Purified, Identified Of The Antiaging And Antioxidative Compounds From The Roots Of I.younghusbandii Sprague

Antioxidative and anti-aging activities in vivo and in vitro of the extracts of the Tibet-herb Incarvillea younghusbandii Sprague were studied systematically here. Two compounds with antioxidative and anti-aging activities were isolated and identificated. Our studies not only demonstrated the practical application value of Incarvillea younghusbandii Sprague but. also showed some theoretical values to the future research.The five extracts from the roots of Incarvillea younghusbandii Sprague were as follows: IYS1 (extract by ethanol, yield 36.8%), IYS2 (residue extract by water, yield 16.9%), IYS3 (extract by water directly, yield 49.7%), IYS4 (supernatant by ethanol from IYS1, yield 7.73%), IYS5 (sediment by ethanol from IYS1, yield 29.0%).Evaluate by O'₂^-, "OH scavenging assay and LPO inhibitory assray, the activities of the five extracts were as follows: IYS4＞IYS1＞IYS3＞IYS5＞IYS2. Compound A and B with antioxidative and antiaging effect were first isolated from the roots of Incarvillea younghusbandii Sprague through ethyl acetate precipitation, silica gel column chromatography, reverse-phase C18 column chromatography and reverse-phase semi-preparative HPLC. Compound A and B had the same molecular weight (624) and the same molecular formula (C₂₉H₃₆O₁₅) which were determined by ESI-MS and high resolution ESI-MS, respectively. Compoung A and B were hydrolyzed and their silylated monosaccharide derivatives were analyzed by GC-MS. Compound A and B consisted a L-rhamnose and a D-glucose. Their structures were identified by 1D-, 2D-NMR and GC-MS as: A,β-D-Glucopyranoside,2-(3,4-dihydroxyphenyl)ethyl-3-O-(6-deoxy-a-L-mannopyran osyl)-,4-[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoate](9CI); B,β-D-Glucopyranoside,2-(3,4-dihydroxyphenyl)ethyl-3-O-(6-deoxy-a-L-mannopyran osyl)-,6-[(2E)-3-(3,4-dihydroxyphenyl)-2-propenoate](9CI). Compound A (CAS registry number, 61276-17-3), a famous compound with various bioactivities and widely commercial use had been reported in 768 literatures (see Chemical Abstracts, USA), but its occurrence in the roots of IYS with a high yield of 43.1mg/g of dry roots was reported here for the first time. Compound B (7.1mg/g in the dry roots) was a novel compound and its structure and bioactivity were reported here for the first time. The UV/PDA spectra of compound A was the same as that of compound B with the maximum absorption at 198nm and 330nm. At the same chromatography conditions, retention time of compoung B was longer than that of compoung A.Compound A and B were used as external standards to quantificated of A and B in IYS1, IYS2, IYS3, IYS4 and IYS5 extracts by analysis HPLC. The level were as follows: A, IYS4 (380.6mg/g)＞IYS1 (113.6mg/g)＞IYS3 (40.4mg/g)＞IYS5 (28.7mg/g)＞IYS2 (7.5mg/g) ; B, IYS4 (76.1mg/g)＞IYS1 (18.9mg/g)＞IYS3 (5.tmg/g)＞IYS5 (3.2mg/g)＞IYS2 (0.7mg/g) ; A+B,IYS4 (456.7mg/g)＞IYS1 (132.5mg/g)＞IYS3 (45.5mg/g)＞IYS5 (31.9mg/g)＞IYS2 (8.2mg/g) .The concentration of compound A (43.1mg/g) in the dry roots of IncarviUea younghusbandii Sprague was far high than that of compound B (7.1rag/g).Result of total phenolic assay were as follows: IYS4 (154.85±2.73rag/g)＞IYS1 (47.94±0.76 mg/g)＞IYS3 (26.18±0.76mg/g)＞IYS5 (20.88±0.76mg/g)＞IYS2 (12.21±1.00 mg/g) . The level of phenolic content in the five extracts were absolutely positive correlation to the level of compound A, or B or A+B. The level of phenolic content of compound A and B were 388.68±0.56 and 353.24±3.55mg/g, respectively.Solvent volume was major factor (range, 672.354) and others were minor factor in yield×phenolic content. See from effect-curve diagram, the best conditions for yield×phenolic content were as follow: extracted at a temperature of 60℃, extracted for 6h, solvent consisted was 50-65% Alcohol in water, solvent volume used for extracting was 9-12 time, stirring speed was 400rpm.In vitro activities, the higher werethe level of phenolic content and the compound A+B, the higher were the scavenging activity of extracts to superoxide radical anion(O'₂^-) and hydroxyl radicals (OH). Positive correlations existed between the level of phenolic content, or the compound A+B and superoxide radical anion(O'₂^-), hydroxyl radicals ('OH) scavenging activity. Both superoxide radical anion(O'₂^-) and hydroxyl radicals ('OH) scavenging activity of Vc were inferiort to those of compound A or B. Vc had not inhibited but raised LPO level in vitro. At a low concentration of 0.0625 mg/mg, compound A and B could intensity protected the mice hepatic tissue from oxidation, while others (such as BHT, Vc) had not this property. The protections of compound A and B at the concentration of 0.2500 mg/ml (medium level) were better than those of at the concentration of 1.0000mg/ml (high level) and 0.0625 mg/ml (low level).With a low level of phenolic content or compound A+B, the prolong lifespan effects in Drosophila melanogaster of IYS2,IYS3,IYS5 were not significant. With a high level of phenolic content or compound A+B, the prolong lifespan effects in Drosophila melanogaster of IYS1,IYS4 were absolutely significant. So positive correlations existed between the level of phenolic content or compound A+B and the prolong lifespan effects in Drosophila melanogaster. However, higher level of phenolic content or compound A+B could not increase the lifespan of Drosophila melanogaster any more. An appropriate dose of IYS4 could increase SOD and CAT activities and inhibit MDA level in Drosophila melanogaster, while a high dose could not. At any dose, Vc could not increase GSH-Px activity or inhibit MDA level in Drosophila melanogaster. At an appropriate dose, the positive correlation between the level of the polyphenol or compound A+B and the life-span extends percent of fruit fly were significant.The oral acute toxicity in mice was actually not toxic. The mouse micronucleus test for IYS1 was negative and the sperm abnormality frequencies were not significantly affected by IYS1.