Composition for preventing and treating mastitis, preparation method and application thereof

Abstract

The invention discloses a composition for preventing and treating mastitis and its preparation method and application, including the following components in parts by mass: 10-12 parts of aloe gel, 3-5 parts of berberine tannate and 2-4 parts of gentiana macrophylla extract. The aloe gel and the gentiana macrophylla extract in the present composition are both plant extract ingredients, and both have anti-inflammatory and bactericidal effects. Berberine tannate is a kind of alkaloid, has significant antibacterial effect, and the three are compounded, and the use of antibiotics can be effectively reduced, thereby reducing the residual of antibiotics in animal products, and increasing the safety of product; in addition, aloe gel, gentiana macrophylla extract and berberine tannate have synergistic synergistic effect, and the three are compounded, and mastitis can be effectively treated.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal mixtures, and in particular to a composition for preventing and treating mastitis, a preparation method thereof and an application thereof. Background Art

[0002] Staphylococcus aureus is a common environmental bacterium, often found on the skin, anterior nares, throat, and perineum of humans and animals. It generally does not cause disease, but can cause infectious diseases only when the host's immune system is compromised. Staphylococcus aureus is a typical Gram-positive bacterium. Once infected, it can cause a variety of diseases, such as endocarditis, pneumonia, osteomyelitis, and bacteremia, seriously endangering human health. Staphylococcus aureus mastitis is a common breast disease not only in dairy cows but also in women. Currently, veterinary practitioners often treat clinical bovine mastitis by increasing the use of antibiotics. This leads to increasing bacterial resistance and often renders treatment ineffective, presenting significant challenges for clinical veterinary practitioners. Furthermore, the overuse of antibiotics has led to the increasingly serious problem of antibiotic residues in livestock products. Antibiotic residues can also cause allergic reactions in consumers, seriously impacting consumer health.

[0003] Traditional Chinese medicine has obvious advantages in the treatment of mastitis, showing significant therapeutic effects with few adverse reactions, among which Coptis chinensis is one of the main medicines. Coptis chinensis is bitter and cold in nature, and enters the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. The main medicinal part is the rhizome, which has the effects of clearing heat and dampness, purging fire and detoxifying. Studies have shown that Coptis chinensis contains a variety of isoquinoline alkaloids, among which berberine has the highest content. Gentiana macrophylla is a traditional Chinese medicine in my country. It was first included in the "Shennong Bencao Jing" and listed as a medium-grade product. "Gentiana macrophylla is used to treat cold and heat evil spirits, cold and dampness, rheumatism, limb pain, diarrhea, and urination." It is introduced in "Yao Xing Lun", "Si Sheng Bencao" and "Compendium of Materia Medica". The tuberous rhizomes, leaves, aerial parts, and flowers of Gentiana macrophylla contain numerous compounds, classified by type as iridoid glycosides, flavonoids, xanthones, alkaloids, triterpenes, and others. These compounds possess hepatoprotective and choleretic properties, anti-inflammatory and analgesic effects, antibacterial and antioxidant effects, antiviral effects, central nervous system effects, anti-tumor effects, and immunomodulatory effects. Aloe vera, a perennial green herb belonging to the Liliaceae family, has triangular thorns on the edges of its leaves and produces pale yellow flowers. Aloe vera contains numerous bioactive compounds, such as flavonoids, terpenoids, fatty acids, anthraquinones, monosaccharides, polysaccharides, tannins, sterols, enzymes, salicylic acid, minerals, and vitamins. Aloe vera is used empirically to treat cuts, burns, inflammation, and other skin conditions.

[0004] Currently, there is no report on the method for effectively utilizing berberine, gentiana macrophylla extract and aloe vera gel. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a composition for preventing and treating mastitis, and a preparation method and application thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides a composition for preventing and treating mastitis, comprising the following components in parts by weight: 10-12 parts of aloe vera gel, 3-5 parts of berberine tannate, and 2-4 parts of gentiana macrophylla extract.

[0008] Preferably, the gentiana macrophylla extract is prepared by the following method:

[0009] (1) crushing the root of Gentiana macrophylla and sieving it to obtain Gentiana macrophylla root powder;

[0010] (2) adding a 65% ethanol solution to the root powder of Gentiana macrophylla, and ultrasonically extracting the powder 1-3 times to obtain an extract;

[0011] (3) Concentrating and drying the extract to obtain the Gentiana macrophylla extract.

