RU2799050C2 - Application of pulsatilla chinensis extract for obtaining a drug for the treatment of viral and/or bacterial diseases - Google Patents

Abstract

FIELD: pharmaceutical industry; veterinary medicine.

SUBSTANCE: group of inventions relates to the use of pulsatilla saponin B4 compound for the preparation of a drug for the treatment of mastitis in dairy cows. Use of the pulsatilla saponin B4 compound represented by formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of dairy cow mastitis:

The use of a medicinal product for the treatment of mastitis of dairy cows, characterized be the following: the medicinal product is a composition containing a pulsatilla saponin B4 compound represented by formula I, and a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable excipients, where the composition is selected from the group consisting of an injection and an intramammary infusion, and wherein the concentration of the pulsatilla saponin B4 compound in the said formulation is 50 mg/mL.

EFFECT: above described pulsatilla saponin B4 compound of formula I is effective for the treatment of mastitis in dairy cows.

7 cl, 5 dwg, 11 tbl, 5 ex

Description

Technical field

The present invention relates to the use of an extract of Pulsatilla Chinensis for the preparation of a medicament for the treatment of viral and/or bacterial diseases.

State of the art

Pulsatilla Chinensis (Bge.) Regel is a pulsatilla plant from the Ranunculaceae family, the main medicinal part of which is its dried roots. The earliest information about pulsatilla is found in Shen Nong's Materia Medica. It has a bitter taste, cooling properties and enters the stomach, kidneys and colon section. The main functions of Pulsatilla are detumescence, cooling of the blood, cessation of diarrhea, reduction of internal heat, etc. Pulsatilla is traditionally used to treat bacillary dysentery, and it also has good therapeutic effects in the symptoms of chills and fever, swelling and pain in the eyes. Pulsatilla has a variety of pharmacological effects, mainly to improve the body's ability to recognize and resist foreign body, reduce the rate of tumor cell proliferation, reduce the number of pathogens, and effectively inhibit the oxidation reaction of free radicals. Among them, the most promising application is the development of new anti-inflammatory and anticancer drugs.

Feng Xiuzhi et al. studied the role of pulsatilla in cell apoptosis and believed that pulsatilla has a good induction effect on gastric cancer cells. In addition, cancer cell proliferation and DNA replication are associated with the active ingredients of pulsatilla, which can suppress the growth of HL260 cells and inhibit the proliferation of breast cancer cells (MCF-7), lung cancer cells (PG), colon cancer cells (SW480), malignant glioma (U87MG) and other tumor cells by inducing apoptosis. Li Wenchao et al. applied an alcoholic extract of Pulsatilla to various pathogenic bacteria and studied the antibacterial effect of the extracts in various solvents. All of these extracts have been found to have antibacterial activity, although the effects vary.

Dairy cow mastitis is one of the three major cow diseases recognized worldwide. It often occurs during lactation. The main cause of its occurrence is a pathogenic microbial infection. Other causes include improper milking, mechanical damage caused by trauma, and damage caused by chemicals. Dairy cow mastitis not only affects milk production and causes economic loss, but also affects milk quality and harms human health.

Dairy cow mastitis can be divided into two main categories: latent mastitis and clinical mastitis. Clinical mastitis is manifested by clear clinical symptoms and visible changes in milk and can be divided into three grades depending on the degree of onset: Grade 1: abnormal milk; Grade 2: abnormal milk, udder becomes hot, painful and enlarged; Grade 3: abnormal milk, udder becomes hot, painful and enlarged, cows show symptoms of a systemic infection. Clinical mastitis is currently the main target of treatment in clinical practice. If clinical mastitis is detected in cows, isolation treatment is necessary. Latent mastitis, also known as subclinical mastitis, is the most common type of mastitis in dairy cows. This type of mastitis usually has no clinical symptoms, the difference between its milk and normal milk cannot be distinguished with the naked eye, and laboratory reagents are required for diagnosis. Also, if this type of mastitis does not reach a certain proportion in the herd, it is usually left untreated. Due to latent morbidity, latent mastitis in dairy cows causes very serious economic losses.

At present, methods such as udder infusion and intravenous injection are mainly used to treat mastitis in dairy cows, and the therapeutic drugs used are mainly antibiotics such as cefkinome, enrofloxacin, penicillin, streptomycin and the like. However, long-term use of antibiotics tends to cause drug resistance and affect the treatment effect; at the same time, when antibiotics are used for treatment, antibiotic residues easily contaminate milk sources and pose a risk to human health. It is worth noting that if mastitis is a Gram-negative bacterial infection, the use of lactam antibiotics causes cell wall breakdown and endotoxin release, causing redness, swelling, heat, and pain throughout the udder area, which will aggravate the condition. Therefore, it is very important to find a safe and effective way to treat mastitis in dairy cows.

There is currently no information on the use of the compounds according to the present invention for the treatment of viral and/or bacterial mastitis in dairy cows.

Brief description of the invention

In order to solve the above technical problem, the present invention proposes the use of a pulsatilla saponin B4 compound represented by formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of viral and/or bacterial diseases:

In a further aspect, the drug is a formulation derived from the above pulsatilla saponin B4 compound or a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable excipients.

In an additional aspect, the composition is an injection, intramammary infusion, powder, ointment, lotion; preferably a formulation for injection, intramammary infusion; more preferably the composition is an injection.

In another aspect, the disease is dairy cow mastitis.

In a further aspect, dairy cow mastitis is clinical mastitis, latent mastitis.

In a further aspect, the clinical mastitis is first-time treated clinical mastitis, persistent clinical mastitis, i. clinical mastitis that has been treated for a long time but not cured.

The present invention also provides a medicament for the treatment of viral and/or bacterial diseases, which is a composition obtained from the above pulsatilla saponin B4 compound or a pharmaceutically acceptable salt thereof as an active ingredient and pharmaceutically acceptable auxiliary materials.

