1、一种中药含药血清及五种杀微生物中药制剂抗HIV-1作用2、乙酰水杨酸铜对HCT-116细胞生长的抑制及诱导凋亡作用
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摘要
目的：测定一种中药含药血清及五种杀微生物中药制剂抗HIV-1活性，并对含药血清药理学研究方法及药物直接灭活HIV活性的检测方法进行探讨和评价。方法：采用合胞体形成抑制实验检测中药含KDX血清及KDX粗制剂抗HIV-1活性；采用MTT法测定KZL-1、2、3，SSJK及表没食子儿茶素没食子酸酯(EGCG)对人T淋巴细胞系(C8166)的细胞毒性，并以PEG6000的盐溶液浓缩病毒，结合合胞体形成抑制实验检测所选样品对HIV-1的直接灭活作用。结果：(1)未经灭活处理的大鼠含KDX血清空白组、低剂量组、高剂量组及KDX粗制剂抑制半数合胞体形成的浓度(EC_(50))分别为1／60(v／v)，1／106(v／v)，1／108(v／v)，0.02mg/ml；经灭活处理的各组含KDX血清对HIV-1诱导合胞体形成的抑制率均小于50％，无法计算EC_(50)。(2)KZL-1、2、3，SSJK及EGCG的半数细胞毒性浓度(CC_(50))分别为1／34(v／V)、1／41(V／V)、1／1698(V／v)、1／633(v／v)、10.27μg／ml；灭活50％HIV-1病毒的浓度(INC_(50))分别为1／96(v／v)、1／513(v／v)、1／14(v／v)、1／50(v／v)、7.29μg／ml。结论：(1)在所选KDX剂量范围内，大鼠含药血清未表现出抗HIV-1活性，而粗制剂有明显的抗HIV-1活性，EC_(50)为0.02mg/ml。(2)KZL-2对HIV-1有很好的灭活作用，KZL-1次之，而KZL-3、SSJK及EGCG在对HIV-1产生灭活作用的剂量范围内均表现出明显的毒性。(3)中药含药血清与粗制剂实验结果并不总是一致，因而，不能简单地依据中药粗制剂体外实验结果来判断药效。(4)中药含药血清药理学实验更接近体内实验，较直接用粗制剂检测更合理。(5)采用PEG6000的盐溶液浓缩病毒，通过合胞体形成抑制实验检测残留病毒感染性是一种简易、快速、敏感的检测杀微生物侯选物潜在灭活HIV-1活性的实验方法。
Objectives: The anti-HIV-1 activity of a kind of drug-containing serum and five kinds of traditional herbal mixtures was detected. Besides, the serologic pharmacologic method and viral inactivation assay were analysed and assessed. Methods: Anti-HIV-1 activity was detected by syncytial formation inhibition assay. KDX direct addition and rats' KDX-containing serum addition were used respectively. In addition, cytotoxicity of KZL-1,2,3, SSJK, EGCG to Human T-lymphocyte lines(C8166) was detected by MTT assay; PEG6000 solution in saline was used to concentrate viruses. Simultaneously, syncytial formation inhibition assay was used to detect the effect of HIV-1 inactivation. Results: (1) The 50% effective concentration(EC5o) of rats' KDX-containing sera (not inactivated) control group, low dose group, high dose group and KDX original reagent are l/60(v/v), l/106(v/v), l/108(v/v), 0.02mg/ml, respectively. The 50% effective concentration(EC50) of all groups of rats' KDX-containing sera (inactivated) are less than 50
%, EC650 can not be calculated. (2)The 50% cytotoxic concentration(CC50) of KZL-1,2,3, SSJK and EGCG are l/34(v/v), l/41(v/v), l/1698(v/v), 1/633(v/v) , 10.27μg/ml, respectively; and the 50% HIV-inactivated concentration(INC50) are l/96(v/v),l/513(v/v), l/14(v/v), l/50(v/v), 7.29 (μg/ml, respectively. Conclusions: (l)The KDX original reagent showed obvious anti-HIV activities, EC50 is 0.02mg/ml. However, the rats' KDX-containing serum didn't showed anti-HIV activities at the dose adopted in this study. (2)The HIV-1 inactivation effect of KZL-2 was more powerful than KZL-1, and other samples all appeared to obvious cytotoxicity around the HIV-inactivated concentration range. (3)The experiment results of drug-containing serum and original reagent aren't always consistent,so we can't draw a conclusion merely according to
the result of the latter. (4)The method of Serologic pharmacologic experiment is more similar to in vivo than that of original reagent direct addition. Apparently, it is more reasonable. (5)PEG6000 solution in saline was used to precipitate viruses, and Syncytial formation inhibition assay was adopted to detect the infection activity of residual viruses,which is a simple, quick and sensitive method to evaluate potential virucidal effect of most microbicide candidates.
