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WO2019149156A1 - 一种白头翁提取物在制备治疗病毒性和/或细菌性疾病的药物中的用途 - Google Patents

一种白头翁提取物在制备治疗病毒性和/或细菌性疾病的药物中的用途 Download PDF

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Publication number
WO2019149156A1
WO2019149156A1 PCT/CN2019/073239 CN2019073239W WO2019149156A1 WO 2019149156 A1 WO2019149156 A1 WO 2019149156A1 CN 2019073239 W CN2019073239 W CN 2019073239W WO 2019149156 A1 WO2019149156 A1 WO 2019149156A1
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Prior art keywords
mastitis
injection
treatment
cows
clinical
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PCT/CN2019/073239
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English (en)
French (fr)
Inventor
杨世林
苏柘僮
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四川英路维特医药科技有限公司
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Priority to CA3089522A priority Critical patent/CA3089522C/en
Priority to EP19746721.0A priority patent/EP3747445B1/en
Priority to RU2020126266A priority patent/RU2799050C2/ru
Priority to ES19746721T priority patent/ES2964618T3/es
Priority to PL19746721.0T priority patent/PL3747445T3/pl
Priority to BR112020015536-5A priority patent/BR112020015536A2/pt
Application filed by 四川英路维特医药科技有限公司 filed Critical 四川英路维特医药科技有限公司
Priority to AU2019214082A priority patent/AU2019214082B2/en
Priority to UAA202005100A priority patent/UA127576C2/uk
Priority to MX2020008047A priority patent/MX2020008047A/es
Priority to US16/966,855 priority patent/US20210038622A1/en
Publication of WO2019149156A1 publication Critical patent/WO2019149156A1/zh
Priority to US17/662,725 priority patent/US11793822B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of a Pulsatilla extract for the preparation of a medicament for the treatment of viral and/or bacterial diseases.
  • Pulsatilla Chinensis (Bge.) Regel is a genus Pulsatilla from the family Ranunculaceae. The main medicinal part is its dry root. The earliest record about Pulsatilla is "Shen Nong's Herbal Classic", which is bitter, cool, and belongs to the stomach, kidney, and large intestine. It is mainly used for eliminating phlegm, cooling blood, stopping diarrhea, and removing fire. It is traditionally used for bacterial dysentery. The treatment also has a good therapeutic effect on cold and fever, eye pain and so on.
  • Pulsatilla has a variety of pharmacological activities, which can improve the body's ability to recognize and resist foreign body invasion, reduce the growth rate of tumor cells, reduce the number of pathogenic microorganisms and effectively inhibit the oxidation of free radicals.
  • the most potential application is anti- Inflammatory anti-tumor new drug development.
  • Pulsatilla chinensis had a good induction effect on gastric cancer cells.
  • cancer cell proliferation and DNA replication are involved in the active constituents of Pulsatilla, which can inhibit the growth of HL260 cells and inhibit breast cancer (MCF-7), lung cancer (PG), and colon cancer (SW480) by inducing apoptosis.
  • Tumor cell proliferation such as malignant glioma cells (U87MG).
  • Li Wenchao et al. applied the alcohol extract of Pulsatilla chinensis to various pathogenic bacteria, and studied the antibacterial effects of different solvent extracts. It was found to have antibacterial effects but different effects.
  • Dairy cow mastitis is recognized as one of the three major diseases of cows in the world. It is often caused by lactation, and the main cause of it is pathogenic microbial infection. In addition, there are improper milking operations, mechanical damage caused by trauma and chemical damage. Mastitis not only affects milk production, causes economic losses, but also affects the quality of milk and endangers people's health.
  • Dairy cow mastitis can be divided into two categories: recessive mastitis and clinical mastitis.
  • Clinical mastitis can be seen with obvious clinical symptoms and visible changes in milk, according to the degree of disease can be divided into three levels: grade 1 mastitis: milk abnormalities; grade 2 mastitis: abnormal milk, breast redness and heat pain; level 3 breast Inflammation, abnormal milk, redness and heat in the breast, and symptoms of systemic infection in the cow.
  • Clinical mastitis is currently the main treatment target in the clinic. When clinical mastitis cows are exposed, isolation treatment is needed.
  • Recessive mastitis also known as subclinical mastitis, is the most common type of mastitis in dairy cows.
  • This type of mastitis generally has no clinical symptoms. The difference between breast and naked eye can not be seen with normal milk. Laboratory reagent testing is required. Can be diagnosed, and if this type of mastitis does not reach a certain proportion in the herd, it is generally not treated. It is because of its concealed concealment that the economic loss caused by recessive mastitis to cows is very serious.
  • the treatment of cow mastitis mostly uses breast perfusion, intravenous injection and other methods, the treatment drugs are mostly antibiotics, commonly cefquinome, enrofloxacin, penicillin, streptomycin and so on.
  • antibiotics commonly cefquinome, enrofloxacin, penicillin, streptomycin and so on.
  • long-term use of antibiotics is prone to drug resistance and affects the therapeutic effect.
  • when antibiotics are used it is easy to cause residual antibiotics, contaminate milk sources, and endanger human health.
  • lactam antibiotics will destroy the release of endotoxin from the cell wall, causing redness, swelling, heat and pain in the entire milk area, aggravating the condition. Therefore, it is important to find a safe and effective method for treating cow mastitis.
  • the present invention provides a use of a compound of the formula I, Pulsatilla saponin B4 or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment of viral and/or bacterial diseases:
  • the drug is a preparation prepared by using the aforementioned compound Pulsatilla saponin B4 or a pharmaceutically acceptable salt thereof as an active ingredient, together with a pharmaceutically acceptable adjuvant.
  • the preparation is an injection solution, a breast injection, a powder, a cream, a lotion; preferably, the preparation is an injection, a breast injection; more preferably, the preparation is an injection.
  • the disease is cow mastitis.
  • cow mastitis is clinical mastitis and recessive mastitis.
  • the clinical mastitis is a clinical type of mastitis that is initially treated, and a long-term clinical mastitis.
  • the present invention also provides a medicament for treating a viral and/or bacterial disease, which is prepared by using the aforementioned compound Pulsatilla saponin B4 or a pharmaceutically acceptable salt thereof as an active ingredient, together with a pharmaceutically acceptable adjuvant. Preparation.
  • the preparation is an injection solution, a breast injection, a powder, a cream, a lotion; preferably, the preparation is an injection, a breast injection; more preferably, the preparation is an injection.
  • the disease is cow mastitis.
  • cow mastitis is clinical mastitis, recessive mastitis; preferably, the clinical mastitis is a clinically-type mastitis for initial treatment, and a long-term clinical mastitis.
  • the extract of Pulsatilla chinensis provided by the present invention is a compound Pulsatilla saponin B4, and its Chinese name is Pulsatilla saponin B4, and its English name is Anemoside B4. Its CAS number is 129741-57-7, its molecular formula is C 59 H 96 O 26 , its molecular weight is 1221.38, its trait is white crystalline powder.
