Investigating high throughput phenotyping based morpho-physiological and biochemical adaptations of indian pennywort (Centella asiatica L. urban) in response to different irrigation regimes.

Indian pennywort (Centella asiatica L. Urban; Apiaceae) is a herbaceous plant used as traditional medicine in several regions worldwide. An adequate supply of fresh water in accordance with crop requirements is an important tool for maintaining the productivity and quality of medicinal plants. The objective of this study was to find a suitable irrigation schedule for improving the morphological and physiological characteristics, and crop productivity of Indian pennywort using high-throughput phenotyping. Four treatments were considered based on irrigation schedules (100, 75, 50, and 25% of field capacity denoted by I100 [control], I75, I50, and I25, respectively). The number of leaves, plant perimeter, plant volume, and shoot dry weight were sustained in I75 irrigated plants, whereas adverse effects on plant growth parameters were observed when plants were subjected to I25 irrigation for 21 days. Leaf temperature (Tleaf) was also retained in I75 irrigated plants, when compared with control. An increase of 2.0 °C temperature was detected in the Tleaf of plants under I25 irrigation treatment when compared with control. The increase in Tleaf was attributed to a decreased transpiration rate (R2 = 0.93), leading to an elevated crop water stress index. Green reflectance and leaf greenness remained unchanged in plants under I75 irrigation, while significantly decreased under I50 and I25 irrigation. These decreases were attributed to declined leaf osmotic potential, increased non-photochemical quenching, and inhibition of net photosynthetic rate (Pn). The asiatic acid and total centellosides in the leaf tissues, and centellosides yield of plants under I75 irrigation were retained when compared with control, while these parameters were regulated to maximal when exposed to I50 irrigation. Based on the results, I75 irrigation treatment was identified as the optimum irrigation schedule for Indian pennywort in terms of sustained biomass and a stable total centellosides. However, further validation in the field trials at multiple locations and involving different crop rotations is recommended to confirm these findings.

In situ phytoremediation characterization of heavy metals promoted by Hydrocotyle ranunculoides at Santa Bárbara stream, an anthropogenic polluted site in southern of Brazil.

Aquatic environments are widely affected by anthropogenic activities and efficient remediation of these areas requires detailed studies for each natural ecosystem. This research aimed to evaluate the natural phytoremediation potential of Hydrocotyle ranunculoides L., a floating aquatic macrophyte located in a polluted aquatic environment in South of the Rio Grande do Sul, Brazil. Nutrients such as P, K, Ca, Mg, and S and heavy metals such as Cu, Zn, Fe, Mn, Na, Cd, Cr, Ni, Pb, Al, As, Co, and V content in the roots and shoots of the plants were evaluated through nitric perchloric acid digestion (HNO3-HClO4) methods and quantified by ICP-OES. Bioconcentration factor (BCF), translocation factor (TF), plant effective number (PEN), and potential phytoremoval (mg m-2) were carried out. H. ranunculoides showed a substantial ability for phytoextracting P, Na, and As, since showed ability of uptake these elements from the water and translocate them to the shoots of the plants. H. ranunculoides also showed potential for application in rhizofiltration of Mg, S, Cu, Zn, Fe, Mn, Cd, Cr, Ni, Pb, Al, and V, since exhibited high potential to uptake higher levels in the roots. The highest potential for bioremoval (mg m-2) of the H. ranunculoides was detected for K, Ca, P (recommending thus the use for phytoextraction), Fe, and Al (highly recommended for rhizofiltration). Therefore, this species under study showed high potential for in situ phytoremediation at Santa Bárbara stream, and as a widespread species, it might be tested for phytoremediation in other sites.

Artificial neural network-based model for the prediction of optimal growth and culture conditions for maximum biomass accumulation in multiple shoot cultures of Centella asiatica.

An artificial neural network (ANN)-based modelling approach is used to determine the synergistic effect of five major components of growth medium (Mg, Cu, Zn, nitrate and sucrose) on improved in vitro biomass yield in multiple shoot cultures of Centella asiatica. The back propagation neural network (BPNN) was employed to predict optimal biomass accumulation in terms of growth index over a defined culture duration of 35 days. The four variable concentrations of five media components, i.e. MgSO4 (0, 0.75, 1.5, 3.0 mM), ZnSO4 (0, 15, 30, 60 µM), CuSO4 (0, 0.05, 0.1, 0.2 µM), NO3 (20, 30, 40, 60 mM) and sucrose (1, 3, 5, 7 %, w/v) were taken as inputs for the ANN model. The designed model was evaluated by performing three different sets of validation experiments that indicated a greater similarity between the target and predicted dataset. The results of the modelling experiment suggested that 1.5 mM Mg, 30 µM Zn, 0.1 µM Cu, 40 mM NO3 and 6 % (w/v) sucrose were the respective optimal concentrations of the tested medium components for achieving maximum growth index of 1654.46 with high centelloside yield (62.37 mg DW/culture) in the cultured multiple shoots. This study can facilitate the generation of higher biomass of uniform, clean, good quality C. asiatica herb that can efficiently be utilized by pharmaceutical industries.

