Studies On Bio-Active Constituents Of Enada Phaseoloides

Entada phaseoloides (Linn.) Merr. is a traditional medicinal plants, medicinal tissues included stem——Entadae Caulis and seed——Entadae Semen. The Entadae Caulis ever was listed in the Chinese Pharmacopoeia (1977 edition). It has the ability of expelling wind, removing damness, promoting blood circulation, freeing channel. It is intended for lumbocrural pain and traumatic injury. The Entadae Semen is listed in the Chinese Pharmacopoeia (2010 edition). It has the ability of invigorating function, invigorating the stomach and promoting digestion, dispel wind and to relieve pain, strengthening the muscles and bones. It is intended for insufficiency of blood, week, pain, poor digestion. However,the insufficient research of E. phaseoloides and the ambiguity of its bioactive components can not effective control the quality of this crude drug. The irregular quality will surely bring adverse effect to its application. In our continuing search for potent antioxidants from natural sources, we discovered that the ethanolic extract of E. phaseoloides had displayed significant antioxidant effects. To the best of our knowledge, this was the first report on the antioxidant properties of E. phaseoloides. We use the bioactivity-guided isolation to study the fractions and compounds of Entadae Caulis; to study on bioactive components of Entadae Semen.Studies on antioxidant constituents of Entadae CaulisThe ethanolic extract of Entadae Caulis displayed potent antioxidant activity when assessed by the DPPH, ABTS radical scavenging, reducing power,β-carotene bleaching, superoxide radical scavenging analyses and lipid peroxidation. Fractionation of the ethanolic extracts showed that the ethyl acetate fraction is the most active followed by the water fraction while n-butanol fraction was least active.Twenty-four compounds were isolated from the active fraction(EAF),and their structures were elucidated based on NMR spectra data. Twenty-one compounds were isolated from genus Entada for the first time, except quercetin,β-sitosterol, daucosterol. The major types of the isolated compounds were flavones. The isolated compounds were 7, 3, 4'-trimethylquercetin, 5-hydroxy-3, 4', 7-trimethoxyflavone, quercetin, (+)-3, 3' ,5', 5, 7-pentahydroflavanone, luteolin, (+)-dihydrokaempferol, dehydrodicatechin A, apigenin (-)-epicatechin, (+)-catechin, 3-deoxysappanchalcone, naringenin, rhamnocitrin, 4', 7-dihydroxyflavone, protocatechuic acid, vanillic acid, 5, 7, 4'-trihydroxy-3'-methoxyflavonol, galangin, rutin, 5, 7, 3', 5'-tetrahydroxyflavanone, 5, 2', 5'-trihydroxy-3, 7, 4'-trimethoxyflavone-2'-O-β-D-glucoside, (-)-epigallocatechin,β-sitosterol, daucosterol.The twenty-two compounds of Entadae Caulis were assessed the antioxidant activity by the DPPH, ABTS radical scavenging, reducing power,β-carotene bleaching, superoxide radical scavenging analyses and lipid peroxidation. The result showed that compounds, (-)-epigallocatechin, (-)-epicatechin (KT-9), (+)-catechin, quercetin, 5, 7, 4'-trihydroxy-3'-methoxyflavonol, (+)-3, 3' ,5', 5, 7-pentahydroflavanone, luteolin, protocatechuic acid are the top three in the activity of different models. Among them, (-)-epigallocatechin showed excellent free radical scavenging activity in the DPPH and ABTS assays, while protocatechuic acid exhibited significant ferric reducing power. Moreover, 5, 7, 4'-trihydroxy-3'-methoxyflavonol demonstrated the highest inhibition of oxidation of theβ-carotene/linoleic acid system, quercetin possessed the strongest superoxide radical-scavenging activity and (-)-epicatechin exhibted the most lipid peroxidation activity.Studies on constituents and quantitative determination of Entadae SemenFive compounds were isolated from n-butanol fraction of ethanolic extract of Entadae Semen,and their structures were elucidated based on NMR spectra data. The isolated compounds were Phaseoloidin, Entadamide A-β-D-glucopyranoside, Entadamide A,β-sitosterol, Daucosterol. A high performance liquid chromatography (HPLC) method was developed for the simultaneous quantification three compounds namely Phaseoloidin, Entadamide A-β-D-glucopyranoside, Entadamide A in Entadae Semen. The methodological study results indicated that the developed assay method was rapid,accurate,reliable and could be readily utilized as a quality control method for Entadae Semen.This assay was successfully applied to the determination of the 3 bioactive compounds in 22 samples. The average total contents of 3 bioactive compounds reach to 80.061 mg/g. In the majority of cases, Phaseoloidin and Entadamide A-β-D-glucopyranoside were the main components, whose contents varied from 64.291 to 96.505 mg/g and from 7.075 to 11.961 mg/g, respectively. But the variation of Entadamide A is large, the contents varied from 0.060 to 1.736 mg/g.Pharmacological activity-based research of Entadae SemenMaximum tolerated dose (MTD) experiments showed the toxic in mice at doses of 20 (ethanolic extract) g/kg body weight; 40 (water extract) g/kg body weight; 45 (processed products extract) g/kg body weight. Fixed-dose procedure acute toxicity of Phaseoloidin and Entadamide A-β-D-glucopyranoside showed innocuity at 2000 mg/kg body weight.The ethanolic extract, Phaseoloidin and Entadamide A-β-D-glucopyranoside were assessed anti-inflammatory and analgesic activity in mice. The ethanolic extract, Phaseoloidin and Entadamide A-β-D-glucopyranoside showed significant anti-inflammatory and analgesic activity. Expecially, on abdominal capillary permeability in mice, the samples exhibited better power than positive control. The ethanolic extract, Phaseoloidin, Entadamide A-β-D-glucopyranoside and Entadamide A showed no significant effect against IFN-γ/STAT1,IL-6/STAT3,TNF-α/NF-κB cell signaling pathway.The ethanolic extract, Phaseoloidin and Entadamide A-β-D-glucopyranoside were assessed gastric empting and gastrointestinal mobility model in mice. The Entadamide A-β-D-glucopyranoside high dosage group showed facilitating effects on gastrointestinal mobility in mice. The ethanolic extrac high dosage group can improve the delayed gastric in mice induced by atropine. The high dosage group of Phaseoloidin and Entadamide A-β-D-glucopyranoside, and low dosage group of ethanolic extract show the significant facilitating effects on delayed gastrointestinal mobility in mice induced by dopamine. The Phaseoloidin dosage group have the significant facilitating effects on gastric empting in mice induced by atropine.In vitro metabolism preliminary study of Entadae SemenThe ethanolic extract, Phaseoloidin, Entadamide A-β-D-glucopyranoside and Entadamide A were incubated in artifical gastric juice and intestinal flora. The results showed no metabolism in artifical gastric juice. The homogentisic acid is the metabolite of constituent from Phaseoloidin. Entadamide A is the metabolite of constituent from Entadamide A-β-D-glucopyranoside. Metabolism of ethanolic extract is occurring in the same as single compounds, but the metabolic rate of entadamide A-β-D-glucopyranoside was 64%.