  • Pulsatilla saponin B4 is a natural active ingredient extracted from the Chinese medicinal material Pulsatilla chinensis. It is non-toxic and non-resistant.
  • the compound of the present invention Pulsatilla saponin B4 has strong biological activity and has excellent therapeutic effect on cow mastitis during treatment. There is no need to abandon milk at the end of treatment, which not only reduces the economic loss for the farmers, but also reduces the risk of drug resistance.
  • Pulsatilla saponin B4 not only has excellent therapeutic effects on clinical mastitis, but also has excellent therapeutic effects on long-term treatment and recessive mastitis.
  • Pulsatilla saponin injection has the characteristics of safe, high-efficiency, no residue, no drug resistance and no toxic side effects in the treatment of cow mastitis.
  • Figure 1 is a standard curve for the determination of the content of Pulsatilla saponin B4.
  • Figure 2 shows the comparison of milk before and after treatment with Pulsatilla saponin B4 injection in dairy cows with clinical mastitis.
  • Figure 3 shows the comparison of milk before and after treatment with Pulsatilla saponin B4 injection in dairy cows with clinical mastitis.
  • Figure 4 shows the treatment of 8558 long-term unhealed cows.
  • Figure 5 shows the treatment of cows with recessive mastitis.
  • Pulsatilla chinensis Take 100kg of Pulsatilla chinensis, add 10 times 70% ethanol and reflux for 2 times.
  • the extract is concentrated under reduced pressure at 75 °C, centrifuged at 4000r/min for 10min, and the supernatant is passed through a polar macroporous adsorption resin column, followed by water, 30% ethanol.
  • Agilent 1260 High Performance Liquid Chromatograph and DAD UV Detector were purchased from Agilent Technologies (China) Co., Ltd., BP211D electronic analytical balance was purchased from Sartorius, Germany, and KQ-400DB CNC ultrasonic cleaner was purchased from Kunshan Ultrasonic Instrument Co., Ltd. .
  • Pulsatilla saponin B4 was prepared in Example 1.
  • Pulsatilla saponin B4 reference substance (batch number: 111766-201702, content: 94.7%), purchased from China Food and Drug Testing Institute.
  • Pulsatilla saponin B4 reference substance Take the appropriate amount of Pulsatilla saponin B4 reference substance, accurately weighed, placed in a 10mL volumetric flask, add mobile phase to dissolve, dilute to the scale, that is, the reference solution (1mg of Pulsatilla saponin B4 per 1mL).
  • Pulsatilla saponin B4 Take the reference material of Pulsatilla saponin B4, accurately weighed, placed in a 10mL volumetric flask, add mobile phase to dissolve, dilute to volume, and then prepare concentrations of about 1mg/mL, 5mg/mL, 10mg/mL, 15mg/mL and 20mg /mL of the reference solution, each precision amount of 20 ⁇ L, into the liquid chromatograph, record the chromatogram.
  • Pulsatilla saponin B4 was prepared as a 1000 mL breast implant.
  • Test Example 1 Effect of different administration routes of the compounds of the present invention on the therapeutic effect of cow mastitis
  • breast perfusion is the most common route of administration, and the therapeutic drug is directly injected into the diseased milk area through the nipple to achieve the effect of local medication and precise treatment.
  • therapeutic drug is directly injected into the diseased milk area through the nipple to achieve the effect of local medication and precise treatment.
  • commercial pharmaceutical preparations for breast perfusion such as Aussie Oriental (Cefosporin Sulfate Mammal Injection) from Oriental Aolong Company, and Milky (Ceficidin Injection) from Pfizer Inc. of the United States.
  • Intramuscular injection is a therapeutic effect by injecting a drug into the muscle tissue of an animal and reaching the diseased milk area through blood circulation. Intramuscular injection is convenient, and it can control the symptoms of systemic inflammation and fever caused by mastitis to some extent.
  • Intravenous injection is to directly inject the drug into the venous blood of the animal, and the blood reaches the diseased milk area through the blood circulation.
  • the drug reaches the effective part quickly, and can treat the systemic symptoms, but has certain requirements for the drug trait, and cannot be a suspension. It is less irritating to blood vessels.
  • Bayer Company of Germany recommends that its products be beneficial (ennofloxacin preparation) to treat mastitis by intravenous injection.
  • the drug can reach a higher concentration in the affected milk area, and it can treat the whole body. .
  • the Pulsatilla saponin B4 injection prepared in Example 3 was at a concentration of 50 mg/mL; the Pulsatilla saponin B4 breast injection prepared in Example 5 was at a concentration of 50 mg/mL.
  • cefquinome sulfate breast injection 8g / support (Foshan Nanhai Dongfang Aolong Pharmaceutical Co., Ltd.).
  • Intramuscular injection of diseased milk area local intramuscular injection of the diseased milk area with Pulsatilla saponin B4 injection, once every morning and evening, for 7 days;
  • Pulsatilla saponin B4 injection is administered intravenously once a day for 7 days;
  • Pulsatilla saponin B4 breast injecting agent and positive drug cefquinome sulfate breast injecting agent are administered by intraductal injection in the affected milk area, once every morning and evening, for 7 days.
  • Each of the administration routes of the Pulsatilla saponin B4 of the present invention is each provided with a blank control group, and administered in the same dose of physiological saline as the administration amount.
  • Clinical cure The clinical symptoms such as redness, swelling, heat and pain in the diseased milk area have basically disappeared, and lactation has basically returned to normal.
  • the cure rate is calculated as:
  • the overall cure rate and cure time of different administration routes of Pulsatilla saponin B4 are shown in Table 4. Different routes of administration have significant effects in the treatment of cow mastitis, and the cure rate of high dose groups of each route is higher than that of positive drugs.
  • Pulsatilla saponin B4 injection prepared by the preparation method of Example 3 was at a concentration of 50 mg/mL.
  • control drug was double Ding injection (10 mL/branch, batch number: DV3180701), Hebei Yuanzheng Pharmaceutical Co., Ltd.
  • test cows were recorded as D0 before the onset of the disease, and the doses were calculated according to the body weight according to the pre-designed drug-administered variety and the dose (Table 5).
  • Table 5 On the D1 day, each group of cows with clinical mastitis began.
  • the daily administration was carried out by intramuscular injection, once a day, the test drug (Pulsatilla saponin B4 injection) and the control drug (double injection) were continued for 4 to 7 days, depending on the cure.
  • the clinical symptoms of each group of cows before and after treatment (D0 ⁇ D12) were observed and scored according to the standard (see Table 6 for details), and the drug administration records were made.
  • D12 the results of the above indicators were combined to determine the efficacy of each cow according to the criteria, and the cure rate and effectiveness of each group of cows were calculated.
  • the milk of the clinical type of mastitis cows was collected in the tested D0, D7, and D12, and the somatic cell number was detected and recorded.
