Assessment of major centelloside ratios in Centella asiatica accessions grown under identical ecological conditions, bioconversion clues and identification of elite lines

Centellosides viz., asiatic acid, madecassic acid, asiaticoside, madecassoside, are the major bioactive molecules in Centella asiatica. In this study madecassic acid:asiatic acid, madecassoside:asiaticoside (C6-hydroxylation versus non-hydroxylation) and asiaticoside:asiatic acid, madecassoside:madecassic acid (C28-glycoside versus aglycone) ratios in 50 C. asiatica accessions originally collected from their natural habitats in south India and grown under identical ecological conditions for six generations were determined using validated HPTLC-densitometry protocols. Asiatic acid, madecassic acid, asiaticoside and madecassoside contents ranged from 0.00–0.29% (average 0.03 ± 0.06%; 28 accessions recorded asiatic acid content as zero), 0.02–0.72% (0.12 ± 0.13%), 0.04–2.41% (0.44 ± 0.52%) and 0.15–5.27% (1.59 ± 1.26%), respectively. Distinctly, C6-hydroxylated (madecassic acid:asiatic acid 4.00, madecassoside:asiaticoside 3.61) and C28-glycosylated (asiaticoside:asiatic acid 14.67, madecassoside: madecassic acid 13.25) centellosides dominated over the respective non-derivatized entities. Our results infer that both C6-hydroxylation by CYP450-dependent monooxygenases and C28-glycosylation by UDP-Glc glucosyltransferases are dominant bioconversion steps in C. asiatica. Besides, this study discovered six elite lines of C. asiatica, with their (asiaticoside + madecassoside) contents above the industrial benchmark (≥ 4%) from south India. Two elite clones with asiaticoside contents ≥ 2% were also identified. Standardization of the agrotechniques of these elite lines could lead to their industrial applications. Further, this study emphasizes the need for standardizing all four centellosides as biomarkers in C. asiatica raw drugs, pharmaceutical and cosmetic products.

www.nature.com/scientificreports/ enzymes 13,14 . CYP716A53v2 is reported to hydroxylate C6 of the dammarane-type tetracyclic sapogenin, protopanaxadiol, in Panax ginseng to form protopanaxatriol 15 . A recent study specifically identified CYP716 enzymes effecting the bioconversion of ursane and oleanane pentacyclic triterpenoid skeletons to their 6β-hydroxy derivatives (C6-hydroxylation) in CA 13,16 . CYP450s and their functional roles in plants are least explored so far. UGTs are pivotal enzymes in the process of glycosylation in plants, contributing to the biosynthesis of medicinally important secondary metabolites. In saponin biosynthesis, UGTs catalyze the transfer of UDP linked sugar moieties to the triterpenoid skeleton 4,9,17 . Glycosides, ASI and MAD, in CA are formed by glycosylation of ASA and MDA, respectively, catalyzed by UGTs which link Glu-Glu-Rha to the C28 carboxyl groups 3,4,9,12 . Hydroxylation and glycosylation change the physicochemical properties and enhance the biological potentials of triterpenoids 12,18,19 . In ASI and MAD, the triterpenoid structures (aglycone) are hydrophobic and are linked to hydrophilic sugar chains (glycone) 12,[18][19][20] . The surface-active properties of saponins (sapo (Latin) = soap; soaplike surfactants that form long-lasting bubbles on shaking an aqueous solution) are distinguishing factors of these amphiphilic compounds from other glycosides. This is of considerable significance in drug design, and in CA, the glycosylated entities (saponins: ASI, MAD) are the prime target molecules in neuroprotection, memory enhancing, wound healing and skin protection. Pentacyclic triterpenes (centellosides) are accumulated in CA in their glycoside (ASI, MAD) forms rather than as aglycones (ASA, MAD); and the glycoside to aglycone ratios influence the efficacy of CA extracts and its pharmaceutical and skin care products [21][22][23] . Several studies quantified the four major centellosides (ASA, MDA, ASI, MAD) by various analytical techniques 20,21,[24][25][26] , whereas a few reports estimated only one or two (not all four) of these terpenoids in CA (examples, Devkota et al. 27 ; Thomas et al. 28 ; Prasad et al. 