CN110898123A

CN110898123A - Traditional Chinese medicine composition for inhibiting growth of pathogenic microorganisms of animal vaginitis and preparation method and application thereof

Info

Publication number: CN110898123A
Application number: CN201911325937.5A
Authority: CN
Inventors: 刘宏生; 刘琦; 赵健; 顾颖; 冯华炜
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-03-24

Abstract

The invention relates to a traditional Chinese medicine composition for inhibiting growth of pathogenic microorganisms of animal vaginitis and a preparation method and application thereof. The Chinese medicinal composition is prepared from fructus Schisandrae chinensis, Coptidis rhizoma, fructus Hippophae, mume fructus, and radix Sophorae Flavescentis. The formula of the invention can effectively inhibit the growth of main pathogenic microorganisms causing candida albicans vaginitis and bacterial vaginitis, and shows strong bacteriostasis and sterilization characteristics. The invention is characterized in that: is prepared from pure Chinese medicaments, has small toxic and side effects and can effectively inhibit the growth of pathogenic bacteria.

Description

Traditional Chinese medicine composition for inhibiting growth of pathogenic microorganisms of animal vaginitis and preparation method and application thereof

Technical Field

The invention belongs to the field of veterinary pharmacy, and particularly relates to a traditional Chinese medicine compound formula for inhibiting pathogenic microorganisms of female mink vaginitis.

Background

The vagina is located at the lowermost end of the genital tract and is the only passage for the connection of the internal genitalia to the outside. Female mink vaginitis mainly occurs in a breeding period, the sanitary condition of a farm is poor, and a large amount of excreta are accumulated in a breeding environment, so that bacteria and fungi are propagated in a large amount, and female minks are susceptible to vaginitis.

Female minks with vaginitis can have symptoms such as dysphoria, difficulty in hybridization, abortion and the like, and if the female minks are not cured for a long time, the female minks can have an influence on the reproductive function, the reproduction rate is reduced, the quality grade of fur is reduced, and the economic income of farmers is seriously influenced.

As for vaginitis, Western antibiotics are mainly used as medicines at present. For example, Liuwenya and the like adopt antibiotic medicines such as penicillin, erythromycin and the like to treat the vaginitis of the female minks, and the cure rate is 84.85 percent. However, there is a great deal of evidence that antibiotics and their related drug resistance genes are entering the natural ecological environment in which humans live with incredible transmission capabilities, becoming recognized environmental pollutants; the major part of these factors is the production or acquisition of resistance genes by microorganisms in animals due to the overuse of a large number of over-prescribed, non-regulated antibiotics in the livestock breeding industry, which are left in the antibiotic-residual environment for a long time. Further reducing the immunity of the animals, causing the pathogenic bacteria to have sudden variation and increasing the death rate. The problem of microbial resistance caused by the improper use of antibiotics has become a serious concern in the livestock breeding industry. Therefore, a pure traditional Chinese medicine compound formula which can effectively inhibit the growth of pathogenic microorganisms of vaginitis, has high bacteriostatic stability and small side effect is needed at present, and a new way is opened up for the treatment of vaginitis.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a Chinese herbal medicine formula for inhibiting the growth of pathogenic microorganisms of vaginitis of animals.

In order to realize the purpose of the invention, the invention adopts the technical scheme that: a traditional Chinese medicine composition for inhibiting growth of pathogenic microorganisms of animal vaginitis is prepared from schisandra chinensis, coptis chinensis, sea buckthorn, dark plum and radix sophorae flavescentis. The physical property of the traditional Chinese medicine composition is gel type.

Preferably, the traditional Chinese medicine composition comprises, by weight, 1-4 parts of schisandra chinensis, 1-2 parts of coptis chinensis, 1-4 parts of sea buckthorn, 1-2 parts of dark plum and 1-3 parts of sophora flavescens.

More preferably, the traditional Chinese medicine composition comprises, by weight, 4 parts of schisandra chinensis, 2 parts of coptis chinensis, 4 parts of sea buckthorn, 2 parts of dark plum and 3 parts of sophora flavescens.

