| Terminalia billerica(Gaertn.)Roxb.is the dried ripening fruit of Terminalia billerica(Gaertn.)Roxb.and widely used in Tibetan medicine.The standard of Tibetan Medicine Department contains dozens of prescriptions including Terminalia billerica(Gaertn.)Roxb..Modern pharmacology has proved that Terminalia billerica has many efficacy,such as antioxidative,treating malaria,antidiabetic,preventing of arteriosclerosis,protecting the Liver Injury Induced by Carbon Tetrachloride,treating diarrhoea and antipyretic-analgesie.Quality control of Terminalia billerica(Gaertn.)Roxb.has rarely reported.In recent years,the research of tannin is more and more extensive.Our team found that tannin in Phyllanthus emblica L.had antitumor activity,the main components of Terminalia billerica(Gaertn.)Roxb.are tannin and triterpenoid compounds.So as the main component of Tibetan medicine,tannin in Terminalia billerica(Gaertn.)Roxb.need more studied in depth.This paper established the extraction technology and the quality control method of Terminalia billerica(Gaertn.)Roxb.and its tannins fraction.The anticancer activity of tannin fraction from Terminalia billerica(Gaertn.)Roxb.was assessed by MTT method,supported scientific theory evidence for the utilization of Terminalia billerica(Gaertn.)Roxb.and the research of new drugs.1 Study on the the extraction and purification technology of the tannins fraction from Terminalia billerica(Gaertn.)Roxb.1.1 The extraction technology of tannins fraction of Terminalia billerica(Gaertn.)Roxb.has been establish.The effects of different particle size on the ratio of extraction were investigated.Methods Using L9(34)orthogonal design to determine the content of tannins,gallic acid,corilagin and ellagic acid as the index of evaluation,study the extraction solvent,extraction times,extraction time,extraction times on the ratio of extraction.According to the results of SPSS analysis,the optimal extraction process of Terminalia billerica(Gaertn.)Roxb.has been establish:use 10 times dosage 50%ethanol,to extract the medical material 3 time,10 hours every time.The results show that the extraction technology is reasonable and stable.1.2 The Purification technology of tannin from Terminalia billerica(Gaertn.)Roxb.by macroporous resin has been establised.Determine the content and metastatic rate of tannins,gallic acid and corilagin index as the index of evaluation to optimization the purification process of macroporous resin.The extraction concentrated to H g·mL-1 and centrifuged at M r·min-1 for N min.According to S the volume ratio of the raw material and the macroporous resin,supernatant liquid absorbed by C macroporous resin(Ratio of diameter to height is R).The absorption speed was L2 ml·min-1.After the adsorption,K2 BV P2%ethanol was used to remove the impurity,the impurity removal speed was L1ml·min-1,then elute with F2 BV Q2%ethanol,the elution rate was L3 ml·min-1.1.3 Enrichment Process is verified by 3 batches tannins extracted from a billeric(Gaertn.)Roxb.The content of tannins is above 50%and the transfer rate is above 70%,which shows that the method is reproducible and stable.2 Study of methods to control the quality of tannins constituent and Terminalia Billerica(Gaertn.)Roxb.2.1Establishment the method of determination of tannins in tannins constituent and Terminalia Billerica(Gaertn.)Roxb.by spectrophotometry.Linear regression examination is used for this study,and foundthat GA has a good linear relationship in the range of 0.0016-0.01289mg·mL-1.The regression equation is Y=99.179X+0.05154,R=0.9991.The method ological study showed that this method had a good repeatability and reproducibility.The content of tannins of herbs was 11.94%,11,63%,11.65%,with RSD=1.47%,The average recovery for gallic was 99.57%,RSD=1.42%(n=6).The contents of total tannins in three batches of the tannins fraction were52.45%、52.48%、51.25%;RSDs were 0.77%,1.04%,1.96%.2.2 For the first time to establish a high performance liquid chromatography(HPLC)method to determine the contents of gallic acid,corilagin,ellagic acid and methyl gallate in Terminalia billerica(Gaertn.)Roxb..The linear regression equation of gallic acid,methyl gallate,corilagin and ellagic acid are Y=62499746.02X+68175.73637(r=0.9997),Y=66947958.74X+13873.18713(r=0.9995),Y=187659620.8X-53516.92953(r=0.9998),Y=39626737.99X+5281.411615(r=0.9999);A good linearity between peak area and the concentration was found in the range of 16.56～207 μg·mL-1,1.38～60.48 μg·mL-1,0.42～21 μg·mL-1,3.57～44.6 μg·mL-1 for gallic acid,methyl gallateare,corilagin,and ellagic acid respectively.The content of gallic acid in Terminalia billerica(Gaertn.)Roxb.was 2.91%,the average percent recovery was 98.08%,RSD was 0.65%;The contend of corilagin in Terminalia billerica(Gaertn.)Roxb.was 0.64%,the average percent recovery was 97.24%,RSD was 2.12%;the content of ellagic acid in Terminalia billerica(Gaertn.)Roxb.was 1.22%,the average percent recovery was 97.51%,RSD was 3.00%;the content of methyl gallate was 0.08%,the average recovery was 98.58%,RSD was 1.31%.The results show that the method is accurate and stable,and it can be used as the method of quality control.There were some differences in the contents of the four components in the different batches,but the contents of the four components in the batches were not significantly higher than those of the other batches.The sum of the four components,the