29 ). But, most of these quantification studies are on limited number of samples from various genetic/ecological origins. Genetic and environmental parameters significantly affect the production of secondary metabolites in plants, viz., centellosides in CA 20,26,28,[30][31][32] . Therefore, the C6-hydroxylation (MDA, MAD) versus non-hydroxylation (ASA, ASI) and C28-glycoside (ASI, MAD) versus aglycone (ASA, MDA) ratios (Scheme 1) in CA are determined by the variations in the genes (and enzymes) involved in their biosynthesis, and to a lesser extent by the ecological parameters 4,5,20 . Otherwise, genetically these ratios depend on the presence and activity of enzymes involved in C6-hydroxylation and C28-glycosylation 20 . In CA cell cultures, high production of centellosides is achieved by growth regulators and elicitors (examples, methyl jasmonate, salicylic acid), and they presumably modulate the expression of certain genes involved in their biosynthesis 10 . CA cell cultures are also capable of converting precursors like α-amyrin into centellosides with very high efficiency 6 . Plant cell cultures are able to carry out regio-and stereoselective hydroxylation, hydrogenation and glycosylation of exogenous substrates, and biotransforming them into other compounds with improved pharmacological actions 10 . Therefore, the (C6-hydroxylation versus non-hydroxylation) and (C28-glycoside versus aglycone) ratios in CA cell cultures are influenced by the biotransformations induced by their growth conditions.
Here, we explore the ratios of the four major centellosides viz., ASA, MDA, ASI, MAD, formed by C6-hydroxylation and/or C28-glycosylation of their precursor (ASA) (Scheme 1), in 50 accessions of CA originally collected from their natural habitats in south India and grown under identical ecological conditions for six generations. The study is conceived to derive bioconversion clues on the four centellosides by nullifying their ecological variations. We also address the significance of using the four centellosides as biomarkers in CA extracts and products. Moreover, these 50 CA accessions under study are scrutinized for elite lines based on industrial benchmarks of the contents of centellosides.
Collection of plant materials, growing conditions. Fifty CA accessions were collected from their natural habitats in various agro-climatic regions of the south Indian states of Kerala, Tamil Nadu and Karnataka (Table 1), taxonomically authenticated by Dr. Mathew Dan, Principal Scientist of Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI), Thiruvananthapuram and a voucher specimen (91008) was preserved at JNTBGRI Herbarium (TBGT) for future reference. These CA accessions were planted in the Field Gene Bank (FGB) of JNTBGRI in an evenly spread potting medium (1:1 top soil-sand) on level ground with uniform spacing of 30 cm apart in a randomized block design, without any external input of organic manure or chemical fertilizer, watered as and when needed, and maintained under uniform environmental conditions for a minimum of 3 years. For phytochemical analysis, aerial parts of four replications of six generation vegetatively propagated plants in flowering stage were collected in May 2019, dried in an oven at 40 °C and powdered. CA collections in this work were made as part of one of the Programme Support projects hosted by JNTBGRI and funded by Department of Biotechnology (DBT), Government of India; and collection of these plant materials is in compliance with relevant institutional, national, and international guidelines and legislation.  Quantitative analysis of ASI and MAD. CA extracts (2 µl each) were applied onto silica gel TLC plates as described in the previous sections ("HPTLC-densitometry analysis, calibration of centellosides" and "Quantitative analysis of ASA and MDA"). Plates were developed up to 80 mm in the twin trough plate development chamber previously saturated with 20 ml organic layer of butanol:ethyl acetate:water (4:1:5, v/v) for 30 min. The twin peaks of ASI and MAD in CA extracts were well resolved in this mobile phase. The plates were derivatised and photo documented.