[0012] Preferably, in step (2), the extraction process is as follows: the volume ratio of Gentiana macrophylla root powder to ethanol solution is 1 g: (10-15) ml, the ultrasonic extraction time is 15-20 min, and the ultrasonic power is 200W-250W.

[0013] The second aspect of the present invention provides use of the above composition in the preparation of a medicament for preventing and treating mastitis.

[0014] The third aspect of the present invention provides use of the above composition in preparing a product for inhibiting Staphylococcus aureus.

[0015] A fourth aspect of the present invention provides a drug for preventing and treating mastitis, comprising the above composition and hydrogen.

[0016] A fifth aspect of the present invention provides a method for preparing the above-mentioned drug, comprising the following steps:

[0017] (1) adding berberine tannate and gentiana macrophylla extract to aloe vera gel to obtain a composition, adding deionized water to the composition, and ultrasonically dispersing for 2-4 hours to obtain a dispersion;

[0018] (2) The dispersion is transferred to a hydrogenation kettle, and hydrogen is added under a pressure of 2.5-4 MPa and continuous stirring to make the final concentration of hydrogen 10-12 mg / L, and then the mixture is filled to obtain the drug.

[0019] Preferably, in step (1), the ultrasonic dispersion condition is: the ultrasonic power is 150W-200W.

[0020] Preferably, in step (1), the amount of deionized water added is 10%-20% by weight of the composition.

[0021] Preferably, in step (2), the hydrogenation flow rate is 10-20 L / min; and the stirring speed is 30-50 rpm.

[0022] In recent years, studies have shown that H2 can react with strong oxidants (such as hydroxyl radicals) in cells, and has application potential for prevention and treatment, which has attracted widespread attention in the industry. First, it is easy to quickly penetrate the cell membrane and diffuse into the cytoplasm or mitochondria that other drugs cannot enter. It has the advantages of rapid response and direct administration, and can exert effective anti-inflammatory properties even at a very low effective concentration. Secondly, the non-cytotoxicity of hydrogen and its metabolites can well avoid the risk of bacterial resistance and drug residues, even at high concentrations of hydrogen. Studies have shown that the use of hydrogen-rich vitamin E glycerol to smear the breasts of mice infected with Staphylococcus aureus reduced the expression of inflammatory factors at the site of infection and alleviated tissue pathological changes. Therefore, the present application introduces hydrogen when preparing the ointment to improve the anti-inflammatory effect of the present composition.

[0023] Beneficial effects of the present invention:

[0024] 1. The aloe vera gel and gentiana macrophylla extract in the composition of the present invention are both plant extracts, both of which have anti-inflammatory and bactericidal effects. Berberine tannate is an alkaloid with significant antibacterial effects. The combination of the three can effectively reduce the use of antibiotics, thereby reducing the residual antibiotics in livestock products and increasing the safety of the products. In addition, aloe vera gel, gentiana macrophylla extract and berberine tannate have a synergistic effect. The combination of the three can effectively treat mastitis.

[0025] 2. The present invention introduces hydrogen during the preparation process, and applies the prepared ointment to the site infected with Staphylococcus aureus, which can reduce the expression of inflammatory factors and increase the anti-inflammatory effect. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0027] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0028] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0029] The aloe vera gel used in the following examples and comparative examples of the present invention was purchased from Guangdong Aiyana Cosmetics Co., Ltd., IMISSMI Curacao Aloe Vera Gel.

[0030] Berberine tannate used in the following examples and comparative examples of the present invention was purchased from Hubei Dibai Chemical Co., Ltd.

[0031] The ELISA kits used in the following examples and comparative examples of the present invention were purchased from Wuhan Elaruite Biotechnology Co., Ltd.

[0032] The gentiana macrophylla extract used in the present invention is obtained by the following method:

[0033] (1) Grinding the root of Gentiana macrophylla and passing it through a 100-mesh sieve to obtain Gentiana macrophylla root powder;

[0034] (2) adding 65% ethanol solution to the root powder of Gentiana macrophylla and ultrasonically extracting the powder three times to obtain an extract;

[0035] The extraction process is as follows: the volume ratio of Gentiana macrophylla root powder to ethanol solution is 1g:10ml, the ultrasonic extraction time is 15min, and the ultrasonic power is 200W;

[0036] (3) The extract was concentrated and dried at 100° C. to obtain a dry powder to obtain a Gentiana macrophylla extract.

