Studies On The Chemical Constituents For Anti-HIV Activity And The Quality Control Of Salvia Yunnanensis

The aqueous extracts of Salviamiltiorrhiza were found to have potenteffect against human immunodeficiency virus type 1 (HIV-1) both in vitroand in vivo in our laboratory. Later, many species of salvia genus wereevaluated for their anti-HIV activity, and Salvia yunnanensis was foundto have the best effect against HIV among them. Further investigation onthe chemical constituents and the quality control of Salvia yunnanensiswere carried out, at the same time, the anti-HIV activity of some phenolicacids were examined. The research results are mainly stated as below:1. The research on active constitutents against HIV in roots of Salviayunnanensis: 40 compounds were isolated from the aqueous extracts of theradix salvia yunnanensis by chromatographic methods (diaion HP20 columnchromatography, sephadex LH-20 column chromatography, ODS columnchromatography). 31 of which were characterised and structurallyelucidated, including 25 polyphenols, 1 fatty acid, 1 triterpenoid, 2sterides, 3 tanshinones, by means of physico-chemical methods andspectroscopic techniques (such as UV or IR spectroscopy, one- andtwo-dimensional NMR techniques as well as MS techniques), or by comparisonwith some authentic samples. Among 31 compounds, five were new compounds,and elucidated as ethyl rosmarinate (8), ethyl salvianolate A (10), methylsalvianolate A (11), cis-lithospermic acid (14) and salvianolic acid N(24), respectively. Other 26 known compounds were protocatechuic dehyde(1), protocatechuic acid (2), caffeic acid (3),R-(+)-β-D-(3, 4-dihydroxyphenyl)-lactic acid (4), ferulic acid (5),rosmarinic acid (6), methyl rosmarinate (7), salvianolic acid A (9),salvianolic acid C (12), lithospermic acid (13), magnesium lithospermate(15), 9'-methyl lithospermate (16), lithospermic acid B (17), magnesiumlithospermate B (18), 9'-methyl lithospermate B (19), 9"'-methyl lithospermate B (20), 9', 9'"-dimethyl lithospermate B (21), 9'-ethyllithospermate B (22), yunnaneic acid E (23), dotriacontanoic acid (25),oleanolic acid (26),β-sitosterol (27), daucosterol (28), tanshinoneâ…¡A (29), tanshinoneâ… (30), cryptotanshinone (31). The compounds 1-5, 7,9, 12, 16-22, 25-28 were first isolated from this plant, and compound 15was first got from Salvia.2. The research on anti-HIV activityof the polyphenols: The polyphenolsisolated from S. yunnanensis were tested for their inhibitory activitieson HIV-1 integrase (IN), reverse transcript (RT) in vitro and HIV-1 P24in MT-4 cell cultures. The HIV-1 IN was detected by ELISA method with apositive control of laboratory proved active polysaccharide sulfate S-y,and the IC₅₀ values were calculated. The HIV-1 RT was detected by ³Hincorporation method with a positive control of clinical usednon-nucleotide nerverapine (NVP), and the IC₅₀ values were calculated.Different concentrations of the polyphenols and a positive controlZeduvidine (AZT) were added to HIV-1 IIIB infected MT-4 cells, after 4days, the supernatants were tested for P24 titers by ELISA methods. Thecells were tested for cytoxicity by MTT methods. Their IC₅₀, TC₅₀ and SIvalues were calculated. The results showed all the polyphenols isolatedfrom S. yunnanensis were active on HIV-1 IN and in MT-4 cell cultures,but weak inhibitors on HIV-1 RT.3. The research on the quality control of S. yunnanensis: A gradientreversed phase HPLC method was developed for the simultaneous separationand determination of five polyphenols, yunnaneic acid E, rosmarinic acid,lithospermic acid, lithospermic acid B and salvianolic acid A in S.yunnanensis and S. miltiorrhiza. All calibration curves showed goodlinearity (r²ï¼ž0.999) within test ranges. The assay was reproducible withoverall intra- and inter-day variation of less than 5ï¼…. The mean recoveryof the method was in the range from 97 to 104ï¼…, with R.S.D. less than 5ï¼…. The assay was successfully applied to the determination of fivepolyphenols in nine resource samples collected in Yunnan Province. Theresults showed the developed HPLC assay was considered to be suitable forthe quality control of S. yunnanensis and for differentiating it frommiltiorrhiza.4. The research on the effects of temperature on the stability oflithospermic acid and lithospermic acid B: The ways that lithospermic acidand lithospermic acid B were transformed into other products aftermaintaining 100℃for 7h were speculated on respectively by LC-MSanalysis and compared with the standard. The results showed that thestructures of lithospermic acid and lithospermic acid B at roomtemperature were steady, but they were both transformed into otherproducts (such as protocatechuic dehyde, salvianolic acid A, salvianolicacid C) after heated over 60℃. All which have provided the basis for thequality control of S. yunnanensis and S. miltiorrhiza and theirpreparations.5. The research on the effects of temperature on the inhibitory activityon HIV-1 integrase (IN) of lithospermic acid and lithospermic acid B: Thetransformed products of lithospermic acid and lithospermic acid B in thedifferent ways were tested for their inhibitory activities on HIV-1 INby ELISA method. The results showed that the inhibitory activity on HIV-1IN of the respective transformed product was better than that oflithospermic acid and lithospermic acid B, and the temperature was animportant factor for the quanilty control.