  • the cure rate for daily treatment on days 2-7 starting at D2, if the mastitis cow meets the sum of the scores ⁇ 1, it is considered to be cured.
  • the cure rate in 7 days is the sum of the cure rates for daily treatment on days 2-7.
  • Effectiveness of daily treatment on days 2-7 At the beginning of D2, if the cow with mastitis meets the sum of 1 ⁇ score ⁇ 2, it is considered to be effective. The effective rate within 7 days is the sum of the effective rates of daily treatment on days 2-7.
  • Recurrence rate During the period from D5 to D12, if the diseased cow was judged to be successful in treatment, the sum of the scores >2 after withdrawal was considered as recurrence.
  • the cure rate of the treatment on the 12th day (total cure rate): At D12, if the cow with mastitis meets the sum of the scores ⁇ 1, it is considered to be cured on the 12th day.
  • Effectiveness of treatment on day 12 (total efficiency): At D12, if the cow with mastitis meets the sum of 1 ⁇ score ⁇ 2, it is considered therapeutically effective.
  • D6-D12 and D0-D3 were significant (p ⁇ 0.05), which indicated that the middle dose group had a significant improvement in D3 and significantly recovered at D6.
  • the clinical symptom scores of high-dose group mastitis cows were not significantly different between D0-D3, D1-D5, D3-D6, and D7-D12 (p>0.05), but in D4-D12.
  • the results showed that the high-dose group of mastitis cows generally began to improve at D4, at D6.
  • the number of cows that have healed has increased significantly, and some cows have recovered again at D7.
  • the clinical symptom scores of the mastitis cows in the control group were not significantly different between D0-D4, D2-D6, and D3-D12 (p>0.05), but between D5-D12 and D0-D4, The difference between D7-D12 and D0-D6 was significant (p ⁇ 0.05). The results showed that the control cows improved at D5 and healed a certain number of cows at D7.
  • the clinical symptom score of the middle dose group at D0 was significantly higher than that of the low dose group and the high dose group (p ⁇ 0.05), indicating that the incidence of cows in the middle dose group was more serious; the score of the clinical symptom check in the middle dose group at D1 was significantly higher than that of the other three groups ( p ⁇ 0.05), the clinical symptom score of the middle dose group was significantly higher than that of the low dose group and the high dose group (p ⁇ 0.05), indicating that in the early stage of treatment, the middle dose group cows have obvious clinical symptoms for improving mastitis cows.
  • the sows in the middle dose group were significantly lower than those in the high dose group and the control group.
  • the average number of days of cure was shorter.
  • the body cells of the cows in the control group were significantly higher than those in the middle dose group, indicating that the treatment of cow mastitis with Pulsatilla saponin B4 injection was better than that of Dingding injection.
  • Pulsatilla saponin B4 injection The effect of Pulsatilla saponin B4 injection on the clinical mastitis of cows, the number of breast cells, clinical cure rate and cure test showed that different concentrations of Pulsatilla saponin B4 injection have obvious therapeutic effects on clinical mastitis in dairy cows.
  • the positive control drug Shuangding injection; and Pulsatilla saponin B4 injection is non-toxic, non-resistant, no need to abandon milk during treatment and treatment, not only reduces the economic loss for the farmers, but also reduces the drug resistance. The risks generated.
  • Figures 2 and 3 show the comparison of milk before and after treatment with Pulsatilla saponin B4 injection in dairy cows with clinical mastitis. It can be seen from Fig. 2 and Fig. 3 that on the first day of treatment, there are more flocs in the milk of clinical mastitis cows. As the treatment time increases, the flocs gradually decrease, and the milk gradually returns to normal. After the sixth day of treatment The milk returned to normal. It shows that Pulsatilla saponin B4 injection has a significant effect on the treatment of clinical mastitis and improving milk quality.
  • Test Example 3 Study on the treatment of long-term treatment of clinical mastitis and recessive mastitis by Pulsatilla saponin B4 of the present invention
  • Pulsatilla saponin B4 injection prepared by the preparation method of Example 3 was at a concentration of 50 mg/mL.
  • Dairy Farm has 8 clinical mastitis cows, which are treated for a month or so of long-term treatment (two close to the milk). These 8 cows have received pasture veterinarians.
  • Therapeutic, therapeutic drugs and methods include cefquinome sulfate breast injection, milk area perfusion, cephalosporin injection intravenous injection and male and female scattered irrigation.
  • the experiment randomly selected 6 cows from the Tianquan dairy cattle pasture mastitis, which were all veterinarians' LMT test results at least one diseased milk area "++" and above.
  • Pulsatilla saponin B4 injection was carried out by the preparation method of Example 3.
  • the treatment of 6 long-term treatments and 2 long-term cures of clinical mastitis cows close to sputum milk were treated with intramuscular injection of 60 mL, regardless of inflammation in several breast areas. Times/day.
  • FIG. 5 shows the treatment of some cows with recessive mastitis after three days of treatment with Pulsatilla saponin B4 injection. As can be seen from Figure 5, the condition of cows with recessive mastitis improved after administration.
  • Pulsatilla saponin B4 has a good effect on recessive mastitis.
  • the sick cows were treated by the veterinarian with cefquinome sulfate breast injection, intravenous injection of cephalosporin injection and male and female scattered dressing for about one month, but no effect, and then turned into chronic mastitis, performance
  • the milk is thin and continues to contain a large amount of floc.
  • the reason may be: on the one hand, the farm uses a single drug for the treatment of mastitis, and only the antibacterial drug is used during the treatment without paying attention to anti-inflammatory; on the other hand, the antibiotic type is single.
  • Recessive mastitis is one of the most common types of mastitis in dairy cows. Because it usually has no clinical symptoms, the difference between the milk and the normal milk can not be seen by the naked eye. It needs to be tested by laboratory reagents to diagnose this type of mastitis. If a certain proportion is not reached in the herd, it is generally not treated, and it is precisely because of its concealed disease that the economic loss to the cow is also the most serious.
  • Pulsatilla saponin B4 injection When using Pulsatilla saponin B4 injection to treat 6 cows with recessive mastitis, 4 cows improved; 1 LMT detected somatic cells decreased, and there was significant effect; 1 cow improved better, the results showed that: Pulsatilla saponin B4 was applied Recessive cow mastitis has a good effect.
  • the randomized sampling test was carried out with the Pulsatilla saponin B4 injection to entrust the Sichuan Provincial Veterinary Drug Surveillance Institute to carry out more than 200 antibiotic tests, and no antibiotics were detected; the second day after the cows were administered, in the cattle farm During the anti-test process, the milk can pass the antibiotic residue detection in the cattle farm; the Pulsatilla saponin B4 has less effect on the immunoglobulin and inflammatory factors in the milk of the cow, which can ensure the stability of the milk composition, no residue, and will not affect the dairy farm. Standing on the quality of fresh milk.
  • Pulsatilla saponin B4 is a natural active ingredient extracted from the Chinese medicinal material Pulsatilla chinensis. It is non-toxic and non-resistant.
