抗猪圆环病毒药物体外筛选研究
摘要
猪圆环病毒感染是由猪圆环病毒引起的一种传染病,猪圆环病毒感染疾病遍及世界各个养猪国家和地区,已然成为严重危害养猪业的重要传染病。虽然有报道猪圆环病毒疫苗研制成功,但是由于上市的时间不长,在临床上使用还不广泛,而且疫苗的效果也有待观察,因此在临床上猪圆环病毒病主要采取药物治疗,但临床用药混乱,疗效不确切,迫切需要筛选高效防治药物。本研究对临床上常用的抗病毒药物金刚烷胺、利巴韦林、盐酸阿比朵尔、黄连、金银花、连翘、板蓝根对猪圆环病毒的活性抑制作用进行了检测,以期筛选出高效药物,为临床防治该病提供选药依据。
MTT染色法能定量检测细胞的增殖活性,来间接反应药物的抗病毒效果。为此我们将MTT作为细胞培养物中病毒增殖情况的指示系统。本研究利用PK-15细胞建立抗猪圆环病毒药物的筛选模型,设立正常细胞对照组。96孔板培养细胞24 h,细胞长成单层细胞后,每孔加入猪圆环病毒液,继续培养6 h。在无毒性浓度范围内加入不同浓度药物,继续培养24 h,加入MTT培养4 h,小心吸取上清液弃去,加入二甲基亚砜,37℃下孵育30 min,用酶标仪在490 nm下测定OD值,根据公式计算病毒的抑制率和药物的TI。
中药提取物抗猪圆环病毒活性结果:黄连的IC50为363.2μg/mL,治疗指数TI为13.8,金银花的IC50为719.7μg/mL,治疗指数TI为4.1,连翘的IC50为1 596.7μg/mL,治疗指数TI为4.6,板蓝根的IC50为1 189.4μg/mL,治疗指数TI为5.0,筛选出了高效的抗病毒中药药物黄连。
化学药物抗猪圆环病毒活性的结果:利巴韦林的IC50为5.53μg/mL,治疗指数TI为33.8,金刚烷胺的IC50为1.19μg/mL,治疗指数TI为124.9,盐酸阿比朵尔的IC50为6.85μg/mL,治疗指数TI为33.1,筛选出了高效抗病毒药物金刚烷胺。
试验结果表明,实验药物对猪圆环病毒均具有抑制作用,中药的抗病毒活性总体上要低于西药;中药中,黄连的抗病毒活性最高;西药中,金刚烷胺的抗病毒活性最高。
Porcine circovirus (PCV) results in porcine dermatitis and nephropathy syndrome (PDNS), postweaning multisystemic wasting syndrome (PMWS), piglet congenital tremor (PT) and other serious diseases. The PCV infection has extended all over the world which has been considered as an important pathogen. It is reported that PCR vaccine has been prepared. Because of a short life time, commercial vaccines are short of clinical application and evaluation. Therefore, the major clinical treatment for CPV is using antivirus medicine. Nowadays, there are sorts of antivirus medicines for clinical application, but efficient medicine is required pressing. In this study, some normal antivirus medicines were detected to screen active medicine anti PCV.
In this study, PK-15 cell stem was used as the model for screening. The blank control and PCV-infected group were set. MTT assay is able to quantity detect alive cells to indirectly evaluation the effect of antivirus medicines. The cells were seeded into 96-well plate and cultured. PCV was added to monolayer cells and incubated for 6 h. the medicines in different dilution were added and the cells were incubated for 24 h. MTT was added and incubated for 4 h. The supernatant was removed, and the wells were added into dimethyl sulfoxide. The wells were incubated at 37℃for 30 min. the OD value at 490 nm was measured by microplate reader. The virus inhibition and medicine therapeutic index was calculated according to the formula. Results of Chinese medicine against Porcine Circovirus in vitro:The half maximal inhibitory concentration (IC50) of rhizoma coptidis, honeysuckle, fructus forsythiae and isatis root were 363.2, 719.7, 1596.7, 1189.4μg/mL; The therapeutic index(TI) of rhizoma coptidis, honeysuckle, fructus forsythiae and isatis root were 13.8, 4.1, 4.6 and 5.0。The results indicate that four Chinese medicine all could inhibit the prolification of porcine circovirus at cell level,but the effect of rhizoma coptidis are the most significant.