A preparation method of a traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of animal vaginitis comprises the following steps:

1) weighing the schisandra chinensis, the coptis chinensis, the sea buckthorn, the dark plum and the radix sophorae flavescentis according to the weight ratio, and soaking the schisandra chinensis, the coptis chinensis, the sea buckthorn, the dark plum and the radix sophorae flavescentis in water for 2-3 hours according to the material ratio of 1: 15-17;

2) boiling with strong fire, then boiling with slow fire for 1-1.5 h, filtering with three layers of gauze, and taking supernatant; adding water into the filtrate according to the material ratio of 1: 13-15, boiling with slow fire for 1-1.5 h, filtering with three layers of gauze, and taking supernatant; merging the two supernatants and cooling for later use;

3) centrifuging the supernatant at 1000rmp for 20min for 2 times, concentrating in rotary evaporator, and sterilizing.

The application of the traditional Chinese medicine composition in preparing the medicine for inhibiting the growth of pathogenic microorganisms of the animal vaginitis.

Preferably, the vaginitis comprises candida albicans vaginitis and bacterial vaginitis.

More preferably, the pathogenic microorganism causing candida albicans vaginitis is candida albicans.

More preferably, the pathogenic microorganisms causing bacterial vaginitis include Staphylococcus aureus, Bacillus pyocyaneus and Escherichia coli.

Preferably, the animal is a female mink.

The invention has the beneficial effects that:

1. the traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of the animal vaginitis can effectively inhibit pathogenic bacteria causing the vaginitis.

2. The traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of the animal vaginitis can effectively inhibit the growth of candida albicans, staphylococcus aureus, bacillus pyocyaneus, escherichia coli and the like.

3. According to the traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of the animal vaginitis, bacteriostasis zone experiments are carried out on strains such as candida albicans, staphylococcus aureus, bacillus pyocyaneus, escherichia coli and the like, and the result shows that the Chinese herbal medicine compound formula has a strong bacteriostasis effect on the strains. The sizes of the bacteriostatic rings of the strains are respectively as follows: staphylococcus aureus 20.25 + -2.06 mm, Escherichia coli 18.25 + -0.96 mm, Pseudomonas aeruginosa 19.50 + -2.08 mm, and Candida albicans 15.00 + -1.41 mm.

4. The traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of the animal vaginitis, which is researched by the invention, is used for carrying out a plurality of times of vagina mucosa stimulation experiments by taking rabbits as test objects, and the result shows that the traditional Chinese medicine composition has no stimulation reaction on vagina mucosa; a skin allergy experiment is carried out by taking a guinea pig as a test object, and the result shows that the sensitization rate of the traditional Chinese medicine composition to the skin is 20 percent, and the sensitization intensity is only causing mild allergy.

5. The traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of the animal vaginitis, which is researched by the invention, can effectively inhibit the growth of pathogenic bacteria causing the vaginitis, relieve the symptoms of the vaginitis, relieve the pain caused by the diseases and reduce the side effects caused by antibiotics.

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the invention in any way, and any modifications or changes that can be easily made by a person skilled in the art to the present invention will fall within the scope of the claims of the present invention without departing from the technical solution of the present invention.

Examples

Formula (I)

A traditional Chinese medicine composition for inhibiting growth of pathogenic microorganisms of animal vaginitis comprises, by weight, 4 parts of Chinese magnoliavine fruit, 2 parts of coptis chinensis, 4 parts of sea buckthorn, 2 parts of dark plum fruit and 3 parts of radix sophorae flavescentis.

(II) preparation method

1. Weighing the schisandra chinensis, the coptis chinensis, the sea buckthorn, the dark plum and the radix sophorae flavescentis according to a formula, and soaking the schisandra chinensis, the coptis chinensis, the sea buckthorn, the dark plum and the radix sophorae flavescentis in water for 2-3 hours according to a material ratio of 1: 15-17;

2. boiling with strong fire, then boiling with slow fire for 1-1.5 h, filtering with three layers of gauze, and taking supernatant; adding water into the filtrate according to the material ratio of 1: 13-15, boiling with slow fire for 1-1.5 h, filtering with three layers of gauze, and taking supernatant; merging the two supernatants and cooling for later use;

3. centrifuging the obtained supernatant at 1000rmp for 20min for 2 times, concentrating to 5g/ml in a rotary evaporator to obtain concentrated preparation, and sterilizing with Co 60-gamma ray for use. The physical property of the prepared traditional Chinese medicine composition is gel type.

