| Erthrophleum fordii Oliver belongs to the genus Erthrophleum in the familyLeguminosae. Erthrophleum fordii is widely distributed in Guangxi, Guangdong, Fujian, Tibet, Zhejiang Provinces, etc, in China, and its barks and seeds are used asChinese traditional medicine with the main actions of "invigoration and promotingblood circulation".In our investigations, the ethanol extract, EtOAc part and n-BuOH part exhibitedcytotoxic activities. Some flavonoids, diterpenoid glycoside, and triterpenoids wereisolated previously from the EtOAc part of the barks of E. fordii. But thesecompounds showed no cytotoxic activities.To develop traditional medicine and find new active lead compounds, we continued tostudy this species. The phytochemical re-investigation of this active extract affordedeight cassaine-type diterpenoid amides, one flavonol, four flavonol-type tannins, twotriterpenoids, one sterol and its one glycoside. Their structures were established as3β-O-tigloyl-nor-erythrophlamide (1), 3β-O-acetyl-nor-erythrophlamide (2), 6α-hydroxy-nor-cassamide (3), 7-dehydro-nor-erythrosuamide (4), nor-erythrosuamide22-O-β-D-galactopyranoside (5), 6α-hydroxy-nor-cassamide 22-O-β-D-galacto-pyranoside (6), nor-cassamide (7), nor-erythrosuamide (8), (-)-robinetinidol (9), robinetinidol-(4→6)-robinetinidol (10), procyanidin B5 (11), fisetinidol-(4β→6)-robinetinidol (12), [4α→8: 4α→6]-bi-fisetinidol-catechins (13), betulinic acid (14), morolic acid acetate (15),β-sitosterol (16), and daucosterol (17). Among them, 1-6are new compounds, and 7, 8, and 10-13 were isolated from this plant for the firsttime.Cytotoxic activities against human cancer cell lines of the compounds were measuredby MTT test in vitro. Compounds 1-8 exhibited selectively cytotoxicities on thegrowth control against A2780, Bel-7402, BGC-823, MCF-7, HCT-8, KB, Hela, PC-3M, A549 and Ketr3 cancer cell lines and exhibited considerable structure-activityrelationship. In addition, compounds 3 and 7 showed activity of inhibiting MAPK pathway in HCT-8 and A549 human cancer cell lines. This is the first time to study thecytotoxic mechanism of cassaine-type diterpenoid alkaloids.The ESI-MS~n fragmentation behaviors of the eight cassaine-type diterpenoid amidesisolated from E. fordii were investigated. The characteristic fragmentation patterns ofthe compounds were discussed in detail to give some valuable conclusions. On thebasis of above conclusions, HPLC-HR-MS, HPLC-DAD-ESI-MS~n and HPLC-NMRof several fractions were measured, and 9 analogous cassaine-type diterpenoid amidesin these fractions were detected. Their structures were characterized as 3β-O-tigloyl-6α-hydroxy-nor-cassamide (T1), 3β-O-tigloyl-11-hydroxy-nor-erythrophlamide (T2), 7-dehydro-nor-cassamidide (T3), 4α, 11-dihydroxy-nor-cassamide (T4), 5-dehydro-6-hydroxy-nor-cassamide (T5), nor-cassamidide (T6), nor-erythrophlamide (T7), 6α-hydroxy-nor-cassamide (T8), and nor-erythrosuamide (T9). Among them, T1-T5are new compounds, and T6 and T7 are known compounds isolated from this plantfor the first time.Using the above analytical method, structures of analogous compounds in same classwere determined. It is important to analyze compounds with molecular diversity inextracts of plants. So we studied on-line on the compounds in various classes from theextract of Lysidicie brevicalyx Wei.The genus Lysidice consists of two species, L. rhodostegia Hance and L. brevicalyxWei. Some phloroglucinols, stilbenes and flavanoids were isolated previously fromthe roots of L. rhodostegia. To identify the constituents in L. brevicalyx of the samegenus rapidly, ESI-MS~n fragmentation behaviors of the isolated six phloroglucinols, five stilbenes and nine flavanoids were investigated. After the characteristicfragmentation patterns and UV spectra of these compounds were summarized, HPLC-HR-MS, HPLC-DAD-ESI-MS~n and HPLC-NMR of some fractions from L.brevicalyx were measured, and 7 analogous compounds of different classes in thesefractions were detected. Their structures were characterized as E-resveratrol-3-O-[6-O-(4-hydroxy)-cinnamyl]-β-D-glucopyranoside (L1), sulphuretin (3', 4', 6-trihydroxy-aurone) (L2), naringenin (L3), E-resveratrol-3-O-[6-O-(3-methoxy-4-hydroxy)-cinnamyl]-β-D-glucopyranoside (L4), lysidiside A (L5), E-resveratrol- 3-O-[6-O-(4-hydroxy)-benzoyl]-β-D-glucopyranoside (L6), E-resveratrol-3-O-[6-O-(3-methoxy-4-hydroxy)-benzoyl]-β-D-glucopyranoside (L7). It suggested that extractof L. brevicalyx Wei contained stilbenes and the analogues mainly.This established analytical method has great potential in structural characterization ofconstituents in same and different classes. It will simplify and accelerate the structuralinvestigation of new trace natural products in plants, and provide the foundation forefficient and rapid chemical screening of trace natural products in plants.
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