Results of drugs against Porcine Circovirus in vitro:The half maximal inhibitory concentration (IC50) of amantadine, ribavirin, arbidol hydrochlorid were 5.53, 1.19, 6.85μg/mL; The therapeutic index(TI) of amantadine, ribavirin, arbidol hydrochlorid were 33.8, 124.9and 33.1。The results indicate that three drugs all could inhibit the prolification of porcine circovirus at cell level,but the effect of ribavirin are the most significant.
The results showed that antivirus activity of chemical drugs is stronger than traditional Chinese medicine. Among the traditional Chinese medicine, rhizoma coptidis is the strongest. Among the chemical medicine, amantadine is the strongest.
MTT染色法能定量检测细胞的增殖活性,来间接反应药物的抗病毒效果。为此我们将MTT作为细胞培养物中病毒增殖情况的指示系统。本研究利用PK-15细胞建立抗猪圆环病毒药物的筛选模型,设立正常细胞对照组。96孔板培养细胞24 h,细胞长成单层细胞后,每孔加入猪圆环病毒液,继续培养6 h。在无毒性浓度范围内加入不同浓度药物,继续培养24 h,加入MTT培养4 h,小心吸取上清液弃去,加入二甲基亚砜,37℃下孵育30 min,用酶标仪在490 nm下测定OD值,根据公式计算病毒的抑制率和药物的TI。
中药提取物抗猪圆环病毒活性结果:黄连的IC50为363.2μg/mL,治疗指数TI为13.8,金银花的IC50为719.7μg/mL,治疗指数TI为4.1,连翘的IC50为1 596.7μg/mL,治疗指数TI为4.6,板蓝根的IC50为1 189.4μg/mL,治疗指数TI为5.0,筛选出了高效的抗病毒中药药物黄连。
化学药物抗猪圆环病毒活性的结果:利巴韦林的IC50为5.53μg/mL,治疗指数TI为33.8,金刚烷胺的IC50为1.19μg/mL,治疗指数TI为124.9,盐酸阿比朵尔的IC50为6.85μg/mL,治疗指数TI为33.1,筛选出了高效抗病毒药物金刚烷胺。
试验结果表明,实验药物对猪圆环病毒均具有抑制作用,中药的抗病毒活性总体上要低于西药;中药中,黄连的抗病毒活性最高;西药中,金刚烷胺的抗病毒活性最高。
Porcine circovirus (PCV) results in porcine dermatitis and nephropathy syndrome (PDNS), postweaning multisystemic wasting syndrome (PMWS), piglet congenital tremor (PT) and other serious diseases. The PCV infection has extended all over the world which has been considered as an important pathogen. It is reported that PCR vaccine has been prepared. Because of a short life time, commercial vaccines are short of clinical application and evaluation. Therefore, the major clinical treatment for CPV is using antivirus medicine. Nowadays, there are sorts of antivirus medicines for clinical application, but efficient medicine is required pressing. In this study, some normal antivirus medicines were detected to screen active medicine anti PCV.
In this study, PK-15 cell stem was used as the model for screening. The blank control and PCV-infected group were set. MTT assay is able to quantity detect alive cells to indirectly evaluation the effect of antivirus medicines. The cells were seeded into 96-well plate and cultured. PCV was added to monolayer cells and incubated for 6 h. the medicines in different dilution were added and the cells were incubated for 24 h. MTT was added and incubated for 4 h. The supernatant was removed, and the wells were added into dimethyl sulfoxide. The wells were incubated at 37℃for 30 min. the OD value at 490 nm was measured by microplate reader. The virus inhibition and medicine therapeutic index was calculated according to the formula. Results of Chinese medicine against Porcine Circovirus in vitro:The half maximal inhibitory concentration (IC50) of rhizoma coptidis, honeysuckle, fructus forsythiae and isatis root were 363.2, 719.7, 1596.7, 1189.4μg/mL; The therapeutic index(TI) of rhizoma coptidis, honeysuckle, fructus forsythiae and isatis root were 13.8, 4.1, 4.6 and 5.0。The results indicate that four Chinese medicine all could inhibit the prolification of porcine circovirus at cell level,but the effect of rhizoma coptidis are the most significant.