Objectives: The anti-HIV-1 activity of a kind of drug-containing serum and five kinds of traditional herbal mixtures was detected. Besides, the serologic pharmacologic method and viral inactivation assay were analysed and assessed. Methods: Anti-HIV-1 activity was detected by syncytial formation inhibition assay. KDX direct addition and rats' KDX-containing serum addition were used respectively. In addition, cytotoxicity of KZL-1,2,3, SSJK, EGCG to Human T-lymphocyte lines(C8166) was detected by MTT assay; PEG6000 solution in saline was used to concentrate viruses. Simultaneously, syncytial formation inhibition assay was used to detect the effect of HIV-1 inactivation. Results: (1) The 50% effective concentration(EC5o) of rats' KDX-containing sera (not inactivated) control group, low dose group, high dose group and KDX original reagent are l/60(v/v), l/106(v/v), l/108(v/v), 0.02mg/ml, respectively. The 50% effective concentration(EC50) of all groups of rats' KDX-containing sera (inactivated) are less than 50
%, EC650 can not be calculated. (2)The 50% cytotoxic concentration(CC50) of KZL-1,2,3, SSJK and EGCG are l/34(v/v), l/41(v/v), l/1698(v/v), 1/633(v/v) , 10.27μg/ml, respectively; and the 50% HIV-inactivated concentration(INC50) are l/96(v/v),l/513(v/v), l/14(v/v), l/50(v/v), 7.29 (μg/ml, respectively. Conclusions: (l)The KDX original reagent showed obvious anti-HIV activities, EC50 is 0.02mg/ml. However, the rats' KDX-containing serum didn't showed anti-HIV activities at the dose adopted in this study. (2)The HIV-1 inactivation effect of KZL-2 was more powerful than KZL-1, and other samples all appeared to obvious cytotoxicity around the HIV-inactivated concentration range. (3)The experiment results of drug-containing serum and original reagent aren't always consistent,so we can't draw a conclusion merely according to
the result of the latter. (4)The method of Serologic pharmacologic experiment is more similar to in vivo than that of original reagent direct addition. Apparently, it is more reasonable. (5)PEG6000 solution in saline was used to precipitate viruses, and Syncytial formation inhibition assay was adopted to detect the infection activity of residual viruses,which is a simple, quick and sensitive method to evaluate potential virucidal effect of most microbicide candidates.
引文
[1] Zhang KL, Ma SJ. Epidemiology of HIV in China: Intravenous drug users,sex workers,and large mobile populations are high-risk groups. BMJ.2002, 324:803-804
[2] 刘成海．中药复方体外药理研究思考．中药新药与临床药理．2000，1(1)：53-56
[3] Lard SL, .Kenneth L. Hastings, and David Feigal. Considerations in the development of vaginal products intended to prevent the sexual transmission of HIV. From: Proceedings of the First Workshop on Antiviral Claims for Topical Antiseptics. 1994.
[4] Stone A. Microbicides: a new approach to preventing HIV and other sexually transmitted infections. Nat Rev Drug Discov.2002,1 (12):977-985.
