WO2016131333A1 - 一种降低家畜体细胞数的复合抗氧化制剂及其制备方法与应用 - Google Patents

一种降低家畜体细胞数的复合抗氧化制剂及其制备方法与应用 Download PDF

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WO2016131333A1
WO2016131333A1 PCT/CN2015/097907 CN2015097907W WO2016131333A1 WO 2016131333 A1 WO2016131333 A1 WO 2016131333A1 CN 2015097907 W CN2015097907 W CN 2015097907W WO 2016131333 A1 WO2016131333 A1 WO 2016131333A1
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somatic cells
preparation
injection
vitamin
cow
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French (fr)
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刘国世
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中国农业大学
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Priority to US15/540,002 priority Critical patent/US10828277B2/en
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Priority to US17/078,531 priority patent/US11931337B2/en

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    • 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
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01J—MANUFACTURE OF DAIRY PRODUCTS
    • A01J7/00—Accessories for milking machines or devices
    • A01J7/04—Accessories for milking machines or devices for treatment of udders or teats, e.g. for cleaning
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K29/00—Other apparatus for animal husbandry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • the invention relates to the field of animal husbandry and veterinary, in particular to a composite antioxidant preparation for reducing the number of somatic cells in livestock, a preparation method and application thereof.
  • Dairy cow mastitis also known as bovine mastitis
  • bovine mastitis is the most common disease in dairy cows. It is mainly caused by the mechanical barrier and immune system abnormalities caused by chemical, physical and microbial stimulation and invasion of the cow's mammary gland. Dairy cow mastitis not only causes certain damage and influence on the body and mental state of the cow, but also reduces the milk yield of the cow and the milk protein, lactose and fat content in the milk. According to reports, the American Mastitis Council estimates that the United States suffers $225/head loss per year due to mastitis, and 26.5% of the cows that are eliminated are suffering from mastitis.
  • MT Melatonin
  • MT is a neuroendocrine hormone produced by the pineal gland of mammals and humans. It has good anti-oxidation and regulates the body's immunity. Since the discovery of MT's regulation of the immune system in the 1980s, the effects of melatonin on the immune function of the body have received increasing attention. Increasing the light significantly reduced the number of white blood cells, the percentage of lymphocytes, and the phagocytic index of neutrophils in mice, but the number of white blood cells, the percentage of lymphocytes, and the phagocytic index of neutrophils were significantly increased after exogenous injection of melatonin. Pick up.
  • melatonin After exposure to continuous light, the number of serum neutrophils decreased and the circadian rhythm of adhesion and phagocytosis disappeared after pinealectomy. In addition to affecting the cellular immune function of the body, melatonin also has an important effect on humoral immunity. Injecting melatonin into older mice, serum IgM and IgG are significantly higher than normal control group, and thymus and spleen weight are significantly increased. The number and activity of B lymphocytes and NK cells also increased accordingly.
  • the application of Ming can reduce the number of somatic cells of high-yield cows with a number of somatic cells between 30 and 1 million/mL by more than 50%, which is significantly higher than that of vitamin E, vitamin C and tea polyphenols (13.9%, Liu Shijun et al., 2010).
  • Biological antioxidant preparations In this study, a composite antioxidant consisting of melatonin and vitamin C (Vitamin C, Vc) was applied to improve the immunity of dairy cows. By subcutaneous injection, the concentration of MT in dairy cows was increased to study the composite antioxidant preparation. The effect on the number of somatic cells in normal dairy cows can improve the immunity of dairy cows to reduce the number of somatic cells, improve the health of dairy cows and improve the quality of milk.
  • the melatonin has a concentration of 2 g/L to 30 g/L.
  • the melatonin concentration is 2.32 g/L.
  • the concentration of the vitamin C is from 1 g/L to 14.3 g/L.
  • the concentration of the vitamin C is preferably 8.8 g/L.
  • the excipient of the formulation is a 75% ethanol solution.
  • the invention further provides a preparation method of the preparation, which comprises dissolving vitamin C in a 75% ethanol solution and then adding melatonin, and the whole operation process is carried out in a dark room (protected from light).
  • the invention also provides for the use of the formulation for improving the quality of livestock milk.