In an additional aspect, the composition is an injection, intramammary infusion, powder, ointment, lotion; preferably the composition is an injection, intramammary infusion; more preferably the composition is an injection.

In another aspect, the disease is dairy cow mastitis.

In an additional aspect, dairy cow mastitis is clinical mastitis, latent mastitis; preferably, the clinical mastitis is first-time treated clinical mastitis, persistent clinical mastitis.

The Pulsatilla extract of the present invention is a Pulsatilla saponin B4 compound, and its English name is Anemoside B4. Its CAS number is 129741-57-7, molecular formula is C ₅₉ H ₉₆ O ₂₆ , molecular weight is 1221.38, and it is a white crystalline powder.

Currently, antibiotics are mainly used to treat mastitis. Among them, broad-spectrum antibiotics that disinfect and damage cell nucleic acids are more often used. During treatment and 4-5 days after treatment, milk must be discarded, resulting in large economic losses for farmers and the risk of drug resistance. Pulsatilla Saponin B4 is a natural active ingredient extracted from the Chinese medicinal herb Pulsatilla, is non-toxic and does not cause drug resistance. The pulsatilla saponin B4 compound of the present invention has a strong biological activity and an excellent therapeutic effect in dairy cow mastitis. There is no need to throw away milk during treatment and at the end of treatment, which not only reduces the economic loss to farmers, but also reduces the risk of drug resistance. In addition, pulsatilla saponin B4 not only has an excellent therapeutic effect in clinical mastitis, but also has an excellent therapeutic effect in persistent clinical mastitis and latent mastitis. At the same time, pulsatilla saponin injection has the advantages of safety, high efficacy, no residue, no drug resistance, and no toxic and side effects in the treatment of dairy cow mastitis.

Obviously, in accordance with the above content of the present invention, in accordance with the usual technical knowledge and conventional technical means in the art, various forms of modification, replacement or change can be made without departing from the above basic technical idea of the present invention.

The above contents of the present invention will be further detailed below by way of specific embodiments in the form of examples. However, it should not be construed that the scope of the above subject matter of the present invention is limited by the following examples. All technologies implemented on the basis of the above content of the present invention are included in the scope of the present invention.

Brief description of the drawings

The figure 1 shows the standard curve for determining the content of saponin B4 pulsatilla.

Figure 2 shows a comparison of milk from cows suffering from clinical mastitis before and after treatment with an injection of pulsatilla saponin B4.

3 shows a comparison of milk from cows suffering from clinical mastitis before and after treatment with pulsatilla saponin B4 injection.

The figure 4 shows the result of treatment of cow No. 8558, which was treated for a long time, but not cured.

The figure 5 shows the result of treatment of cows with latent mastitis.

Implementation Options

Example 1 Preparation of a compound according to the present invention

100 kg of pulsatilla drug material was withdrawn, 10 times 70% ethanol was added and extracted twice by heating and refluxing. The extract was concentrated under reduced pressure at 75°C, centrifuged at 4000 rpm for 10 minutes. The supernatant was passed through a polar macroporous adsorption resin column and then eluted successively with water, 30% ethanol and 70% ethanol. The fraction eluted with 70% ethanol was concentrated under reduced pressure at 75° C. and then spray dried to obtain 3450 g of the total extract of pulsatilla saponins. After dissolving the extract in water and filtering, the filtrate was loaded into a 200 dynamic axial preparative chromatography system (filled with 10 μm ODS), eluted with 50% methanol, the corresponding eluent was collected according to the chromatographic peak, concentrated under reduced pressure and lyophilized to obtain 1700 g of pulsatilla saponin B4 .

Example 2 Determination of the content of the compound according to the present invention

1. Tools and reagents

The Agilent 1260 high performance liquid chromatograph and DAD UV detector were purchased from Agilent Technologies (China) Co., Ltd., the BP211D electronic analytical balance was purchased from Sartorius of Germany, and the KQ-400DB CNC ultrasonic cleaner was purchased from Kunshan Ultrasonic Instrument Co.,Ltd.

Pulsatilla saponin B4 sample prepared in Example 1.

Pulsatilla saponin B4 reference material (batch number: 111766-201702, content: 94.7%) was purchased from the National Institute of Food and Drug Administration.

2. Chromatography conditions

Chromatographic column: Sepax Bio-C18 (4.6×250 mm, 5 µm); mobile phase: methanol-water (64:36); detection wavelength: 201 nm; flow rate: 1.0 ml/min.

3. Preparation of a solution of a standard substance

Accurately weigh the required amount of Pulsatilla Saponin B4 standard, place it in a 10 ml volumetric flask, add mobile phase to dissolve, and make up to the mark to obtain a standard solution (containing 1 mg ml of Pulsatilla Saponin B4).

4. Preparation of the sample solution

A 10 mg sample of pulsatilla saponin B4 obtained in Example 1 was accurately weighed, placed in a 10 ml volumetric flask, mobile phase was added to dissolve, and the volume was adjusted to the mark to obtain a sample solution.

5. Definition

Accurately measured 20 μl of the standard solution and sample solution, introduced them into the liquid chromatograph, recorded the chromatogram, and calculated the content in accordance with the external standard method.

6. Standard Curve and Linear Regression Equation

Pulsatilla saponin B4 standard was taken, weighed accurately and placed in a 10 ml volumetric flask, mobile phase was added to dissolve, the volume was adjusted to the mark to prepare solutions of the standard at a concentration of approximately 1 mg/mL, 5 mg/mL, 10 mg /ml, 15 mg/ml and 20 mg/ml, respectively. Accurately measured 20 μl of each solution, injected it into a liquid chromatograph and recorded the chromatogram.

