| Objective:In this study,osteoblast solid phase chromatographic method was established to research on active ingredients of cortex moutan for fracture healing,to provide experimental basis for cortex moutan in clinical treatment of fracture,and the basis for further development of Guzhe mixture.Methods:The high-performance liquid chromatography fingerprint of cortex moutan water extraction was established,and quantitative study on three essential component including gallic acid,paeoniflorin and paeonol by this HPLC method.A Agilent 5 TC-C18 chromatographic column was used at 30?.The binary gradient consisted of acetonitrile and 0.4%formic acid/water.Peaks were detected at 254 nm.The flow rate was 1.0 mL·min-1 and the sample injection volume was 5 ?L.MC3T3 E1 Osteoblast solid phase chromatography was established:using high temperature sterilization cortex moutan water extraction,and medicine and MC3T3-E1 osteoblastic cells were incubated for 4 h,eluted 4 times to inactivation,made the active ingredients from down into dissociation liquid,finally to cell lysis,and analyzed the composition of isolation solution and lysates by HPLC and HPLC/MS to verify the quasi active ingredients.MTT method and ALP activity assay were used to study the effects of the selected quasi active ingredients on the activity of osteoblasts.Results:The control fingerprints of 10 batches of cortex moutan water extraction was established and 30 peaks were regarded as the characteristic peaks.Among them,the peak 4 was gallic acid,the peak 7 was methyl gallate,the peak 14 was albiflorin,the peak 16 was paeoniflorin,the peak 21 was 1,2,3,4,6-penta-O-?-galloyl-D-glucose(PGG)and the peak 30 was paeonol.According to the similarity,the similarity of different samples were greater than 0.9.The results of the methodological investigations showed that:gallic acid,paeoniflorin and paeonol had a good linearity at 1.691?270 ?g·mL-1(R2=0.9997),1.847?250?g·mL-1(R2=0.9999)and 0.181?29.0 ?g·mL-1(R2=0.9999),respectively.The average recoveries(n=3)were 94.12%?100.01%.The average content of gallic acid,paeoniflorin and paeonol in cortex moutan were 0.34%,0.54%and 0.0091%,respectively,and the RSD were 5.01%,5.40%and 2.41%,respectively.MC3T3-E1 Osteoblast solid phase chromatography was established,screened and analyzed four main quasi active ingredients in cortex moutan water extraction.Among them,the composition of the cell membrane were gallic acid and PGG,and the components into the cell were methyl gallate and albiflorin.The pharmacology experiments showed that,0.6?1.6 mg/mL gallic acid had obvious effect on promoting the proliferation of osteoblasts(P<0.05,0.001)and played a significant role in promoting the differentiation of osteoblasts(P<0.05);0.5?1.0 mg/mL methyl gallate significantly promoted the proliferation of osteoblasts(P<0.001)and had obvious promoting effect on osteoblast differentiation(P<0.05,0.01,0.001);4 mg/mL albiflorin had obvious effect on promoting the proliferation of osteoblasts(P<0.05),but 0.5?4.0 mg/mL had an inhibitory effect on the differentiation of osteoblasts.1.2 mg/mL PGG had a significant role in promoting the proliferation(P<0.05)and played a significant role in promoting the differentiation of osteoblasts(P<0.01).Conclusion:1)This research was developed to establish the fingerprint of cortex moutan water extraction.The method was simple and reproducible.The results of the methodological investigations showed that the method was stable and reliable,and providing the standard of charge for the extraction of cortex moutan for this subject.2)The feasibility of the method was verified by the solid phase chromatography of MC3T3-E1 osteoblast,which can be used for the screening of the active ingredients of promoting fracture healing in cortex moutan.3)Through pharmacological experiments,it was found that the effective substance of promoting fracture healing in cortex moutan were gallic acid,methyl gallate and PGG. |
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