Isolation,Structure Elucidation And α-Glucosidase Inhibitory Activity Of The Constituents From The Roots Of Dendrobium Huoshanense

Dendrobium huoshanense C.Z.Tang et S.J.Cheng,also known as"Mihu",is a perennial herb of Dendrobium species in the Orchidaceae family.It was first recorded in the"Shennong Herbal Classic"and has the reputation of"the first of the nine immortal herbs".The geographical distribution of Dendrobium huoshanense is narrow,which is limited to Yingshan in Hubei,Nanzhao in western Henan and Huoshan,Jinzhai and Shucheng in Anhui in Dabie Mountains,meanwhile,its unique growth environment makes it a valuable Chinese herbal medicine.During the collection and processing of Dendrobium huoshanense,in addition to the main medicinal part-stem,the roots of the same plant are often discarded,resulting in a waste of resources.Therefore,in this paper,The root of Dendrobium huoshanense was taken as the main research object,and 80%methanol water was used as the extraction solvent to obtain 2.2 kg of root extract,then,the alcohol extract was separated and purified by liquid-liquid extraction,normal phase silica gel and macroporous adsorption resin column chromatography,and combined with recrystallization and preparative thin layer chromatography,14 monomer compounds were obtained from the root of Dendrobium huoshanense,and the structure of the monomer compounds were determined by mass spectrometry（MS）,hydrogen spectroscopy（¹H-NMR）and carbon spectroscopy(¹³C-NMR).To screenα-glucosidase inhibitors and explore the interaction modes and the binding energy between monomer compounds andα-glucosidase,molecular docking simulation was carried out,and then theα-glucosidase inhibitory activity of monomer compounds with confirmed structure in vitro was determined to validate the docking results.The main results of this paper are as followed:1.The 80%methanol aqueous extract of Dendrobium huoshanense root was extracted by liquid-liquid extraction to obtain four parts:petroleum ether phase,ethyl acetate phase,n-butanol phase and water phase.Through normal phase silica gel column chromatography,macroporous adsorption resin and preparative thin layer chromatography,14 monomer compounds were finally isolated.The structures of the obtained monomer compounds were identified by MS,¹H-NMR and ¹³C-NMR,and the structures of 14 compounds were confirmed:Huoshandendrol（1）,Dibutyl phthalate（2）,Ethyl 3-（4-hydroxyphenyl）propanoate（3）,Apigenin（4）,4-Hydroxybenzoic acid（5）,Methyl cinnamate（6）,2,4-Di-tert-butylphenol（7）,β-Sitosterol（8）,Naringenin（9）,（3β,5α,6β）-Stigmastane-3,5,6-triol（10）,β-sitosterol-3-O-β-D-glucopyranoside（11）,Quercetin（12）,Tristin（13）,Gigantol（14）;Among them,compound 1 is a novel compound,and other monomer compounds both are isolated from the roots of Dendrobium huoshanense for the first time.2.The molecular docking study took the compounds whose structures have been determined as small molecular ligands and theα-glucosidase as protein receptor to explore the interaction modes and affinity between each compound and receptor protein,11 compounds performed promising binding energy,the results were as followed:Huoshandendrol（Minimum binding energy-9.8 kcal/mol）,Dibutyl phthalate（Minimum binding energy-7.6 kcal/mol）,Ethyl 3-（4-hydroxyphenyl）propanoate（Minimum binding energy-7.8 kcal/mol）,Apigenin（Minimum binding energy-8.8 kcal/mol）,β-Sitosterol（Minimum binding energy-6.9 kcal/mol）,Naringenin（Minimum binding energy-8.6 kcal/mol）,（3β,5α,6β）-Stigmastane-3,5,6-triol（Minimum binding energy-7.5 kcal/mol）,β-sitosterol-3-O-β-D-glucopyranoside（Minimum binding energy-7.4kcal/mol）,Quercetin（Minimum binding energy-8.9 kcal/mol）,Tristin（Minimum binding energy-7.0 kcal/mol）,Gigantol（Minimum binding energy-6.6 kcal/mol）;Taking acarbose as the positive control,first of all,theα-glucosidase inhibition activity of ethanol extract of Dendrobium huoshanense roots and each extraction phase were determined in vitro:Ethanol extract of Dendrobium huoshanense root IC₅₀=46.08μg/m L;Petroleum ether phase IC₅₀=47.94μg/m L;Ethyl acetate phase IC₅₀=3.42μg/m L;N-butanol phase IC₅₀=5.91μg/m L;Aqueous phase IC₅₀=102.31μg/m L,of which the ethyl acetate phase possessed the best inhibitory activity;The results ofα-glucosidase inhibitory activity of monomer compounds:Huoshandendrol(IC₅₀=131.94μg/m L),Dibutyl phthalate(IC₅₀=177.09μg/m L),Ethyl3-（4-hydroxyphenyl）propanoate(IC₅₀=109.46μg/m L),Apigenin(IC₅₀=18.72μg/m L),β-sitosterol(IC₅₀=271.28μg/m L),Naringenin(IC₅₀=24.54μg/m L),β-sitosterol-3-O-β-D-glucopyranoside(IC₅₀=208.99μg/m L),Quercetin(IC₅₀=2.26μg/m L),Gigantol(IC₅₀=347.97μg/m L),of which the quercetin exhibited the best inhibitory activity,however,the inhibition rate of（3β,5α,6β）-Stigmastane-3,5,6-triol was only 14.4%at the concengration of 600μg/m L.3.Based on UPLC-Q-TOF-MS,14 compounds were identified and analyzed by retention time and secondary ion fragmentation.Semi-quantitative analysis of the distribution of these chemical components in the roots,rhizomes and leaves of D.huoshanense using high-resolution mass spectral peak area integration,the results showed that all 14 compounds were distributed to varying degrees in the root and stem,with compound 10 being the most abundant in the root and compound 6 the most abundant in the stem.This article focused on the Dendrobium huoshanense roots,14 monomeric compounds were isolated from the ethyl acetate phase of Dendrobium huoshanense root extract.Molecular docking and in vitro validation experiments have shown that flavonoids such as quercetin,naringin,and apigenin have good inhibitory activity and could be used as potentialα-Glucosidase inhibitors,and UPLC-Q-TOF-MS analysis showed differences in the content of different compounds in roots,stems,and leaves.This study has made certain contributions to the comprehensive utilization of Dendrobium huoshanense and provided reference for the development and utilization of Dendrobium huoshanense roots.