Heterogeneous water supply affects growth and benefits of clonal integration between co-existing invasive and native Hydrocotyle species.

Spatial patchiness and temporal variability in water availability are common in nature under global climate change, which can remarkably influence adaptive responses of clonal plants, i.e. clonal integration (translocating resources between connected ramets). However, little is known about the effects of spatial patchiness and temporal heterogeneity in water on growth and clonal integration between congeneric invasive and native Hydrocotyle species. In a greenhouse experiment, we subjected severed or no severed (intact) fragments of Hydrocotyle vulgaris, a highly invasive species in China, and its co-existing, native congener H. sibthorpioides to different spatial patchiness (homogeneous and patchy) and temporal interval (low and high interval) in water supply. Clonal integration had significant positive effects on growth of both species. In the homogeneous water conditions, clonal integration greatly improved the growth in fragments of both species under low interval in water. However, in the patchy water conditions, clonal integration significantly increased growth in both ramets and fragments of H. vulgaris under high interval in water. Therefore, spatial patchiness and temporal interval in water altered the effects of clonal integration of both species, especially for H. vulgaris. The adaptation of H. vulgaris might lead to invasive growth and potential spread under the global water variability.

Seasonal Variation of Triterpenes and Phenolic Compounds in Australian Centella asiatica (L.) Urb.

INTRODUCTION: Specific triterpenes, phenolic acids and flavonoids in Centella asiatica have been found to be bioactive. Harvesting the plant when these putative bioactive compounds are at their highest concentrations would provide consistency in their chemical profile, thus ensuring the quality and efficacy of derived medicinal products. OBJECTIVE: The aim of the study was to determine the impact of harvesting time on the contents of major triterpenoid and phenolic compounds in C. asiatica. METHODOLOGY: Australian C. asiatica was collected from a designated area in different months. The principal triterpenes (asiaticoside, madecassoside, asiatic acid and madecassic acid), flavonoid compounds (rutin, quercetin and kaempferol) and chlorogenic acid were quantitatively determined by HPLC-DAD analysis. RESULTS: Triterpenoid, kaempferol and chlorogenic acid content showed significant variation (p < 0.05) in different collecting months. The total content of the four triterpenes reached its highest levels in January and February (83.15 ± 0.16 mg/g and 78.41 ± 0.16 mg/g, respectively), the summer season of the southern hemisphere, and their lowest values in winter (June) and spring (October) seasons (35.65 ± 0.20 and 35.50 ± 0.55 mg/g, respectively). Similarly, the contents of chlorogenic acid and kaempferol were the highest in December and January (1.62 ± 0.01 and 0.33 ± 0.01 mg/g, respectively), and the lowest in June (0.06 ± 0.01 and 0.09 ± 0.01 mg/g, respectively). CONCLUSION: The results indicate that harvesting C. asiatica in summer returns the highest yield of the target triterpenoids, kaempferol and chlorogenic acid.

Ecologically relevant UV-B dose combined with high PAR intensity distinctly affect plant growth and accumulation of secondary metabolites in leaves of Centella asiatica L. Urban.

We investigated the effects of environmentally relevant dose of ultraviolet (UV)-B and photosynthetic active radiation (PAR) on saponin accumulation in leaves on the example of Centella asiatica L. Urban. For this purpose, plants were exposed to one of four light regimes i.e., two PAR intensities with or without UV-B radiation. The experiment was conducted in technically complex sun simulators under almost natural irradiance and climatic conditions. As observed, UV-B radiation increased herb and leaf production as well as the content of epidermal flavonols, which was monitored by non-destructive fluorescence measurements. Specific fluorescence indices also indicate an increase in the content of anthocyanins under high PAR; this increase was likewise observed for the saponin concentrations. In contrast, UV-B radiation had no distinct effects on saponin and sapogenin concentrations. Our findings suggest that besides flavonoids, also saponins were accumulated under high PAR protecting the plant from oxidative damage. Furthermore, glycosylation of sapogenins seems to be important either for the protective function and/or for compartmentalization of the compounds. Moreover, our study revealed that younger leaves contain higher amounts of saponins, while in older leaves the sapogenins were the most abundant constituents. Concluding, our results proof that ambient dose of UV-B and high PAR intensity distinctly affect the accumulation of flavonoids and saponins, enabling the plant tissue to adapt to the light conditions.