  • the livestock is a cow, a dairy cow or a dairy goat; preferably a cow.
  • the subject is a Chinese Holstein cow.
  • the invention finds in the animal experiment that 2 mL of the composite anti-oxidation preparation of the invention is injected subcutaneously into the neck of the Holstein cow for 4 consecutive days, thereby reducing the number of somatic cells in the milk-producing sample of the cow, and reducing the somatic cells of the cow for further anti-oxidation preparation.
  • the number of studies provides a useful reference.
  • the active antioxidant of the composite antioxidant provided by the invention is melatonin and vitamin C, and naturally no There is no problem of veterinary drug residues, drug withdrawal period and abandonment period, which not only saves production costs but also reduces the workload of the cattle farm staff.
  • the number of somatic cells in the cow is higher than 100,000/ml
  • the number of somatic cells in the milk can be significantly reduced by about 50% by using the composite antioxidant provided by the present invention. It is fully illustrated that the composite antioxidant preparation of the present invention can effectively reduce the number of somatic cells in dairy cows.
  • Fig. 1 is a graph showing changes in the number of somatic cells at 1-6 days after injection of a composite antioxidant preparation in a cow having a somatic cell count of 60 to 1 million/mL in Experimental Example 1.
  • Fig. 2 is a graph showing changes in the number of somatic cells 6-16 days after the injection of the composite antioxidant preparation in the cows having the number of somatic cells in the experimental example 1 of the present invention.
  • Fig. 3 is a graph showing changes in the number of somatic cells at 1-6 days after injection of a composite antioxidant preparation in a cow having a somatic cell number of 300,000 to 500,000/mL in Experimental Example 1.
  • Fig. 4 is a graph showing the change in the number of somatic cells 6-16 days after the injection of the composite antioxidant preparation in the cows having the number of somatic cells of 300-500,000/mL in the experimental example 1 of the present invention.
  • Fig. 5 is a graph showing changes in the number of somatic cells from 1 to 16 days after injection of a composite antioxidant preparation in a cow having a somatic cell number of 100,000 to 200,000/mL in Experimental Example 1.
  • Fig. 6 is a graph showing changes in the number of somatic cells from 1 to 16 days after injection of a composite antioxidant preparation in a cow having a number of somatic cells of 0 to 100,000/mL in Experimental Example 1.
  • Fig. 7 is a graph showing changes in cell number of cells 1 to 16 days after injection of vitamin C and vitamin E (Vitamin E, Ve) preparations in cows with a somatic cell count of 60 to 1 million/mL in Comparative Example 1.
  • Fig. 8 is a graph showing the change in the number of cells in the first to sixth days after the injection of the different ratios of the preparations of the present invention in the cows of 60-100,000/mL in the second embodiment of the present invention.
  • Raw materials melatonin, vitamin C
  • Preparation method accurately weigh 880mg vitamin C, 232mg melatonin, first dissolve 880mg vitamin C in a 100mL volumetric flask with 75% ethanol solution, stir evenly, then add 232mg melatonin, stir evenly, to a volume of 100mL, Finally, the injection dose was 2 mL, and all operations were completed in a dark room.
  • Example 2 The same as Example 1, except that the melatonin concentration was 2 g/L, and the vitamin C concentration was 1 g/L.
  • Example 2 The same as Example 1, except that the melatonin concentration was 30 g/L, and the vitamin C concentration was 14.3 g/L.
  • concentrations of melatonin and vitamin C in the compound antioxidant were 2g/L-30g/L and 1g/L-14.3g/L, respectively, and the method of subcutaneous injection in the neck was used.
  • test cattle feeding method, milking method and daily management are all carried out according to the existing regulations of the dairy farm. That is, all the test cattle are kept in the bar, fed with the mixed mixed diet, free drinking water, and milked three times a day.
  • the collected milk samples were added to three drops of saturated potassium dichromate solution, placed in a refrigerator at 4 ° C for refrigeration, and then sent to the National Dairy Cow Production Performance Determination Standard Preparation Laboratory for determination of the number of somatic cells.
