Asiaticoside

ECa 233 is an off-white extracted powder of Centella asiatica. The compounds of the ECa 233 extract were analyzed by using HPLC ESI-QTOF-MS/MS technique compared with analytical standards, and the quantitative analysis was performed with a triple quadrupole LC-MS/MS system^{40 (link)}. Two major components corresponding to the two major peaks in the analysis were madecassoside and Asiaticoside. In addition, the detected minor components were centellasaponins. However, the specific types of centellasaponins (A, C or D) could not be determined due to the lack of commercially available centellasaponins. Moreover, two other minor components were madecassic and asiatic acids. These triterpenic acids were accounted for less than 1.0% of ECa 233. The obtained ECa 233 extract contained 51% madecassoside and 38% Asiaticoside (Siam Herbal Innovation Co., Ltd., Thailand, prepared by a patent-pending method). Asiaticoside (>98.5%) and madecassoside (>95.0%) were purchased from Sigma-Aldrich Corp. Glycyrrhetinic acid (an internal standard, >99.0%) was obtained from Wako Pure Chemical Industries, Ltd.

Standardized extract ECa 233 powder was provided by Siam Herbal Innovation Co., Ltd., Bangkok, Thailand. The standardized extract contained 50% madecassoside and 40% asiaticoside, as determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Analytical standard asiaticoside (purity > 98.5%) was purchased from Sigma-Aldrich Corp, USA and madecassoside (purity > 96.7%) from Chromadex Corp, USA. Glycyrrhetinic acid (purity > 98.0%) as internal standard (IS) for LC-MS/MS analysis was purchased from Wako Pure Chemical Industries, Ltd., Japan. Dimethyl sulfoxide (DMSO) and carboxymethyl cellulose (CMC) used as a suspending agent were purchased from Sigma-Aldrich Corp., USA.

Aβ_1–42 (human, 1–42) was purchased from the Peptide Institute (Osaka, Japan). Asiaticoside was purchased from Sigma-Aldrich. The reference dye 5-carboxytetramethylrhodamine (TAMRA) was purchased from Olympus America Inc, whereas TAMRA-Aβ_1–42 was obtained from ANASPEC Inc. CA. Other chemicals were of analytical grade. Uranyl acetate was obtained from BDH. All experiments were carried out with the approval of an appropriate ethics committee of Shimane University compiled from the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science.

The Centell-S (purity ≥ 86.7%), piperine (purity ≥ 98.0%) and curcumin (purity ≥ 95.0%) used in the pharmacokinetic experiment were provided by Siam Herbal Innovation Co. Ltd. Analytical grade madecassoside (purity ≥ 96.7%) and madecassic acid (purity ≥ 97.5%) were purchased from Chromadex Corp. Analytical standards of asiaticoside (purity ≥ 98.5%) and asiatic acid (purity ≥ 97.0%) were purchased from Sigma-Aldrich Inc. The internal standards glycyrrhizin (purity ≥ 90.0%) and glycyrrhetinic acid (purity ≥ 98.0%) were purchased from Wako Pure Chemical Industries Ltd.

Nitro blue tetrazolium chloride (NBT) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma, Germany. Hanks' balanced salt solution (HBSS) was purchased from Gibco, USA. Lipopolysaccharide (LPS, from Escherichia coli) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma, USA. RPMI medium powder 1640 (RPMI 1640), DMEM medium powder, fetal bovine serum (FBS), penicillin-streptomycin (P/S), trypsin-EDTA, and trypan blue were purchased from Gibco, USA. Phosphate buffer saline (PBS) was from Amresco (USA) and other chemicals were from Sigma and Merck. α-Mangostin, asiaticoside and thymoquinone were purchased from Sigma, USA. Glycyrrhizin was purchased from Tokyo chemical industry (TCI), Singapore.