复方中药免疫增强剂“增康灵”对动物免疫功能的影响
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摘要
中药免疫增强剂的开发是现代中药学和免疫学的研究热点,本研究旨在通过一系列体内外试验研制开发一种疗效确实的中药免疫增强剂。
(1)复方的筛选及对小鼠非特异性免疫功能的影响
通过小鼠体内试验,从六个中药复方中筛选有效的中药免疫增强剂。结果表明：复方A能显著增加小鼠的负重游泳时间,比对照组提高20-27.5%；复方B能显著增加小鼠的负重游泳时间、单核细胞数量、补体总活性,比对照组分别提高28.4-39.5%、2.8-94.4%和0.8%-39.9%；复方C能显著增加小鼠的负重游泳时间、白细胞数量、中性粒细胞数量、单核-巨噬细胞吞噬活性、LZM酶活力、血清中IFN-α含量,比对照组分别提高6.4-25.0%、2.4-89.6%、12.2-27.9%、4.1-23.4%、45.8-65.0%和18.0-40.6%；复方D能显著增加小鼠的负重游泳时间、白细胞数量、淋巴细胞数量、单核细胞数量、中性粒细胞数量、单核-巨噬细胞吞噬活性、LZM酶活力,与对照组相比能够分别提高22.6-39.5%、15-94.8%、11.1-111.1%、56-88%、8.7-107.5%、5.7-34.3%和9.84-233.9%；复方E能显著增加小鼠的负重游泳时间、白细胞数量、淋巴细胞数量、单核细胞数量、中性粒细胞数量、单核-巨噬细胞吞噬活性、LZM酶活力,与对照组相比能够分别提高15.8-25.6%、5.2-110%、2.7-88.4%、10-132%、18-114.1%、6.2-26.9%和108.4-152.3%；复方E能显著增加小鼠的负重游泳时间、免疫器官指数、LZM酶活力、补体总活力,与对照组相比能够分别提高9.6-35.6%、31.3-108.4%、39.7-166.7%、7.1-8.4%。综合分析,选择复C作为“增康灵”的最佳处方。
(2)对特异性免疫功能的影响
研究“增康灵”对新城疫疫苗和猪瘟疫苗免疫效果的影响：将试验动物分成高、中、低三个剂量组和一个空白对照组,检测血清中的抗体效价水平。结果表明：鸡在第一次接种NDV疫苗后,高剂量组的抗体效价在12d后达到峰值,效价水平在100以上的持续时间达7d；在第二次接种后,高剂量组在10d后达到峰值,效价水平在100以上的持续时间达21d。仔猪在接种猪瘟疫苗后,各试验组在2w后抗体效价达到峰值,血清OD值大于0.4(抗体水平)的持续时间以高剂量组最长,达5w。提示“增康灵”能显著提高动物的特异性免疫功能。
(3)体外抗菌试验
用试管法测得“增康灵”对大肠杆菌、沙门氏菌、巴氏杆菌、金黄色葡萄球菌的最低抑菌浓度(MIC)分别为875μg·mL-1、218.75μg·mL-1、437.5μg·mL-1、875μg·mL-1；最低杀菌浓度(MBC)分别为875μg·mL-1、437.5μg·mL-1、875μg·mL-1、875μg·mL-1。表明“增康灵”具有一定的体外抗菌作用。
(4)体外抗病毒试验：
用先接种病毒后加药物、先加药物后加病毒、病毒和药物体外作用2小时后再同时加入三种方式测定“增康灵”的体外抗PRV效果,通过观察PRV引起的PK-15细胞病变,用Reed-Muench法计算“增康灵”对PK-15细胞的最大无毒浓度(TDo)为138.6μg/mL、半数中毒浓度(TDso)为353.6μg/mL、抑制50%细胞病变的药物浓度(IC50)分别为2.8、5.5、12.8μg/mL、治疗指数(TI)分别为：126、64、28。提示：“增康灵”具有一定的体外抗病毒作用。
研究表明：“增康灵”能显著提高动物机体免疫功能,也有一定的抗菌、抗病毒作用,是一种具有多重药理活性的免疫增强剂。
The exploitation of Chinese herb immunopotentiators is a hot spot of modern pharmacy of Chinese herb medicine and modern immunology. This research's aim is to develop an effective Chinese herb immunopotentiator based on a series of vivo and. vitro tests.