[0037] The strains used in the experiment were Staphylococcus aureus, purchased from Hangzhou Baosai Biotechnology Co., Ltd., with the accession number being ATCC 33593.

[0038] Example 1: Preparation of a composition for preventing and treating mastitis

[0039] 1. Raw materials: aloe vera gel 100g, berberine tannate 30g, gentiana macrophylla extract 20g.

[0040] 2. Preparation steps: add berberine tannate and gentiana macrophylla extract to aloe vera gel, mix well, and obtain a composition.

[0041] Example 2: Preparation of a composition for preventing and treating mastitis

[0042] 1. Raw material composition: aloe vera gel 110g, berberine tannate 40g, gentiana macrophylla extract 30g.

[0043] 2. Preparation steps: add berberine tannate and gentiana macrophylla extract to aloe vera gel, mix well, and obtain a composition.

[0044] Example 3: Preparation of a composition for preventing and treating mastitis

[0045] 1. Raw material composition: aloe vera gel 120g, berberine tannate 50g, gentiana macrophylla extract 40g.

[0046] 2. Preparation steps: add berberine tannate and gentiana macrophylla extract to aloe vera gel, mix well, and obtain a composition.

[0047] Example 4 Preparation of a drug for preventing and treating mastitis

[0048] 1 Preparation steps:

[0049] (1) Weigh 120 g of the composition prepared in Example 1, add 24 g of deionized water to the composition, and ultrasonically disperse it at an ultrasonic power of 150 W for 2 h to obtain a dispersion;

[0050] (2) The dispersion was transferred to a hydrogenation reactor, and hydrogenation was performed under the conditions of a pressure of 2.5-4 MPa, a rotation speed of 30 rpm, and a hydrogenation flow rate of 20 L / min to make a final hydrogen concentration of 10 mg / L. The mixture was then filled to obtain the drug.

[0051] Example 5: Preparation of a drug for preventing and treating mastitis

[0052] 1. Preparation steps:

[0053] (1) Weigh 120 g of the composition prepared in Example 2, add 24 g of deionized water to the composition, and ultrasonically disperse it at an ultrasonic power of 150 W for 2 h to obtain a dispersion;

[0054] (2) The dispersion was transferred to a hydrogenation reactor, and hydrogenation was performed under the conditions of a pressure of 2.5-4 MPa, a rotation speed of 30 rpm, and a hydrogenation flow rate of 20 L / min to make a final hydrogen concentration of 10 mg / L. The mixture was then filled to obtain the drug.

[0055] Example 6: Preparation of a drug for preventing and treating mastitis

[0056] 1. Preparation steps:

[0057] (1) Weigh 120 g of the composition prepared in Example 3, add 24 g of deionized water to the composition, and ultrasonically disperse it at an ultrasonic power of 150 W for 2 h to obtain a dispersion;

[0058] (2) The dispersion was transferred to a hydrogenation reactor, and hydrogenation was performed under the conditions of a pressure of 2.5-4 MPa, a rotation speed of 30 rpm, and a hydrogenation flow rate of 20 L / min to make a final hydrogen concentration of 10 mg / L. The mixture was then filled to obtain the drug.

[0059] Test Example 1: Antibacterial Test

[0060] 1. Test method:

[0061] According to 1.2.1 Indoor toxicity determination in the literature "Inhibitory effects of several fungicides on Fusarium solani" (Zhang Heng, He Jing, Zhang Xiaoyan, et al. Journal of Gansu Agricultural University, 2021, 56(05):77-82.DOI:10.13432 / j.cnki.jgsau.2021.05.011.), experiments were conducted on the compositions prepared in Examples 1-3, aloe vera gel, berberine tannate and Gentiana macrophylla extract.

[0062] The test bacteria was Staphylococcus aureus.

