| Rheum lhasaense A.J.Li et P.K.Hsiao belongs to Section Rheum,the roots of which are an important source of the Tibetan medicine "Ya-Da-Huang".At present,there are few studies on it,with only 12 chemical constituents reported,and there is also a lack of studies related to biological activity.Current studies have shown that R.lhasaense differs greatly from other Rheum plants in terms of chemical composition,with its absence of anthraquinones and anthrones.In order to gain a deeper understanding of R.lhasaense and its interspecific relationships with other Rheum plants,this paper presents a systematic study of R.lhasaense in terms of phytochemistry,bioactivity and chloroplast genome.I.Study of the chloroplast genome of R.lhasaenseThe chloroplast DNA of R.lhasaense was sequenced by high-throughput sequencing,and a complete chloroplast genome sequence of 161767 bp in length was obtained,which displayed a typical tetrad structure,i.e.a large single-copy region,a small single-copy region and two inverted repeat regions.A total of 130 genes,including 85 protein-coding genes,37 transfer RNAs and 8 ribosomal RNAs,were annotated through using the online software GeSeq,and the chloroplast genome sequence of R.palmatum was used as a reference.Among them,26 genes contained introns,21 genes contained single introns,and 2 genes contained double introns.The codon preference analysis showed that the leucine codon had the highest content,accounting for 9.74%of the total codons,while the tryptophan codon had the lowest content,accounting for only 1.22%.There is a preference in A/U-encoding codons in R.lhasaense.Thirty-nine repetitive sequences greater than 20 bp in length and 72 simple repeats(SSRs)were identified from the chloroplast genome of R.lhasaense.In addition,the differentiation and the IR/SC boundary of the chloroplast genome in R.lhasaense were similar to those of other Rheum plants.In order to determine the phylogenetic position of R.lhasaense in the genus Rheum,the phylogenetic tree was established via using the neighbour-joining method and the maximum likelihood method based on 34 chloroplast genome sequences,and the results indicated that R.lhasaense was more closely related to R.hotaoense and R.franzenbachii,which belongs to the same section with R.lhasaense in the traditional classification.II.Isolation,identification and determination of the active ingredients of R.lhasaenseNineteen compounds were isolated and purified from 95%ethanol extracts of roots and rhizomes through employing kinds of chromatogramphic methods,containing middle chromatography isolated gel(MCI),Sephadex LH-20,Hw-40F and C18 reversed-phase chromatography.These components were identified based on various spectroscopic techniques such as 600M NMR,IR spectroscopy,UV spectroscopy,and high resolution mass spectrometry.14 stilbenes were identified as deoxyrhapontigenin(1),desoxyrhaponticin(2),gnetin C(3),maximol A(4),4’-methoxy-scirpusin A(5),rheumlhasol A(6),piceatannol-3’-O-glucoside(7),ε-viniferin(10),polydatin(12),piceatannol-3’-O-[2"-(3,5-dihydroxy-4-methoxybenzoyl)]-β-D-glucopyranoside(13),piceatannol-3’-O-(2"-galloyl)-β-D-glucopyranoside(14),resveratrol(15),piceatannol-3’O-(6"-p-coumaroyl)-β-D-glucopyranoside(16)and piceatannol(17);2 hydrolysed tannins were identified as 1,6-di-O-galloyl-β-D-glucose(11),1,2,6-tri-O-galloyl-β-D-glucose(18);other three components were determined as catechin(8),dibutyl phthalate(9)and gallic acid(19),respectively.Of these,compounds 5,13,14 and 16 were new compounds and compounds 1,8,9,11,18 and 19 were isolated for the first time from R.lhasaense.In addition,a UPLC-DAD method was established for the simultaneous determination of six stilbene components(1,2,7,12,13,14)in R.lhasaense.desoxyrhaponticin and piceatannol-3’-O-glucoside were the two components with the highest contents,with 35.4 mg/g and 64.7 mg/g,respectively.The contents of the other four stilbene components were as follows:polydatin with 9.4 mg/g5 piceatannol-3’-O-(2"-galloyl)-3-D-glucopyranoside with 10.8 mg/g,piceatannol-3’-O-[2"-(3,5-dihydroxy-4-methoxybenzoyl)]-β-Dglucopyranoside with 17.8 mg/g,and deoxyrhapontigenin with 2.5 mg/g.In addition,some compounds were subjected to screened for antioxidant activity and acetylcholinesterase inhibitory activity,and several compounds exhibited better activity,especially the new compound 4’-methoxy-scirpusin A.III.Identification of chemical constituents in R.lhasaense by UPLC-Q-TOF-MS2 methodThe cleavage patterns of five reference components were firstly analyzed,including two hydrolytic tannins:1,6-digalloyl-β-D-glucose and 1,2,6-tri-O-galloyl-β-D-glucose,three stilbene components:piceatannol-3’-O-glucoside,piceatannol-3’-O-[2"-(3,5dihydroxy-4-methoxybenzoyl)]-β-D-glucopyranoside,piceatannol-3’-O-(2"-galloyl)-β-Dglucopyranoside,and the results showed that the cleavage sites of these components occurred mainly at the glycosidic bonds,and the galloyl-containing components were observed in the secondary mass spectra with mass numbers of 169 Da fragment ions.A total of 45 components,were identified from the methanol extraction of R.lhasaense based on the cleavage pattern of the five standards and literature reports,which included 26 stilbenes,13 hydrolytic tannins and 6 other components.Among them,the stilbenes were mainly stilbene glycosides,and most of the glycosides were acylated with gallic acid.This research indicated that the main components of R.lhasaense were tannins and stilbenes,and anthraquinones and anthrone components were not found from R.lhasaense.Ⅳ.Screening the in vitro activity of the main components in R.lhasaenseIn this study,the main components isolated from R.lhasense were screened for in vitro activities related to antioxidant,acetylcholinesterase inhibition and hypoglycemia.The results suggested that two hydrolyzed tannins,4’-methoxy-scirpusin A,piceatannol-3’-O[2"-(3,5-dihydroxy-4-methoxybenzoyl)]-β-D-glucopyranoside,piceatannol-3’-O-(2"galloyl)-β-D-glucopyranoside and catechin exhibited good antioxidant effect;piceatannol3’-O-[2"-(3,5-dihydroxy-4-methoxybenzoyl)]-β-D-glucopyranoside,piceatannol-3’-O(2"-galloyl)-β-glucopyranoside and 4’-methoxy-scirpusin A exhibited moderate inhibitory effects on acetylcholinesterase;resveratrol,piceatannol and polydatin exhibited potential hypoglycemic activities.In summary,the traditional method and UPLC-Q-TOF-MS2 technique were employed to analyze the phytochemical components of R.lhasaense,indicating that its main components are stilbenes and tannins,and that anthraquinones and anthraquinones are absent.Analyses of chloroplast genome and phylogenetic relationship showed that R.lhasense belonged to the genus Rheum.In addition,related biological activity studies indicated that its chemical constituents possessed the activities.In conclusion,this study clarified the material basis of R.lhasaense and laid the foundation for the in-depth study and application of R.lhasaense herbs as well as the study of the genus Rheum. |
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