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The Study of Gene Marker Related to Cassia Angustifolia Vahi and Correlation Analysis between the Clinical Efficient of Senna Leaf Cleaning Intestines and the Content of Sennoside A and Sennoside B

Author: FengLiFang
Tutor: LaiRenSheng
School: Nanjing University of Traditional Chinese Medicine
Course: Chinese and Western medicine combined with the basis
Keywords: Senna leaf gene LC-MS/MS sequencing PAL CHS
CLC: R285
Type: Master's thesis
Year: 2009
Downloads: 55
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Abstract


Objective1 The analysis on Gene sequencing and bioinformatics of partial nucleotide sequences of phenylalanine ammonia-lyase(PAL) gene and chalcone synthase(CHS) gene,which is the key enzyme gene of biosynthesis metabolic pathway of anthrone in Cassia angustifolia Vahi.2 Research on the clinical condition of intestine,clicnical purgation and side effects,the content of sennoside A,B in intestine fluid,compared with pathologic morphology of intestinal mucosa in subjects took Cassia angustifolia Vahi,the biological correlation were investigated between clinical performance and PAL,CHS gene marker.Methods1 DNA was extracted from Cassia angustifolia Vahi and primers were designed for PCR and bidirectional sequencing.The sequencing results were searched by NCBI BLAST program,as well as were compared with PAL and CHS gene sequences to model plants with pairwise alignment to get Similarity Index under DNAStar Megalign software.2 Observed and scored on clinical performance of 60 cases in HPLC test group and 5 cases in LC-MS/MS test group.All cases were divided into four groups and made with statistical analysis of significant difference.Sennoside A,sennoside B with HPLC,and sennoside B with LC-MS/MS in intestinal fluid samples were analyzed from the two groups,respectively. Moreover,it was statistically analyzed the correlation of the results and clicnical performance and carried on research to the relationship of gene marker and six pathologic morphology indexes of intestinal mucosa of each group.The statistical software is SPSS16.0 is available. Results1 Three sennoside gene sequence fragments were obtained,733bp,573bp and 262bp by size. The similary sequences searched were short and no similary sequences of PAL gene or CHS gene of senna leaf in NCBI were found of NCBI Map Viewer plant structure trees.The Similarity Index of the three gene sequences compared with PAL and CHS gene sequences of Arabidopsis thaliana and Glycine max were 29.7~37.1.2 The 60 cases in HPLC test group were divided into four groups:1 case of group without diarrhea,25 cases of light group,27 cases of moderate group and 7 cases of severe group.Total scores of side effects were analyzed with group t test,the significant difference was:light group compared with moderate group(P=0.008,P<0.05).The Retention Time of sennoside A,B detected in senna leaf and sennoside A,B reference substances was uniform with HPLC analysis, but could not be detectable in intestinal fluid samples of the 60 cases.The 5 cases in LC-MS/MS test group belong to three groups:light,moderate and severe group,total scores of side effects of all groups were above 4,because of the number of cases,correlation of the side effects and clinical effect was unclear yet.Absorption peaks of sennoside B appeared in 7/16 tubes of intestinal fluid samples of moderate and severe group by LC-MS/MS analysis.3 According to gene marker of this research to use this product,there were no significant difference between each group of population rates with pathologic morphology,but significant difference was found between moderate and light group of not and light congestive and hemorrhage phenomenon(P=0.041,P<0.05).Most cases were moderate inflammation,light congestive and hemorrhage,without edema(P>0.05).Conclusion1 Three unidentified sequences of Cassia angustifolia Vahi were found with resemblance to PAL and CHS gene sequences of Arabidopsis thaliana than that of Glycine max.Gene markers of this research were found for the initial basic tendency in correlation analysis of the further pharmacodynamical effect and gene.2 Cases in light and moderate group outnumbered cases in other groups.The total scores of side effects of moderate group were higher than that of light group.The intestine cleared was fine. The Retention Time of sennoside A,B in senna leaf and reference substances analyzed with HPLC is uniform in the range of 1.5%error,but could not be detected in samples of test group. Intestinal fluid of control group have noninterference to HPLC analysis of sennoside A,B.The more diarrheas in subjects,the more content of sennoside in intestine fluid analyzed with LC-MS/MS.Furthermore,the content of sermoside increased gradually with the passage of time, which suggested that LC-MS/MS method could get experiment data reliably.The metabolism of sennoside need intestinal environment and discomposition of intestinal microflora,because of the number of cases,correlation of the content of sennoside and the side effects was unclear yet.3 There were no significant differences between each group of pdpulation rates about pathologic morphology based on the PAL and CHS gene marker of Cassia angustifolia Vahi,which suggested high safety.The extent of congestive and hemorrhage of light group was lighter than that of moderate group,implied that the severe diarrhea compared with the possibility of congestive and hemorrhage should be cared when performed enteroscopy and taking senna leaf.

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