Table 1. Linear regression equation for determining the content of the standard substance saponin B4 of pulsatilla

Sample Concentration (mg/ml) Peak area Regression Equation 1 0.98488 115.7 Y=100.07+10.721
R=0.9999 2 4.92440 529.5 3 9.84880 1057.8 4 14.77320 1560.3 5 19.69760 2097.9

From the experimental results, it can be seen that pulsatilla saponin B4 has a good linear relationship in the concentration range of 1.04-20.80 mg / ml, R = 0.9999, and is suitable for determining the content of pulsatilla saponin B4.

7. Determination of Pulsatilla Saponin B4 Content in a Sample

In accordance with the method described in paragraph "3" above, 2 parts of the standard substance of pulsatilla saponin B4, 10 mg each, were accurately weighed, placed in a 10 ml volumetric flask, the mobile phase was added to dissolve, and the volume was adjusted to the mark to obtain a standard solution 1 and standard solution 2.

In accordance with the method described in paragraph "4" above, 2 parts of a sample of pulsatilla saponin B4, 10 mg each, were accurately weighed, placed in a 10 ml volumetric flask, mobile phase was added to dissolve, and the volume was adjusted to the mark to obtain a sample solution 1 and sample solution 2.

In accordance with the above chromatography conditions, 20 μl of each solution was accurately measured, injected into a liquid chromatograph, and a chromatogram was recorded.

Table 2. Results of determining the content of saponin B4 of pulsatilla

Sample Mass (mg) Peak area Conc. (mg/l) Content (%) Average content (%) Standard solution 1 10.22 1039.6 0.968 // // 1044.4 Standard solution 2 10.05 1025.6 0.952 // // 1022.5 Sample solution 1 10.13 1081.8 1.013 99.7 99.4 1092.6 Sample solution 2 10.10 1075.1 1.010 99.1 1079.2

According to the test results, the content of Pulsatilla saponin B4 obtained in this experiment was 99.4%.

Example 3 Preparation of Pulsatilla Saponin B4 Injection

1) Composition: 1000 ml solution for injection prepared from 50 g of pulsatilla saponin B4 with water.

2) Preparation method: Weighed 50 g of pulsatilla saponin B4 obtained in Example 1, added 800 ml of water for injection to completely dissolve it, filtered, adjusted pH to 7.0, added water for injection to a volume of 1000 ml, filtered on a fine filter cleaning, placed in a container, closed and sterilized at 100°C for 30 min.

Example 4 Preparation of Pulsatilla Saponin B4 Powder

1) Composition: 1000 g of powder was obtained from 1000 g of pulsatilla saponin B4.

2) Production method: 1000 g of pulsatilla saponin B4 obtained in Example 1 was weighed, ground into a fine powder, passed through a 100 mesh sieve, and mixed well.

Example 5 Preparation of Intramammary Infusion of Pulsatilla Saponin B4

1) Composition: 1000 ml intramammary infusion was prepared from 50 g pulsatilla saponin B4.

2) Production method: Weighed 50 g of pulsatilla saponin B4 obtained in example 1, added 800 ml of distilled water to completely dissolve it, filtered, adjusted the pH to 7.0, added distilled water to a volume of 1000 ml, filtered on a fine filter, placed in a container, closed, sterilized at 100°C for 30 min.

The beneficial effects of the present invention will be described below by means of experimental examples.

Experimental Example 1 Effect of Different Routes of Administration of the Compound of the Invention on Therapeutic Effect in the Treatment of Dairy Cow Mastitis

Presently, routes of administration for the treatment of mastitis include udder infusion, intramuscular injection, and intravenous injection.

интрамаммарная инфузия сульфата цефкинома) от Eastern Along Pharmaceutical Co., Ltd., Ruchang (инъекция цефотифура) от Pfizer и т. д.Among them, udder infusion is the most common route of administration. Drugs are injected directly into the affected area of the udder through the teats to achieve the effect of topical application and precision therapy. There are now many commercial pharmaceutical drugs for udder infusion, such as Aonaikang (

cefkinome sulfate intramammary infusion) from Eastern Along Pharmaceutical Co., Ltd., Ruchang (cefotifur injection) from Pfizer, etc.

Intramuscular injection is the introduction of drugs into the muscle tissue of animals so that the drugs reach the affected area of the udder through the bloodstream and have a therapeutic effect. The intramuscular injection is easy to administer and can to some extent control the symptoms of systemic inflammation and fever caused by mastitis.

Intravenous injection consists of injecting the drug directly into the venous blood of the animal so that the drug enters the affected area of the udder through the bloodstream. When injected intravenously, the drug reaches the site of action quickly and can treat systemic symptoms. However, intravenous injection places certain requirements on the properties of the drug, which cannot be a suspension and have little irritation of the blood vessels. Bayer (Germany) recommends the treatment of mastitis by intravenous administration of its product Baytrli (enrofloxacin), which can reach a higher concentration in the affected area of the udder and have a therapeutic effect there, and at the same time can control systemic infections.

The above three routes of administration for the treatment of mastitis in dairy cows have their own advantages and disadvantages. This experiment aims to investigate the therapeutic effect of pulsatilla saponin B4 on mastitis in dairy cows and determine the most appropriate route of administration. The results of this study will provide important information on how to achieve the best therapeutic effect of pulsatilla saponin B4 and for future drug development and packaging.

1. Tested drugs

Injection of saponin B4 of pulsatilla obtained in example 3 at a concentration of 50 mg/ml; intramammary infusion of pulsatilla saponin B4, obtained in example 5, at a concentration of 50 mg/ml.

Positive Control: Intramammary infusion of cefkinome sulfate, 8 g/unit (Eastern Along Pharmaceutical Co., Ltd., Nanhai, Foshan).

2. Test animals

Were selected 130 sick cows (clinical mastitis grade 2) and randomly divided into 13 groups of 10 cows in each group.