  • the change curve of the number of somatic cells in the milk cows with the number of somatic cells between 60 and 1 million/mL is shown in Fig. 1. From the day of injection, the number of somatic cells decreased significantly. The reduction of the test group was significantly higher than that of the control group, and there was no increase during the injection period. The largest decrease was from the pre-injection of 757,000/mL. Dropped to 215,000 / mL. However, the duration of this reduction was not long. Shortly after stopping the injection of the composite antioxidant preparation, the number of somatic cells in the cows appeared to be undulating like the control group (Fig. 2).
  • the change in the number of somatic cells in the cows with the number of somatic cells between 100,000 and 200,000/mL is shown in Fig. 5.
  • the number of somatic cells decreased significantly after the injection of the composite antioxidants in cows with a cell number of 100,000-200,000.
  • the number of somatic cells decreased from 150,000/mL before injection to 60,000/mL after injection, and was maintained after stopping the injection. 5-10 million / mL.
  • the number of somatic cells in the control group also showed a downward trend, but the decline was compared. Slowly, the number of somatic cells is finally maintained at 10-15 million/mL.
  • the change in the number of somatic cells in the cows with the number of somatic cells between 0 and 100,000/mL is shown in Fig. 6.
  • the number of somatic cells in the number of somatic cells ranging from 0 to 100,000/mL decreased, but there was a significant increase after stopping the injection, and finally maintained at 2-4 million/mL.
  • the number of somatic cells in the control cows was small, and was maintained at 2-4 million/mL during the experiment.
  • the cow may develop mastitis, but whether the pathogenic microorganism can cause mastitis after invading the mammary gland Depending on the virulence of the pathogen, it is also closely related to the immunity of the body. Russian scholars use surfactants, immunomodulators and microbial cell enzymes to develop biological agents, and improve the immune system of cows to treat recessive mastitis. A good result. In recent years, studies have found that melatonin can increase the weight of immune organs, increase the percentage of lymphocytes in peripheral blood, promote antibody formation and proliferation of T and B lymphocytes, stimulate the production of cytokines, and participate in the regulation of neuro-endocrine-immune networks. And melatonin plays an important role in mediating the circadian rhythm of immune function.
  • the number of lactating days and the number of sows with similar milk production in the 60-100 million/mL Holstein cows were randomly divided into two groups, namely, 60-100,000/mL and 10 mice per group.
  • the test cattle feeding method, milking method and daily management are all carried out according to the existing regulations of the dairy farm. That is, all the test cattle are kept in the bar, fed with the mixed mixed diet, free drinking water, and milked three times a day. Only the first milk sample was collected every day, and 2 ml of vitamin C and vitamin E preparations were injected after sampling for 4 consecutive days. The injection was stopped from the fifth day, only the milk samples were collected, and the sixth, eighth, ten, and tenth thereafter. Continue to collect milk samples for three and sixteen days. Add three drops to the collected milk sample The saturated potassium dichromate solution was placed in a refrigerator at 4 ° C for refrigeration, and then sent to the National Dairy Cow Production Performance Standard Preparation Laboratory to determine the number of somatic cells.
  • the injection was stopped from the fifth day, only the milk samples were collected, and the milk samples were collected on the sixth, eighth, tenth, thirteenth and sixteenth days thereafter.
  • the collected milk samples were added to three drops of saturated potassium dichromate solution, placed in a refrigerator at 4 ° C for refrigeration, and then sent to the National Dairy Cow Production Performance Determination Standard Preparation Laboratory for determination of the number of somatic cells.
  • the invention discloses a composite antioxidant preparation for reducing the number of somatic cells in livestock, which can improve the milk of livestock Juice quality.
  • 2 mL of the compound antioxidant of the present invention was injected subcutaneously into the neck of the Holstein cow for 4 consecutive days, which reduced the number of somatic cells in the milk sample of the cow, and reduced the number of somatic cells in the cow for further antioxidant preparation.
  • the active antioxidant of the composite antioxidant preparation provided by the invention is melatonin and vitamin C, which is natural and harmless, and has no problem of veterinary drug residue, drug withdrawal period and abandonment period, which not only saves production cost but also reduces cattle work. The workload of the staff.

