Isolation, purification and quantitative determination of Asiaticoside, Madecassoside, Asiatic acid and Madecassic acid in Varieties of Centella Asiatica (L.) URB.

The fractional crystallization method was developed to isolate and purify asiaticoside (AS) and madecassoside (MS) from the extract of Centella asiatica (CA); The maximum percentage recoveries of AS and MS were 78 and 67, respectively. AS and MS were subjected to alkali hydrolysis to yield asiatic acid (AA) and madecassic acid (MA) with percentage recoveries 89.6 and 63.1, respectively. The pure isolated substances were identified by the spectroscopic method compared to reference substances. A comparative study of three extraction methods: refluxing, ultrasonic-assisted extraction (UAE) and microwave-assisted extraction (MAE) has been investigated in order to determine the time to achieve maximum content of asiaticoside, madecassoside, asiatic acid and madecassic acid in CA. Both UAE and MAE were found significantly decreased in total extraction time and were more effective for extraction of the triterpenes from CA than the conventional refluxing method. The amount of triterpenes in CA collected from various locations of Thailand were evaluated by the HPLC using Alltech column (Alltima C18, 4.6x150mm, 5µm) as stationary phase, a mixture of acetonitrile: 10 mM phosphate buffer pH 7.1 (29:71) as mobile phase, a photodiode array at wavelength 210 nm as detector. All accessions of CA exhibited chemical variations in percentage content of triterpenes. The maximum contents were obtained as followed: asiaticoside 3.47%, w/w (Rayong province), madecassoside 5.48%, w/w (Phitsanulok province), asiatic acid 0.39%, w/w (Trat province) and madecassic acid 0.91%, w/w (Chiang Mai province). Furthermore, the content of these compounds was also annually determined in CA that collected from the commercial crops of Nakhon Pathom, Ubon Ratchathani and Nakhon Si Thammarat province. The maximum glycosides of CA in Nakhon Pathom and Nakhon Si Thammarat province were also observed in CA that collected on May and the maximum glycosides of CA in Ubon Ratchathani province were observed in CA that collected on March. Information obtained may be utilized as criteria for plant varieties selection and breeding programs for commercial cultivars of optimal triterpenes content for further isolation of pure triterpenes.