Results of drugs against Porcine Circovirus in vitro:The half maximal inhibitory concentration (IC50) of amantadine, ribavirin, arbidol hydrochlorid were 5.53, 1.19, 6.85μg/mL; The therapeutic index(TI) of amantadine, ribavirin, arbidol hydrochlorid were 33.8, 124.9and 33.1。The results indicate that three drugs all could inhibit the prolification of porcine circovirus at cell level,but the effect of ribavirin are the most significant.
The results showed that antivirus activity of chemical drugs is stronger than traditional Chinese medicine. Among the traditional Chinese medicine, rhizoma coptidis is the strongest. Among the chemical medicine, amantadine is the strongest.
引文
[1]陈溥言.兽医传染病学(第五版)[M].北京:中国农业出版社, 2006: 231– 234.
[2]Segales J, Domingo M, Chianini F, et al. Immunosuppression in postweaning multisystemic wasting syndrome affected pigs[J]. Vet Microbiol, 2004, 98(2): 151 - 158.
[3]王庆波,李昭春,袁冲.猪圆环病毒病及其防治策略[J].中国兽医杂志, 2010, 46(11): 88 - 89.
[4]郎洪武,张广川,吴发权等.断奶猪多系统衰弱综合征血清抗体检测[J].中国兽医科技, 2000, 30(3): 3-5.
[5]王忠田,杨汉春.规模化猪场PCV2感染的流行病学调查[J].中国兽医杂志, 2002, 38(10): 3-6.
[6]张国玲,康晓冬.宁夏规模化猪场猪圆环病毒和蓝耳病的流行病学和血清学调查[J].黑龙江畜牧兽医,2011,11:76-77.
[7]郎利敏,王克领,游一等.猪圆环病毒病的血清学调查及防治措施[J].河南农业科学, 2007, 11, 115-116.
[8]Allan G M, Ellis J A. Porcine circoviruses: A review[J]. J Vet Diagn Invest, 2000, 12(1): 3-14
[9]Chae C. A review of porcine circovirus 2-associated syndromes and diseases[J]. Vet J, 2005, 169 (3): 326-336.
[10]琚春梅,陈焕春,吴斌等.猪圆环病毒病的研究进展[J].养殖与饲料,2003,10:13-15.
[11] Madec F, Rose N, Grasland B, et al. Post-weaning multisystemic wasting syndrome and other PCV2-related problems in pigs: A 12-year experience[J].Transboundary and Emerg Dis,2008,55(7): 273-283.
[12]Fort M, Sibila M, Pérez-Martín E, et al. One dose of a porcine circovirus 2 (PCV2) subunit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model[J]. Vaccine, 2009, 27(30): 4031-4037.
[13]Opriessnig T, Patterson A R, Madson D M, et al. Comparison of efficacy of commercial one dose and two dose PCV2 vaccines using a mixed PRRSV-PCV2-SIV clinical infection model 2-3-months post vaccination[J]. Vaccine, 2009, 27(7): 1002-1007.
[14] Madson D M, Patterson A R, Ramamoorthy S, et al. Effect of porcine circovirus type 2 (PCV2) vaccination of the dam on PCV2 replication in utero[J]. Clin Vaccine Immunol, 2009, 16(6): 830-834.
[15]孙婉,李敏.药物筛选技术的最新进展—高内涵筛选[J].中国新药杂志,2006,15(1):12-16
[16]陈凯先,蒋华良,罗小民等.后基因组时代的药物发现,趋势和实践[J].中国天然药物,2004,2(5):257- 260.
