Study Of The Demonstration Of Dendrobium Officinale From Guangxi Based On The Quality From Guangnan Area Was The Best

This study was based on the former research by our research group.Through textual criticism,we had found the evidence of the basis that the quality of the Dendrobium officinale from Guangnan area was the best.We used HPLC to determine the content and establish the characterize chromatography in order to study the quality difference of the Dendrobium officinale from different habitat in Guangxi province,in order to figure out the reason why the ancients would consider that the quality of the Dendrobium officinale from Guangnan area was the best.Objective:1.Textual criticism of the Dendrobium officinale from the Guangnan area.2.The peak area ratio of the mannose and glucose,the content of mannose and the content of polysaccharide of the Dendrobium officinale from different habitat in Guangxi province was determined and compared with each other.3.To establish the HPLC characterize chromatography of the Dendrobium officinale from Guangxi province.To compare the characterize chromatography of the Dendrobium officinale from Guangdong province,Yunnan province,Wenzhou and Taizhou from Zhejiang province with the Guangxi province.To study the quality difference of Dendrobium officinale from Guangxi province.4.To establish the HPLC characterize chromatography of the flavones from Dendrobium officinale from Guangxi province.And compare it with the fingerprint chromatography of the Dendrobium officinale from Guangdong province,Yunnan province,Wenzhou and Taizhou from Zhejiang province.Preliminary analyze the characteristic components of Dendrobium officinale from Guangxi province in order to provide certain theoretical evidence for resource development of the Dendrobium officinale from Guangxi province.5.To study the antitumor and antioxidant activities of D.officinale polysaccharides with different molecular weight.In order to find the molecular weight range of D.officinale polysaccharides with high antitumor activity.6.To analyze the genes associated with D.officinale polysaccharides.Method:1.To determine the peak area ratio of the mannose and glucose,mannose content of the Dendrobium officinale from different habitat in Guangxi province according to the Ch.P methods.2.To establish the HPLC characterize chromatography method for Dendrobium officinale from Guangxi province and the flavones from it.Apply the similarity evaluation software for chromatographic fingerprint of the Ch.P to determine the similarity.3.The qualitative analysis of the flavones from Dendrobium officinale from different habitat in Guangxi province were done by HPLC-MS.4.The antioxidant activities of the polysaccharides in different molecular weight from Dendrobium officinale using were evaluated by free radical scavenging method in vitro.5.The antitumor activities of the polysaccharides in different molecular weight from Dendrobium officinale were evaluated by MTT assay and flow cytometric analysis of cell apoptosis and the qPCR analysis of expression of Bax,Bal-2mRNA of Hela cells.6.Transcriptome analysis of polysaccharide biosynthesis related genes in D.officinale from Guiping,Guangxi.Results:1.The distribution of Dendrobium officinale from Guangnan area present some sort of provenance regularity.2.There was a huge difference between Dendrobium officinale from different habitat in Guangxi province in the peak area ratio of the mannose and glucose,mannose and glucose content,ranging from 0.33 to 5.91.Cluster analysis was applied to group 23 batches of the samples and they were clustered into 5 categories.We can conclude as below:(1)Exclude the effects of the harvest time,the peak area ratio of mannose and glucose of the samples from different habitats including Guangxi province,Fujian province,Shaoguan in Guangdong province and Yunnan province ranges from 0.33to 1.95.It was obvious that the influence of the habitat and species on the ratio of mannose and glucose in not significant.However,the influence of harvest time is more obvious.The ratio of the samples harvest in winter is higher than those harvested in other seasons.