  • the compound of the present invention Pulsatilla saponin B4 has strong biological activity and has excellent therapeutic effect on cow mastitis during treatment. There is no need to abandon milk at the end of treatment, which not only reduces the economic loss for the farmers, but also reduces the risk of drug resistance.
  • Pulsatilla saponin B4 not only has excellent therapeutic effects on clinical mastitis, but also has excellent therapeutic effects on long-term treatment and recessive mastitis.
  • Pulsatilla saponin injection has the characteristics of safe, high-efficiency, no residue, no drug resistance and no toxic side effects in the treatment of cow mastitis.

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Abstract

一种式（Ⅰ）所示的化合物白头翁皂苷B4或其药学上可接受的盐在制备治疗病毒性和/或细菌性疾病的药物中的用途，以及以所述式（Ⅰ）所示的白头翁皂苷B4或其药学上可接受的盐为活性成分的药物，所述疾病为奶牛乳房炎。

Description

一种白头翁提取物在制备治疗病毒性和/或细菌性疾病的药物中的用途 技术领域
本发明涉及一种白头翁提取物在制备治疗病毒性和/或细菌性疾病的药物中的用途。
背景技术
白头翁Pulsatilla Chinensis(Bge.)Regel是毛莨科(Ranunculaceae)白头翁属植物，主要的药用部位是其干燥根。最早关于白头翁的记录是《神农本草经》，其味苦，性凉，归胃、肾、大肠经，功效以消赘、凉血、止泻、去火等为主，传统用于细菌性痢疾的治疗，对寒热温症、眼目赤痛等也有较好的治疗效果。白头翁的药理活性多样，主要可提高机体识别和抵御外来异物入侵的能力，降低肿瘤细胞的繁殖速率，减少病原微生物的数量及有效抑制自由基的氧化反应等，其中，最具有潜力应用是在抗炎抗肿瘤的新药开发方面。
冯秀芝等对白头翁在细胞凋亡方面的作用做了研究，认为白头翁对胃癌细胞有较好的诱导作用。此外，癌细胞增生及DNA复制与白头翁有效成分有关，可以对HL260细胞的生长起到抑制作用，并通过诱导凋亡来抑制乳腺癌(MCF-7)、肺癌(PG)、结肠癌(SW480)、恶性胶质瘤细胞(U87MG)等肿瘤细胞增生。李文超等将白头翁醇提物作用于各致病菌种，并研究了不同溶剂萃取物的抑菌作用，发现均有抑菌作用但效果不同。
奶牛乳房炎是世界公认的奶牛三大疾病之一，泌乳期多发，其发生的主要原因是病原微生物感染，此外还有挤奶操作不当、外伤造成的机械性损伤和化学试剂损伤等。乳房炎不仅影响产奶量，造成经济损失，而且影响乳的品质，危害人的健康。
奶牛乳房炎可分为隐性乳房炎和临床型乳房炎两大类。临床型乳房炎可见明显的临床症状和乳汁的肉眼可见变化，根据发病程度又可分为三级：1级乳房炎：牛奶异常；2级乳房炎：牛奶异常，乳房红肿热痛；3级乳房炎，牛奶异常，乳房红肿热痛，奶牛出现全身感染症状。临床型乳房炎是目前临床中主要的治疗对象，当揭发出临床型乳房炎奶牛时，需进行隔离治疗。隐性乳房炎又称为亚临床乳房炎，是奶牛发病最普遍的乳房炎类型，该类型乳房炎一般无临床症状表现，乳汁肉眼不可看出与正常乳汁的区别，需进行实验室试剂检测才可诊断，且该类乳房炎如果在牛群中未达到一定比例，一般不予治疗，正是因为其发病隐蔽，隐性乳房炎对奶牛造成的经济损失十分严重。
目前，对于奶牛乳房炎的治疗多采用乳房灌注、静脉注射等方法，治疗药物多为抗生素类药物，常见有头孢喹肟、恩诺沙星、青霉素、链霉素等。但长期使用抗生素容易产生耐药性，影响治疗效果；同时，使用抗生素治疗时，容易造成抗生素残留，污染奶源，危 害人体健康。值得注意的是，若乳房炎为革兰氏阴性菌感染时，使用内酰胺类抗生素治疗，会破坏细胞壁释放内毒素，造成整个乳区的红、肿、热、痛，加重病情。因此，寻找一种安全且有效的治疗奶牛乳房炎的方法具有重要意义。
目前，未见关于本发明化合物用于治疗病毒性和/或细菌性奶牛乳房炎的报道。
发明内容
为了解决上述技术问题，本发明提供了一种如式Ⅰ所示的化合物白头翁皂苷B4或其药学上可接受的盐在制备治疗病毒性和/或细菌性疾病的药物中的用途：
Figure PCTCN2019073239-appb-000001
进一步地，所述药物是以前述化合物白头翁皂苷B4或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
进一步地，所述制剂为注射液、乳房注入剂、散剂、膏剂、洗剂；优选的，所述制剂为注射液、乳房注入剂；更优选的，所述制剂为注射液。
进一步地，所述疾病为奶牛乳房炎。
进一步地，所述奶牛乳房炎为临床型乳房炎、隐性乳房炎。
进一步地，所述临床型乳房炎为初次治疗的临床型乳房炎、久治不愈的临床型乳房炎。
本发明还提供了一种治疗病毒性和/或细菌性疾病的药物，它是以前述化合物白头翁皂苷B4或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
进一步地，所述制剂为注射液、乳房注入剂、散剂、膏剂、洗剂；优选的，所述制剂为注射液、乳房注入剂；更优选的，所述制剂为注射液。
进一步地，所述疾病为奶牛乳房炎。
进一步地，所述奶牛乳房炎为临床型乳房炎、隐性乳房炎；优选的，所述临床型乳房炎为初次治疗的临床型乳房炎、久治不愈的临床型乳房炎。
本发明提供的白头翁提取物为化合物白头翁皂苷B4，其中文别名为白头翁皂甙B4，其英文名称为Anemoside B4。其CAS号为129741-57-7，其分子式为C 59H 96O 26，其分子量为1221.38，其性状为白色结晶状粉末。