[17]杜冠华.高通量药物筛选与中药现代化研究[J].中成药,1999 ,21( 5):268-270
[18]胡娟娟,杜冠华.药物筛选模型研究进展[J].基础医学与临床, 2001,21 (4):302-305
[19]杜冠华,胡娟娟,夏丽娟等.药物筛选的发展与现状[J].药学学报, 1998, 33( 11) 876- 879
[20]Enslein K1. An overview of structure2activity relationships as an alternative to testing in animalsfor carcinogenicity, mutagenicity,dermal and eye irritation , and acute oral toxicity[J]. Toxicol And Health, 1988, 4:479 - 498.
[21]黄家学,胡娟娟,杜冠华. 52HT受体结合物的高通量筛选[J].药学学报,2000, 35(增刊):53- 56.
[22]刘亚刚,刘世贵,刘晓钢.动物病毒性传染病的基本特征[J].四川畜牧兽医,2002,(7)
[23]张国红,章海欧,韩玉国.动物抗病毒药物的开发与应用[J].中国牧业通讯, 2000,(12).
[24]李巧如,任健康,刘宗智等. 18种中草药抗菌作用的筛选[J].陕西中医, 2002, 23 (6) : 555 - 556.
[25]匡铁吉,董梅,宋萍,等.黄连素对结核分枝杆菌的体外抑菌作用[J].中国中药杂志, 2001, 26 (12) :867 - 868.
[26]翟华强,王双艳,张硕峰等.黄连、丁香外用药理作用研究[J].中国实验方剂学杂志, 2011,17(11):192-194.
[27]马伏英.黄连等中药抗试验性小鼠柯萨奇B3病毒性心肌炎的实验研究[J].武警医学,1998,9(4):187-190.
[28]李凡,易世红,赵春艳等.双黄连粉针剂抗病毒作用[J].中草药, 2002, 33(1):52-55
[29]耿东升,刘发,刘学彬,等.硫酸黄连素对免疫系统的影响[J].中国药理学通报,1996,12(6) :536.
[30]何贤辉,曾耀英,徐丽慧,等.黄连素对T淋巴细胞活化和增殖的抑制作用[J].中国病理生理杂志, 2002,18(10) : 1 183 - 1 186.
[31]王睿,顾月荣.黄连降糖胶囊与二甲双胍治疗对2型糖尿病疗效比较[J].中医药学刊, 2003, 21(7) : 1189 -1190.
[32]康巧真,叶菲.黄连素治疗充血性心力衰竭17例疗效观察[J].中国自然医学杂志,2002, 4(2): 83 - 84 .
[33]Lin J G, Chung J G, Wu L T , et al. Effects of berberine on arylamine N - acetyltransfernse activity in human colon tuorcells [J] . Chin Med ,1999,27 (2):265 - 275.
[34]Fukuda K, Hibiya Y, Mutoh M , et al. Inhibition of activator protern 1 activity by berberine in human hepatoma cells[J] . Planta Med ,1999 ,65(4):381 - 383.
[35]王利津,徐强.黄连解毒汤的抗氧化作用研究[J].中国药科大学学报, 2001,1: 51-53.
[36]孙廷波.金银花对口腔病源性微生物体外抑菌实验的研究[J].中国中药志, 1996, 21(4): 242.
[37]陈晨华.四种中草药抗病毒研究[J].山东中医学院学报, 1993 , 17 (4) :46-48.
[38]阎明,王英才,夏德昭.复方金银花提取液抗Ⅰ型单纯疱疹病毒的实验研究[J].中国实用眼科杂志,1998,16(2):82.
[39]张素梅,杨明凡,王学斌.黄芪、金银花体外对伪狂犬病病毒作用研究[J].中兽医医药杂志,2003,(2):8-10.
[40]王国霞,邹海棠,梅春升.黄芪、金银花提取物体外抗禽流感病毒的试验研究[J].中兽医学杂志,2005,3, 4-6.
[41]王林青,崔保安,张红英.金银花、山银花黄酮类提取物体外抗伪狂犬病病毒作用研究[J].中国畜牧兽医, 2012,3,183-188.
[42]梁晓天.常用中药基础研究,第二卷[M].北京,科学出版社,2004:287-300.