(2)The content of mannose and glucose of the samples from different habitats ranged from 51.13%to 13.21%.It is obvious that the polysaccharides content in different habitats is vastly different.The content of polysaccharides in the samples from Lipu county,Guilin city in Guangxi province is the highest.3.We had established the HPLC characterize chromatography of the Dendrobium officinale from Guangxi province.4.We had established the HPLC characterize chromatography of the flavones in Dendrobium officinale from Guangxi province and preliminary confirmed the characterize flavones components of the samples from different habitat in Guangxi province.5.The D.officinale polysaccharides in different molecular weight exhibit certain scavenging ability to DPPH and hydroxyl radical.This kind of activity exhibit dose-dependent manner,with the increase of the concentration of the polysaccharides,the activity became stronger.The ability varied significantly from different molecular weight.The clearance of the DOP is the strongest.(1)The clearance of the DOP is stronger than the lower molecular weight polysaccharides includes DOP4,DOP5,DOP6.It can be concluded that the clearance of the polysaccharides was stronger when the molecular weight was lower in low concentration.(2)When the concentration of the polysaccharides was 0.5-3.0 mg/mL,the scavenging ability of DOP,DOP1,DOP2,DOP3,DOP4 and DOP5 on DPPH was vastly different(p<0.05).It showed that the clearance of polysaccharides in different molecular weight differ from each other.The high molecular weight polysaccharide DOP was stronger than the others in clearance.6.The D.officinale polysaccharides in different molecular weight all exhibit certain anti-proliferation activity to the A549,PC9,HepG2,HT-29,HeLa,SGC7901 cells.The survival rate of these cells decreased as the concentration of the polysaccharides increased.Amount all these cancer cells,the anti-proliferation of the polysaccharides on SGC7901,Hela and HT-29 cells were more obvious.This activities of polysaccharides in different molecular weight differed with each other.It is certain that the polysaccharides can inhibit proliferation of HepG2,A549 and PC9 cells,although it is not so significant.And the activities of the polysaccharides in different molecular weight were not so different.The ability inducing apoptosis of Hela cells of DOPI(176.29kDa),DOP5(28.48kDa)was more powerful than DOP with high molecular weight(652.29 Kda)and low molecular weight(11.10kDa).Forthermore,The ratio of Bax/Bct-2 which was a key factor regulating apoptosis was increased which was related to the molecular weight of D.officinale Polysaccharides.Compared with the DOP group,The anti-apoptotic gene Bel-2 was down-regulated and the pro-apoptotic gene Bax was up-regulated after the cells were treated with DOP(p<0.01).Compared with the DOP6 group,The anti-apoptotic gene Bel-2 was down-regulated and the pro-apoptotic gene Bax was up-regulated after the cells were treated with DOP5((p<0.01)).7.In this study,the transcriptome of D.officinale was sequenced by high-throughput sequencing,and 45012 Unigenes were obtained.These Unigenes were annotated into Nr,Swissprot,KOG and KEGG,and the annotated Unigenes were 23685,17925,14887,and 9100,Respectively.3.27%(298 Unigenes)of the 9100 Unigenes which were annotated in KEGG were involved in the biosynthesis and metabolism of plant polysaccharides pathways(Starch and sucrose metabolism and Fructose and mannose metabolism),and 52 proteins were encoded.Forthermore,3887 SSR loci were obtained from 45012 Unigenes by SSR analysis.Conclusion:1.The content of mannose in Dendrobium officinale harvested in winter(October to September)is the highest while the content of glucose harvested in March is the highest2.The HPLC characterize chromatography of Dendrobium officinale and its flavones can reveal the difference of chemical category and relative content of the Dendrobium officinale in different habitat in Guangxi province in a certain extent.3.The polysaccharides with different molecular weight from Dendrobiul officinale exhibit certain scavenging ability to DPPH and hydroxyl free radical.The clearance of DOP(652.29kDa)is the highest which shows that it has significant antioxidant activity in vitro.4.The polysaccharides in different molecular weight from Dendrobium officinale have certain antitumor activity on different types of tumor cells.This activity is the strongest on HeLa,HT-29 and SGC7901cells in adequate molecular weight(176.29?28.48kDa).5.We have got 45012 Unigenes by high-throughput sequencing of D.officinale from Guiping,Guangxi.3.27%(298 Unigenes)of the 9100 Unigenes annotated in KEGG are involved in the biosynthesis and metabolism of plant polysaccharides pathways(Starch and sucrose metabolism and Fructose and mannose metabolism),and 52 proteins are encoded.And the Dendrobium officinale which come from Guiping,Guangxi,have abundant SSR loci.