3. Dosage and mode of administration

Table 3. Dosage and mode of administration

Group Dosage (ml/udder area affected/time) Mode of administration Blank control group normal saline / Positive control group 1 unit intraductal infusion Pulsatilla Saponin B4 Injection, Intramuscular Injection, Low Dose Group 10 Intramuscular injection Pulsatilla Saponin B4 Injection, Intramuscular Injection, Medium Dose Group 20 Intramuscular injection Pulsatilla Saponin B4 Injection, Intramuscular Injection, High Dose Group thirty Intramuscular injection Pulsatilla Saponin B4 Injection, IV Injection, Low Dose Group 40 intravenous injection Pulsatilla Saponin B4 Injection, IV Injection, Medium Dose Group 60 intravenous injection Pulsatilla Saponin B4 Injection, Intravenous Injection, High Dose Group 80 intravenous injection Intramammary Infusion of Pulsatilla Saponin B4, Low Dose Group 20 intraductal infusion Intramammary infusion of pulsatilla saponin B4, medium dose group thirty intraductal infusion Intramammary infusion of pulsatilla saponin B4, high dose group 40 intraductal infusion

Administration by intramuscular injection into the affected area of the udder: Pulsatilla saponin B4 injection was used for local intramuscular injection into the affected area of the udder once every morning and evening for 7 days;

Administration by intravenous injection: Pulsatilla saponin B4 was administered intravenously once a day for 7 days;

Intramammary infusion administration: Pulsatilla saponin B4 intramammary infusion and a positive control (cephkinome sulfate intramammary infusion) were administered via intraductal infusion into the affected area of the udder once every morning and evening for 7 days.

For each route of administration of the pulsatilla saponin B4 of the present invention, a blank control group was provided to which the same volume of saline was administered.

4. Evaluation guide

Before each administration, the cow's body temperature, respiration, mental state, feeding status and other parameters were checked to observe changes in the udder and milk.

Complete cure: Clinical symptoms such as redness, swelling, heat and pain in the affected area of the udder completely disappeared and lactation was restored to normal levels.

Clinical cure: Clinical symptoms such as redness, swelling, heat and pain in the affected area of the udder have essentially disappeared and lactation has almost returned to normal levels.

No effect: No significant improvement or even worsening of clinical symptoms in the affected area of the udder, or relapse within two weeks of discontinuation of the drug, both should be considered no effect.

The cure rate was calculated as follows:

Degree of cure

5. Results

Table 4 Cure rates and cure times for mastitis in dairy cows treated by different routes of administration

Group Cure rate (%) Healing time Blank control group 0 / Positive control group 60 5-7 Pulsatilla Saponin B4 Injection, Intramuscular Injection, Low Dose Group 50 5-7 Pulsatilla Saponin B4 Injection, Intramuscular Injection, Medium Dose Group 60 5-6 Pulsatilla Saponin B4 Injection, Intramuscular Injection, High Dose Group 80 4-6 Pulsatilla Saponin B4 Injection, IV Injection, Low Dose Group 40 5-7 Pulsatilla Saponin B4 Injection, IV Injection, Medium Dose Group 60 5-6 Pulsatilla Saponin B4 Injection, Intravenous Injection, High Dose Group 70 4-7 Intramammary Infusion of Pulsatilla Saponin B4, Low Dose Group 40 6-7 Intramammary infusion of pulsatilla saponin B4, medium dose group 50 5-6 Intramammary infusion of pulsatilla saponin B4, high dose group 70 4-7

The overall cure rate and cure time of pulsatilla saponin B4 administered by different routes are shown in Table 4: infusion of cefkinome sulfate). Among them, pulsatilla saponin B4 intramuscular injection has the best curative effect and the shortest treatment period.

Experimental Example 2 Study of Pulsatilla Saponin B4 According to the Present Invention for the Treatment of Clinical Mastitis

1. Tested drugs

Pulsatilla saponin B4 injection, obtained by the production method according to example 3, at a concentration of 50 mg/ml;

(10 мл/единица, номер партии: DV3180701), Hebei Yuanzheng Pharmaceutical Co., Ltd.Control drug: SHUANGDING Injection

(10 ml/unit, batch number: DV3180701), Hebei Yuanzheng Pharmaceutical Co., Ltd.

2. Test animals

We selected 60 Chinese Holstein cows weighing approximately 612 ± 47 kg, aged 3-5 years, number of births 2-4, healthy at clinical examination and with clinical mastitis that arose naturally during lactation, and randomly divided into 5 groups, 12 in each group.

3. Testing method

3.1 Dosage and mode of administration

Table 5. Dosage and mode of administration

Group Dosage (ml/kg body weight) Mode of administration Blank control group normal saline Intramuscular injection Positive control group 0.1 Intramuscular injection Pulsatilla Saponin B4 Injection, Intramuscular Injection, Low Dose Group 0.025 Intramuscular injection Pulsatilla Saponin B4 Injection, Intramuscular Injection, Medium Dose Group 0.05 Intramuscular injection Pulsatilla Saponin B4 Injection, Intramuscular Injection, High Dose Group 0.1 Intramuscular injection

After cows were tested, the day before administration was recorded as D0. The dosed amount was calculated based on body weight in accordance with the pre-designed dosed substance and dosage (Table 5). Starting from day D1, each group of cows with clinical mastitis was administered the test drug (pulsatilla saponin B4 injection) or the control drug (SHUANGDING injection) by intramuscular injection once a day for 4-7 days, depending on the cure of the disease. Clinical symptoms of cows in each group were monitored before and after drug administration (D0~D12), evaluated according to the standard (see Table 6 for details), and the dosage was recorded. At the end of the trial (D12), based on the results of observing the above indicators, the therapeutic effect for each cow was evaluated according to the standard, and the cure rate and the efficacy rate for each group of cows were calculated.