目前，乳房炎治疗大多采用抗生素，其中又以广谱杀菌、破坏细胞核酸类抗生素较多常用，在治疗期间和治疗结束后4～5天需要弃奶，为养殖户造成较大经济损失，并有产生耐药性的风险。白头翁皂苷B4是从中药材白头翁中提取分离的天然有效成分，无毒，无耐药性，本发明的化合物白头翁皂苷B4具有较强的生物活性，对奶牛乳房炎具有优异的治疗效果，在治疗期间和治疗结束均不需弃奶，不仅为养殖户减少了经济损失，同时也减少了耐药性产生的风险。此外，白头翁皂苷B4不仅对临床型乳房炎具有优异的治疗效果，对久治不愈和隐性乳房炎也有优异的治疗效果。同时，白头翁皂苷注射液应用于奶牛乳房炎的治疗时具有安全、高效、无残留、无耐药性、无毒副作用的特点。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为白头翁皂苷B4含量测定的标准曲线。
图2为患临床型乳房炎的奶牛使用白头翁皂苷B4注射液治疗前后乳汁对比。
图3为患临床型乳房炎的奶牛使用白头翁皂苷B4注射液治疗前后乳汁对比。
图4为8558号久治不愈奶牛的治疗情况。
图5为隐性乳房炎奶牛的治疗情况。
具体实施方式
实施例1、本发明化合物的制备
取白头翁药材100kg，加10倍量70％乙醇加热回流提取2次，提取液75℃减压浓缩，4000r/min离心10min，上清液过极性大孔吸附树脂柱，依次用水、30％乙醇、70％乙醇洗脱，取70％乙醇洗脱部位75℃减压浓缩后，喷雾干燥得白头翁总皂苷提取物3450g；取提取物加水溶解后，滤液上动态轴向200制备色谱系统(填料为10μm的ODS)，采用50％ 甲醇洗脱，根据色谱峰收集相应的洗脱液，减压浓缩后，冷冻干燥得白头翁皂苷B4 1700g。
实施例2、本发明化合物的含量测定
1、仪器及试药
Agilent 1260型高效液相色谱仪、DAD紫外检测器购于安捷伦科技(中国)有限公司，BP211D型电子分析天平购自德国Sartorius公司，KQ-400DB型数控超声波清洗器购于昆山市超声仪器有限公司。
白头翁皂苷B4样品，实施例1制备。
白头翁皂苷B4对照品(批号：111766-201702，含量：94.7％)，购于中国食品药品检定研究院。
2、色谱条件
色谱柱：Sepax Bio-C18(4.6×250mm，5μm)；流动相：甲醇-水(64：36)；检测波长为201nm；流速：1.0mL/min。
3、对照品溶液的配制
取白头翁皂苷B4对照品适量，精密称定，置于10mL容量瓶中，加流动相溶解，定容至刻度，即得对照品溶液(每1mL含1mg白头翁皂苷B4)。
4、样品溶液的配制
取实施例1制备的白头翁皂苷B4样品10mg，精密称定，置于10mL容量瓶中，加流动相溶解，定容至刻度，即得样品溶液。
5、测定法
分别精密量取对照品溶液和样品溶液各20μL，注入液相色谱仪，记录色谱图，按外标法计算含量。
6、标准曲线和线性回归方程
取白头翁皂苷B4对照品，精密称定，置于10mL容量瓶中，加流动相溶解，定容至刻度，依次配制浓度约为1mg/mL、5mg/mL、10mg/mL、15mg/mL和20mg/mL的对照品溶液，各精密量取20μL，注入液相色谱仪，记录色谱图。
表1 白头翁皂苷B4对照品含量测定线性回归方程
Figure PCTCN2019073239-appb-000002
由实验结果可知，白头翁皂苷B4在浓度为1.04～20.80mg/mL范围内，线性关系良好，R＝0.9999，适用于白头翁皂苷B4的含量测定。
7、白头翁皂苷B4样品含量测定
按照前述“3”所述方法，取白头翁皂苷B4对照品2份，各10mg，精密称定，置于10mL容量瓶中，加流动相溶解，定容至刻度，即得对照品溶液1和对照品溶液2。
按照前述“4”所述方法，取白头翁皂苷B4样品2份，各10mg，精密称定，置于10mL容量瓶中，加流动相溶解，定容至刻度，即得样品溶液1和样品溶液2。
按照上述色谱条件各精密量取20μL，注入液相色谱仪，记录色谱图。
表2 白头翁皂苷B4含量测定结果
Figure PCTCN2019073239-appb-000003
由试验结果可知，本实验所得白头翁皂苷B4的含量为99.4％。
实施例3、本发明白头翁皂苷B4注射液的制备
1)处方：50g白头翁皂苷B4加水制备成1000mL的注射液。
2)制备方法：称取实施例1制备的白头翁皂苷B4 50g，加注射用水800mL，使其完全溶解，过滤，调pH至7.0，加注射用水至1000mL，精滤，灌装，封口，100℃灭菌30min，即得。
实施例4、本发明白头翁皂苷B4散剂的制备
1)处方：1000g白头翁皂苷B4制备成1000g的散剂。
2)制备方法：称取实施例1制备的白头翁皂苷B4 1000g，粉碎成细粉，过100目筛，混匀，即得。
实施例5、本发明白头翁皂苷B4乳房注入剂的制备
1)处方：50g白头翁皂苷B4制备成1000mL的乳房注入剂。
2)制备方法：称取实施例1制备的白头翁皂苷B4 50g，加蒸馏水800mL，使其完全溶解，过滤，调pH至7.0，加蒸馏水至1000mL，精滤，灌装，封口，100℃灭菌30min， 即得。
以下通过试验例的方式来说明本发明的有益效果。
试验例1、本发明化合物不同给药途径对奶牛乳房炎治疗效果的影响
目前治疗乳房炎的给药途径有乳房灌注、肌内注射和静脉注射。
其中，乳房灌注是最常见的给药途径，将治疗药物通过乳头直接注入到患病乳区，达到局部用药，精准治疗的效果。现已有多种用于乳房灌注的商品化药物制剂，如东方澳龙公司的澳乃康(硫酸头孢喹肟乳房注入剂)、美国辉瑞公司的乳畅(头孢噻呋注入剂)等。
肌内注射为将药物通过注入到动物肌肉组织中，经血液循环到达患病乳区，起到治疗效果。肌内注射给药方便，且能够在一定程度上控制由乳房炎引起的全身性的炎症、发热等症状。
静脉注射为将药物直接注入到动物静脉血中，经血液循环到达患病乳区，药物到达有效部位速度快，且能够治疗全身症状，但对药物的性状有一定要求，不能为悬浊液，对血管刺激性小。德国拜尔公司推荐其产品拜有利(恩诺沙星制剂)通过静脉注射的方式治疗乳房炎，药物在患病乳区能达到较高的浓度，起到治疗的效果，同时可控制全身的感染。
以上三种治疗奶牛乳房炎的给药途径各有优缺点，本试验旨在探究白头翁皂苷B4对奶牛乳房炎的治疗作用及最适宜的给药途径，此研究结果将会对如何使白头翁皂苷B4发挥最佳治疗效果、及药物之后的研发和包装提供重要的参考。
1、受试药物
实施例3制备的白头翁皂苷B4注射液，浓度为50mg/mL；实施例5制备的白头翁皂苷B4乳房注入剂，浓度为50mg/mL。
阳性药物：硫酸头孢喹肟乳房注入剂，8g/支(佛山市南海东方澳龙制药有限公司)。
