Study On The Orientation Of Dendrobium Officinale “Medicinal Materials”

Before the eighties of the last century,almost all of the medicinal materials of Dendrobium used wild resources.Nowadays,the market mainly uses greenhouse cultivation products.According to field investigations conducted by the research group,there is a large difference between wild and cultivated products.Wild products grow for many years,including one-,two-,and three-year and above（since it is convenient to draw and write later,the latter is expressed as three years）,which stems are distinctly different.There are many records of Dendrobium,medicinal use,and indications of efficacy in ancient books,but the medicinal years of sarcophagus are not clearly defined.According to the research of the research group,the recorded in Tang and Song periods mainly refer to Dendrobium officinale Kimura et Migo（D.officinale）or Dendrobium huoshanense.Due to the high price of Dendrobium huoshanense,D.officinale was selected in this study.The medicinal parts of D.officinale in Chinese Pharmacopoeia are dry stems,and the specific growth period has not been defined.In the early stage of the our research group,characteristic fingerprint,flavonoid characteristic fingerprint,water-soluble polysaccharides,salt-soluble polysaccharides and degraded polysaccharides in tissue culture and shed D.officinale were conducted.There was an empirical study of D.officinale with“Guangnan as its best”,which including a large number of documents verifying that D.officinale planted in Guangxi is an authentic medicinal material,quality evaluation of more than twenty species of D.officinale（shelf planting and imitation wild planting）in different producing areas of Guangxi,but not different growth periods of the same origin.Therefore,this study is to evaluate the quality and activity in vitro of imitation wild D.officinale planted in Guiping,Guangxi（one or two and three years old）,determine the medical years of D.officinale,that is,the"positioning"study of D.officinale and provide a certain references for medicinal selection,location,development and application,and cultivation mode selection of D.officinale.Objective:To clarify the specific parts of the medicinal Dendrobium medicinal materials and the quality of D.officinale,research the texts of the Chinese herbal medicine.To analyze the quality of imitation wild D.officinale（one,two,and three years old）and compare their polysaccharide content,monosaccharide composition,molecular weight,and characteristic fingerprint.To compare the antioxidant and antitumor activity of imitation wild D.officinale（one,two,and three years old）crude polysaccharides and alcohol extracts.To determine the appropriate duration of medicinal growth of imitating wild D.officinale by comparing the study on the components and activity difference of imitaton wild D.officinale（one-,two-and three-years）.Methods:1.Mainstream herbs,medical historical materials and local records were used to verify the medicinal sites,quality standards and efficacy of Dendrobium.2.Traditional identification method was used to identify the imitating wild D.officinale.Phenol-sulfuric acid method was used to detect the polysaccharide content of imitating wild D.officinale（one-year,two-year-old,three-year-old）.3.HPLC method was used to compare the monosaccharide composition of crude polysaccharides（GPC1,GPC2,GPC3）and the characteristic fingerprints of alcohol extracts of one-,two-and three-year-old imitation wild D.officinale.4.ELSD-UHPLC was used to detect the molecular weight of GPC1,GPC2,and GPC3.5.The antioxidant activity in vitro(the clearance rates of DPPH^-,·OH^-,and Cu²⁺)of crude polysaccharides and alcohol extracts（GPE1,GPE2,and GPE3）of imitation wild D.officinale in one-year-old,two-year-old,three-year-old were tested.6.MTT assay was used to detect the effect of crude polysaccharides and alcohol extracts of imitation wild D.officinale on the proliferation of Hela,Hep G2,SGC7901,A540 and B16 tumors cells with different growth years.Flow cytometry,fluorescence staining and q RT-PCR were used to analyze the apoptosis of crude polysaccharides or alcohol extracts in Hela cells.7.GC-MS method was used to analysis of the primary metabolites of imitation wild D.officinale in the first,second and third year.Results:1.The use of parts of wild Dendrobium was recorded in Materia Medica was stems,which“disposed root”,“abandoned seedlings”and“abandoned dark”.The habitat and origin of wild Dendrobium was“on the stone”and“with Guangnan are good”.The well quality of wild Dendrobium were“The folded flesh is in the flesh”,“Dendrobium short and medium solid,Hu wood long and false”and“Tielan is color blue and black···To fat ze fat is better than Tilan”.The