Evaluation Of Active Ingredients And Accumulation Mechanism Of Dendrobium Officinale Cultivated With Trees

Dendrobium officinale Kimura et Migo（DOKM）is considered a precious herb in traditional or folk medicine,and enjoys the name of"panda in medicine",with the efficacy of benefiting the stomach,nourishing Yin,and clearing heat.It is a popular medicinal herb among many consumers.Due to the low germination rate of DOKM seeds under natural conditions and the strict growth environment,the wild resources of DOKM are on the verge of extinction.To meet the market demand for DOKM herbs,artificial cultivation has become a trend.The rich forest resources in Guizhou provide a strong guarantee for the development of the imitation wild DOKM plantation industry,which has significant economic and ecological advantages for planting imitation wild DOKM in the forest.At present,the research on imitation wild DOKM mainly focuses on the effects of cultivation modes on the growth and polysaccharide of imitation wild DOKM.However,the effects of epiphytic tree species on the accumulation of DOKM metabolites and how different tree species regulate the DOKM metabolic pathway are still unclear.In this paper,the quality of imitation wild DOKM and the biological activity and molecular regulation level of epiphytic tree species in vitro were studied.The main research contents and results are as follows:1.Identification of chemical constituents of imitation wild Dendrobium officinale and establishment of quantitative determination methodThe stem of Dendrobium officinale was used as the research object,and the UPLC-ESI-MS/MS method was used to compare the quasi-molecular ion peak,fragment ion peak,and relative retention time（TR）of the reference substance and the sample.In the negative ion mode,9 phenolic compounds were identified from DOKM simply and quickly,mainly ferulic acid,chrysin,naringenin,luteolin,epicatechin,quercetin,isorhamnetin,luteoloside,and naringin.An UPLC-ESI-MS/MS method was established for the simultaneous determination of 9 phenolic compounds in imitation wild DOKM.The results showed that the method was accurate,reliable,rapid,and simple.2.Simultaneous determination of phenolic content and quality evaluation of imitation wild Dendrobium officinale by UPLC-MS/MSThe contents of phenolic components in different provenances,different canopy densities,and different epiphytic tree species under the Pinus massoniana forest were determined by the established method.The accumulation of polysaccharides,mannose,and phenolic substances was used to evaluate the provenances of Pinus massoniana forest（Zhejiang,Fanjingshan,Anlong,Guangxi,and Fujian）.Three canopy densities（0.55～0.65,0.70～0.80 and 0.80～0.90）;the effects of simulated wild DOKM on the growth of five epiphytic tree species（Pinus massoniana,Betula platyphylla,Salix psammophila,Acer davidii,and Magnolia denudata）.The polysaccharide content of Zhejiang provenance was significantly higher than that of other provenances,and higher than that of Zhejiang provenance planted in the greenhouse.Under the canopy density of 0.55～0.65,the polysaccharide content of DOKM was（53.81±0.85）%,and the polysaccharide content decreased with the increase in canopy density.The content of DOKM polysaccharide was the highest（51.37±0.84）%under Acer davidii and the lowest（27.89±0.68）%under Magnolia.Through TOPSIS comprehensive analysis,it was found that Zhejiang provenances had certain advantages over the other four provenances.The comprehensive score index was the highest when the canopy density was 0.55～0.65.Therefore,the Zhejiang provenance with a canopy density of 0.55～0.65 was used as a habitat to further study the DOKM growing under the five tree species.Cluster analysis,PCA analysis,and OPLS-DA analysis showed that the cultivation methods of five epiphytic tree species could be divided into three categories.The first category was D3,D5,and D1,the third category was D4,and the third category was D2.Through TOPSIS comprehensive analysis and evaluation of DOKM quality differences under five epiphytic tree species,it was found that the DOKM quality of Pinus massoniana（CI=0.6243）epiphytic cultivation was better,and the DOKM quality of Acer davidii（CI=0.3430）was poor,which was consistent with the PCA comprehensive score.3.Study on in vitro biological activity of ethanol extracts from different epiphytic tree species of Dendrobium officinaleTo evaluate the in vitro biological activity of DOKM alcohol extracts from five epiphytic tree species and greenhouse cultivation.The in vitro biological activities of DOKM ethanol extracts were different under different cultivation methods.The specific performance was that the DOKM ethanol extract cultivated by Pinus massoniana（D4）had better DPPH free radical,ABTS free radical,and hydroxyl radical scavenging rate,and its IC₅₀value was lower than other groups（0.22±0.04）,（0.003±0.002）,and（2.06±0.07）mg·m L^-1,and the antioxidant activity was better.The IC₅₀values ofα-amylase,α-glucosidase,and pancreatic lipase were（1.02±0.04）,（0.47±0.15）,and（0.03±0.02）mg·m L^-1,respectively.Correlation analysis showed that nine phenolic components in the ethanol extract of DOKM cultivated by different epiphytic tree species were negatively correlated with DPPH free radical scavenging rate.Among them,DPPH free radical scavenging rate was strongly negatively correlated with isorhamnetin and epicatechin,and strongly negatively correlated with naringenin.The higher the content of isorhamnetin,epicatechin,and naringenin,the lower the IC₅₀value and the higher the DPPH clearance rate.The phenolic content was negatively correlated with DPPH,ABTS,and hydroxyl radical scavenging rates,indicating that the more the alcohol extract content,the stronger the antioxidant capacity.However,α-amylase,α-glucosidase and pancreatic lipase were moderately negatively correlated,which may be the inhibitory effect of other substances in the ethanol extract onα-amylase,α-glucosidase,and pancreatic lipase.4.Based on transcriptional sequencing,the effects of epiphytic tree species on the regulation of wild-like DOKM metabolites were analyzed.Transcriptome sequencing of fresh stems and leaves of DOKM cultivated under two epiphytic tree species yielded a total of 18.09 G Clean Data.Gene function annotation and expression analysis total of 1876 differentially expressed genes（DEGs）were identified,including 40 DEGs involved in flavonoid and phenylpropanoid biosynthesis,and 14 DEGs involved in starch and sucrose metabolism.The combined analysis of transcriptome and metabolome showed that the biosynthesis of flavonoid metabolites was regulated by the transcription of DFR,ANS,CYP75B1,F3H,and CHS genes.There were 19 phenylpropanoid biosynthesis pathway-related genes regulating ferulic acid biosynthesis,of which 15 were up-regulated,mainly 4CL,HRP,F5H,COMT,CCR,CSE,CAD,REF1,and PAL gene families regulate ferulic acid synthesis.HST,CCo AOMT,and YP73A4 family genes were strongly correlated with flavonoid metabolites and phenolic acid metabolites ferulic acid.A total of 14 genes were related to polysaccharides and mannose.Among them,five genes in the EGLC,TPP and Ptr BAM families were up-regulated,and six genes in the SUS,TPS,endoglucanase,beta-glucosidase,ISA and TREH families were down-regulated.The combined analysis of metabolites and differential genes of the epiphytic wild-like DOKM of the two tree species can not only explain the way influence of cultivated tree species on the quality of DOKM at the molecular level,but also provide a reference for future research on the development mechanism of cultivated wild-like DOKM and the regulation of metabolic synthesis pathways.