[5] 吕繁，刘中夫等．中国艾滋病监测现状及近期监测工作要点．中国性病艾滋病防治．2002，8(6)：321-324
[6] Yamaguchi K, Honda M, Ikigai H, et al. Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1). Antiviral Research.2000,53 (2002): 19-34
[7] 韩俭，于红娟，吴勇杰，等．中药血清药理学方法学研究-抗菌试验含药血清处理方案研究．中药药理与临床．2002，18(1)：47-48
[8] 杜平，范中善，郭葆玉．病毒在细胞上的感染性滴定．医学实验病毒学[M]．人民军医出版社．1995，102-109
[9] Kohno T, Mohan S, Goto T, et al. A new improved method fbr the concentration of HIV- 1 infective particles.J Virol Methods. 2002,106(2): 167-173.
[10] 杨奎，张德波，史焱，等．含黄芩血清及黄芩甙影响内生致热原产生的研究．中药药理与临床．1994，6：13-14
[11] 李振光，王净净．关于中药血清药理学方法的思考．中国中医药信息杂志．2002，9(2)：5-6
[12] 吴水生，张小如，阮时宝．血清药理学研究中含药血清是否需灭活的探讨．福建医学院学报．2001，11(2)：45-46
[13] 徐海波，李彩君．中药血清药理学方法探讨．中国中医基础医学杂志．1999，5(8)：13-15
[14] 崔晓兰，贺玉琢，高英杰，等．中药复方血清药理研究方法学探讨-1．中国方剂学杂志．1998，4(2)：13-15
[15] 俞仲毅，汪鸿宇，胡月娟，等．小青龙汤整体给药与含药血清作用的比较研究．中国中医药科技．2001，8(4)：233-234
[16] Van de Wijgert J, Coggins C. Microbicides to prevent heterosexual transmission of HIV: ten years down the road. BETA.2002,15(2):23-28.
[17] Niruthisard S, Roddy RE, Chutivongse S. The effects of frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis. 1991,18(3): 176-179.
[18] Kreiss J, Ngugi E, Holmes K, et al. Efficacy of nonoxynol-9 contraceptive sponge use in preventing heterosexual acquisition of HIV in Nairobi prostitutes. JAMA. 1992,268 (4):477-482
[19] Resnick L, Veren K, Salahuddin SZ et al. Stability and inactivation of HTLV-Ⅲ/LAV under clinical and laboratory environments. JAMA.1986,255(14): 1887-1891. JAMA. 1992,268 (4):477-482
[20] Aranda-Anzaldo A, Viza D, Busnel RG. Chemical inactivation of human immunodeficiency virus in vitro. J Virol Methods. 1992,37(1):71-81.
[21] Howett MK, Neely EB, Christensen ND, et al. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob Agents Chemother. 1999,43(2):314-21.
[22] Krebs FC, Miller SR, Malamud D, et al. Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate. Antiviral Res. 1999,43(3): 157-73.
[23] Greene RA, Japour AJ, Brewster F, et al. Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-flee RT assay. Clin Diagn Virol. 1996,7 (2): 111 - 119.
[24] Sarmati L, Ercoli L, Parisi SG, et al. High rate of HIV isolation from plasma of asymptomatic patients through polyethylene glycol (PEG) treatment.J Acquir Immune Defic Syndr.1994,7 (1):10-14.
[25] Sanyal D, Kudesia G, Corbitt G. Comparison of ultracentrifugation and polyethylene glycol precipitation for concentration of hepatitis B virus (HBV) DNA for molecular hybridisation tests and the relationship of HBV-DNA to HBe antigen and anti-HBe status. J Med Microbiol. 1991,35 (5):291-293.
[2] 刘成海．中药复方体外药理研究思考．中药新药与临床药理．2000，1(1)：53-56
[3] Lard SL, .Kenneth L. Hastings, and David Feigal. Considerations in the development of vaginal products intended to prevent the sexual transmission of HIV. From: Proceedings of the First Workshop on Antiviral Claims for Topical Antiseptics. 1994.