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Abstract

本发明提供一种降低家畜体细胞数的复合抗氧化制剂及其制备方法与应用。所述制剂的有效成分为浓度为2g/L-30g/L的褪黑素和浓度为1g/L-14.3g/L的维生素C。本发明提供的复合抗氧化制剂可以有效降低奶牛体细胞数，提高牛奶质量。

Description

一种降低家畜体细胞数的复合抗氧化制剂及其制备方法与应用 技术领域
本发明涉及畜牧兽医领域，具体地说，涉及一种降低家畜体细胞数的复合抗氧化制剂及其制备方法与应用。
背景技术
奶牛乳房炎又名牛乳腺炎，是奶牛最常见的疾病，主要是由于奶牛乳腺受化学、物理以及微生物等刺激和侵袭导致机体的机械性屏障和免疫系统异常而引起的。奶牛乳房炎不仅对患牛机体和精神状态造成一定的损伤和影响，而且会降低奶牛的产奶量以及牛乳中乳蛋白、乳糖和脂肪的含量。据报道，美国乳房炎理事会估计美国每年因为乳房炎造成225美元/头的损失，被淘汰的奶牛26.5％是因为患有乳房炎。在芬兰、挪威、雅典等国，因为乳房健康问题而被淘汰的奶牛分别占35％、19％和22％。在我国，报道的奶牛乳房炎阳性率在46.4％～85.7％，乳区阳性率在28％～59％；一般隐性乳房炎的发病率达到20％～40％以上，甚至高达50％～80％。近年来奶牛隐性乳房炎发病率还呈上升趋势。全世界每年因奶牛乳房炎导致奶产量的损失量在380万吨左右。病原微生物入侵乳腺后能否引起乳房炎除了取决于病原体的毒力以外，还与机体的本身免疫力密切相关。
褪黑素(Melatonin，MT)是由哺乳动物和人类的松果体产生的一种神经内分泌激素，具有很好的抗氧化和调节机体免疫力的作用。自从20世纪80年代MT对免疫系统有调节作用被发现以后，褪黑素对机体免疫功能的影响也越来越受到关注。增加光照会使小鼠血浆内白细胞数量、淋巴细胞百分率、中性粒细胞的吞噬指数明显降低，但外源注射褪黑素后，白细胞数量，淋巴细胞百分率和中性粒细胞的吞噬指数均显著回升。暴露于持续光照下的大鼠，松果体摘除后，血清中性粒细胞的数量减少，其粘附与吞噬的昼夜节律也消失。褪黑素除了影响机体的细胞免疫功能外，对体液免疫也有重要影响，给大龄小鼠注射褪黑素，其血清IgM、IgG较正常对照组显著升高，胸腺和脾脏重量明显增加，T、B淋巴细胞、NK细胞的数量和活性也相应的增加。本发 明的应用可以使体细胞数在30-100万/mL的高产奶牛的体细胞数降低50％以上，明显高于维生素E、维生素C和茶多酚等(13.9％，刘仕军等，2010)其它生物抗氧化制剂。本研究将由褪黑素和维生素C(Vitamin C，Vc)组成的复合抗氧化制剂应用到提高奶牛机体免疫力的研究中，通过皮下注射的方式，提高奶牛体内的MT浓度，研究复合抗氧化制剂对正常奶牛体细胞数的影响，提高奶牛机体免疫力从而达到降低体细胞数量、提升奶牛健康程度和提高牛奶质量的目的。
发明内容
本发明的目的是提供一种降低家畜体细胞数的复合抗氧化制剂及其制备方法与应用。
为了实现本发明目的，本发明的技术方案如下：
一种降低家畜体细胞数的复合抗氧化制剂，所述制剂为针剂，其有效成分为褪黑素和维生素C。
进一步地，所述褪黑素的浓度为2g/L-30g/L。
作为优选，所述褪黑素的浓度为2.32g/L。
进一步地，所述维生素C的浓度为1g/L-14.3g/L。
作为优选，作为优选，所述维生素C的浓度为8.8g/L。
更进一步地，所述制剂的辅料为75％乙醇溶液。
本发明进一步提供了所述制剂的制备方法，所述方法为：将维生素C溶于75％乙醇溶液，然后添加褪黑素，整个操作过程需在暗室内(避光)进行。
本发明还提供了所述制剂在提高家畜乳汁质量上的应用。