Centelloside accumulation in leaves of Centella asiatica is determined by resource partitioning between primary and secondary metabolism while influenced by supply levels of either nitrogen, phosphorus or potassium.

In the present study we aimed to investigate the relevance of either N, P or K supply for herb and leaf yield and for centelloside concentrations in Centella asiatica L. Urban leaves. In this regard, we elucidated the causal relationship between assimilation rate, leaf N, P and K concentrations, herb and leaf production, and centelloside accumulation. The experiments were conducted consecutively in a greenhouse where C. asiatica was grown in hydroponic culture and fertigated with nutrient solutions at either 0, 30, 60, 100 or 150% of the N, P or K amount in a standard Hoagland solution. In general, the increase in N, P or K supply enhanced assimilation rate and herb and leaf yield. However, exceeding specific thresholds, the high availability of one single nutrient caused lower leaf N concentrations and a decline in assimilation rate and plant growth. Irrespective of N, P and K supply, the leaf centelloside concentrations were negatively associated with herb and leaf yield, which is in accordance with the assumptions of the carbon/nutrient balance and the growth differentiation balance hypotheses. Moreover, we found strong negative correlations between saponins and leaf N concentrations, while the respective sapogenins were negatively correlated with K concentrations. Using C. asiatica as model system, our experiments reveal for the first time that the accumulation of saponins and sapogenins is affected by resource allocation between primary and secondary metabolism and that besides carbon, also nutrient availability is relevant for the regulation of the centelloside synthesis. Finally, our results highlight the huge potential of optimized and carefully controlled mineral nutrition of medicinal plants for steering the bio-production of high-quality natural products.

Growth and asiaticoside production in multiple shoot cultures of a medicinal herb, Centella asiatica (L.) Urban, under the influence of nutrient manipulations.

Growth and in vitro asiaticoside accumulation in multiple shoot cultures of Centella asiatica (L.) Urban was studied as a function of nutrient manipulations in the culture media. Shoot cultures raised in liquid Murashige and Skoog medium supplemented with 2.5 mg/l kinetin attained a growth index (GI) of 6.06 along with the highest asiaticoside content of 3.8 mg/g dry weight on the 35th day of the culture cycle. The shoot growth and asiaticoside accumulation were found to be influenced by the relative proportions of NH(4)(+)-N:NO(3)(-)-N or Cu(2+) concentration in the medium. Asiaticoside content in shoots increased from 5.3 to 8.9 and 8.7 mg/g dry weight when total nitrogen concentration of 60 mM in the control medium was reduced to 50 and 40 mM with a corresponding change in NH(4) (+):NO(3)(-) ratio from 20:40 to 20:30 or 20:20, respectively. Total nitrogen level higher than 60 mM drastically reduced the asiaticoside concentration in these in vitro shoot cultures. Medium devoid of Cu(2+) significantly favored higher asiaticoside accumulation in the cultured tissue (7.05 mg/g dry weight) along with an improved biomass production (GI = 7.7) when compared with shoots reared on the control medium with 0.10 µM Cu(2+) (GI = 5.8; asiaticoside content = 4.4 mg/g dry weight). Carbohydrate enrichment of the medium by increasing the sucrose concentration from 3.0 to 5.0 or 7.0% was also beneficial for biomass and asiaticoside production with GI = 17.1 and 16.9 and asiaticoside content = 7.2 and 5.2 mg/g dry weight, respectively, in comparison to control cultures maintained on medium containing 3.0% sucrose. The procedure described here provides a viable production platform for generating clean and quality material from Centella with high bioactive content.

Discrimination of three Pegaga (Centella) varieties and determination of growth-lighting effects on metabolites content based on the chemometry of 1H nuclear magnetic resonance spectroscopy.

The metabolites of three species of Apiaceae, also known as Pegaga, were analyzed utilizing (1)H NMR spectroscopy and multivariate data analysis. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) resolved the species, Centella asiatica, Hydrocotyle bonariensis, and Hydrocotyle sibthorpioides, into three clusters. The saponins, asiaticoside and madecassoside, along with chlorogenic acids were the metabolites that contributed most to the separation. Furthermore, the effects of growth-lighting condition to metabolite contents were also investigated. The extracts of C. asiatica grown in full-day light exposure exhibited a stronger radical scavenging activity and contained more triterpenes (asiaticoside and madecassoside), flavonoids, and chlorogenic acids as compared to plants grown in 50% shade. This study established the potential of using a combination of (1)H NMR spectroscopy and multivariate data analyses in differentiating three closely related species and the effects of growth lighting, based on their metabolite contents and identification of the markers contributing to their differences.

[Studies on fingerprints of Centella asiatica by HPLC].