(1) selection of the complex prescription and the effect on the non-specific immunofunction in mice
Vivo tests in mice were done to select the most effective prescription of Chinese medicine immunopotentiators from 6 given complex Chinese herb medicine prescription. The results showed that:prescription A could extend the mice's weight-carrying swimming time significantly,20-27.5% more than the control groups; prescription B could extend the mice's weight-carrying swimming time, increase the number of mononuclear cells and the CH50 significantly, and the increasing amounts were respectively 28.4-39.5%,2.8-94.4% and 0.8%-39.9% than the control groups; prescription C could extend the mice's weight-carrying swimming time, increase the number of white blood cells and neutrophilic granulocytes, the phagocytosis activity of mononuclear macrophage, the LZM enzyme's activity and the content of IFN-αin serum significantly, and respectively increased 6.4-25.0%,2.4-89.6%,12.2-27.9%,4.1-23.4%,45.8-65.0%,18.0-40.6% than control groups; prescription D could extend the mice's weight-carrying swimming time, increase the number of white blood cells, lymphocytes, mononuclear cells and neutrophilic granulocytes, improve the phagocytosis activity of mononuclear macrophages and the LZM enzyme's activity significantly, and the increasing amounts were respectively 22.6-39.5%,15-94.8%,11.1-111.1%,56-88%,8.7-107.5%,5.7-34.3% and 9.84-233.9% compared with the control groups; prescription E could extend the mice's weight-carrying swimming time, increase the number of white blood cells, lymphocytes, mononuclear cells, and neutrophilic granulocytes, improve the phagocytosis activity of mononuclear macrophages and the LZM enzyme's activity significantly, and the increasing amounts were respectively 15.8-25.6%,5.2-110%,2.7-88.4%,10-132%,18-114.1%,6.2-26.9%, and 108.4-152.3% compared with control groups; while, prescription F could extend the mice's weight-carrying swimming time, increase the index of immune organ, improve the LZM enzyme's activity and the CH50 significanlyt, and the increasing amounts were respectively 9.6-35.6%,31.3-108.4%,39.7-166.7% and 7.1-8.4% compared with the control groups. Thus prescription C was selected as the best prescription of the "Zengkangling" after comprehensive analysis.
(2) Effect on specific immunofunction
In order to study the effect of "Zengkangling" on the immunofunction of Newcastle disease(ND) vaccine and hog cholera(HC) vaccine, the experimental animals were divided into four groups:high, medium, low dose and control groups, and detected the valence of corresponding antibody in serum. The results showed that: the chickens of the high dose group reached the peak of valence of antibody at 12th day after inoculated with ND vaccine for the first time, and the continued period of high level of antibody valence above 100 could reach 7 days; and the time when the valence of antibody reach the peak was the 10th day after inoculated the second time, and the corresponding continued period could last for 21 days. Meanwhile, The piglets of all trial groups inoculated the HC vaccine for the first time, reached the peak of valence of antidody after 2 weeks, the high dose group's continued period of high OD(level of valence, OD≥0.4) was the longest one lasting for 5 weeks. The results indicated that "Zengkangling" could enhance the animal's specific immunofunction significantly.
(3) Antibacterial tests in vitro
The MIC of "Zengkangling" to Escherichia coli, Salmonella, Pasteurella and Staphylococcus aureus were respectively 875μg·mL-1,218.75μg·mL-1,437.5μg·mL-1 and 875μg·mL-1 and the MBC to those were respectively 875μg·mL-1,437.5μg·mL-1, 875μg·mL-1 and 875μg·mL-1 through test tube method. The results indicated that "Zengkangling" have certain antibacterial activity in vitro.
(4) Anti-virus test in vitro.
To evaluated the anti-PR V effect of "Zengkangling" in vitro,3 ways were adopted:one way was PRV added alone onto the PK-15 cells before"Zengkangling", the second way was "Zengkangling" added before PRV, and the third way was PRV and "Zengkangling" added together after mixed and Interacting for 2 hours. With Observing the cytopathic effect of PK-15 cells originated by PRV, and calculating the results with Reed-Muench Method, the TDo was 138.6μg/mL; TD50 was 353.6μg/mL, IC50 were 2.8,5.5 and 12.8μg/mL, and TI were 126,64 and 28. The results indicated that "Zengkangling" have certain anti-virus activity in vitro.
The results of above-mentioned showed that Chinese herb complex immunopotentiators "Zengkangling" could enhance the immunofuction of animals, and possessed certain antibacterial activity together with anti-virus activity in vitro. Thus it was an immunopotentiator with multi-pharmacological activities.