(III) bacteriostatic test

1. Culture of test strains

Taking the slant fresh culture (18-24 h) of the nutrient agar culture medium (bacteria)/Sabouraud's agar culture medium (fungi) of the 3 rd-14 th generation of the strain, adding 3.0-5.0 ml of diluent into a slant test tube by using a liquid transfer gun, repeatedly blowing and sucking, and washing off the lawn. Subsequently, the wash solution was transferred to another sterile test tube using a pipette and mixed for 20 seconds with shaking using an electric mixer to prepare a bacterial suspension. The bacterial suspension is diluted to 5 x 10 by diluent after the concentration is measured by turbidimetry⁵cfu/ml。

Preparing a diluent: tryptone 1.0g, sodium chloride 8.5g, adding distilled water to 1000ml, and autoclaving at 121 ℃ for later use.

2. Zone of inhibition experiment

Preparing the bacteriostatic tablets: making the filter paper into a round piece with the diameter of 5mm, sterilizing under high pressure, and drying for later use. Dripping 20 μ l of the Chinese medicinal concentrated preparation prepared in step two into each tablet, placing the filter paper sheet in a clean sterile plate, opening the cover, and oven drying at 37 deg.C.

Preparation of negative control sample: taking a sterile dry filter paper sheet, dripping 20 mu l of sterile distilled water into each sheet, and drying for later use.

Inoculation of test bacteria: dipping with sterile cotton swab to 5 × 10 concentration⁵cfu/ml test bacterial suspension is evenly smeared on the surface of a nutrient agar culture medium or a saxashi agar culture medium plate for 3 times. The plate was covered and dried at room temperature for 5 min.

Sticking bacteriostatic agent sample pieces: each test is stuck with 1 bacteria staining plate, and each plate is stuck with 3 bacteriostatic tablets containing the traditional Chinese medicine composition, 1 negative control sample tablet and 1 positive control sample tablet. And (5) placing the mixture in a 37 ℃ incubator, and culturing for 16-18 h to observe results. The diameter of the antibacterial ring (including the patch) was measured with a vernier caliper and recorded. The experiment was repeated 3 times.

Judging the bacteriostatic action:

if the diameter of the bacteriostatic ring is larger than 7mm, the bacteriostatic ring is judged to have bacteriostatic action.

If the diameter of the antibacterial ring is less than or equal to 7mm, the antibacterial ring is judged to have no antibacterial effect.

The results of the pathogen inhibition experiments are shown in table 1.

TABLE 1 experiment of zone of inhibition of Chinese medicinal composition

As can be seen from the table 1, the traditional Chinese medicine composition disclosed by the invention is prepared from 4 parts of schisandra chinensis, 2 parts of coptis chinensis, 4 parts of sea buckthorn, 2 parts of dark plum and 3 parts of sophora flavescens, is finally concentrated to 5g/ml, and has a strong bacteriostatic effect on pathogenic bacteria of vaginitis.

(IV) multiple vagina mucosa irritation experiments with the Chinese medicinal composition

The purpose is as follows: and (3) detecting the stimulation effect and the strength of the traditional Chinese medicine composition on the vaginal mucosa of the experimental animal.

Experimental animals: 6 healthy and early-adult female white rabbits with the weight of 2.0 kg-2.5 kg are selected. The vaginal orifice of the animal was checked for the absence of secretions, congestion, edema and other lesions prior to the experiment.

Grouping tests: two groups, i.e. the infected group and the control group, were divided into 3 individuals each.

The operation procedure is as follows:

1. a Chinese medicinal preparation prepared from the Chinese medicinal composition is used as a test solution. The control group was saline.

2. A disposable blunt tube about 8cm in length was connected to a 2ml disposable syringe.

3. The animal is fixed on its back to expose perineum and vaginal opening. The catheter is inserted into the vagina, 2ml of test solution is injected, and the catheter is drawn out to finish the contamination. Control animals were treated with saline as well. The infection is repeated every 24h for 5 days continuously.

4. And 24h after the last infection, taking the vaginal tissue, and observing whether congestion, edema and other characteristics exist. The tissues were then fixed with 4% paraformaldehyde and the vaginal tissue was sectioned at both ends and 3 central sites, and HE stained and histopathological examination was performed.

5. The vaginal mucosal irritation response scores were made according to table 2, and the average score of the vaginal mucosal irritation response of the test group was determined by adding the stimulation response scores of 3 sites of 3 animals in the test group and dividing by the total number of observations (number of animals x 3). Control group scores were as above.

