Identification Of Active Components In Shenmai Injection Differentially Effected Midazolam1'-hydroxylation And4-hydroxylation Mediated By CYP3A4

Background:In our previous study, shenmai injection (SMI) had been shown differentialeffect on the metabolic pathways of midazolam (MDZ), which could significantlyenhance MDZ1′-hydroxylation but inhibit MDZ4-hydroxylation in human livermicrosomes (HLM), rat liver microsomes (RLM), recombinant human CYP3A4andrats. It is not clear that what components in SMI should be responsible for thedifferential effect of MDZ metabolism and its mechanism, and the primary problem isto identify the material base differentially effecting MDZ1'-hydroxylation and4-hydroxylation.Objectives:In present study, it will be conducted to identify the active components present inSMI differentially effected1′-hydroxylation and4-hydroxylation of MDZ bygradually separating using SPE (solid phase extraction) system and HPLCcombined with in vitro incubation systems and spectrum analysis, providingexperimental basis for further analysis the mechanism being responsible for thedifferential effect of CYP3A4-mediated MDZ metabolism of "Shenmai Fang", andproviding new ideas for further analysis the interaction mechanism of Chinesemedicine complex components based on different combination points of CYP3A4.Methods and Results:1. Using solid phase extraction sepatation, fractions were collected after each1mlof SMI was loaded on each SPE cartridge and separated by differentconcentrations of methanol (10%~100%) respectively. Than, fingerprints of SMIand different fractions were determined by HPLC, at the same time, weinvestigated the effect of each fraction on the metabolism of MDZ using RLMsystem. At last, the material basis and its properties were estimated by thedifferential effect of these fractions combined with comparing each fingerprintswith SMI. The results found that after pretreatment with those fractions eluted with60%of methanol or above, the differential effects of these fractions on themetabolism of MDZ were similar to SMI. Instead, there was no effect happenedwhen the methanol concentrations below50%. The above results demonstratedthat the possible active components might be mainly attributed to thelipid-soluble constituents in SMI.2. Using HPLC separation method, different fractions were collected as follows:SM-1(0-15min), SM-2(15-30min), SM-3(30-45min), SM-4(45-60min),SM-5(60-72min), SM-6(72-87min), SM-7(87-97min), according to the HPLCfingerprint of SMI and retention time of components peaks. Then, the fingerprintof each fraction was determined in order to make sure that each fraction wascollected sufficiently compared with the fingerprint of SMI. At last, weinvestigated the effect of each fraction on the metabolism of MDZ using RLMsystem. The results found that only the fraction of SM-5(60-72min) showeddifferent effect similar to SMI, which could enhance MDZ1′-hydroxylation andinhibit MDZ4-hydroxylation.3. In the above HPLC primary separation basis, chromatographic conditions werechanged and the component SM-5(60-72min) was separated again. Under theoptimized chromatographic, different fractions were collected as follows:SM-5-1(0-10min), SM-5-2(10-24min), SM-5-3(24-32min), and SM-5-4(32-45min). Then, the fingerprint of each fraction was determined. At last, weinvestigated the effect of each fraction on the metabolism of MDZ using RLMsystem. The results found that the fraction of SM-5-3(24-32min) was the activecomponents, had differential effect on the metabolism of MDZ, and appearedsingle peak.4. In view of the above positive component, active component Fr. SM-5-a (about6.0mg) was collected through a series of extraction, isolation and preparation.Based on it, a series of spectrum diagram were got, such as1H-NMR,13C-NMR,DEPT135°, COSY and LC-MS/MS scan. At last, the active component Fr.SM-5-a was confirmed to be panaxytriol (PXT) with molecular weight278.19byspectrum analysis and structure identification, and its molecular formula isC17H26O3. After comparing the chromatographys of the above positive component and PXT standard, they had same chromatographic behaviors, whichfurther confirmed that the active component was PXT.5. At last, in order to confirm the differential effect of PXT on the metabolism ofMDZ, and compare the difference with the overall effect of SMI withcorresponding concentrations, it was conducted to study the effect of PXT atdifferent concentrations on the MDZ metabolism in RLM, HLM and recombinanthuman CYP3A4incubation systems. The results found that PXT had significantdifferential effect on the MDZ metabolism in the above incubation systemssimilar to SMI, which could enhance MDZ1'-hydroxylation and inhibit MDZ4-hydroxylation.Conclusions:Panaxytriol (PXT) was the active component in SMI being responsible for thedifferential effect of MDZ metabolism, and the PXT had significant differential effecton the MDZ metabolism in the above incubation systems similar to SMI, which couldenhance MDZ1′-hydroxylation and inhibit MDZ4-hydroxylation. The study resultscould provide experiment basis and perspective for further clarify the interactionmechanism of complicated components of "Shenmai Fang" based on the differentcombination points of CYP3A4.