3.2 Clinical symptom assessment standard for dairy cows with clinical mastitis

In D0 (after clinical mastitis is detected before administration), D1 (after first administration, before second administration), D2~D7 (cured cows stop receiving the drug and non-cured cows continue to receive the drug), D8~D12 (all cows stop receiving medication), regularly checked the clinical and udder symptoms of each cow, such as mental state, appetite and drinking, etc., and scored according to the following table (see Table 6). Recorded clinical symptoms (assessment of local symptoms relating to the udder).

Table 6 Standard for scoring clinical symptoms in dairy cows with clinical mastitis

Observations Grade Assessment standard Mental condition 0 Good 1 A little bit worse 2 Excited or depressed 3 Lying on the floor or even unconscious Appetite and drink 0 A good appetite; moderate drinking 1 Slightly reduced food intake; slightly reduced or increased drinking 2 Significantly reduced food intake; significantly reduced or increased drinking 3 Loss of appetite; drinks a lot of water or refuses to drink Volume of lactation 0 normal milk production 1 Reduced milk production 2 Significantly reduced milk production 3 Almost complete cessation of lactation Milk quality 0 normal milk quality 1 Milk has a small amount of flakes and lumps 2 Lots of lumps in milk 3 The milk has traces of blood, pus and an obvious smell Local symptoms concerning the udder 0 Normal 1 Slight redness and swelling, without obvious thermal pain reaction on palpation 2 Moderate redness and swelling, with thermal pain reaction on palpation 3 Great redness and swelling, with obvious thermal pain reaction on palpation

3.3 Method for determining the number of somatic cells in cow's milk

Collected milk from cows suffering from clinical mastitis, D0, D7 and D12 test, determined and recorded the number of somatic cells.

3.4 Performance evaluation

Carried out according to the assessment of the examination of clinical symptoms

; степень излечения в течение 7 дней представляет собой сумму суточных степеней излечения при лечении в дни 2-7.Daily Cure Rate on Days 2-7: Starting at D2, if a cow suffering from mastitis met the "sum of scores <1", she was considered cured. Daily cure rate on days 2-7=

; the cure rate for 7 days is the sum of the daily cure rates for treatments on days 2-7.

; степень эффективности в течение 7 дней представляет собой сумму суточных степеней эффективности при лечении в дни 2-7.Daily Efficacy Rate on Days 2-7: From D2, if a cow with mastitis was "1 ≤ sum of scores ≤ 2", the treatment was considered effective. Daily treatment success rate on days 2-7=

; the rate of effectiveness for 7 days is the sum of the daily rates of effectiveness for treatment on days 2-7.

.Relapse Rate: During the period D5 to D12, if a diseased cow judged to be successfully treated met a "sum of scores >2" after discontinuation of the drug, this was considered a relapse. Recurrence rate=

.

.Day 12 cure rate (overall cure rate): In D12, if a cow with mastitis was "score <1", she was considered cured on day 12. Day 12 cure rate=

.

.Day 12 Treatment Efficacy Rate (Overall Efficacy Rate): In D12, if a cow with mastitis was "1 ≤ sum of scores ≤ 2", the treatment was considered effective. Degree of effectiveness per day 12=

.

Using the degree of cure as the main indicator and efficacy, evaluation of clinical symptoms and somatic cell count as secondary indicators, the therapeutic effect of the test drug (pulsatilla saponin B4 injection) on clinical mastitis in naturally infected cows was fully evaluated and compared with the therapeutic effect of the control drug (SHUANGDING injection) to determine the clinical recommended dosage of the test drug.

4. Test results

4.1 Results of treatment of cows with clinical mastitis in each group

Table 7 shows that when Pulsatilla Saponin B4 Injection was used to treat clinical mastitis in dairy cows, regardless of low, medium, and high doses, the 7-day cure rate and overall cure rate were higher than those of the SHUANGDING injection control group, and the mean cure time is shorter than the control group, indicating that pulsatilla saponin B4 injection is superior to SHUANGDING injection in the treatment of clinical mastitis. The overall cure rate in the high dose group is higher than in the medium and low dose groups. The overall cure rate in the medium dose group is the same as in the low dose group, but there are no relapses and the mean time to cure is shorter than in the low dose group, demonstrating a certain dose-dependent effect.

Table 7. 4.1 Results of therapeutic effects for cows with clinical mastitis in each group

Group Cure rate in 7 days Recurrence rate Overall cure rate Overall degree of effectiveness Average cure time Low dose group 83.33% 8.33% 75.00% 100% 6.33 days Medium dose group 66.67% 0 75.00% 100% 6.11 days High dose group 75.00% 0 91.67% 100% 6.91 days Control group 41.67% 0 58.33% 66.67% 7.00 days

4.2 Evaluation of the examination of the clinical symptoms of dairy cows with mastitis in each group

It can be seen from Table 8 that the clinical symptom score of dairy cows with mastitis in the low dose group did not differ significantly between D0-D4 and D5-D12 (p>0.05), but significantly differed between D5-D12 and D0-D2, between D7-D12 and D0-D4 (p<0.05), indicating that in the low dose group, cows with mastitis improved significantly at D5 and a significant number were cured at D7. Survey scores for clinical symptoms of dairy cows with mastitis in the medium dose group did not differ significantly between D0-D1, between D2-D5, and between D4-D12 (p>0.05), but differed significantly between D3-D12 and D0-D2, between D6-D12 and D0-D3 (p<0.05), indicating that in the medium dose group cows showed a significant improvement at D3 and a significant number were cured at D6. Survey scores of clinical symptoms in dairy cows with mastitis in the high dose group did not differ significantly between D0-D3, between D1-D5, between D3-D6, and between D7-D12 (p>0.05), but differed significantly between D4-D12 and D0, between D6-D12 and D0-D2, and between D7-D12 and D0-D6 (p<0.05), indicating that cows with mastitis in the high dose group generally improved at D4, the number of cows cured increased significantly at D6, and several cows were cured at D7. Survey scores of clinical symptoms in dairy cows with mastitis in the control group did not differ significantly between D0-D4, D2-D6 and D3-D12 (p>0.05), but significantly differed between D5-D12 and D0-D4 and between D7- D12 and D0-D6 (p<0.05), indicating that in the control group cows improved at D5 and a certain number of cows were cured at D7.