(1)复方的筛选及对小鼠非特异性免疫功能的影响
通过小鼠体内试验,从六个中药复方中筛选有效的中药免疫增强剂。结果表明：复方A能显著增加小鼠的负重游泳时间,比对照组提高20-27.5%；复方B能显著增加小鼠的负重游泳时间、单核细胞数量、补体总活性,比对照组分别提高28.4-39.5%、2.8-94.4%和0.8%-39.9%；复方C能显著增加小鼠的负重游泳时间、白细胞数量、中性粒细胞数量、单核-巨噬细胞吞噬活性、LZM酶活力、血清中IFN-α含量,比对照组分别提高6.4-25.0%、2.4-89.6%、12.2-27.9%、4.1-23.4%、45.8-65.0%和18.0-40.6%；复方D能显著增加小鼠的负重游泳时间、白细胞数量、淋巴细胞数量、单核细胞数量、中性粒细胞数量、单核-巨噬细胞吞噬活性、LZM酶活力,与对照组相比能够分别提高22.6-39.5%、15-94.8%、11.1-111.1%、56-88%、8.7-107.5%、5.7-34.3%和9.84-233.9%；复方E能显著增加小鼠的负重游泳时间、白细胞数量、淋巴细胞数量、单核细胞数量、中性粒细胞数量、单核-巨噬细胞吞噬活性、LZM酶活力,与对照组相比能够分别提高15.8-25.6%、5.2-110%、2.7-88.4%、10-132%、18-114.1%、6.2-26.9%和108.4-152.3%；复方E能显著增加小鼠的负重游泳时间、免疫器官指数、LZM酶活力、补体总活力,与对照组相比能够分别提高9.6-35.6%、31.3-108.4%、39.7-166.7%、7.1-8.4%。综合分析,选择复C作为“增康灵”的最佳处方。
(2)对特异性免疫功能的影响
研究“增康灵”对新城疫疫苗和猪瘟疫苗免疫效果的影响：将试验动物分成高、中、低三个剂量组和一个空白对照组,检测血清中的抗体效价水平。结果表明：鸡在第一次接种NDV疫苗后,高剂量组的抗体效价在12d后达到峰值,效价水平在100以上的持续时间达7d；在第二次接种后,高剂量组在10d后达到峰值,效价水平在100以上的持续时间达21d。仔猪在接种猪瘟疫苗后,各试验组在2w后抗体效价达到峰值,血清OD值大于0.4(抗体水平)的持续时间以高剂量组最长,达5w。提示“增康灵”能显著提高动物的特异性免疫功能。
(3)体外抗菌试验
用试管法测得“增康灵”对大肠杆菌、沙门氏菌、巴氏杆菌、金黄色葡萄球菌的最低抑菌浓度(MIC)分别为875μg·mL-1、218.75μg·mL-1、437.5μg·mL-1、875μg·mL-1；最低杀菌浓度(MBC)分别为875μg·mL-1、437.5μg·mL-1、875μg·mL-1、875μg·mL-1。表明“增康灵”具有一定的体外抗菌作用。
(4)体外抗病毒试验：
用先接种病毒后加药物、先加药物后加病毒、病毒和药物体外作用2小时后再同时加入三种方式测定“增康灵”的体外抗PRV效果,通过观察PRV引起的PK-15细胞病变,用Reed-Muench法计算“增康灵”对PK-15细胞的最大无毒浓度(TDo)为138.6μg/mL、半数中毒浓度(TDso)为353.6μg/mL、抑制50%细胞病变的药物浓度(IC50)分别为2.8、5.5、12.8μg/mL、治疗指数(TI)分别为：126、64、28。提示：“增康灵”具有一定的体外抗病毒作用。
研究表明：“增康灵”能显著提高动物机体免疫功能,也有一定的抗菌、抗病毒作用,是一种具有多重药理活性的免疫增强剂。
The exploitation of Chinese herb immunopotentiators is a hot spot of modern pharmacy of Chinese herb medicine and modern immunology. This research's aim is to develop an effective Chinese herb immunopotentiator based on a series of vivo and. vitro tests.
