The Study Of Anti-LPS Material Basis And Biological Activity Within Allium Sativum L.

Objective:Lipopolysaccharide (LPS) is the main constitution of G~- bacterial outmembrane. LPS is recognized as a main promotor in the pathophysiology of sepsis. Preventing the Lipid A binding to its receptors is considered as the most promising strategy. Clinical experiences and studies had suggested that many medicine-food plants possess the function of anti-sepsis. But there are complicated chemical constitutes in the plants, and the material basis of the anti-sepsis activity is not clear. Based on the biosensor by target of Lipid A, we screened the active components extreated from medicine-food plants, and studied their biological activities against sepsis。Methods:(1) The biosensor technology was applied to screen the medicine-food plants, then isolate the active fractions from one of the medicine-food plants selected by the target of Lipid A. (2) The active fractions were isolated by silica gel chromatography, ion-exchange HPLC. The fraction with the best affinity to Lipid A was choosed to futher study its anti-sepsis activities. (3) In vitro, studing the LPS-neutralizing effect of the fraction; observing the inhibition of TNF- α and IL-6 release in RAW264.7 cells induced by LPS. (4) In vivo, observing the protection of the fraction for mice from lethal challenge of LPS or hot-inactivated G~- and G~+ bacteria. (5) Then studing the main components of the active fraction by RP-HPLC. Isolating the active monomes from ASLA, identifing their purities,and analyed their structure by NMR and MS. In addition, studing their LPS-neutralizing effect and inhibition of TNF-α release in RAW264.7 cells induced by LPS.Results :(1) Among the five medicine-food plants, the garlic (Allium Sativum L.) had the best affinity to Lipid A and was selected to isolate the active substance. (2) Then the activefraction with the best affinity was separated by HPLC and be named of Allium Sativum L. fraction A (ASLA). (3) The experiment showed that ASLA had significant LPS-neutralizing effect in vitro and in vivo; It could markedly inhibite TNF-a and IL-6 release in RAW264.7 cells induced by LPS; It also protected mice from lethal challenge of LPS or hot-inactivated G" and G+ bacteria in a dose-dependent manner. (4) The constituent analysis of ASLA showed that there were two main components in ASLA. And their purities were more than 99%. The NMR analysis showed that ASLA1# had nine C atoms , ASLA2# had seven C atoms ,both of them had -OH and -C=C- in molecule constitution. There both had 176 baisc MS peak and ASLA1# had 245 and 267 peaks more than ASLA2#. We can't find the same components in present data bank. The analysis of the two monomes' constitutions is going on uptoday. The study showed both of them had significant biological activities against sepsis. Conclusions:(1) The biosensor technique was a rapid and orientable method in isolating the active components binding to Lipid A. (2) The ASLA had significant activity aganst sepsis in vitro and in vivo. (3) The main components of ASLA were two monomers. And they had significant activities against sepsis.