The clinical symptom survey score in the medium dose group on D0 was significantly higher than in the low dose group and in the high dose group (p<0.05), indicating that the disease in cows in the medium dose group was more severe; the score of clinical symptoms in the middle dose group on D1 was significantly higher than in the other 3 groups (p<0.05), the score of clinical symptoms in the middle dose group on D2 was significantly higher than in the low dose group and in the high dose group ( p<0.05), indicating that early in the treatment the medium dose group had a clear effect on improving clinical symptoms in cows with mastitis; differences in cow clinical symptom scores between the four groups on D3-D5 and D7 were not significant (p>0.05); the D12 clinical symptom score of cows in the control group was significantly higher than cows in the high dose group (p<0.05), indicating that the therapeutic effect in the control group is inferior to that in the group treated with pulsatilla.

Table 8. Evaluation of the examination of the clinical symptoms of dairy cows in each group

G N D0 D1 D2 D3 D4 D5 D6 D7 D12 L 12 1.58±0.67 ^A _B 1.42±0.51 ^A _B 1.17±0.58 ^AB _B 1.08±0.29 ^AB _A 1.08±0.29 ^AB _A 0.83±0.58 ^BC _A 0.83±0.83 ^BC _AB 0.33±0.89 ^C _{A 0.50} ±1.00 ^CAB M 12 2.92±2.11 ^A _A 2.67±1.61 ^A _A 2.17±1.11 ^AB _A 1.42± _0.90V ^A 1.08±0.67 ^BC _A 1.00±0.74 ^BC _A 0.50±0.52 ^C _B 0.33±0.49 ^C _A 0.25± _0.45 ^CAB H 12 1.50±0.67 ^A _B 1.33±0.65 ^AB _B 1.42±0.67 ^AB _B 1.17±0.58 ^ABC _A 1.00±0.43 ^BC _A 1.00±0.60 ^BC _A 0.83± _0.58 ^CAB 0.33±0.65 ^D _A 0.08± _0.29 ^DB C 12 _2.08 ±0.67A ^AB 1.83±0.72 ^AB _B 1.75±0.75 ^AB _AB 1.50±0.67 ^ABC _A 1.42±0.90 ^ABC _A 1.33±0.78 ^BC _A 1.17±0.83 ^BC _A 0.92±1.00 ^C _A 0.92±1.24 ^C _A

Note: "G" stands for "group", "N" stands for "number of cows", "L" stands for "low dose group", "M" stands for "medium dose group", "H" stands for "high dose group"; "C" stands for "control group"; And

In the same row, superscripts with different capital letters indicate significant differences (p<0.05), and superscripts with the same capital letters indicate no significant difference (p>0.05); in the same column, subscripts with different capital letters indicate significant differences (p<0.05) and subscripts with the same capital letters indicate no significant difference (p>0.05). The same applies to the table below.

4.3 Results of detection of somatic cells in cow's milk in each group

Table 9. Results of the detection of somatic cells in the milk of cured cows in each group (10 ⁴ /ml)

Group Number of cows D0 D7 D12 Low dose group 9 255.8±38.9 ^A _{A 90.5} ±55.8 ^BAB _47.5 ±35.6 ^BAB Medium dose group 9 244.6±42.0 ^A _A 65.8±51.5 ^B _B 38.1±23.0 ^B _B High dose group eleven 310.6±132.4 ^A _A 182.3±151.7 ^B _{A 48.8} ±58.9 ^CAB Control group 7 267.9±123.7 ^A _A 180.7±119.2 ^A _A 88.8±67.4 ^B _A

Table 9 shows that the number of somatic cells in the milk of cows in each group showed a downward trend by D0-D7. The difference between D7 and D12 in the low dose group, the medium dose group and the control group was not significant, but D7 was significantly different from D0, indicating that the number of somatic cells in milk improved significantly at D7 during drug treatment, and thereafter did not a relapse occurred. In the high dose group, D0, D7 and D12 were all significantly different, which, on the one hand, showed a significant improvement in the number of somatic cells in milk, and on the other hand, was also associated with a longer average recovery time for cows in this group. The difference in somatic cells in milk of the four groups of cows in D0 was not significant, indicating that in terms of somatic cells in milk, cows in each group had similar disease status at onset. At D7, somatic cell counts in milk were significantly lower in medium dose cows than in high dose and control cows, which was associated with a shorter mean cure time in the medium dose group. At D12 day, the number of somatic cells in the milk of cows in the control group was significantly higher than that of cows in the medium dose group, indicating that the therapeutic effect of injection of pulsatilla saponin B4 for mastitis of dairy cows is better than that of injection of SHUANGDING.

From the therapeutic effect of injection of pulsatilla saponin B4 in clinical dairy cow mastitis, the number of somatic cells in milk, the degree of clinical cure and observations of cure tests, it can be seen that all injections of pulsatilla saponin B4 at various concentrations have a clear therapeutic effect on clinical mastitis of dairy cows. , and this effect is better than the positive control, SHUANGDING injection. In addition, the injection of pulsatilla saponin B4 is non-toxic and does not cause drug resistance, and there is no need to throw away milk during treatment and at the end of treatment, which not only reduces the economic loss of farmers, but also reduces the risk of drug resistance.

Figures 2 and 3 show a comparison of milk from cows with clinical mastitis before and after treatment with pulsatilla saponin B4 injection. As can be seen from figures 2 and 3, cows with clinical mastitis had more flakes in their milk on the first day of treatment. As the treatment time increased, the amount of flakes gradually decreased and the milk gradually returned to normal. After the sixth day of treatment, the milk returned to normal. This demonstrated that pulsatilla saponin B4 injection had a significant effect on the treatment of clinical mastitis and improved milk quality.