[43]何显忠,兰荣德.金银花的药理作用与临床应用[J].时珍国医国药,2004,15(12):865-867.
[44]王本祥.现代中药药理学[M].天津:天津科学技术出版社,1997. 204– 208.
[45]雷志均,周日宝,曾嵘等.灰毡毛忍冬与正品金银花解热作用的比较研究[J].湖南中医学院学报,2005,25(5):14-15.
[46]侯冬岩,回瑞华,杨梅等.金银花中总黄酮的光谱分析及抗氧化性能测定[J].分析试验室,2004,23(11):52-55.
[47]孙德梅.金银花叶提取物的抗氧化活性和抑菌作用研究[J].河南科学,2002, 20(5): 511-513.
[48]张泽生,乌兰.金银花中绿原酸的体外抑菌和抗氧化性的研究[J].天津科技大学学报,2005,20(2):5-8.
[49]李晓燕.中药连翘抗菌活性的考察[J].山东医药工业,1997,16(2):46.
[50]马振亚.连翘种子挥发油抗流感病毒等病原微生物作用的实验研究[J].陕西新医药,1980,9(11):51.
[51]董杰德,陈晨华,仇素英.四种中草药抗柯萨奇及爱埃柯病毒的实验研究[J].山东中医学院学报,1993,17(4):46.
[52]于起福,孙非.四种中草药水煎剂抗柯萨奇B5病毒的细胞学实验研究[J].吉林中医药,1995,(1):35.
[53]张海燕.连翘化学成分及药理活性的研究进展[J].中药材,2000,23(10):657.
[54]刘静,杨建雄.连翘叶茶对小鼠非特异性免疫及应激作用的实验研究[J].榆林学院学报,2006,(2): 45-47.
[55]柴渭莉,杨建雄,周俊芳等.连翘叶苷类成分对力竭游泳小鼠心肌和骨骼肌超氧化物歧化酶活性、丙二醛含量及超微结构的影响[J].中国运动医学杂志,2007,(5): 609-610.
[56]杨建雄,侯改霞,李发荣等.连翘叶提取物对高脂血症的影响[J].中华中西医杂志, 2003; 24 (11)∶1621-1623.
[57]郑剑玲,王美惠,杨秀珍等.大青叶和板蓝根提取物的抑菌作用研究[J].中国微生态学杂志, 2003, 15 (1) : 18 - 19.
[58]刘盛,陈万生,乔传卓等.不同种质板蓝根和大青叶的抗甲型流感病毒作用[J].第二军医大学学报, 2000, 21 (3) : 204 - 206.
[59]张宸豪,高梅,马爱新等.板蓝根对柯萨奇病毒抑制作用的研究[J].第四军医大学吉林军医学院学报, 2003, 25 ( 3) : 125 -126.
[60]王学斌,魏战勇,崔保安等.板蓝根、黄芪对猪繁殖与呼吸综合征病毒的体外抑制作用[J].畜牧与兽医, 2006, 38(4): 11– 14.
[61]许益民,陆平成,王永珍等.板蓝根多糖促进免疫宫灯的实验研究[J].中西医结合杂志,1991,11(6):357-359.
[62]秦箐,贺海平, Soren B C,等.中药板蓝根两个高极流分的分离及活性的研究[J].中国药理学通报, 2001, 17 (6) : 714 - 715.
[63]胡兴昌,黄旭冬,许燕.板蓝根凝集素对小鼠胸腺发育的影响[J].解剖学杂志, 2000, 23 (6) : 559 - 562.
[64]单风平. 50%板蓝根注射液对小鼠Friend红白血细胞3CL-8体内外的杀伤作用[J].中草药,1994,25(8)417-418.
[65]游松,姚新生,陈英杰等.中药板蓝根中活血有效成分的研究[J].中药通报,1988,13(2):31-31.
[66]李丹,向华,黄良宗等.中药体外对猪繁殖与呼吸综合征病毒的影响[J].广东农业科学, 2009, 5: 140– 143.