2、受试动物
选取患病奶牛(2级临床型乳房炎)130头，随机分成13组，每组10头奶牛。
3、给药剂量与方式
表3 给药剂量与给药方式
Figure PCTCN2019073239-appb-000004
Figure PCTCN2019073239-appb-000005
患病乳区肌内注射给药：用白头翁皂苷B4注射液对患病乳区进行局部肌内注射，每天早晚各一次，连用7天；
静脉注射给药：将白头翁皂苷B4注射液通过静脉注射给药，每天一次，连用7天；
乳房注入给药：白头翁皂苷B4乳房注入剂和阳性药硫酸头孢喹肟乳房注入剂采用患病乳区乳管内注入给药，每天早晚各一次，连用7天。
本发明白头翁皂苷B4的每种给药途径各设置一组空白对照组，以给药量同等剂量生理盐水给药。
4、评价指南
每次给药前，检查奶牛体温、呼吸、精神、采食状况等指标，观察乳房和乳汁的变化。
完全治愈：患病乳区红、肿、热、痛等临床症状完全消失，泌乳恢复正常。
临床治愈：患病乳区红、肿、热、痛等临床症状基本消失，泌乳基本恢复正常。
无效：患病乳区临床症状无明显改善甚至加重，或者停药两周内复发，两者均视为无效。
治愈率的计算方式为：
Figure PCTCN2019073239-appb-000006
5、实验结果
表4 不同给药途径治疗奶牛乳房炎的治愈率及治愈时间
Figure PCTCN2019073239-appb-000007
Figure PCTCN2019073239-appb-000008
白头翁皂苷B4不同给药途径总体治愈率及治愈时间结果见表4：不同给药途径用于治疗奶牛乳房炎时均有显著效果，且各给药途径的高剂量组治愈率均高于阳性药物硫酸头孢喹肟乳房注入剂，其中白头翁皂苷B4注射液肌内注射治愈效果最好，治疗周期最短。
试验例2、本发明白头翁皂苷B4治疗临床型乳房炎的研究
1、受试药物
采用实施例3制备方法制备的白头翁皂苷B4注射液，浓度为50mg/mL。
对照药物为双丁注射液(10mL/支，批号：DV3180701)，河北远征药业有限公司。
2、受试动物
选择体重612±47kg左右，年龄3-5周岁，2-4胎次，临床检查健康、泌乳期自然发生临床型乳房炎的中国荷斯坦奶牛60头，随机分为5组，每组12只。
3、试验方法
3.1给药剂量与方式
表5 给药剂量与给药方式
Figure PCTCN2019073239-appb-000009
试验奶牛发病后给药前记为D0，根据预先设计的给药品种和给药剂量(表5)，按体重计算给药量，在D1日开始对各组患有临床乳房炎的奶牛，每日按肌内注射的方法给药，1次/日，试验药物(白头翁皂苷B4注射液)和对照药物(双丁注射液)持续用药4～7天，视治愈情况而定。观察用药前后(D0～D12日)各组奶牛的临床症状，并按标准进行评分(详见表6)，并做好给药记录。在试验结束时(D12日)，综合上述各项指标观测结果，按标准判定每头奶牛的疗效，计算各组奶牛的治愈率和有效率。
3.2患临床型乳房炎的奶牛临床症状检查评分标准
分别在D0(揭发临床乳房炎后，给药前)、D1(初次给药后，第二次给药前)、D2～D7(痊愈的奶牛停药，未痊愈的奶牛继续给药)、D8～D12(所有奶牛停药)，定时检查各奶牛的临床症状和乳房局部症状，如精神状态、食欲饮欲等，按下表标准进行评分(见表 6)，做好临床症状记录(乳房局部症状评分)。
表6 患临床型乳房炎的奶牛临床症状检查评分标准表
Figure PCTCN2019073239-appb-000010
3.3奶牛乳汁体细胞数检测方法
分别在试验的D0、D7、D12采集试验患临床型乳房炎奶牛的乳汁，进行体细胞数检测并做好记录。
3.4疗效判定
根据临床症状检查评分标准判断
第2-7天每日治疗的治愈率：在D2开始，如果乳房炎奶牛满足评分总和＜1，则视为治疗痊愈。
Figure PCTCN2019073239-appb-000011
7天内治愈率为第2-7天每日治疗的治愈率之和。
第2-7天每日治疗的有效率：在D2开始，如果患乳房炎的奶牛满足1≤评分总和≤2，则视为治疗有效。
Figure PCTCN2019073239-appb-000012
7天内有效率为第2-7天每日治疗的有效率之和。
复发率：在D5～D12期间，如果判定为治疗成功的患病奶牛，停药后满足评分总和＞2，则视为复发。
Figure PCTCN2019073239-appb-000013
第12天治疗的治愈率(总治愈率)：在D12，如果患乳房炎的奶牛满足评分总和＜1，则视为第12天治愈。
Figure PCTCN2019073239-appb-000014
第12天治疗的有效率(总有效率)：在D12，如果患乳房炎的奶牛满足1≤评分总和≤2，则视为治疗有效。
Figure PCTCN2019073239-appb-000015
以治愈率为主要指标，以有效率、临床症状评分、体细胞数为次要指标，综合评价受试药物(白头翁皂苷B4注射液)对自然感染的奶牛临床乳房炎的治疗效果，并与对照药物双丁注射液的治疗效果进行比较，确定受试药物的临床推荐用药剂量。
4、试验结果
4.1各组临床型乳房炎奶牛治疗效果结果
由表7可见，白头翁皂苷B4注射液治疗奶牛临床乳房炎，不论低、中、高剂量，其七天内治愈率和总治愈率均大于使用双丁注射液的对照组，平均治愈时间均短于对照组，说明白头翁皂苷B4注射液对临床乳房炎的治疗效果优于双丁注射液。高剂量组总治愈率高于中、低剂量组，中剂量组与低剂量组总治愈率相同，但未见复发，且平均治愈时间短于低剂量组，表现出一定的量效关系。
表7 各组患临床型乳房炎的奶牛的治疗效果结果
Figure PCTCN2019073239-appb-000016
4.2各组试验奶牛临床症状检查评分结果
由表8可见，低剂量组乳房炎奶牛临床症状检查评分在D0-D4之间、D5-D12之间差异均分别不显著(p＞0.05)，但在D5-D12与D0-D2之间、D7-D12与D0-D4之间差异均分别显著(p＜0.05)，表明：低剂量组乳房炎奶牛在D5时发生显著好转，且在D7时发生较显著痊愈。中剂量组乳房炎奶牛临床症状检查评分在D0-D1之间、D2-D5之间、D4-D12之间差异均分别不显著(p＞0.05)，但在D3-D12与D0-D2之间、D6-D12与D0-D3之间差异均分别显著(p＜0.05)，表明：中剂量组奶牛在D3时发生较为显著的好转，且在D6时发生显著痊愈。高剂量组乳房炎奶牛临床症状检查评分在D0-D3之间、D1-D5之间、D3-D6之间、D7-D12之间异均分别不显著(p＞0.05)，但在D4-D12与D0之间、D6-D12与D0-D2之间、D7-D12与D0-D6之间差异显著(p＜0.05)，结果显示：高剂量组乳房炎奶牛在D4时普遍开始好转，在D6时痊愈奶牛头数已明显增多，且在D7时有再次有部分 奶牛痊愈。对照组乳房炎奶牛临床症状检查评分在D0-D4之间、D2-D6之间、D3-D12之间差异均分别不显著(p＞0.05)，但在D5-D12与D0-D4之间、D7-D12与D0-D6之间差异均分别显著(p＜0.05)，结果显示：对照组奶牛在D5时发生好转，在D7时痊愈一定数量奶牛。