[4] Stone A. Microbicides: a new approach to preventing HIV and other sexually transmitted infections. Nat Rev Drug Discov.2002,1 (12):977-985.
[5] 吕繁，刘中夫等．中国艾滋病监测现状及近期监测工作要点．中国性病艾滋病防治．2002，8(6)：321-324
[6] Yamaguchi K, Honda M, Ikigai H, et al. Inhibitory effects of (-)-epigallocatechin gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1). Antiviral Research.2000,53 (2002): 19-34
[7] 韩俭，于红娟，吴勇杰，等．中药血清药理学方法学研究-抗菌试验含药血清处理方案研究．中药药理与临床．2002，18(1)：47-48
[8] 杜平，范中善，郭葆玉．病毒在细胞上的感染性滴定．医学实验病毒学[M]．人民军医出版社．1995，102-109
[9] Kohno T, Mohan S, Goto T, et al. A new improved method fbr the concentration of HIV- 1 infective particles.J Virol Methods. 2002,106(2): 167-173.
[10] 杨奎，张德波，史焱，等．含黄芩血清及黄芩甙影响内生致热原产生的研究．中药药理与临床．1994，6：13-14
[11] 李振光，王净净．关于中药血清药理学方法的思考．中国中医药信息杂志．2002，9(2)：5-6
[12] 吴水生，张小如，阮时宝．血清药理学研究中含药血清是否需灭活的探讨．福建医学院学报．2001，11(2)：45-46
[13] 徐海波，李彩君．中药血清药理学方法探讨．中国中医基础医学杂志．1999，5(8)：13-15
[14] 崔晓兰，贺玉琢，高英杰，等．中药复方血清药理研究方法学探讨-1．中国方剂学杂志．1998，4(2)：13-15
[15] 俞仲毅，汪鸿宇，胡月娟，等．小青龙汤整体给药与含药血清作用的比较研究．中国中医药科技．2001，8(4)：233-234
[16] Van de Wijgert J, Coggins C. Microbicides to prevent heterosexual transmission of HIV: ten years down the road. BETA.2002,15(2):23-28.
[17] Niruthisard S, Roddy RE, Chutivongse S. The effects of frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis. 1991,18(3): 176-179.
[18] Kreiss J, Ngugi E, Holmes K, et al. Efficacy of nonoxynol-9 contraceptive sponge use in preventing heterosexual acquisition of HIV in Nairobi prostitutes. JAMA. 1992,268 (4):477-482
[19] Resnick L, Veren K, Salahuddin SZ et al. Stability and inactivation of HTLV-Ⅲ/LAV under clinical and laboratory environments. JAMA.1986,255(14): 1887-1891. JAMA. 1992,268 (4):477-482
[20] Aranda-Anzaldo A, Viza D, Busnel RG. Chemical inactivation of human immunodeficiency virus in vitro. J Virol Methods. 1992,37(1):71-81.
[21] Howett MK, Neely EB, Christensen ND, et al. A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses. Antimicrob Agents Chemother. 1999,43(2):314-21.
[22] Krebs FC, Miller SR, Malamud D, et al. Inactivation of human immunodeficiency virus type 1 by nonoxynol-9, C31G, or an alkyl sulfate, sodium dodecyl sulfate. Antiviral Res. 1999,43(3): 157-73.
[23] Greene RA, Japour AJ, Brewster F, et al. Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-flee RT assay. Clin Diagn Virol. 1996,7 (2): 111 - 119.
[24] Sarmati L, Ercoli L, Parisi SG, et al. High rate of HIV isolation from plasma of asymptomatic patients through polyethylene glycol (PEG) treatment.J Acquir Immune Defic Syndr.1994,7 (1):10-14.
[25] Sanyal D, Kudesia G, Corbitt G. Comparison of ultracentrifugation and polyethylene glycol precipitation for concentration of hepatitis B virus (HBV) DNA for molecular hybridisation tests and the relationship of HBV-DNA to HBe antigen and anti-HBe status. J Med Microbiol. 1991,35 (5):291-293.