[67]王林青,崔保安,张红英,等.金银花、山银花黄酮类提取物体外抗伪狂犬病病毒作用研究[J].中国畜牧兽医, 2011, 38(3): 183– 186.
[68]王英燕,王海.抗病毒中药的研究进展[J].中医药学报,2006,34(1):25-26.
[69]储怡士,王月雷.中药抗病毒合剂对小鼠巨噬细胞吞噬能力的影响[J].中医药学报,2006,34(1):25-26.
[70]邓海英,杨占秋,石丽桥等.盐酸阿比朵尔体外抗汉坦病毒药效学研究[J].中国药学杂志,2009,44(3):183-186.
[2]Segales J, Domingo M, Chianini F, et al. Immunosuppression in postweaning multisystemic wasting syndrome affected pigs[J]. Vet Microbiol, 2004, 98(2): 151 - 158.
[3]王庆波,李昭春,袁冲.猪圆环病毒病及其防治策略[J].中国兽医杂志, 2010, 46(11): 88 - 89.
[4]郎洪武,张广川,吴发权等.断奶猪多系统衰弱综合征血清抗体检测[J].中国兽医科技, 2000, 30(3): 3-5.
[5]王忠田,杨汉春.规模化猪场PCV2感染的流行病学调查[J].中国兽医杂志, 2002, 38(10): 3-6.
[6]张国玲,康晓冬.宁夏规模化猪场猪圆环病毒和蓝耳病的流行病学和血清学调查[J].黑龙江畜牧兽医,2011,11:76-77.
[7]郎利敏,王克领,游一等.猪圆环病毒病的血清学调查及防治措施[J].河南农业科学, 2007, 11, 115-116.
[8]Allan G M, Ellis J A. Porcine circoviruses: A review[J]. J Vet Diagn Invest, 2000, 12(1): 3-14
[9]Chae C. A review of porcine circovirus 2-associated syndromes and diseases[J]. Vet J, 2005, 169 (3): 326-336.
[10]琚春梅,陈焕春,吴斌等.猪圆环病毒病的研究进展[J].养殖与饲料,2003,10:13-15.
[11] Madec F, Rose N, Grasland B, et al. Post-weaning multisystemic wasting syndrome and other PCV2-related problems in pigs: A 12-year experience[J].Transboundary and Emerg Dis,2008,55(7): 273-283.
[12]Fort M, Sibila M, Pérez-Martín E, et al. One dose of a porcine circovirus 2 (PCV2) subunit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model[J]. Vaccine, 2009, 27(30): 4031-4037.
[13]Opriessnig T, Patterson A R, Madson D M, et al. Comparison of efficacy of commercial one dose and two dose PCV2 vaccines using a mixed PRRSV-PCV2-SIV clinical infection model 2-3-months post vaccination[J]. Vaccine, 2009, 27(7): 1002-1007.
[14] Madson D M, Patterson A R, Ramamoorthy S, et al. Effect of porcine circovirus type 2 (PCV2) vaccination of the dam on PCV2 replication in utero[J]. Clin Vaccine Immunol, 2009, 16(6): 830-834.
[15]孙婉,李敏.药物筛选技术的最新进展—高内涵筛选[J].中国新药杂志,2006,15(1):12-16
[16]陈凯先,蒋华良,罗小民等.后基因组时代的药物发现,趋势和实践[J].中国天然药物,2004,2(5):257- 260.
[17]杜冠华.高通量药物筛选与中药现代化研究[J].中成药,1999 ,21( 5):268-270
[18]胡娟娟,杜冠华.药物筛选模型研究进展[J].基础医学与临床, 2001,21 (4):302-305
[19]杜冠华,胡娟娟,夏丽娟等.药物筛选的发展与现状[J].药学学报, 1998, 33( 11) 876- 879
[20]Enslein K1. An overview of structure2activity relationships as an alternative to testing in animalsfor carcinogenicity, mutagenicity,dermal and eye irritation , and acute oral toxicity[J]. Toxicol And Health, 1988, 4:479 - 498.
[21]黄家学,胡娟娟,杜冠华. 52HT受体结合物的高通量筛选[J].药学学报,2000, 35(增刊):53- 56.