6. Vaginal mucosal irritation intensity grading was performed according to table 3.

TABLE 2 vaginal mucosal irritation response Scoring criteria

Note: stimulation response integral ═ A + B + C + D

TABLE 3 vaginal mucosal irritation Strength grading

The experimental results are shown in table 4 below.

TABLE 4 multiple vaginal mucosal irritation scores for rabbits

Note: a: test group (sample prepared from the traditional Chinese medicine composition) B: control group (physiological saline)

As can be seen from Table 4, the Chinese medicinal composition of the invention does not produce irritation reaction on the tested animals in multiple times of mucosa irritation experiments, so that the Chinese medicinal herb compound formula is judged to have no irritation on the vaginal mucosa.

(V) carrying out skin allergy experiment on the traditional Chinese medicine composition:

the purpose is as follows: detecting the possibility and intensity of skin allergy of experimental animal after repeated contact of sample prepared from Chinese medicinal composition.

Experimental animals: 15 healthy white guinea pigs with intact skin are selected, and the weight of the guinea pigs is 200 g-300 g.

Grouping tests: guinea pigs were randomly divided into test, negative control and positive groups of 5 animals each.

Operating procedure

1. To the test group of guinea pigs, the sample prepared with the Chinese medicinal composition was given induction and stimulation treatments. The positive control group was given 2, 4-dinitrochlorobenzene induction and challenge treatment. The negative control group was given only the test substance challenge treatment.

2. Depilating the left side of the back of guinea pig within 3cm × 3cm 24h before test, applying 0.5g sample prepared by Chinese herbal medicine compound formula on gauze covering skin for 6 h. The same procedure is repeated for 7d and 14 d.

3. At 14d after the last induction, 0.5g of the prepared sample (one) was spread on gauze covering the right depilated skin and after 6h the sample was washed off. The skin was observed after 24h and 48h, scored according to table 5, and the sensitization intensity was evaluated according to table 6.

TABLE 5 Scoring criteria for skin reactions

TABLE 6 sensitization Strength grading Standard

The experimental results are shown in tables 7 and 8:

TABLE 7 skin allergy test results of guinea pigs

Note: the inducer and the excitant of the negative group and the test group are prepared sample stock solution I, and the inducer and the excitant of the positive group are dinitrochlorobenzene. Two observations were made at 24h and 48h post-infection.

TABLE 8 sensitization intensity in skin allergy experiments

As can be seen from tables 7 and 8, the traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of the animal vaginitis has the skin sensitization rate of 20 percent and the sensitization intensity of causing only mild allergy.

Claims

1. A traditional Chinese medicine composition for inhibiting growth of pathogenic microorganisms of animal vaginitis is characterized by being prepared from schisandra chinensis, coptis chinensis, sea buckthorn, dark plum and radix sophorae flavescentis.

2. The traditional Chinese medicine composition according to claim 1, wherein the composition comprises, by weight, 1-4 parts of schisandra chinensis, 1-2 parts of coptis chinensis, 1-4 parts of sea buckthorn, 1-2 parts of dark plum and 1-3 parts of sophora flavescens.

3. The traditional Chinese medicine composition according to claim 2, wherein the composition comprises, by weight, 4 parts of schisandra chinensis, 2 parts of coptis chinensis, 4 parts of sea buckthorn, 2 parts of dark plum and 3 parts of sophora flavescens.

4. A preparation method of a traditional Chinese medicine composition for inhibiting the growth of pathogenic microorganisms of animal vaginitis is characterized by comprising the following steps:

1) weighing the schisandra chinensis, the coptis chinensis, the sea buckthorn, the dark plum and the radix sophorae flavescentis according to the weight proportion of the materials in the claim 2 or 3, and soaking the materials in water for 2-3 hours according to the material ratio of 1: 15-17;

5. Use of the composition of claim 1, 2 or 3 in the preparation of a medicament for inhibiting the growth of pathogenic microorganisms in vaginitis in an animal.

6. Use according to claim 5, wherein the vaginitis comprises Candida albicans vaginitis and bacterial vaginitis.

7. Use according to claim 6, characterized in that the pathogenic microorganisms causing Candida albicans vaginitis are Candida albicans.

8. Use according to claim 6, wherein the pathogenic microorganisms causing bacterial vaginosis comprise Staphylococcus aureus, Bacillus pyocyaneus and Escherichia coli.

9. Use according to claim 5, wherein the animal is a female mink.