## 3 Active Constituents of _C. asiatica_  
Inconsistent pharmacological activities of _C. asiatica_ extracts harvested from various origins were reported; these extracts displayed substantial variations in the quality and quantity of their bioactive compounds [11]. Previous analytical studies showed _C. asiatica_ to contain triterpenoids, amino acids and essential oils. The most important bioactive compounds are triterpene glycosides (saponins), such as asiaticoside and madecassoside, along with their respective aglycones (sapogenins), asiatic acid and madecassic acid [5; 8; 10; 30; 31; 32]. These four major compounds, also known as pentacyclic triterpenes, are claimed to be responsible for the biological effects of _C. asiatica_ extracts (Figure 2). Other constituents include oxyasiaticoside, centelloside, brahmoside, brahminoside, thankunoside, isothankunoside, brahmic acid, isobrahmic acid, betulic acid, \(\beta\)-sitosterol, hexacosan octanoate, kaempfer, quercetin, daucosterol, vanillic acid and succinic acid [2; 7; 9; 33; 34]. Madecassoside and asiaticoside are identified as the biomarker components of this plant extract, due to their high contents relative to other constituents, with the content of madecassoside (C\({}_{48}\)H\({}_{78}\)O\({}_{20}\)) being the highest [35].  
Several methods have been used to quantify the triterpene content of Centella extracts. These include thin-layer chromatography and mass spectrometry (TLC-MS) [36], TLC coupled to high-speed counter-current chromatography [37; 38], ultraviolet-visible spectroscopy (UVS) [39], high performance liquid chromatography (HPLC) [40; 41; 42], HPTLC [37; 42; 43; 44; 45], HPLC-electrospray ionization-mass spectrometry (HPLC-ESI-MS) [6], HPLC-ultraviolet (HPLC-UV) [5; 41; 46], and HPLC-paired with evaporative light scattering detector (HPLC-ELSD) [5; 42]. Among these methods, some are comprised of multiple-step extraction and/or purification processes. These techniques have limited sensitivity and specificity, while necessitating long analysis cycles to acquire satisfactory chromatographic separation. High-performance anion-exchange chromatography combined with pulsed amperometric detection (HPAEC-PAD) is often used to quantify carbohydrates in plant extracts, allowing absolute detection of carbohydrates with great sensitivity [5]. Glycos have previously been analyzed using reversed phase HPLC-PAD, where the compounds  
Figure 2: Chemical structures of madecassoside, asiaticoside, madecassic acid and asiatic acid (Glu: glucose, Rha: rhamno).  
were detected following chromatographic separation. For example, madecassoside and asaaticoside were detected within 5 min of sample pre-treatment by combination with a post-column sodium hydroxide solution using the reversed phase HPLC-PAD method [5].  
Although the quantification of _C. asaatica_ triterpenes have been corroborated by numerous researchers using HPLC-UV and HPTLC, variations in the triterpene content of extracts were found, depending on the location and the diverse environmental conditions for the growth of the plant. An investigation of the chemotypic variations of active biomarkers in relation to morphotypic changes in _C. asaatica_ found no distinct disparity in botanical descriptors, physicochemical parameters or in bioactive metabolites quantified through HPTLC; this led the team to conclude that morphotypic variations do not always lead to chemotypic variations in _C. asaatica_[33].  
A different approach using a polyclonal antibody against madecassoside, ELISA and Eastern blotting was used to quantitatively analyze triterpene glycosides in _C. asaatica_. ELISA was used to determine total madecassoside and asaaticoside content, whereas Eastern blotting was used to separate and quantify madecassoside and asaaticoside. These methods are faster, more straightforward and cost effective compared to HPLC [4].