[0063] The antibacterial effects of the compositions prepared in Examples 1-3, aloe vera gel, berberine tannate, and Gentiana macrophylla extract against Staphylococcus aureus were tested using a mycelial growth rate assay. Under sterile conditions, the compositions prepared in Examples 1-3, aloe vera gel, berberine tannate, and Gentiana macrophylla extract were each prepared with sterile water to a concentration of 20 mg / mL. After the sterilized PDA culture medium was cooled to 45-50°C, 2 mL of the drug was added and shaken thoroughly to prepare the drug-containing culture medium for later use. A PDA plate containing an equal amount of sterile water served as a control. Use a hole puncher (D = 6 mm) to punch a bacterial cake in an area 2 to 3 cm away from the center of the test strain colony. Use tweezers (dip in alcohol and heat on a flame for disinfection before each inoculation) to pick up the bacterial cake and inoculate it in the center of the drug-containing PDA plate. Place it in a 25°C constant temperature box and culture it upside down for 6 to 7 days. When the colony of the control group grows to 3 / 4 of the entire plate, use the cross method to measure the colony diameter (mm), calculate the average value, and calculate the mycelial growth inhibition rate of the test agent at different mass concentrations according to the following formula.

[0064] S(%)=(LR) / L×100

[0065] Where: S is the inhibition rate, L is the colony diameter of the control group (mm), and R is the colony diameter of the treatment group (mm).

[0066] 2. Test results:

[0067] Compositions 1-3, antibacterial rates of each active ingredient against Staphylococcus aureus

[0068]

[0069] As can be seen from Table 1, the present invention combines aloe vera gel, berberine tannate, and gentiana macrophylla extract, and the antibacterial rate against Staphylococcus aureus is above 93%. Aloe vera gel alone has an antibacterial rate of 25.32% against Staphylococcus aureus; berberine tannate alone has an antibacterial rate of 35.27% against Staphylococcus aureus; and gentiana macrophylla extract alone has an antibacterial rate of 28.96% against Staphylococcus aureus. The sum of the antibacterial rates of the three alone against Staphylococcus aureus is lower than the antibacterial rate of the combination of the three. Therefore, the present invention combines aloe vera gel, berberine tannate, and gentiana macrophylla extract to achieve a synergistic effect in inhibiting Staphylococcus aureus.

[0070] Test Example 2: Mouse Mastitis Test

[0071] The experiment was conducted with reference to the materials and methods in the literature "Effects of Curcumin on Cellular Immunity in Mastitis Model Mice" (Xu Jin et al. Chinese Journal of Veterinary Medicine, 2019, 39(04):774-781. DOI: 10.16303 / j.cnki.1005-4545.2019.04.29). This experiment was commissioned by a laboratory institution. Mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0072] 1. Construction of Mouse Mastitis Model

[0073] The purified Staphylococcus aureus was cultured at 37°C for 18-24 hours until the logarithmic phase, and the concentration of the bacterial solution was adjusted to 5.0×10 8 CFU / mL.

[0074] The female mice that had given birth for 6-7 days were anesthetized with sodium pentobarbital, and the area around the nipples was disinfected with iodine tincture cotton. 100 μL (5.0×10 8 CFU / mL) model, 50 μL per nipple.

[0075] 2. Mouse Blood Test

[0076] Forty mice were divided equally into four groups. Three days after modeling, the T1 treatment group was treated with 0.1 g of the drug prepared in Example 6 applied to the fourth pair of nipples of the mice. The T2 treatment group was treated with 0.1 g of a mixture prepared by dissolving the composition prepared in Example 3 in deionized water, where the deionized water was added at a concentration of 20% by weight of the composition, applied to the fourth pair of nipples of the mice. The T3 treatment group was treated with 0.1 g of a mixture prepared by dissolving hydrogen in deionized water, where the concentration of hydrogen in the deionized water was 10 mg / L, applied to the fourth pair of nipples of the mice. The control group was treated with 0.1 g of deionized water applied to the fourth pair of nipples of the mice. All four treatment groups were treated with the drug for seven consecutive days. All groups were treated with identical care and husbandry.

[0077] After seven consecutive days of smearing, the mice were euthanized, blood was collected from the mouse eyeballs, and the serum was separated (stored at -80°C). The procedures in the ELISA kit were followed, and the levels of IL-1, IL-6, and TNF-α in the supernatants of the three groups were detected in a microplate reader.

[0078] Table 2 Effects of different treatment groups on inflammatory factors in mouse blood

[0079]

[0080] As shown in Table 2, the levels of IL-1β, IL-6, and TNF-α in the serum of mice in group T1 were significantly lower than those in the control group, indicating that the compounding of aloe vera gel, berberine tannate, gentiana macrophylla extract, and hydrogen in the present invention can reduce the mastitis reaction caused by Staphylococcus aureus.

[0081] Test Example 3: Cow Mastitis Test

[0082] Forty dairy cows suffering from mastitis were selected from a ranch of Shandong Hi-Speed ​​Group.