Experimental Example 3 Study of Pulsatilla Saponin B4 According to the Present Invention for the Treatment of Persistent Clinical Mastitis and Latent Mastitis

1. Tested drugs

Pulsatilla saponin B4 injection, obtained by the production method according to example 3, at a concentration of 50 mg/ml;

2. Tested cows

и т.д.In this test, 8 cows suffering from clinical mastitis were selected from Shijiazhuang Tianquan Dairy Cows Co., Ltd. These 8 cows were treated for about a month, but were not cured (2 of them were almost lactated, i.e. almost did not produce milk). These 8 cows were treated by farm veterinarians, drugs and treatments included cefkinome sulfate infusion into the udder, intravenous injection of cephalosporins, and gastric tube with Gongying San

etc.

In this test, 6 cows with latent mastitis at Tianquan Dairy Farm were randomly selected, all of whom had at least one udder area of "++" or higher in the last LMT test performed by a veterinarian.

3. Content of the study

To study the effect of pulsatilla saponin B4 injection on persistent clinical mastitis and the effect of pulsatilla saponin B4 injection on latent mastitis.

4. Tested medicinal products, dosage and mode of administration

In this test, the injection of pulsatilla saponin B4, obtained by the preparation method of Example 3, was used to treat 6 cows with persistent clinical mastitis and 2 cows with persistent clinical mastitis and close to cessation of lactation. Regardless of how many areas of the udder were inflamed, an intramuscular injection (60 ml) was given once a day.

6 randomly selected cows with latent mastitis, regardless of how many areas of the udder were inflamed, were given an intramuscular injection (30 ml) once a day.

Table 10. Tested drugs, dosage and mode of administration

Test cow Number of cows Injection volume Mode of administration Persistent clinical mastitis 6 60 ml Intramuscular injection, once a day Almost complete cessation of lactation 2 60 ml Intramuscular injection, once a day Latent mastitis 6 30 ml Intramuscular injection, once a day Total 15 // Intramuscular injection, once a day

5. Test results

5.1 Outcome of treatment of cows with persistent clinical mastitis

In this test, 8 cows with clinical mastitis, which had not received treatment for a long time, were selected and treated. After 3 injections, four cows were cured, one cow showed a significant improvement, and one cow showed no significant effect; it was not effective in two cows close to lactation stop. The results of the study showed that saponin B4 of pulsatilla has a good effect on mastitis in dairy cows, the treatment of which with antibiotics or other means is not effective.

Results of treatment of some cows that have not received treatment for a long time: For the cow with ear tag number 8558, the cow's milk before treatment was thin and contained a small amount of flakes, and the result of the LMT test was "+++"; after a single intramuscular injection of 60 ml of pulsatilla saponin B4, there were no visible flakes in the milk, and the color of the milk was slightly yellowish. After the second injection, the milk returned to normal, and the result of the LMT test performed by the veterinarian was "++". The veterinarian then moved the cow to the stall.

The figure 4 shows the treatment of cow number 8558, which has not received treatment for a long time. From figure 4 it can be seen that as the treatment time increases, the secretion of milk in cow number 8558 gradually normalizes. After a 2-time injection, the milk returned to normal, the cow was clinically cured.

5.2 Treatment of cows with latent mastitis

According to the results of the LMT test performed by the farm veterinarian, 6 cows with latent mastitis were randomly selected, all of which had at least one udder area with "++" or higher. An injection of 30 ml of pulsatilla saponin B4 was administered by intramuscular injection. After three days, the results of the LMT test in 5 cows improved significantly. The other cow (#532006) improved slightly, but did not worsen.

Table 11. Latent Mastitis Test Results

Ear tag number Latest LMT results Somatic cells before the test LMT results three days after administration 131213 Both front right and rear right +++ 1323 Front right -, rear right ++ 0909041 Rear left ++, front right +++ 1294 Rear left +, front right - 131208 Front right +++, rear right ++ 847 Front right -, rear right - 160128 Four udder areas +++ 5450 Four udder areas +++ 532006 Four udder areas +++ 2041 Four udder areas +++ 1106021 Front left ++, rear left +++ - Front left -, rear left ++

Note: Since the farm does not have a somatic cell detector, the test must be performed on other farms. The dairy farm veterinarian did not perform somatic cell testing, but LMT test results after three days of administration showed significant improvement in 5 cows with latent mastitis.

Notes on individual cows: For cow #1106021, prior to administration, in which two areas of the udder on the left were affected by latent mastitis, the LMT test results on the anterior left and posterior left were "++" and "+++", respectively; rear right lactation completely stopped and milk was not allocated; the milk of the first three pressures on the right front was thick and yellow in color, and after several pressures, a bloody mucous liquid appeared without signs of milk. Pulsatilla saponin B4 30 ml was administered by intramuscular injection once a day, and after 3 days of administration there was no improvement in the anterior right, the results of the LMT test in the anterior left and posterior left were "-" and "++" respectively, the number of somatic cells decreased significantly and the effect was significant.

Another 5 cows suffering from latent mastitis were injected intramuscularly with 30 ml of pulsatilla saponin B4 once a day. After three days of treatment, the condition of 4 cows improved, and the results of LMT showed that the number of somatic cells decreased; the improvement in another cow (#532006) was not significant. In FIG. 5 shows the results of treating some cows suffering from latent mastitis after three days of treatment with an injection of pulsatilla saponin B4. From FIG. 5 shows that the condition of cows suffering from latent mastitis improved after taking the drug.

The results of the study showed that saponin B4 of pulsatilla has a good effect on latent mastitis.