所述家畜为奶牛、乳肉兼用牛或奶山羊；优选为奶牛。
在本发明的一个具体实施方式中，研究对象为中国荷斯坦奶牛。
本发明的有益效果在于：
本发明通过动物实验发现，在荷斯坦奶牛连续4天颈部皮下注射2mL本发明复合抗氧化制剂，降低了奶牛产奶样中的体细胞数，并为进一步开展抗氧化制剂对降低奶牛体细胞数的研究提供有益的参考。
本发明提供的复合抗氧化制剂的有效成分为褪黑素和维生素C，天然无 害，不会存在兽药残留、休药期和弃奶期的问题，不仅节约了生产成本而且减轻了牛场工作人员的工作负担。
当奶牛体细胞数高于10万/ml时，使用本发明提供的复合抗氧化制剂，可以使牛奶中的体细胞数显著降低50％左右。充分说明了本发明所述的复合抗氧化制剂可以有效降低奶牛体细胞数。
附图说明
图1为本发明实验例1中体细胞数在60-100万/mL的奶牛注射复合抗氧化制剂后1-6天体细胞数变化曲线。
图2为本发明实验例1中体细胞数在60-100万/mL的奶牛注射复合抗氧化制剂后6-16天体细胞数变化曲线。
图3为本发明实验例1中体细胞数在30-50万/mL的奶牛注射复合抗氧化制剂后1-6天体细胞数变化曲线。
图4为本发明实验例1中体细胞数在30-50万/mL的奶牛注射复合抗氧化制剂后6-16天体细胞数变化曲线。
图5为本发明实验例1中体细胞数在10-20万/mL的奶牛注射复合抗氧化制剂后1-16天体细胞数变化曲线。
图6为本发明实验例1中体细胞数在0-10万/mL的奶牛注射复合抗氧化制剂后1-16天体细胞数变化曲线。
图7为本发明对比例1中体细胞数在60-100万/mL的奶牛皮下注射维生素C和维生素E(Vitamin E，Ve)制剂后1-16天的细胞数变化曲线。
图8为本发明对比例2中体细胞数在60-100万/mL的奶牛皮下注射本发明不同配比的制剂后1-16天的细胞数变化曲线。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中使用的试剂和材料均为市售商品。
褪黑素要求纯度达到99.9％。
实施例1复合抗氧化制剂及其制备方法
原料：褪黑素、维生素C；
制备方法：准确称量880mg维生素C、232mg褪黑素，先将880mg维生素C用75％乙醇溶液溶解于100mL容量瓶，搅拌均匀，然后再加入232mg褪黑素，搅拌均匀，定容至100mL，最后分装成2mL的注射剂量，所有操作均在暗室中完成。
实施例2复合抗氧化制剂及其制备方法
同实施例1，区别在于，所述褪黑素的浓度为2g/L，所述维生素C的浓度为1g/L。
实施例3复合抗氧化制剂及其制备方法
同实施例1，区别在于，所述褪黑素的浓度为30g/L，所述维生素C的浓度为14.3g/L。
实验例1复合抗氧化制剂在降低奶牛体细胞数和提高牛奶质量上的应用
1材料与方法
1.1实验时间与地点
2013年9月1日—2013年9月30日在北京规模牛场。
1.2复合抗氧化制剂的制备与用法
复合抗氧式化剂中褪黑素和维生素C的浓度分别为2g/L-30g/L和1g/L-14.3g/L，采用颈部皮下注射的方法。
1.3实验动物
选择泌乳天数、产奶量相近的荷斯坦奶牛80头，根据体细胞数多少分4组，即60-100万/mL、30-50万/mL、10-20万/mL、0-10万/mL，每组20头。试验牛饲喂方式、挤奶方式及日常管理均按奶牛场现有规程运行，即所有试验牛均采用散栏饲养、饲喂全混合日粮、自由饮水，日挤奶三次。
1.4实验设计
每天只采集第一次挤奶的奶样，连续4天在采样后注射复合抗氧化制剂2ml，从第五天开始停止注射，只采集奶样，之后的第六、八、十、十三和十六天继续采集奶样。
1.5体细胞数量的测定
