Secondary Metabolites of Medicinal Plants. Bharat Singh

asiaticoside B, [O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)]-O-β-D-glucopyranose ester of 2α,3β,6β,23α-tetrahydroxy-urs-12-ene-28-oic acid and [O-α-spl-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)]-O-β-D-glucopyranose ester of 2α,3β,6β,23α-tetrahydroxyolean-12-ene-28-oic acid (Sahu et al. 1989), and brahmic acid have been isolated from C. asiatica (Singh and Rastogi 1968).

Similarly, asiaticosides C, D, E, and F asiaticoside, madecassoside, and scheffuroside B were isolated from the butanol fraction of the EtOH extract of C. asiatica (Jiang et al. 2005). The (2α,3β,4α)-23-(acetyloxy)-2,3-dihydroxyurs-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, (2α,3β)-2,3-dihydroxyurs-12-en-28-oic acid, O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester, asiatic acid 6-O-β-D-glycopyranosyl-β-D-glucopyranosyl ester, (3β,4α)-3,23-dihydroxyurs-12-en-28-oic acid, and O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester were separated from this plant species (Xing et al. 2009). The ursane- and oleanane-type triterpene oligoglycosides; centellasaponins B, C, and D; madecassoside; asiaticoside; asiaticoside B; and sceffoleoside A have been isolated from Centella of Sri Lanka. The chemical structures of centellasaponins B, C, and D, madecassic acid 28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside, madasiatic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside, 3β,6β,23-trihydroxyolean-12-en-28-oic acid, 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (Matsuda et al. 2001), 3-O-[α-L-arabinopyranosyl] 2α,3β,6β,23α-tetrahydroxyurs-12-ene-28-oic acid, 6β-hydroxyasiatic acid (Shukla et al. 2000), 2α,3β,23-trihydroxyurs-20-en-28-oic acid and 2α,3β,23-trihydroxyurs-20-en-28-oic acid, O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl ester (Yu et al. 2007), 23-acetyloxy-2α,3β-dihydroxyurs-12-en-28-oic acid, 28-O-αL-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-6-D-glucopyranosyl ester, 2α,3β,6β-trihydroxyurs-12-en-28-oic acid, 3β,6β,23-trihydroxyurs-12-en-28-oic acid, 2α,3β,6β-trihydroxyolean-12-en-28-oic acid, and 3β,6β,23-trihydroxyolean-12-en-28-oic acid were determined by spectral data analysis (Kuroda et al. 2001). The 11,12-dehydroursolic acid lactone, ursolic acid, pomolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 3-epimaslinic acid, asiatic acid, corosolic acid, 8-acetoxy-1,9-pentadecadiene-4,6-diyn-3-ol, β-sitosterol 3-O-β-glucopyranoside, and rosmarinic acid were isolated from C. asiatica and showed antiproliferative activity against human uterine carcinoma (HeLa) and murine melanoma (B16F10) cells (Yoshida et al. 2005). The centellin, asiaticin, centellicin, asiaticoside, kaempferol, madecassoside, madecassic acid, and quercetin were identified and estimated from C. asiatica (Siddiqui et al. 2007; Hashim et al. 2011), but the isolated triterpenes did not exhibit significant cytotoxic activity against human leukemia-60 (HL-60), human squamous cells-2 (HSC-2), HSG, and human gingival fibroblasts (HGF) cell lines (Kuroda et al. 2001; Rafamantanana et al. 2009). The β-caryophyllene, α-humulene, and germacrene-D along with 41 sesquiterpenes were separated and identified by GC–MS analysis from the aerial parts of C. asiatica (Wong and Tan 1994; Verma et al. 1999). Similarly, the triterpenoid saponins such as asiaticoside and madecassoside, collectively called centelloids, were also identified from aqueous extract (Azerad 2016; Kim et al. 2009).

2.24.2 Culture Conditions

Due to its versatile medicinal properties, C. asiatica has been increasingly required in pharmaceutical industries, thus leading to the over exploitation of this herb (Zheng and Qin 2007). The callus cultures of H. asiatica was established on MS medium with supplementation of 2,4-D and kinetin for enhancing the production of asiaticoside (Josekutty 1998). The cell growth and asiaticoside accumulation reached maximum peak after 24 days of suspension culture at an agitation speed of 150 r/min and aeration rate of 2.5 l/min in bioreactors (Loc and Nhat 2014). Alkaloid content was increased higher in the callus cultures of C. asiatica developed in MS medium with supplementation of 2,4-D + BAP (Rao et al. 2015). Maximum growth of callus was achieved on days 21 and 28 of inoculation on MS medium with addition of 2,4-D and BAP (Nath and Buragohain 2005). The maximum centelloside production was observed in the stationary growth phase at day 25 of the culture. The methyl jasmonate as elicitor did not change the centelloside pattern, with madecassoside being the main compound, followed by asiaticoside. The maximum production of centellosides was obtained at day 15, with a time lag between gene activation and centelloside biosynthesis (Bonfill et al. 2011). The centelloside profile in the elicited cell suspension cultures was the same as in calli (Mangas et al. 2009). Thus, the main compound was madecassoside