в течение примерно месяца, но лечение было неэффективным, и болезнь перешла в хронический мастит, который проявлялся жидким молоком и постоянным скоплением хлопьев в молоке. Причина такой ситуации может заключаться в том, что: во-первых, разнообразные лекарственные средства для лечения мастита, применяемые на ферме, были простыми, в процессе лечения применяли только антибактериальные лекарственные средства, не уделяя внимания противовоспалительным; во-вторых, разнообразные применяемые антибиотики были простыми, и патогенные бактерии не выделяли и не идентифицировали, что приводило к устойчивости бактерий и отсутствию чувствительности к лекарственным средствам; в-третьих, длительное лечение приводило к гиперплазии ткани молочной железы, вызывая хроническое воспаление, что, в свою очередь, затрудняет попадание лекарственного средства в эффективную область, что делает состояние устойчивым. Когда инъекцию сапонина B4 применяли для лечения 8 коров, задействованных в эксперименте, после 3 введений четыре коровы были вылечены, у одной коровы наблюдали значительное улучшение, и одна корова не продемонстрировала значительного эффекта; он не был эффективным для двух коров, близких к прекращению лактации. Результаты исследования показали, что сапонин B4 пульсатиллы хорошо влияет на мастит у молочных коров, лечение которого с применением антибиотиков или других средств не является эффективным.Farm veterinarians treated sick cows with udder cefkinome sulfate infusion, intravenous cephalosporins, and Gongying San gavage

for about a month, but the treatment was ineffective, and the disease turned into chronic mastitis, which was manifested by liquid milk and a constant accumulation of flakes in the milk. The reason for this situation may be that: firstly, the variety of mastitis drugs used on the farm were simple, only antibacterial drugs were used in the treatment process, not paying attention to anti-inflammatory drugs; second, the variety of antibiotics used were simple and pathogenic bacteria were not isolated or identified, leading to bacterial resistance and lack of drug sensitivity; thirdly, long-term treatment resulted in breast tissue hyperplasia, causing chronic inflammation, which in turn makes it difficult for the drug to reach the effective area, making the condition stable. When saponin B4 injection was used to treat 8 cows involved in the experiment, after 3 injections, four cows were cured, one cow showed a significant improvement, and one cow showed no significant effect; it was not effective in two cows close to lactation stop. The results of the study showed that saponin B4 of pulsatilla has a good effect on mastitis in dairy cows, the treatment of which with antibiotics or other means is not effective.

Latent mastitis is one of the most common types of mastitis in dairy cows. This type of mastitis usually has no clinical symptoms, the difference between its milk and normal milk cannot be distinguished with the naked eye, and laboratory reagents are required for diagnosis. If this type of mastitis does not reach a certain proportion in the herd, it is usually left untreated. Due to latent morbidity, latent mastitis in dairy cows causes very serious economic losses. When pulsatilla saponin B4 injection was used to treat 6 cows suffering from latent mastitis, four cows improved; one cow showed a significant effect and reduction in somatic cell count in the LMT test; one cow had no significant improvement. The results of the study showed that saponin B4 of pulsatilla has a good effect on latent mastitis.

In addition, during the treatment of mastitis in dairy cows, a tested injection of pulsatilla saponin B4 was randomly selected, and the Sichuan Institute of Medicines Control was instructed to conduct more than 200 antibiotic tests, and the antibiotic component was not found. On the second day after administration to dairy cows, all milk can pass the on-farm antibiotic residue test. Pulsatilla saponin B4 has little effect on immunoglobulin and inflammatory factors in cow's milk, thus can guarantee the stability of the milk composition. In addition, the absence of residues will not affect the quality of fresh milk on dairy farms.

Currently, antibiotics are mainly used to treat mastitis. Among them, broad-spectrum antibiotics that disinfect and damage cell nucleic acids are more often used. During treatment and 4-5 days after treatment, milk must be discarded, resulting in large economic losses for farmers and the risk of drug resistance. Pulsatilla Saponin B4 is a natural active ingredient extracted from the Chinese medicinal herb Pulsatilla, is non-toxic and does not cause drug resistance. The pulsatilla saponin B4 compound of the present invention has a strong biological activity and an excellent therapeutic effect in dairy cow mastitis. There is no need to throw away milk during treatment and at the end of treatment, which not only reduces the economic loss to farmers, but also reduces the risk of drug resistance. In addition, pulsatilla saponin B4 not only has an excellent therapeutic effect in clinical mastitis, but also has an excellent therapeutic effect in persistent clinical mastitis and latent mastitis. At the same time, pulsatilla saponin injection has the advantages of safety, high efficacy, no residue, no drug resistance, and no toxic and side effects in the treatment of dairy cow mastitis.

Claims (11)

1. Use of the pulsatilla saponin B4 compound represented by formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of dairy cow mastitis:

2. The use according to claim 1, characterized in that the drug is a composition containing the pulsatilla saponin B4 compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient, and pharmaceutically acceptable excipients. 3. Use according to claim 2, characterized in that the composition is selected from the group consisting of injection, intramammary infusion, powder, ointment, lotion; preferably the formulation is selected from the group consisting of injection, intramammary infusion; more preferably the composition is an injection. 4. Use according to claim 3, characterized in that the mastitis of dairy cows is clinical mastitis, latent mastitis. 5. The use according to claim. 4, characterized in that the clinical mastitis is clinical mastitis, which is treated for the first time, persistent clinical mastitis. 6. The use of a drug for the treatment of mastitis in dairy cows, characterized in that the drug is a formulation containing a pulsatilla saponin B4 compound represented by formula I,

or its pharmaceutically acceptable salt as an active ingredient and pharmaceutically acceptable excipients, wherein the formulation is selected from the group consisting of injection and intramammary infusion, and wherein the concentration of pulsatilla saponin B4 compound in said formulation is 50 mg/mL. 7. Use according to claim 6, characterized in that the mastitis of dairy cows is clinical mastitis, latent mastitis; preferably, the clinical mastitis is first-time treated clinical mastitis, persistent clinical mastitis.

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