[22]刘亚刚,刘世贵,刘晓钢.动物病毒性传染病的基本特征[J].四川畜牧兽医,2002,(7)
[23]张国红,章海欧,韩玉国.动物抗病毒药物的开发与应用[J].中国牧业通讯, 2000,(12).
[24]李巧如,任健康,刘宗智等. 18种中草药抗菌作用的筛选[J].陕西中医, 2002, 23 (6) : 555 - 556.
[25]匡铁吉,董梅,宋萍,等.黄连素对结核分枝杆菌的体外抑菌作用[J].中国中药杂志, 2001, 26 (12) :867 - 868.
[26]翟华强,王双艳,张硕峰等.黄连、丁香外用药理作用研究[J].中国实验方剂学杂志, 2011,17(11):192-194.
[27]马伏英.黄连等中药抗试验性小鼠柯萨奇B3病毒性心肌炎的实验研究[J].武警医学,1998,9(4):187-190.
[28]李凡,易世红,赵春艳等.双黄连粉针剂抗病毒作用[J].中草药, 2002, 33(1):52-55
[29]耿东升,刘发,刘学彬,等.硫酸黄连素对免疫系统的影响[J].中国药理学通报,1996,12(6) :536.
[30]何贤辉,曾耀英,徐丽慧,等.黄连素对T淋巴细胞活化和增殖的抑制作用[J].中国病理生理杂志, 2002,18(10) : 1 183 - 1 186.
[31]王睿,顾月荣.黄连降糖胶囊与二甲双胍治疗对2型糖尿病疗效比较[J].中医药学刊, 2003, 21(7) : 1189 -1190.
[32]康巧真,叶菲.黄连素治疗充血性心力衰竭17例疗效观察[J].中国自然医学杂志,2002, 4(2): 83 - 84 .
[33]Lin J G, Chung J G, Wu L T , et al. Effects of berberine on arylamine N - acetyltransfernse activity in human colon tuorcells [J] . Chin Med ,1999,27 (2):265 - 275.
[34]Fukuda K, Hibiya Y, Mutoh M , et al. Inhibition of activator protern 1 activity by berberine in human hepatoma cells[J] . Planta Med ,1999 ,65(4):381 - 383.
[35]王利津,徐强.黄连解毒汤的抗氧化作用研究[J].中国药科大学学报, 2001,1: 51-53.
[36]孙廷波.金银花对口腔病源性微生物体外抑菌实验的研究[J].中国中药志, 1996, 21(4): 242.
[37]陈晨华.四种中草药抗病毒研究[J].山东中医学院学报, 1993 , 17 (4) :46-48.
[38]阎明,王英才,夏德昭.复方金银花提取液抗Ⅰ型单纯疱疹病毒的实验研究[J].中国实用眼科杂志,1998,16(2):82.
[39]张素梅,杨明凡,王学斌.黄芪、金银花体外对伪狂犬病病毒作用研究[J].中兽医医药杂志,2003,(2):8-10.
[40]王国霞,邹海棠,梅春升.黄芪、金银花提取物体外抗禽流感病毒的试验研究[J].中兽医学杂志,2005,3, 4-6.
[41]王林青,崔保安,张红英.金银花、山银花黄酮类提取物体外抗伪狂犬病病毒作用研究[J].中国畜牧兽医, 2012,3,183-188.
[42]梁晓天.常用中药基础研究,第二卷[M].北京,科学出版社,2004:287-300.
[43]何显忠,兰荣德.金银花的药理作用与临床应用[J].时珍国医国药,2004,15(12):865-867.
[44]王本祥.现代中药药理学[M].天津:天津科学技术出版社,1997. 204– 208.
[45]雷志均,周日宝,曾嵘等.灰毡毛忍冬与正品金银花解热作用的比较研究[J].湖南中医学院学报,2005,25(5):14-15.
[46]侯冬岩,回瑞华,杨梅等.金银花中总黄酮的光谱分析及抗氧化性能测定[J].分析试验室,2004,23(11):52-55.