OBJECTIVE: To study the HPLC fingerprints and establish a sensitive and specific method for controlling the quality of Centella asiatica. METHODS: HPLC gradient elution was applied for the fingerprints of Centella asiatica. All 16 samples are collected from different habitats of China. The columni was Alltech C18 column (250 mm x 4.6 mm, 5 microm), mobile phase was acetonitrile-water, flow rate was 1.0 ml/min, wavelength was 205 nm. RESULTS: The fingerprint of Centella asiatica was established, 16 samples of different areas of Centella asiatica were detected. There were 15 common peaks in the HPLC fingerprints of Centella asiatica. By comparison with the reference standards and using LC-ESI-MS(n) to corroborate the structure, 5-10 peaks were identified as madecassoside, asiaticoside, quercetin, kaemperol, madecassic acid and asiatic acid respectively. After calculating the similarity of the HPLC fingerprints of 16 habitants, the similarity of different habitats has been bad quite. CONCLUSION: The method is accurate, reliable and good repeatability. This chromatographic fingerprint method can be used to controll the quality of Centella asiatica.

Optimization of an efficient semi-solid culture protocol for sterilization and plant regeneration of Centella asiatica (L.) as a medicinal herb.

The present study investigates the effects of different concentrations, as well as type of plant growth regulators (PGRs) and medium (MS, Duchefa) on the growth and development of Centella asiatica in semi-solid culture. In addition, a protocol for successful sterilization of C.asiatica explants prepared from field-grown plants highly exposed to fungal and bacterial contamination was determined. Results for sterilization treatments revealed that applying HgCl2 and Plant Preservative Mixture (PPM) with cetrimide, bavistin and trimethoprim which were included after washing with tap water, followed by the addition of PPM in the medium, produced a very satisfactory result (clean culture 90 ± 1.33%) and TS5 (decon + cetrimide 1% + bavistin 150 mg/L + trimethoprim 50 mg/L + HgCl20.1% + PPM 2% soak and 2 mL/L in medium) was hence chosen as the best method of sterilization for C.asiatica. The synergistic combination of 6 benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) in concentrations of 2 mg/L and 0.1 mg/L, respectively, in Duchefa medium compared with MS induced the most optimal percentage of sprouted shoots (93 ± 0.667), number of shoots (5.2 ± 0.079) and nodes (4 ± 0.067) per explant, leaf per explant (14 ± 0.107) and shoot length (4.1 ± 0.67 cm). Furthermore, optimum rooting frequency (95.2 ± 0.81%), the number of roots/shoot (7.5 ± 0.107) and the mean root length (4.5 ± 0.133 cm) occurred for shoots that were cultured on full-strength MS medium containing 0.5 mg/L indole-3-butyric acid (IBA). In this study, the acclimatized plantlets were successfully established with almost 85% survival. The findings of this study have proven an efficient medium and PGR concentration for the mass propagation of C.asiatica. These findings would be useful in micropropagation and ex situ conservation of this plant.

Triterpenoid saponin content and the expression level of some related genes in calli of Centella asiatica.

Centella asiatica has been extensively studied but there has been no report to date that relates gene expression and centelloside production in non-differentiated tissues. We have determined the content of the four principal triterpenoid bioactive compounds of C. asiatica (asiaticoside, madecassoside, asiatic acid and madecassic acid) in calli grown in different media and checked the expression level of some of the genes in the centelloside biosynthetic pathway. The results when compared with data from in vitro plant cultures showed a significantly lower expression of the gene encoding beta-amyrin synthase in calli, which is consistent with the observed lower production of centellosides (less than 900 microg/g DW), while in the plants the production was around 1.5-2 mg/g DW. Moreover, we find an efficient housekeeping gene for this plant. The biosynthesis of phytosterols is also discussed.

Stimulation of asiaticoside accumulation in the whole plant cultures of Centella asiatica (L.) urban by elicitors.

The effects of a number of different elicitors on asiaticoside production in whole plant cultures of Centella asiatica were studied, including yeast extract, CdCl(2), CuCl(2) and methyl jasmonate (MJ). Only MJ and yeast extract stimulated asiaticoside production--1.53 and 1.41-fold, respectively. Maximum asiaticoside production was achieved following treatment with 0.1 mM MJ (116.8 mg/l). The highest asiaticoside production (342.72 mg/l) was obtained after 36 days of elicitation in cultures treated with 0.1 mM MJ and 0.025 mg/l 1-phenyl-3-(1,2,3-thidiazol-5-yl)urea (TDZ). Interestingly, MJ not only stimulated the production of asiaticoside but also had an important role in the senescence of C. asiatica. Although asiaticoside content did not change when TDZ was added to medium containing an elicitor, TDZ did increase shoot growth of C. asiatica. We discuss the interactive roles of MJ and TDZ in secondary metabolic production and biomass in whole plants of C. asiatica.