Research On The Germplasm Resources And Biologically Active Components Of Fei Cai

Sedu,m aizoon Linn.is a perennial herb within the family Crassulaceae,and is widely distributed in Shaanxi,Hubei,Gansu,Ningxia,Jiangsu and Fujian in China.The earliest record of S.aizoon can date back to Jiuhuang Bencao compiled during China's Ming Dynasty.It was also recorded in Zhiwu Mingshi Tukao,in which it was described as 'Tusanqi'.S.aizoon is not only a vegetable,but also a traditional medicinal herb which has been employed to clear away heat and toxic and to improve scattered stasis and hemostatic ability for hundreds of years.In the project,I collected germplasm of S.aizoon across its wild distribution range,and investigated its morphological and genetic characteristics systematically.In addition,its bioactive components were determined and assayed using GC-MS techniques;the effect of the phenolic compounds from S.aizoon was also evaluated against hypoxia/reoxygenation injury in neonatal rat cardiomyocytes,and its antimicrobial properties were characterized.The findings provide meaningful references for the further exploitation and utilization of this valuable germplasm resource.The major findings are summarized as follows:1.A total of 140 germplasm samples of S.aizoon were collected from eight provinces/municipalities,including Shaanxi,Henan,Jiangsu,Shandong,Ningxia,Gansu and Beijing in China.A technical system has been established for collecting,analyzing and conserving the germplasm.Effective strategies and suggestions are proposed by analyzing the status quo of the utilization of this medicinal germplasm resource.2.Morphological and genetic characteristics of S.aizoon germplasm were also investigated.Results showed that considerable genetic variability occurred within the germplasm,and that there occurred highly significant morphological variation,especially in leaf,stem and petal.3.The germplasm of S.aizoon was also genetically characterized using the amplified fragment length polymorphism(AFLP)technique.In all,eight primer combinations produced 1467 loci from 60 germplasm samples,of which 1443 loci proved polymorphic(i.e.PPB = 98.36%);out of these loci,1443 were shared by all samples.A cluster analysis was conducted using Nei's genetic distance matrix and neighbor-joining(NJ)method;all 60 samples were clustered into four major clades.The Mantel test revealed a significant association between genetic and geographic distances in S.aizoon(P<0.05).4.The contents of total polyphenols,total flavonoids and proanthocyanidin were determined in different organs(i.e.root,stem,leaf and fruit)of S.aizoon,and were 33.97?96.25 mg/g,11.77?40.52 mg/g and 2.97-17.35mg/g,respectively.The root possessed the highest contents of total polyphenols and proanthocyanidin,sequentially followed by the leaf,.fruit and stem.5.The fat-soluble components in S.aizoon were extracted by soxlet extraction,and then esterified and analyzed by the GC-MS technique.The unsaturated fatty acids consituted the main fat-soluble components,accounting for 42.59%,52.57%,37.04%and 80.06%in root,stem,leaf and fruit,respectively.The relative percentage contents of fatty acids differed among different organs of S.aizoon.However,linoleic acid and linolenic acid are always the two fatty acids with the highest contents in different organs,ranging from 15.44%to 44.27%.6.The compositions and contents of amino acids in the root,stem,leaf and seeds of S.aizoon were analyzed with the amino acid auto analyzer.Results indicated that the contents of amino acids were abundant.There were remarkable differences in contents of amino acids among different organs of S.aizoon.The percentages of the dry weight of amino acids in the root,stem,leaf and seed of Sedum aizoon L.ranged from 4.44%to 7.20%.The analytical results of essential and non-essential amino acids indicated that the leaf possessed higher nutritional values.7.I established a hypoxia/reoxygenation model in neonatal rat cardiomyocytes and gave the phenolic compounds from S.aizoon before hypoxia.At the end of reoxygenation,cell viability was determined by the MTT assay.Culture medium was collected for measurement of total nitric oxide(NOx)production and LDH,and hoechst33342 staining was used to identify apoptotic cells.Akt and eNOS expression and phosphorylation,PI3K,Bcl-2,Bax were determined by Western blot.It was found that H/R induced significant decrease in cell viability and Bcl-2/Bax ratio compared to control cells(P<0.01),and that H/R induced significant increase in LDH(P<0.01),apoptosis(P<0.01),PI3K,Akt and eNOS phosphorylation,NO level and caspase-3 activity(P<0.01).The phenolic compounds from S.aizoon protected cell viability of neonatal rat cardiomyocytes treated with H/R(P<0.01),and decreased the LDH(P<0.05)and apoptosis(P<0.01).The phenolic compounds from S.aizoon activated PI3K/Akt,increased eNOS phosphorylation and induced NO increase in neonatal rat cardiomyocytes under H/R(P<0.01).The effect was abolished by PI3K inhibitor wortmannin(100 nmol/L).The phenolic compounds from S.aizoon increased the Bcl-2/Bax ratio(P<0.01),and decreased caspase-3 activity(P<0.01)in neonatal rat cardiomyocytes cultured in H/R.The effect was abolished by wortmannin.These results support that the phenolic compounds from S.aizoon protect cardiomyocytes from H/R-induced apoptosis via enhancing the activation of PI3K/Akt-eNOS pathways.Our data also suggest the potential role of Akt and eNOS as cardioprotective targets in H/R.8.The antimicrobial activity of phenolic extracts from S.aizoon was evaluated using the Oxford cup method and 96-well plates to measure the size of the zone of inhibition,minimum inhibitory concentration(MIC)and minimun bactericidal concentration(MBC).Results showed that the phenolic extracts was active against almost all tested microbial species.Staphylococcus aureus was most inhibited by the extracts,with the MIC and MBC being 6.25mg/mL and 25mg/mL,respectively.