efficacy of wild Dendrobium was“make up the five organs and consumptive labor”,“Kidney”etc.2.The results of traditional identification methods showed that the stem of imitating wild D.officinale was similar to the stem length of wild Dendrobium recorded in ancient books.The content of polysaccharides in the first,second and third year imitation wild D.officinale herbs were 51.49%,35.31%and 33.52%respectively.The average molecular weights of one-,two-and three-year-old imitation wild D.officinale were 2 148 k Da,1 450k Da and 1 688 k Da.The ratios of mannose to glucose peak areas of GPC1,GPC2,and GPC3 were 6.48:1,7.12:1,10.80:1.Initially explored characteristic fingerprints of 80%ethanol extract of imitation wild D.officinale were more complex.3.The percentage of DPPH free radicals scavenged by the crude polysaccharide solution of imitation wild D.officinale was 71.73%-87.01%.The·OH^-scavenging ability of the crude polysaccharide pair were GPC3>GPC2>GPC1.The reduction of copper ion by crude polysaccharides was weak.4.The general trend of anti-proliferative effect of GPC on Hela cells were GPC3>GPC1>GPC2.Hep G-2 cells in high concentrations of crude polysaccharide solution showed for 48 h were GPC1<GPC2<GPC3,the difference between low concentration performance is not obvious.Anti-proliferative effect of SGC7901,GPC3 was better than GPC1,GPC2.The inhibitory effect on the activity of A549 and B16 cells,GPC1 was worse than that of GPC2 and GPC3.The crude polysaccharide solution could induce the apoptosis of Hela cells,such as chromosome shrinkage,apoptotic bodies and nuclear fragmentation,apoptosis Hela cells,and up-regulation of caspase-8,p38,et al m RNA.5.The alcohol extract solutions at 0.5 mg/m L and 1.0 mg/m L·OH^-clearance rates were GPE3>GPE1>GPE2.At 5.0 mg/m L,·OH^-scavenging capacity was GPE1>GPE3>GPE2.Reducing capacity for copper ions were GPE1>GPE3>GPE2.Different concentrations of alcohol extract samples have little effect on DPPH radicals at the same years.DPPH radical scavenging trend at the concentration of 2.0-5.0 mg/m L were GPE1>GPE3>GPE2,and clearance rate was higher than that of positive drug Vc.6.The effect of alcohol extract of imitation wild D.officinale on tumor cells(GPE（PBS dissolved,then diluted with medium）,GPED（first DMSO assisted solution,then diluted with the medium）]on Hela cells were GPE1<GPE2<GPE3.The effect of anti proliferation on Hep G-2 cells in 125-500μg/m L was GPE1,GPE2 better than GPE3.Inhibition effect on SGC7901 was GPE2,GPE3 better than GPE1.GEP2 inhibits A549cells better than GPE1 and GPE3.For B16 melanoma cells,cells in the concentration range of 62.5-1 000μg/m L in GPE solution for 48 and 72 h,respectively,GPE2,GPE3 solution inhibits B16 better than GPE1.GPED solution promoted the apoptosis of Hela cells were GPED3>GPED2>GPED1 and induced Hela cell morphological changes.7.GC-MS analysis results showed that GC-MS detected 134 constituents containing in imitation wild D.officinale,such as monosaccharides,amino acids and so on,which number of compounds were one-year-old>two-year-old>three-year-old.Conclusions:1.The ancient Medicinal Dendrobium was used for"stems".It needed to"abandoned roots","remove seedlings","go black",seedlings and old stems.“Fold to the flesh”and“short and real”,there ware suggest that stems are rich in polysaccharides and that the stems of the wild Dendrobium are shorter.2.The stems of imitation wild D.officinale（annual,biennial,and three year old）are similar to those of wild Dendrobium recorded in ancient books.The polysaccharide content of imitation wild D.officinale meets the requirements of the Chinese Pharmacopoeia.The average molecular weight of crude polysaccharide:one-year>three-year>two-year-old.The ratio of the mannose and glucose peak area of the crude polysaccharide increases with the growth period and the ratio are larger.3.There are some differences in the antioxidant activity of the crude polysaccharides of imitation wild D.officinale.GPC3 has the best antioxidant activity.There have some inhibition effect of crude polysaccharide solutions respectively act on Hela,Hep G-2,SGC7901,A549 and B16 tumor cells and the strongest effect is GPC3.4.The anti-oxidation activity general trend of 80%ethanol extract of imitation wild D.officinale are GPE1>GPE3>GPE2.It has certain anti-proliferative effects on Hela,Hep G-2,SGC-7901,A549 and B16 cells.Comprehensive ancient literature,as well as anti-tumor and anti-oxidant activities of crude polysaccharides and alcohol extracts,imitation wild D.officinale（first,second and third year）has certain effects in tumor cells.Imitation wild D.officinale can be used in daily health,in response to the therapeutic effect,especially in the tumors treatment of three year old.