Validation methods. HPTLC method was validated in terms of accuracy, precision, repeatability, reproducibility, linearity, limits of detection (LOD), limits of quantification (LOQ) and % recovery [33][34][35] . Calibration curves were generated by plotting amounts of analytes (standards: ASA, MDA, ASI, MAD) against peak response(s) ( Table 2). Intra-day precision was performed by repeating the same assay four times on the same day (of each standard). Inter-day precision was performed by repeating the assay twice for five consecutive days. were added to pre-analyzed CA extracts and re-analyzed (Table 3). Instrumental precision was determined by applying a sample solution (CA extract, ASA and MDA 6 µl each, ASI and MAD 2 µl each) on a TLC plate, developed as per the protocols described in previous sections, track(s) were scanned repeatedly (ten times each) and % coefficient(s) of variations were determined (Table 4).   1A) and MAD and ASI (Fig. 1B). The quantitative estimation data of the four centellosides are shown in Table 1. HPTLC-densitometry method was validated in terms of linearity,  Table 4. Intra-day and inter-day precision of ASA, MDA, ASI and MAD. a Mean SD of four trials on same day. b Mean SD of two trials for consecutive 5 days; % CV: Coefficient of variation (SD*100/mean). www.nature.com/scientificreports/  Biosynthetic clues. In CA, MDA and MAD are formed by hydroxylation at the C6 position (controlled by CYP450-dependent monooxygenases) of ASA and ASI, respectively. ASI and MAD are formed by C28-glycosylation of ASA and MDA (Scheme 1), respectively, catalyzed by UGTs which link two glucose units and one rhamnose unit to the C28 carboxyl group 3,4,12 . MAD also could be formed by C6-hydroxylation and C28-glycosylation of ASA (Scheme 1). In the present study, quantitative estimation of the four major centellosides in 50 CA accessions revealed significant variability in their contents, and the average C28-glycoside:aglycone ratios ASI:ASA and MAD:MDA are 14.67 (n = 50) and 13.25 (n = 50), respectively (Table 1). C6-hydroxylation:nonhydroxylation ratios in 50 CA accessions are MDA:ASA 4.00 (n = 50), MAD:ASI 3.61 (n = 50) ( Table 1).
In previous studies, the C6-hydroxylation:non-hydroxylation ratios in CA accessions from natural habitats were ( 39 . In fact, in these studies no consistent patterns were observed in C6-hydroxylation:non-hydroxylation and C28-glycoside:aglycone ratios in CA samples both from natural habitats or grown in vitro; moreover the sample sizes are limited, and their growing conditions, plant part, sample preparation, extraction and analytical parameters varied substantially. In the present study, 50 CA accessions collected from various agroclimatic conditions were grown under identical ecological conditions and their C6-hydroxylation:non-hydroxylation (MDA:ASA; MAD:ASI) and C28-glycoside:aglycone (ASI:ASA; MAD:MDA) ratios were analyzed under standardized (identical) parameters. Therefore, these ratios viz., MDA:ASA (4.00, n = 50, including the 28 accessions with zero ASA content), MAD:ASI (3.61, n = 50), ASI:ASA (14.67, n = 50), MAD:MDA (13.25, n = 50), are genetically determined (controlled), and reliable bioconversion and pharmaceutical clues can be derived from these data. Thereby, our study indicates that the C6-hydroxylation and C28-glycosylation driven by CYP450-dependent monooxygenases and UGTs, respectively, are leading bioconversion steps in CA.
In www.nature.com/scientificreports/ decade ago 28 , 60 CA accessions were originally collected from a wide range of locations in south India and the Andaman Islands (as in the present study) and grown under identical ecological conditions for 3 generations. ASI and MAD contents of these 60 CA accessions were quantified by similar HPTLC-densitometry protocol, and the average MAD:ASI ratio was 1.86:0.37 (i.e., 3.96, n = 60, which is close to our current ratio of 3.61). Only one of these accessions showed higher ASI content (0.80%) compared to MAD (0.29%). Of these 60 CA accessions, one showed absence of both the glycosides (ASI, MAD) and two other accessions showed the absence of ASI 28 .
