| Si-wu decoction (SDE) is a classic prescription in Traditional Chinese Medicine.Investigation suggested si-wu decoction might accelerate proliferation of marrowmesenchymal stem cells (MSCs) in vitro these years. Some research also showed thatit could induced MSCs differentiating into the nerve cells, bone cells andhematopoietic cells in given conditions. But no thorough reports of chemicalconstituents in si-wu decoction which accelerated proliferation of marrowmesenchymal stem cells(MSCs) in vitro yet. This text used a variety of seperation andidentification methods to carry out the analysis.In our research, si-wu decoction was extracted by petroleum ether, ethyl acetate,chloroform and redistillation water in order to gain different polarity solventextractions. The cytoactive of extractions was carry out by MTT assay. Resultsdemonstrated ethyl acetate extraction (A-2) might accelerate proliferation of marrowmesenchymal stem cells (MSCs) in vitro. As a consequence, the further separation,composition analysis and activity experiments were mainly concerned to A-2.Chemical constituents and bioactivity fingerprints were introduced in theseresearch. Modern analytical technologies such as GC-MS, HPLC and HPLC-MS~nwere also been used in constituent analysis. As regards to bioactivity analysis, MTTmethod and flow cytometry were mutual corroboration.There were 42 volatile components in A-2 detected by GC-MS, than gradientelution by column chromatography was introduced to separate A-2, twenty sampleswere marked with F-1～F-20. Ligustilide (F-4) isolated from SDE showed the bestproliferation-promoting effect which detected by HPLC, IR spectrum and massspectrum. 6,7-dihydroxy-3-(-prop-1-enyl)isobenzofuran-1(3H)-one, identified for thefirst time by HPLC-MS~n whose structure was similar with ligustilide, main constituentin F-11, the relative peak area was 84.47%. MTT assay manifested F-11's bioactivitywas merely inferior to Ligustilide.The qualitative and quantitative analysis demonstrated there were large contentof fatty esters in F-7 and F-10. We choosed four fatty esters as standards to evaluate the bioactivity by flow cytometry. The proliferation index (PI) of palmitic acid methylester (S-1) and stearic acid ethyl ester (S-4) were higher than control group, whichshowed proliferation on MSCs, while palmitic acid ethyl ester (S-2) and stearic acidmethyl ester (S-3) were lower than control group, which showed inhibition. Thequantitative analysis on F-7 manifested that S-1(3.04%), S-2(3.92%), S-4(9.61%),while F-10 showed that S-1(14.46%), S-2(17.47%), S-3(3.19%), S-4(1.03%).Bioactivity suggested F-7 was better than F-10. We conjected S-1 and S-4 wereresponsible for MSCs proliferation. Based on the results of standards evaluated byMTT assay, S-1 and S-4 could promote the proliferation on MSCs at the concentration0.15μg/μL. S-4 showed the highest optical density (O.D.). While the four standards'O.D. value were lower than the control group, which suggested inhibition bioactivitiesat the con. 1.5μg/μL.Owing to the multitarget and synergism of different components in TraditionalChinese Medicine formula, the bioactivity fingerprints were analyzed by GC-MS. Thebioactivity fingerprints on MSCs proliferation was obtained.The chemical conponents in si-wu decoction were Ligustilide, palmitic acidmethyl ester, stearic acid ethyl ester that accelarated MSCs proliferation. It is not therole of single isolated but the result of cooperation of an ensemble molecules.Considering the synergism, there were proliferous constituents as well as prohibitiveconstituents in the same concentration. This result provided the experiment basis, forthe conclusion, that si-wu decoction could promote proliferation of MSCs, but notincrease overly, and also offered new chemical sunstances for the research ofTraditional Chinese Medicine. |
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