[0083] The symptoms of these cows are: redness and swelling of the affected area of ​​the mammary gland, increased surface temperature, sensitivity to touch, increased volume, significantly reduced or stopped milk secretion, abnormal milk quality, light yellow or light red, thin or thick milk, or the presence of flocs.

[0084] The 40 cows were divided into four groups. Treatment group T1 applied 5g of the drug prepared in Example 6 to the affected milk area; treatment group T2 applied 5g of a mixture prepared by dissolving the composition prepared in Example 3 in deionized water, where the deionized water was added at a concentration of 20% by weight of the composition; treatment group T3 applied 5g of a mixture prepared by dissolving hydrogen in deionized water, where the concentration of hydrogen in the deionized water was 10mg / L; and the control group applied 5g of deionized water to the affected milk area. All four treatment groups cleaned the affected milk area with warm water before applying the drug, wiped it dry with a dry towel, and then applied the drug twice daily for a course of seven days. Severe cases were treated with the drug for two consecutive courses to observe the therapeutic effect. The efficacy of the drug was determined based on clinical symptoms and changes in milk quality, and the cure rate, effective rate, and ineffective rate were calculated. SCC was measured using a milk composition analyzer (Fossomatic 5000, Shanghai Fosso Co., Ltd.).

[0085] Cure: Redness, swelling, heat and pain in the milk area disappear, the cow is in good spirits and has a normal appetite, milk properties, color and milk production return to normal, and the somatic cell count in the milk is less than 500,000 / mL;

[0086] Effective: Redness, heat, and pain in the breast area are significantly reduced, a small amount of clots remain in the milk, milk production recovers, milk quality improves, and the somatic cell count in the milk is more than 500,000 / mL;

[0087] Ineffective: After two courses of treatment, the redness, swelling, heat and pain of the breast are still not significantly relieved, the milk is still thin or yellow, the floccules are still obvious, or the course of the disease has not been reduced during the treatment and other treatment methods are used.

[0088] Table 3 Clinical efficacy of different treatment groups

[0089]

[0090] As can be seen from Table 3, the present invention compounds aloe vera gel, berberine tannate, gentiana macrophylla extract composition and hydrogen, and the cure rate of the T1 treatment group for cow mastitis is 90%, which is 90% higher than that of the blank control group, and has excellent preventive and therapeutic effects on cow mastitis.

[0091] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A composition for preventing and treating mastitis, characterized in that: The invention is composed of the following components in parts by mass: 10-12 parts of aloe vera gel, 3-5 parts of berberine tannate and 2-4 parts of gentiana macrophylla extract; The gentiana macrophylla extract is prepared by the following method: (1) Grinding the root of Gentiana macrophylla and sieving it to obtain Gentiana macrophylla root powder; (2) Adding 65% ethanol solution to the root powder of Gentiana macrophylla and ultrasonically extracting the mixture 1-3 times to obtain an extract; (3) concentrating and drying the extract to obtain a Gentiana macrophylla extract; In step (2), the extraction process is as follows: the volume ratio of Gentiana macrophylla root powder to ethanol solution is 1 g: (10-15) ml, the ultrasonic extraction time is 15-20 min, and the ultrasonic power is 200 W-250 W.

2. Use of the composition according to claim 1 in the preparation of a medicament for preventing and treating mastitis.

3. Use of the composition according to claim 1 in the preparation of a medicament for inhibiting Staphylococcus aureus.

4. A drug for preventing and treating mastitis, characterized in that: The medicine consists of the composition according to claim 1 and hydrogen; The preparation method of the medicine comprises the following steps: (1) adding berberine tannate and gentiana macrophylla extract to aloe vera gel to obtain a composition, adding deionized water to the composition, and ultrasonically dispersing for 2-4 hours to obtain a dispersion; (2) The dispersion is transferred to a hydrogenation kettle, and hydrogen is added under a pressure of 2.5-4 MPa and continuous stirring to achieve a final hydrogen concentration of 10-12 mg / L. The mixture is then filled to obtain the drug.

5. The drug for preventing and treating mastitis according to claim 4, characterized in that In step (1), the amount of deionized water added is 10%-20% by weight of the composition.

6. The drug for preventing and treating mastitis according to claim 4, characterized in that: In step (2), the hydrogenation flow rate is 10~20L / min.

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