In another recent study, we screened 106 CA accessions collected from various natural habitats in south India (i.e., directly from different ecological conditions) using similar protocol, and the observed MAD:ASI average ratio was 1.22:0.55 = 2.71 (unpublished data). This ratio (2.71) is considerably different from the two screening studies viz., current study MAD:ASI 3.61, Thomas and co-workers MAD:ASI 3.96 28 , under identical ecological conditions. In these 106 CA accessions, 6 showed higher ASI contents compared to MAD levels (unpublished data). These extensive data (under identical and varying ecological conditions) demonstrate MAD as the prominent constituent of the four centellosides in CA. These results clearly support the bioconversion possibilities portrayed in Scheme 1.
On further evaluation of the data, among the four major centellosides in CA, ASA which is formed by terpenoid biosynthesis has three bioconversion probabilities, viz., ASA to MDA (C6-hydroxylation), ASA to ASI (C28-glycosylation) and ASA to MAD (C6-hydroxylation + C28-glycosylation) (Scheme 1), whereas MDA and ASI have only one (each) possibility of getting converted to MAD by C28-glycosylation and C6-hydroxylation, respectively. In 50 CA accessions under identical environmental conditions, the average contents of ASA, MDA, ASI and MAD are 0.03, 0.12, 0.44 and 1.59%, respectively. As anticipated, ASA (aglycone) which has highest probability of enzymatic conversion(s), showed the lowest content in CA. MDA (aglycone) % content is 4 times that of ASA, and the highest content was displayed by MAD (1.59%) which has no further conversion prospects (Scheme 1). The average content of MAD (1.59%, n = 50) is 2.69 times the total average contents of the other three centellosides (ASA + MDA + ASI = 0.59%, n = 50). These data are inferring high rates (probabilities) of one or more transformations of the three centellosides as depicted in Scheme 1. The enzymes involved in C6-hydroxylation 13,16 and C-28 glycosylation 3,4,12 reactions in CA are already elucidated by biosynthetic studies.
ASI and (ASI + MAD) have been widely assigned as the major biomarkers for quality evaluation of CA raw drugs, pharmaceutical and cosmetic products 23,28 . But the four centellosides, ASA, MDA ASI and MAD, exert varying effects in the biological (neuroprotection, wound healing, skin protection) activities of CA, and thereby display disproportionate influences (roles) in its pharmaceutical applications. In our study, CA accessions with their ecological variations nullified, showed considerable fluctuations (even absence) in their ASA, ASI, MDA and MAD contents. Therefore, efficient quality control practices of CA raw drugs warrant the quantification of a set of major triterpenoids (four centellosides) as biomarkers 23 .
Apart from deductions on centelloside bioconversions, this study discovered six elite clones of CA from south India with their (ASI + MAD) contents above the industrial benchmark (≥ 4%). Two elite clones with ASI contents ≥ 2% were also identified. The agricultural practices of these CA elite lines can be standardized, and they can utilized for industrial purposes. All six elite accessions of CA discovered in this study are from high altitude locations (800-1332 m MSL) in the Idukki district in the south Indian state of Kerala. In our previous search (screening) for CA elite lines over a decade ago, two of the highest bioactive (ASI + MAD) yielding accessions were from high altitudes in Idukki district 28 . Our studies discovered a hotspot of high-yielding CA accessions in south India.
The high demand of CA is leading to its overexploitation at an uncontrolled rate and destruction of its wild genotypes 1 . Our study discovered six elite lines of CA from south India; the agro-practices of these high yielding genotypes can be standardized and utilized for its pharmaceutical and cosmetic purposes. The four major centellosides viz., ASA, ASI, MDA and MAD, have uneven effects on the bioactivities of CA based extracts/drugs. In this study, ASA was below detectable levels in 28 of the 50 screened CA accessions. These facts emphasize the need for quantifying the contents of all the four centellosides (as biomarkers) in CA extracts, pharmaceutical and cosmetic products. www.nature.com/scientificreports/