(1) selection of the complex prescription and the effect on the non-specific immunofunction in mice
Vivo tests in mice were done to select the most effective prescription of Chinese medicine immunopotentiators from 6 given complex Chinese herb medicine prescription. The results showed that:prescription A could extend the mice's weight-carrying swimming time significantly,20-27.5% more than the control groups; prescription B could extend the mice's weight-carrying swimming time, increase the number of mononuclear cells and the CH50 significantly, and the increasing amounts were respectively 28.4-39.5%,2.8-94.4% and 0.8%-39.9% than the control groups; prescription C could extend the mice's weight-carrying swimming time, increase the number of white blood cells and neutrophilic granulocytes, the phagocytosis activity of mononuclear macrophage, the LZM enzyme's activity and the content of IFN-αin serum significantly, and respectively increased 6.4-25.0%,2.4-89.6%,12.2-27.9%,4.1-23.4%,45.8-65.0%,18.0-40.6% than control groups; prescription D could extend the mice's weight-carrying swimming time, increase the number of white blood cells, lymphocytes, mononuclear cells and neutrophilic granulocytes, improve the phagocytosis activity of mononuclear macrophages and the LZM enzyme's activity significantly, and the increasing amounts were respectively 22.6-39.5%,15-94.8%,11.1-111.1%,56-88%,8.7-107.5%,5.7-34.3% and 9.84-233.9% compared with the control groups; prescription E could extend the mice's weight-carrying swimming time, increase the number of white blood cells, lymphocytes, mononuclear cells, and neutrophilic granulocytes, improve the phagocytosis activity of mononuclear macrophages and the LZM enzyme's activity significantly, and the increasing amounts were respectively 15.8-25.6%,5.2-110%,2.7-88.4%,10-132%,18-114.1%,6.2-26.9%, and 108.4-152.3% compared with control groups; while, prescription F could extend the mice's weight-carrying swimming time, increase the index of immune organ, improve the LZM enzyme's activity and the CH50 significanlyt, and the increasing amounts were respectively 9.6-35.6%,31.3-108.4%,39.7-166.7% and 7.1-8.4% compared with the control groups. Thus prescription C was selected as the best prescription of the "Zengkangling" after comprehensive analysis.
(2) Effect on specific immunofunction
In order to study the effect of "Zengkangling" on the immunofunction of Newcastle disease(ND) vaccine and hog cholera(HC) vaccine, the experimental animals were divided into four groups:high, medium, low dose and control groups, and detected the valence of corresponding antibody in serum. The results showed that: the chickens of the high dose group reached the peak of valence of antibody at 12th day after inoculated with ND vaccine for the first time, and the continued period of high level of antibody valence above 100 could reach 7 days; and the time when the valence of antibody reach the peak was the 10th day after inoculated the second time, and the corresponding continued period could last for 21 days. Meanwhile, The piglets of all trial groups inoculated the HC vaccine for the first time, reached the peak of valence of antidody after 2 weeks, the high dose group's continued period of high OD(level of valence, OD≥0.4) was the longest one lasting for 5 weeks. The results indicated that "Zengkangling" could enhance the animal's specific immunofunction significantly.
(3) Antibacterial tests in vitro
The MIC of "Zengkangling" to Escherichia coli, Salmonella, Pasteurella and Staphylococcus aureus were respectively 875μg·mL-1,218.75μg·mL-1,437.5μg·mL-1 and 875μg·mL-1 and the MBC to those were respectively 875μg·mL-1,437.5μg·mL-1, 875μg·mL-1 and 875μg·mL-1 through test tube method. The results indicated that "Zengkangling" have certain antibacterial activity in vitro.
(4) Anti-virus test in vitro.
To evaluated the anti-PR V effect of "Zengkangling" in vitro,3 ways were adopted:one way was PRV added alone onto the PK-15 cells before"Zengkangling", the second way was "Zengkangling" added before PRV, and the third way was PRV and "Zengkangling" added together after mixed and Interacting for 2 hours. With Observing the cytopathic effect of PK-15 cells originated by PRV, and calculating the results with Reed-Muench Method, the TDo was 138.6μg/mL; TD50 was 353.6μg/mL, IC50 were 2.8,5.5 and 12.8μg/mL, and TI were 126,64 and 28. The results indicated that "Zengkangling" have certain anti-virus activity in vitro.
The results of above-mentioned showed that Chinese herb complex immunopotentiators "Zengkangling" could enhance the immunofuction of animals, and possessed certain antibacterial activity together with anti-virus activity in vitro. Thus it was an immunopotentiator with multi-pharmacological activities.
引文
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