| AIDS is the most seriously disease for human health. During the struggle against AIDS,people developed many antiviral drugs, which reduced the incidence of AIDS and mortalityrate. Today, more than 90％of new HIV infections are spread through unprotectedintercourse in whole world. To develop a microbicides used under women control has beenrecognized as an important method as vaccine of HIV to control transmission of HIV-1.There has been significant progress in microbicide research and development in the past 15years. Over 30 compounds or formulations are undergoing preclinical or clinical studies.However, the number of potential products is not large enough. Discovery of novelcompounds for use as potential microbicide candidates is a long-term task. In this paper, wefocus on the developing novel microbicides from natural products.1. In vitro anti-HIV activity and safety of sodium rutin sulfate as a microbicidecandidateRutin or quercetin rutinoside, is a flavonol glycoside. Its sulfated compound, sodiumrutin sulfate (SRS), has activity of anti-HIV found by our research group. From the view ofmicrobicide development, in present study, we evaluated its activity of anti-HIV and -HSV,its mechanism and safety in vitro. For detection of anti-HIV-1 activity of SRS, severallaboratory adapted HIV-1 strains (including the viruses using either coreceptor CXCR 4(HIV-lain) or CCR5 (HIV-1Ba-L and HIV-1Ada-M) for entry into host cells) and primaryHIV-1 isolates (particularly CCR5- used viruses) were used for this study. Anti-HIV activitywas detected with syncytial formation assay and quantitative P24 enzyme-linkedimmunosorbent assay (ELISA).The results showed that the IC50s of SRS for anti-HIV-1ⅢB,-HIV-1Ba-L and -HIV - 1Ada-M activities were 2.3±0.2μM, 8.5±3.8μM and 4.5+2.0μM,respectively, and the CC50s of SRS to corresponding virus host cells C8166, TZM-b1 and PBMC were 1295.4±269.8μM, 2800.0±204.5μM and 2589.5±204.5μM, respectively. Theselective indexes (SI) of SRS for the above paired viruses and cells were 563,329 and 575,respectively. However, the non-sulfated compound rutin did not show any anti-HIV-1ⅢBactivity. For primary R5 viruses, the IC50s of SRS were 13.1±5.5μM, and SI of SRS was197. Time-of-addition experiment indicated that if SRS was added after viral entry (2h postinfection), its inhibitory activity completely lost. SRS may prevent the cell fusion of C8166cells and chronically infected H9 cells. Furthermore, when HIV-1ⅢB virus was pre-treatedby SRS, virus lost infectivity; when C8166 was pre-treated by SRS, SRS did not showantiviral activity. Taken together, the above three experiments provided direct evidence forconfirming interaction between SRS and virus. In vitro test also showed that SRS has noeffect on HIV-1 RT and protease. Cytotoxicities were tested with MTT colorimetric assay,the half inhibition concentrations (CC50) for HeLa and ME180 cell lines and humanforeskin fibroblast cells was more than 3.0 mM. Anti-vaginal bacteria activity were testedwith microdilution method, minimal inhibition concentration (MIC) for vaginallactobacteria was more than 3.0 mM. Conclusion: These results suggest that.SRS had highanti-HIV activities and low cytotoxicity, and to vaginal lactobacteria and could be furtherstudied as a potential candidate of microbicides.2.Preliminary evaluation on in vitro anti-HIV activities and safety ofpolysaccharide sulfate PS20Many polysaccharides derived from numerous species have been found to exert apotent inhibitory effect against HIV-1 in vitro. We evaluated in vitro anti-HIV and -otherSTD pathogen activities and safety of polysaccharide sulfate PS20. Methods: anti-HIVactivity was detected with syncytial formation assay and quantitative P24 enzyme-linkedimmunosorbent assay (ELISA); anti-HSV activity was detected with plaque reduction assay;eytotoxieities were tested with MTT colorimetric assay and anti-bacteria activity was testedwith microdilution method. Antiviral mechanism was investigated with fusion inhibitionand time of addition experiments. The results showed that the half inhibition concentrations(IC50) of PS20 for HIV- 1Ⅲa, HIV- 1Ada-m HIV- 1Bal, HSV- I were 0.261μM, 0.46μM, 0.90μM 3.45μM, respectively; their selective indexes (SI = IC50 / CC50)) were 653, 50, 39, 85,respectively. The 50％inhibition concentrations (CC50) of PS20 for HeLa and ME180 cellsand human foreskin fibroblast cells were more than 3.0 mM. No effect for candida albicansand neisseria gonorrhoeae. Minimal inhibition concentration (MIC) for vaginallactobacteria was more than 1 mM. Conclusion: These results suggest that PS20 had highanti-HIV and -HSV activities and low cytotoxicity to genital cell, and to vaginallactobacteria and could be further studied as a potential candidate of microbicides.3. In vitro anti-HIV activity and safety of lanthanum citrate and lanthanumnitrate as a microbicide candidateAn ideal microbicide should be a combination of compounds which could blockinfection of HIV at different stages of life cycle as highly active antiretroviral therapy(HAART) for AIDS. We evaluated in vitro anti-HIV and -other STD pathogen activitiesand safety of lanthanum citrate and lanthanum nitrate. The results showed that the halfinhibition concentrations (IC50)of lanthanum nitrate for HIV- 1ⅢB, HIV- 1Ada-M, HIV- 1Bal andprimary HIV-1 isolates were 67.3±2.5μM, 438±2.6μM, 368.7±2.5μM and 592±4.3μM,respectively; its selective indexes (SI = IC50 / CC50) were 111, 16, 19,15, respectively;IC50s of lanthanum citrate for the above viruses were3.08±0.98μM, 407.0±2.2μM, 52.7±3.2μM and 51.4±5.4μM, respectively; its selective indexeswere 293, 7, 58, 161, respectively. Lanthanum citrate and lanthanum nitrate did not showany anti-HSV activity. The half inhibition concentrations (CC50) for HeLa and ME 180 celllines were more than 10 mM. Minimal inhibition concentration (MIC) for vaginallactobacteria was more than 30mM. Time-of-addition experiment indicated that iflanthanum citrate was added alter viral entry (2h post infection), its inhibitory activitycompletely lost. Lanthanum citrate may prevent the cell fusion of C8166 cells and H9 cellsfrom chronical infection. When HIV-1ⅢB virus was pre-treated by lanthanum citrate, thevirus could infect cells. When C8166 cells were pre-treated by lanthanum citrate, viruscould not infect the C8166 cells. Taken together, the above experiments provided directevidence for confirming interaction between lanthanum citrate and cell. Lanthanum citrate may be useful in combination with compounds which had different anti-HIV mechanisms.These results suggest that lanthanum citrate has good anti-HIV activity, and merits furtherstudy as a potential candidate of microbicides. |
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