Development and validation of a high-performance liquid chromatography method for standardization of the bioactive ethyl acetate fraction of Alstonia scholaris (Linn.) R. Br. growing in Egypt.

Bio-guided fractionation of the ethanolic extract of the leaves of Alstonia scholaris (Apocynaceae) growing in Egypt was carried out to evaluate its antihyperglycemic activity in alloxan-induced diabetic rats and its hepatoprotective activity against CCl4-induced hepatotoxicity in rats. The ethyl acetate fraction of the ethanolic extract showed the highest antihyperglycemic [(133.6 +/- 4.2) mg/mL, relative to metformin with (92.3 +/- 2.7) mg/mL] and hepatoprotective [(37.9 +/- 1.4) U/L, relative to silymarin with (29.7 +/- 0.8) U/L] activities. Four compounds were isolated from this fraction, and identified by spectroscopic techniques and by comparison with reported data: caffeic acid and isoquercitrin for the first time from this plant, in addition to quercetin 3-O-beta-D-xylopyranosyl (1''' --> 2")-beta-D-galactopyranoside (major compound) and chlorogenic acid. A validated reversed phase-high-performance liquid chromatography (RP-HPLC) method was developed for the standardization of the bioactive ethyl acetate fraction. The calibration curve showed good linearity (r2 > 0.999) within tested ranges. The relative standard deviation of the method was less than 3% for intra- (0.4-2.0%) and inter-day (1.9-2.8%) assays. Mean recovery of the method was within the range of 98.5-102.5%. The minimum detectable concentration of the analyte (LOD) was found to be 0.04 microg/mL. This developed HPLC method was shown to be simple, rapid, precise, reproducible, robust, specific, and accurate for quality assessment of the bioactive fraction.

Flavonoids and terpenoids from Helichrysum forskahlii.

Three new flavonoids, namely helichrysone A (1), helichrysone B (2) and helichrysone C (3) were isolated from the aerial parts of Helichrysum forskahlii, together with 10 known flavonoids, three triterpenes, and one sesquiterpene. The structures of the new flavonoids 1-3 were established by 1D and 2D NMR spectral data. In addition, the antimicrobial activities of the isolated compounds were determined.

Bioguided isolation of pentacyclic triterpenes from the leaves of Alstonia scholaris (Linn.) R. Br. growing in Egypt.

Ethanolic and aqueous extracts of the leaves and flowers of Alstonia scholaris were evaluated for their antioxidant activity by investigating their effect on blood glutathione levels in alloxan-induced diabetic rats. The ethanolic extract of the leaves was the most active; therefore, its cytotoxicity against HepG2 cells was also tested. Promising GI50 values of 1.96, 4.34 and 4.65 µg mL⁻¹ were observed for the extract, its chloroform and ethyl acetate fractions, respectively. The chloroform active subfraction I (GI50 = 2.97 µg mL⁻¹) yielded betulin (1), betulinic acid (2) and ursolic acid (3) upon purification. Compounds 1-3 were identified using spectroscopic techniques and by comparison with reported data. GLC of unsaponifiable and saponifiable fractions of the hexane extract revealed ß-sitosterol (7.37%) and n-tetracosane (54.4%) to be the major sterol and hydrocarbon components, respectively. Linoleic acid (48.89%) was the predominant fatty acid.