D0时中剂量组临床症状检查评分显著高于低剂量组和高剂量组(p＜0.05)，说明中剂量组奶牛发病较为严重；D1时中剂量组临床症状检查评分显著高于其余3组(p＜0.05)，D2时中剂量组临床症状检查评分显著高于低剂量组和高剂量组(p＜0.05)，说明在治疗初期，中剂量组奶牛对改善乳房炎奶牛临床症状有较明显的效果；D3-D5和D7时四组间奶牛临床症状检查评分差异均不显著(p＞0.05)；D12时对照组奶牛临床症状检查评分显著高于高剂量组(p＜0.05)，说明对照组的治疗效果较白头翁治疗组差。
表8 各组奶牛临床症状检查评分结果
Figure PCTCN2019073239-appb-000017
注：同行上标有不同大写字母表示差异显著(p＜0.05)，上标有相同大写字母表示差异不显著(p＞0.05)。同列下标有不同大写字母表示差异显著(p＜0.05)，下标有相同大写字母表示差异不显著(p＞0.05)。下表同。
4.3各组试验奶牛乳汁体细胞检测结果
表9 各组痊愈奶牛乳汁体细胞结果(万个/mL)
Figure PCTCN2019073239-appb-000018
Figure PCTCN2019073239-appb-000019
由表9可见，各组奶牛乳汁体细胞D0-D7呈下降趋势，低剂量组、中剂量组和对照组D7与D12之间差异不显著，但与D0差异显著，说明随着药物的治疗，乳汁体细胞在D7时发生显著改善，并在之后没有复发。而高剂量组D0、D7与D12三个时间点间均差异显著，一方面说明乳汁体细胞的显著好转，另一方面也与该组奶牛平均治愈时间较长有关。D0时四组奶牛乳汁体细胞差异不显著，说明各组奶牛发病时从乳汁体细胞的角度来看病情相似；D7时中剂量组奶牛体细胞显著低于高剂量组和对照组，这与中剂量组平均治愈天数较短有关；D12时对照组奶牛乳汁体细胞显著高于中剂量组，说明白头翁皂苷B4注射液治疗奶牛乳房炎较双丁注射液恢复好。
通过白头翁皂苷B4注射液对奶牛临床型乳房炎疗效、乳汁体细胞数量、临床治愈率及治愈试验观察发现，不同浓度白头翁皂苷B4注射液对奶牛临床型乳房炎均有明显治疗作用，效果优于阳性对照药双丁注射液；并且白头翁皂苷B4注射液无毒，无耐药性，在治疗期间和治疗结束均不需弃奶，不仅为养殖户减少了经济损失，同时也减少了耐药性产生的风险。
图2和图3为患临床型乳房炎的奶牛使用白头翁皂苷B4注射液治疗前后乳汁对比。由图2和图3均可知：治疗第一日，患临床型乳房炎奶牛的乳汁中絮状物较多，随着治疗时间增加，絮状物逐渐减少，乳汁逐渐恢复正常，治疗第六日后，乳汁恢复正常。说明白头翁皂苷B4注射液治疗临床型乳房炎，改善牛奶质量有明显的作用。
试验例3、本发明白头翁皂苷B4治疗久治不愈临床型乳房炎和隐性乳房炎的研究
1、受试药物
采用实施例3制备方法制备的白头翁皂苷B4注射液，浓度为50mg/mL。
2、试验奶牛
试验选取石家庄天泉良种奶牛有限公司奶牛牧场现有全部临床型乳房炎奶牛8头，这8头为治疗一个月左右的久治不愈病例(2头接近瞎奶)，这8头奶牛曾接受过牧场兽医治疗，治疗药物及方法有硫酸头孢喹肟乳房注入剂乳区灌注、头孢类注射液静脉注射和公英散灌服等。
试验随机选取天泉奶牛牧场隐性乳房炎奶牛6头，均为兽医上一次LMT检测结果至少一个患病乳区“++”及以上。
3、试验内容
研究白头翁皂苷B4注射液对久治不愈临床型乳房炎的疗效；以及白头翁皂苷B4注射液对隐性乳房炎的疗效。
4、试验药物及用量用法
试验采用实施例3制备方法制备白头翁皂苷B4注射液，对6头久治不愈及2头久治不愈接近瞎奶的临床型乳房炎奶牛进行治疗，不论几个乳区发生炎症，均采取肌内注射60mL，1次/日。
对随机选取的6头隐性乳房炎奶牛，不论几个乳区发生炎症，均采取肌内注射30mL，1次/日。
表10 试验药物及用量用法
Figure PCTCN2019073239-appb-000020
5、试验结果
5.1久治不愈临床型乳房炎奶牛治疗情况
试验收治8头患临床型乳房炎且久治不愈的奶牛，给药3次后，痊愈4头，明显好转1头，效果不明显1头；2头接近瞎奶奶牛无效。研究结果表明：白头翁皂苷B4对于采用抗生素或其他手段治疗无效的奶牛乳房炎依然有很好的疗效。
部分久治不愈奶牛治疗情况：耳号8558奶牛，该奶牛用药前检查乳汁稀薄，含有少量絮状物，LMT检测结果为“+++”；肌内注射白头翁皂苷B4注射液60mL一次后，乳汁中无肉眼可见絮状物，颜色略黄，用药第二次后乳汁恢复正常，兽医进行LMT检测，结果为“++”，随后牧场兽医转移圈舍。图4为8558号久治不愈奶牛的治疗情况，由图4可知随着治疗时间增加，8558号奶牛分泌乳汁逐渐正常，用药2次后，乳汁恢复正常，达到临床治愈。
5.2隐性乳房炎奶牛治疗情况
依据牧场兽医LMT检测结果，随机选取发生隐性乳房炎奶牛6头，均为至少一个乳区“++”及以上，肌内注射白头翁皂苷B4注射液30mL，三天后，5头奶牛LMT检测结果均明显好转，另一头奶牛(532006号)好转不明显，但未发生加重现象。
表11 隐性乳房炎试验结果
Figure PCTCN2019073239-appb-000021
Figure PCTCN2019073239-appb-000022
注：由于牧场没有体细胞检测仪，需送至其他牧场检测，奶牛场兽医还未完成试验后体细胞检测工作，但给药三天后LMT检测结果表示5头隐性乳房炎奶牛出现明显好转。
特殊牛只说明：1106021号奶牛，给药前左边两个乳区患有隐性乳房炎，LMT检测结果左前为“++”，左后为“+++”，右后为完全瞎奶，无法挤出乳汁，右前头三把奶乳汁粘稠，颜色发黄，再挤出数把后，出现带血黏液状液体，无乳汁特性。每天肌内注射1次白头翁皂苷B4注射液30mL，给药3天后，右前未见好转，左前LMT检测结果为“－”，左后为“++”，体细胞明显降低，有显著疗效。
其他5头患隐性乳房炎的奶牛每天肌内注射1次白头翁皂苷B4注射液30mL，治疗三天后，4头发生好转，LMT检测结果说明体细胞降低，另一头奶牛(532006号)好转不明显。图5为部分患隐性乳房炎的奶牛通过白头翁皂苷B4注射液治疗三天后的治疗情况，由图5可知，通过给药治疗后，患隐性乳房炎的奶牛病情出现好转。