Tibet 1 is 6.27%and the Tibet 2 is 5.33%slightly higher,the rest are between 4.28%and 4.88%,indicating that the ingredients are relatively stable.2.3 A high performance liquid chromatography(HPLC)method to determine the contents of gallic acid,corilagin and ellagic acid in tannin fraction were firstly established by using HPLC.The method ology study show that the method of HPLC to determine the contents of gallic acid,corilagin and ellagic acid in tannin fraction was precise,valid and satisfactory,it can be a method for quality control of tannin fraction.The content of gallic acid in tannin fraction was 7.35%,the average percent recovery was 99.27%RSD was 2.14%;The contend of corilagin in tannin fraction was 2.10%,the average percent recovery was 101.54%,RSD was 2.44%;the content of ellagic acid in tannin fraction was 2.18%,the average percent recovery was 101.15%,RSD was 2.20%.2.4 The HPLC fingerprint of Terminalia billerica(Gaertn.)Roxb.was established.The similarity evaluation method of chromatographic fingerprints in Terminalia billerica(Gaertn.)Roxb.was studied.The results showed the similarity of 11 baches of Terminalia billerica(Gaertn.)Roxb.was more than 0.900,which means the chemical composition were relatively stable.There were 20 common peaks in the diagram,and 4 common peaks were identified by adding reference substances to the sample.Four peak were identified as peak12(chebulagic acid),peak 3(gallic acid),peak 9(corilagin),peak 14(ellagic acid).Cluster analysis divided the medicinal materials into three categories,principal component analysis revealed that there are 6 principal components and total variance explained was 93.213%.The 6 principal components can represent the quality of Terminalia billerica(Gaertn.)Roxb..It can be seen from the PCA score chart that the result agrees well with the cluster analysis.PLS-DA found that peak 5,17,6,16 and 12 may be the main chromatographic peaks to identificate Terminalia billerica(Gaertn.)Roxb..2.5 The HPLC fingerprint of tannin fraction was established.There were 21 common peaks in the diagram and the similarity analysis of 11 samples revealed that the similarities were between 0.973 and 0.832 with 10 regions above 0.900.Cluster analysis divided the medicinal materials into three categories,Principal component analysis revealed that there are 6 principal components and total variance explained was 91.814%.The 6 principal components can represent the quality of tannins fraction.It can be seen from the PCA score chart that the result agrees well with the cluster analysis.PLS-DA found that peak 1,13 and 14 may be the main chromatographic peaks to identificate tannins fraction.2.6 Analysis of components in Terminalia billerica(Gaertn.)Roxb.by HPLC-MSn technology.Terminalia billerica(Gaertn.)Roxb.was analyzed by by HPLC-MSn technology for the first time,the information of the 38 common peaks in Terminalia billerica(Gaertn.)Roxb.was summarized and identified 22 components and it can provide a reliable basis for the quality control of Terminalia billerica(Gaertn.)Roxb..The common peaks in the fingerprints were characterized.The total peaks can reflect the fingerprints and chemical profiles of the medicinal materials in a more comprehensive way,and they can provide a reliable basis for the quality control of Terminalia billerica(Gaertn.)Roxb.in different places.3 Anticancer Activity of Terminalia billerica(Gaertn.)Roxb.and tannins fractionAntitumor experiment in vitro was done by applying MTT assay to Terminalia billerica(Gaertn.)Roxb.and tannins fraction from Terminalia billerica(Gaertn.)Roxb.on HeLa,MCF-7,A549 and HepG2.This study find that the antitumor activity in vitro of tannins fraction from Terminalia billerica(Gaertn.)Roxb.inhibited the growth of HeLa,MCF-7,A549 and HepG2.With the cancentration elevated,inhibiting action increased.It’s indicated that Terminalia billerica(Gaertn.)Roxb.and tannins fraction was with a good antitumor activity and especially to MCF-7.4 Summary and Innovation4.1 The extraction and purification technology of the tannins fraction from Terminalia billerica(Gaertn.)Roxb.was established for the first time.The content of tannin fraction was beyond 50%,the transfer rate was beyond 78%or more.Antitumor experiment in vitro was done to tannins fraction from Terminalia billerica(Gaertn.)Roxb4.2 The method of determination of tannins in tannins fraction and Terminalia Billerica(Gaertn.)Roxb.by spectrophotometry was established for the first time.For the first time to establish a high performance liquid chromatography(HPLC)method to determine the contents of gallic acid,corilagin,ellagic acid and methyl gallate in Terminalia billerica(Gaertn.)Roxb.,A high performance liquid chromatography(HPLC)method to determine the contents of gallic acid,corilagin and ellagic acid in tannin fraction were firstly established by using HPLC.4.3 The HPLC fingerprints of Terminalia billerica(Gaertn.)Roxb.and tannin fraction were established for the first time.There were 21 common peaks in the diagram in Terminalia billerica(Gaertn.)Roxb.,the similarity of the fingerprints was over 0.9 in all 11 batches.There were 20 common peaks in the diagram in tannin fraction,the similarity of the fingerprints were between 0.832～0.973 in all 11 batches.And the common peaks were analyzed by chemical pattern identification. |
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