收集到的奶样加入三滴饱和重铬酸钾溶液，放置冰箱4℃冷藏，之后送至全国奶牛生产性能测定标准物质制备实验室进行体细胞数的测定。
1.6数据处理
所有试验数据均采用SPSS软件进行统计分析。
2结果与分析
2.1皮下注射实施例1复合抗氧化制剂对体细胞数在60-100万/mL的奶牛体细细胞数量变化的影响
注射复合抗氧化制剂后，体细胞数在60-100万/mL的奶牛奶牛体细胞数变化曲线见图1。奶牛从注射之日起，体细胞有一个明显降低的趋势，试验组的降低幅度明显高于对照组，而且在注射期内没有升高的现象，降幅最大的由注射前的75.7万/mL，降到21.5万/mL。但这种降低的持续时间不长，在停止注射复合抗氧化制剂不久，奶牛体细胞数又出现像对照组一样起伏不定的规律(图2)。
2.2皮下注射实施例1复合抗氧化制剂对体细胞数在30-50万/mL的奶牛体细胞数量变化的影响
注射复合抗氧化制剂后，体细胞数在30-50万/mL的奶牛体细胞数变化曲线见图3。连续注射复合抗氧化制剂后，体细胞数在30-50万/mL的奶牛体细胞数呈现一个连续下降的趋势，与对照组的上升的趋势形成鲜明对比。降幅最大的由注射前的37.5万/mL，降到14.7万/mL。在停止注射复合抗氧化制剂后，试验奶牛的体细胞数有一个小幅度的回升，但最终会长时间维持在20-30万/mL，而对照组的奶牛的体细胞数此时呈现一个体细胞数下降的趋势，最终维持在30-40万/mL(图4)。
2.3皮下注射实施例1复合抗氧化制剂对体细胞数在10-20万/mL的奶牛体细细胞数量变化的影响
注射复合抗氧化制剂后，体细胞数在10-20万/mL的奶牛体细胞数变化曲线见图5。体细胞数在10-20万的奶牛在注射复合抗氧化制剂后，体细胞数量下降趋势明显，体细胞数由注射前的15万/mL降到注射后6万/mL，停止注射后维持在5-10万/mL。对照组奶牛的体细胞数也呈下降的趋势，但下降的比较 缓慢，体细胞数最终维持在10-15万/mL。
2.4皮下注射实施例1复合抗氧化制剂对体细胞数在0-10万/mL的奶牛体细细胞数量变化的影响
注射复合抗氧化制剂后，体细胞数在0-10万/mL的奶牛体细胞数变化曲线见图6。注射复合抗氧化制剂后，体细胞数在0-10万/mL的奶牛体细胞数有下降的趋势，但在停止注射后有一个明显的回升，最终维持在2-4万/mL。对照组奶牛的体细胞数变化较小，实验期间一直维持在2-4万/mL。
3小结
体外注射抗氧化制剂可以起到降低奶牛的体细胞数量作用，对体细胞数在10万/ml以上的奶牛作用尤为明显。褪黑素对机体免疫能力的调节作用可能是造成奶牛体细胞数降低的原因。国际上将50万/ml体细胞数定为(临床)乳房炎的基准。病原微生物突破第一道防线角蛋白后，就会引起奶牛炎症，如果嗜中性粒细胞未能及时进入乳腺，奶牛就可能会发生乳房炎，但病原微生物在入侵乳腺后能否引起乳房炎除了取决于病原体的毒力以外，也与机体的免疫力密切相关，俄罗斯学者利用表面活性剂、免疫调节剂和微生物细胞酶研制出生物制剂，通过提高奶牛机体免疫能力来治疗隐性乳房炎，取得了较好的效果。近年来研究发现，褪黑素可增加免疫器官的重量，提高外周血中淋巴细胞百分数，促进抗体形成及T、B淋巴细胞增殖反应，刺激细胞因子产生，参与神经—内分泌—免疫网络的调控，且褪黑素在介导免疫功能昼夜节律发挥重要作用。
对比例1
1、皮下注射维生素C和维生素E(Vitamin E，Ve)制剂(2mL)
选择泌乳天数、产奶量相近体细胞数在60-100万/mL荷斯坦奶牛20头，随机分成2组，即60-100万/mL每组10头。试验牛饲喂方式、挤奶方式及日常管理均按奶牛场现有规程运行，即所有试验牛均采用散栏饲养、饲喂全混合日粮、自由饮水，日挤奶三次。每天只采集第一次挤奶的奶样，连续4天在采样后注射维生素C和维生素E制剂2ml，从第五天开始停止注射，只采集奶样，之后的第六、八、十、十三和十六天继续采集奶样。收集到的奶样加入三滴 饱和重铬酸钾溶液，放置冰箱4℃冷藏，之后送至全国奶牛生产性能测定标准物质制备实验室进行体细胞数的测定。