研究结果表明：白头翁皂苷B4对于隐性乳房炎具有很好的疗效。
患病奶牛经牧场兽医用硫酸头孢喹肟乳房注入剂乳区灌注、头孢类注射液静脉注射和公英散灌服等方法治疗一个月左右，但未取得效果，进而转归为慢性乳房炎，表现为乳质稀薄，持续含有大量絮状物，究其原因可能为：一方面该牧场治疗乳房炎使用药物单一，治疗过程中仅使用抗菌药物而未注意抗炎；另一方面使用抗生素种类单一，且未进行致病菌分离鉴定，产生细菌耐药性，对药物不敏感；再一方面同时长期治疗，乳腺组织增生，造成慢性炎症，药物更难到达有效部位，病情顽固。采用白头翁皂苷B4注射液对试验收治8头奶牛进行治疗时，给药3次后，痊愈4头，明显好转1头，效果不明显1头；2头接近瞎奶奶牛无效。研究结果表明：白头翁皂苷B4对于采用抗生素或其他手段治疗无效的奶牛乳房炎依然有很好的疗效。
隐性乳房炎是奶牛发病最普遍的乳房炎类型之一，由于其通常无临床症状表现，乳汁肉眼也不可看出与正常乳汁的区别，需进行实验室试剂检测才可诊断，该类乳房炎如果在牛群中未达到一定比例，一般不予治疗，而正是因为其发病隐蔽，对奶牛造成的经济损失也最严重。应用白头翁皂苷B4注射液治疗6头隐性乳房炎奶牛时，4头奶牛发生好转；1 头LMT检测体细胞降低，有显著疗效；1头奶牛好转不明显，研究结果表明：白头翁皂苷B4应用于隐性奶牛乳房炎时具有很好的疗效。
此外，在奶牛乳房炎治疗过程中，随机抽取试验用白头翁皂苷B4注射液委托四川省兽药监察所进行200多种抗生素检测，未检测出抗生素成分；奶牛给药后第二天，在牛场对乳汁进行过抗检测过程中，均能通过牛场抗生素残留检测；白头翁皂苷B4对奶牛乳汁中免疫球蛋白和炎性因子影响较小，能够保证乳汁成份稳定，无残留，不会对奶牛养殖场站鲜乳品质造成影响。
目前，乳房炎治疗大多采用抗生素，其中又以广谱杀菌、破坏细胞核酸类抗生素较多常用，在治疗期间和治疗结束后4～5天需要弃奶，为养殖户造成较大经济损失，并有产生耐药性的风险。白头翁皂苷B4是从中药材白头翁中提取分离的天然有效成分，无毒，无耐药性，本发明的化合物白头翁皂苷B4具有较强的生物活性，对奶牛乳房炎具有优异的治疗效果，在治疗期间和治疗结束均不需弃奶，不仅为养殖户减少了经济损失，同时也减少了耐药性产生的风险。此外，白头翁皂苷B4不仅对临床型乳房炎具有优异的治疗效果，对久治不愈和隐性乳房炎也有优异的治疗效果。同时，白头翁皂苷注射液应用于奶牛乳房炎的治疗时具有安全、高效、无残留、无耐药性、无毒副作用的特点。

Claims (10)

  1. 一种如式Ⅰ所示的化合物白头翁皂苷B4或其药学上可接受的盐在制备治疗病毒性和/或细菌性疾病的药物中的用途：
    Figure PCTCN2019073239-appb-100001
  2. 根据权利要求1所述的用途，其特征在于：所述药物是以权利要求1所述化合物白头翁皂苷B4或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
  3. 根据权利要求2所述的用途，其特征在于：所述制剂为注射液、乳房注入剂、散剂、膏剂、洗剂；优选的，所述制剂为注射液、乳房注入剂；更优选的，所述制剂为注射液。
  4. 根据权利要求1所述的用途，其特征在于：所述疾病为奶牛乳房炎。
  5. 根据权利要求4所述的用途，其特征在于：所述奶牛乳房炎为临床型乳房炎、隐性乳房炎。
  6. 根据权利要求5所述的用途，其特征在于：所述临床型乳房炎为初次治疗的临床型乳房炎、久治不愈的临床型乳房炎。
  7. 一种治疗病毒性和/或细菌性疾病的药物，其特征在于：它是以权利要求1所述化合物白头翁皂苷B4或其药学上可接受的盐为活性成分，加上药学上可接受的辅料制备而成的制剂。
  8. 根据权利要求7所述的药物，其特征在于：所述制剂为注射液、乳房注入剂、散剂、膏剂、洗剂；优选的，所述制剂为注射液、乳房注入剂；更优选的，所述制剂为注射 液。
  9. 根据权利要求7所述的药物，其特征在于：所述疾病为奶牛乳房炎。
  10. 根据权利要求9所述的药物，其特征在于：所述奶牛乳房炎为临床型乳房炎、隐性乳房炎；优选的，所述临床型乳房炎为初次治疗的临床型乳房炎、久治不愈的临床型乳房炎。
PCT/CN2019/073239 2018-01-31 2019-01-25 一种白头翁提取物在制备治疗病毒性和/或细菌性疾病的药物中的用途 WO2019149156A1 (zh)

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ES19746721T ES2964618T3 (es) 2018-01-31 2019-01-25 Saponina B4 de pulsatilla para el tratamiento de la mastitis de la vaca lechera
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BR112020015536-5A BR112020015536A2 (pt) 2018-01-31 2019-01-25 uso de extrato de pulsatilla chinensis na preparação de um medicamento para o tratamento de doenças virais e/ou bacterianas
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AU2019214082A AU2019214082B2 (en) 2018-01-31 2019-01-25 Uses of Pulsatilla Chinensis extract in preparing drug for treating viral and/or bacterial diseases
UAA202005100A UA127576C2 (uk) 2018-01-31 2019-01-25 Застосування екстракту pulsatilla chinensis в одержанні лікарського препарату, призначеного для лікування маститу молочних корів
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