| Based on an improved HPLC-DAD and HPLC-ESI-MS/MS method, twelve indoline amides were tentatively identified in traditional Chinese medicine Portulaca oleracea L., among which eight were new compounds (oleracein H-O) and four were known compounds (oleracein A-D). A standardized HPLC fingerprint of indoline amides was generated for the first time through analyzing 11 batches of P. oleracea. Simultaneously, the contents of two major amides oleracein A (OA) and B (OB), and amounts of total indoline amides (TID) were determined. As a result, the contents of OA and OB in 11 batches of P. oleracea were in the range of 35.00-151.93 μg/g and 40.00-150.44 μg/g, respectively, and TID was in the range of 314.16-928.60 μg/g (calculated by OA), or 475.83-1393.00μg/g (calculated by OB).To further identify the structures of new hydrosoluble indoline amides in P. oleracea, polymide column chromatography was applied to accumulate indoline amides alkaloids, and other column chromatographic methods including ODS-C18, sephadex LH-20, MCI and semi-preparative HPLC, were used for isolation of eluted fractions of polyamide column chromatography. As a result, nineteen compounds were isolated and identified based on 1D-NMR,2D-NMR, HRMS, CD and UV spectra. Among them, fourteen were hydrolysable indoline amides:oleracein H (2S-5-hydroxy-1-p-coumaric acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1â†'6)-β-D-gluco-pyranoside,1), oleracein I (2S-5-hydroxy-1-ferulic acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranoside,2), oleracein N (2S-5-hydroxy-1-p-coumaric acyl-2,3-dihydro-1H- indole-2-carboxylic acid-6-O-[2-O-ferulic acyl-β-D-glucopyranosyl]-(1â†'6)-β-D-glucopyranoside,3), oleracein O (2S-5-hydroxy-1-ferulic acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[2-O-ferulic acyl-β-D-glucopyranosyl]-(1â†'6)-β-D-gluco-pyranoside,4), oleracein K (2S-5-hydroxy-1-p-coumaric acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[2-O-caffeic acyl-β-D-glucopyranosyl]-(1â†'6)-β-D-gluco-pyranoside,5), oleracein L (2S-5-hydroxy-l-ferulic acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[2-O-caffeicacyl-β-D-glucopyranosyl]-(1â†'6)-β-D-gluco-pyranoside,6), oleracein P (2S-5-hydroxy-1-(p-coumaric acyl-7’-O-β-D-gluco-pyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranoside,7), oleracein Q (2S-5-hydroxy-l-(ferulic acyl-7’-O-β-D-gluco-pyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-β-D-glucopyranosyl-(1â†'6)-β-D-glucopyranoside,8), oleracein R (2S-5-hydroxy-l-(p-coumaric acyl-7’-O-β-D-glucopyranose)-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[2-O-ferulic acyl-β-D-glucopyranosyl]-(1â†'6)-β-D-glucopyranoside,9), oleracein S (2S-5-hydroxy-1-p coumaric acyl-2,3-dihydro-1H-indole-2-carboxylic acid-6-O-[6-O-ferulic acyl-β-D-glucopyranosyl]-(1â†'6)-β-D-glucopyranoside,10), oleracein A (11), oleracein B (12), oleracein C (13), oleracein D (14); four were phenolic acids:(2R)-(+)-feruloyl malate (15),p-hydroxybenzoic acid (16), ferulic acid (18),p-coumaric acid (19); one was coumarin:6,7-dihydroxy coumarin (17). Among them, compounds 1-10 were new indoline amide alkaloids and 15,16 were isolated from P. oleracea for the first time. In addition, DPPH radical scavenging activities of indoline amide alkloids 1-14 were examined and their structure-activities relationships were studied.Moreover, distribution of these indoline amide alkaloids was detected by HPLC method in the fresh plants of genus Portulaca, including P. oleracea, P. grandiflora and P. oleracea var. granatus, as well as in the stem and leaf of fresh P. oleracea. Preliminary results showed that the four indoline amides oleraceins A, B, H, I were only discovered in P. oleracea, and proved to be the characteristic chemical constituents of P. oleracea. Furthermore, their contents in leaf were higher than in stem. Above results may provide some theoretical basis for the chemotaxonomy of genus Portulaca. |
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