2、实验结果
皮下注射维生素C和维生素E制剂后，奶牛体细胞数降低约15％，效果明显不如褪黑素与维生素C的制剂，而且作用效果持续时间不长，停止注射后4天开始，就会出现像对照组一样起伏不定的规律(图7)。
对比例2
1、皮下注射MT与Vc不同配比的制剂(2mL)
选择泌乳天数、产奶量相近，体细胞数在60-100万/mL的荷斯坦奶牛40头，随机分为5组，连续4天注射本发明所述不同配比的制剂2mL，主要有MT(1g/L)+Vc(0.5g/L)；MT(1g/L)+Vc(1g/L)；MT(2.32g/L)+Vc(8.8g/L)；MT(23.2g/L)+Vc(14.3g/L)；MT(50g/L)+Vc(25g/L)5种。从第五天开始停止注射，只采集奶样，之后的第六、八、十、十三和十六天继续采集奶样。收集到的奶样加入三滴饱和重铬酸钾溶液，放置冰箱4℃冷藏，之后送至全国奶牛生产性能测定标准物质制备实验室进行体细胞数的测定。
2、实验结果
不同配比的制剂均有降低奶牛细胞数的作用，但效果差异很大，MT(1g/L)+Vc(1g/L)、MT(2.32g/L)+Vc(8.8g/L)和MT(23.2g/L)+Vc(14.3g/L)三种制剂的作用效果基本一致，起到了明显降低高产奶牛体细胞的作用，降低幅度约50％，MT(1g/L)+Vc(0.5g/L)和MT(50g/L)+Vc(25g/L)两种制剂，降低体细胞数的效果较差，降低幅度在5％-16％，并且作用效果持续时间不长，停止注射后4-6天开始，就会出现像对照组一样起伏不定的规律(图8)。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明公开的降低家畜体细胞数的复合抗氧化制剂，能够提高家畜的乳 汁质量。通过动物实验发现，在荷斯坦奶牛连续4天颈部皮下注射2mL本发明复合抗氧化制剂，降低了奶牛产奶样中的体细胞数，并为进一步开展抗氧化制剂对降低奶牛体细胞数的研究提供有益的参考。本发明提供的复合抗氧化制剂的有效成分为褪黑素和维生素C，天然无害，不会存在兽药残留、休药期和弃奶期的问题，不仅节约了生产成本而且减轻了牛场工作人员的工作负担。

Claims (10)

  1. 一种降低家畜体细胞数的复合抗氧化制剂，其特征在于，所述制剂为针剂，其有效成分为褪黑素和维生素C。
  2. 根据权利要求1所述的制剂，其特征在于，所述褪黑素的浓度为2g/L-30g/L。
  3. 根据权利要求2所述的制剂，其特征在于，所述褪黑素的浓度为2.32g/L。
  4. 根据权利要求1-3任一项所述的制剂，其特征在于，所述维生素C的浓度为1g/L-14.3g/L。
  5. 根据权利要求4所述的制剂，其特征在于，所述维生素C的浓度为8.8g/L。
  6. 根据权利要求5所述的制剂，其特征在于，所述制剂的辅料为75％乙醇溶液。
  7. 权利要求1-6任一项所述制剂的制备方法，其特征在于，所述方法为：将维生素C溶于75％乙醇溶液，然后添加褪黑素，操作过程避光进行。
  8. 权利要求1-6任一项所述制剂在提高家畜乳汁质量上的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述家畜为奶牛、乳肉兼用牛或奶山羊。
  10. 根据权利要求9所述的应用，其特征在于，所述家畜为奶牛。
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