[47]孙德梅.金银花叶提取物的抗氧化活性和抑菌作用研究[J].河南科学,2002, 20(5): 511-513.
[48]张泽生,乌兰.金银花中绿原酸的体外抑菌和抗氧化性的研究[J].天津科技大学学报,2005,20(2):5-8.
[49]李晓燕.中药连翘抗菌活性的考察[J].山东医药工业,1997,16(2):46.
[50]马振亚.连翘种子挥发油抗流感病毒等病原微生物作用的实验研究[J].陕西新医药,1980,9(11):51.
[51]董杰德,陈晨华,仇素英.四种中草药抗柯萨奇及爱埃柯病毒的实验研究[J].山东中医学院学报,1993,17(4):46.
[52]于起福,孙非.四种中草药水煎剂抗柯萨奇B5病毒的细胞学实验研究[J].吉林中医药,1995,(1):35.
[53]张海燕.连翘化学成分及药理活性的研究进展[J].中药材,2000,23(10):657.
[54]刘静,杨建雄.连翘叶茶对小鼠非特异性免疫及应激作用的实验研究[J].榆林学院学报,2006,(2): 45-47.
[55]柴渭莉,杨建雄,周俊芳等.连翘叶苷类成分对力竭游泳小鼠心肌和骨骼肌超氧化物歧化酶活性、丙二醛含量及超微结构的影响[J].中国运动医学杂志,2007,(5): 609-610.
[56]杨建雄,侯改霞,李发荣等.连翘叶提取物对高脂血症的影响[J].中华中西医杂志, 2003; 24 (11)∶1621-1623.
[57]郑剑玲,王美惠,杨秀珍等.大青叶和板蓝根提取物的抑菌作用研究[J].中国微生态学杂志, 2003, 15 (1) : 18 - 19.
[58]刘盛,陈万生,乔传卓等.不同种质板蓝根和大青叶的抗甲型流感病毒作用[J].第二军医大学学报, 2000, 21 (3) : 204 - 206.
[59]张宸豪,高梅,马爱新等.板蓝根对柯萨奇病毒抑制作用的研究[J].第四军医大学吉林军医学院学报, 2003, 25 ( 3) : 125 -126.
[60]王学斌,魏战勇,崔保安等.板蓝根、黄芪对猪繁殖与呼吸综合征病毒的体外抑制作用[J].畜牧与兽医, 2006, 38(4): 11– 14.
[61]许益民,陆平成,王永珍等.板蓝根多糖促进免疫宫灯的实验研究[J].中西医结合杂志,1991,11(6):357-359.
[62]秦箐,贺海平, Soren B C,等.中药板蓝根两个高极流分的分离及活性的研究[J].中国药理学通报, 2001, 17 (6) : 714 - 715.
[63]胡兴昌,黄旭冬,许燕.板蓝根凝集素对小鼠胸腺发育的影响[J].解剖学杂志, 2000, 23 (6) : 559 - 562.
[64]单风平. 50%板蓝根注射液对小鼠Friend红白血细胞3CL-8体内外的杀伤作用[J].中草药,1994,25(8)417-418.
[65]游松,姚新生,陈英杰等.中药板蓝根中活血有效成分的研究[J].中药通报,1988,13(2):31-31.
[66]李丹,向华,黄良宗等.中药体外对猪繁殖与呼吸综合征病毒的影响[J].广东农业科学, 2009, 5: 140– 143.
[67]王林青,崔保安,张红英,等.金银花、山银花黄酮类提取物体外抗伪狂犬病病毒作用研究[J].中国畜牧兽医, 2011, 38(3): 183– 186.
[68]王英燕,王海.抗病毒中药的研究进展[J].中医药学报,2006,34(1):25-26.
[69]储怡士,王月雷.中药抗病毒合剂对小鼠巨噬细胞吞噬能力的影响[J].中医药学报,2006,34(1):25-26.
[70]邓海英,杨占秋,石丽桥等.盐酸阿比朵尔体外抗汉坦病毒药效学研究[J].中国药学杂志,2009,44(3):183-186.