3 4 di o caffeoylquinic acid

The methanol extract of the stem and leaves of Guettarda speciosa Linn. (MGS; voucher #: FBM224–095) was obtained from the International Biological Material Research Center at the Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Fingerprinting MGS was conducted with Agilent 1200 series high-performance liquid chromatographic (HPLC) system. MGS (15 μl in methanol) was injected onto a column (CapcellPAK MGII C18, Shiseido, Japan, 4.6 × 150 mm ID, 3 μm). The column temperature was 35 °C, and the flow rate was 0.5 mL/min. Samples were eluted in a gradient of 0.1% formic acid and incremental acetonitrile in water. Standards and samples were detected at wavelengths of 254 ~ 320 nm. Index chemicals, including 3-O-caffeoylquinic acid, quercetin 3-O-galactoside, quercetin 3-O-glucoside, 3,4-di-O-caffeoylquinic acid, apigenin 7-O-glucuronide, and quercetin rutinoside, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemstation software (Agilent Corporation, Germany) was used for data acquisition. The chemical constituents of MGS were identified by comparing their retention times to those of standard chemicals under identical analysis conditions and the UV spectra.

Folin–Ciocalteu reagent; catechin; gallic acid; oleanolic acid; vanillin; 2,2-diphenyl-1-picrylhydrazl (DPPH); chlorogenic acid; caffeic acid; 3,4-di-O-caffeoylquinic acid; 3,5-di-O-caffeoylquinic acid; and 4,5-di-O-caffeoylquinic acid were purchased from Sigma-Aldrich (Bangalore, India). Methanol was purchased from Honeywell (Hamburg, Germany). Sodium acetate trihydrate and 70% perchloric acid were purchased from V.W.R. (Leuven, Belgium). Ferric chloride, sodium carbonate, and acetic acid were purchased from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) was obtained from Carlo Erba (Val-de-Reuil, France). Formic acid and high-performance liquid chromatography (HPLC) solvents (acetonitrile and Methanol) were obtained from Fisher Scientific (Merelbeke, Belgium).