Elicitation with methyl jasmonate combined with cultivation in the Plantform™ temporary immersion bioreactor highly increases the accumulation of selected centellosides and phenolics in Centella asiatica (L.) Urban shoot culture

Centella asiatica (L.) Urban is an important pharmacopoeial plant used not only in medicine but also in cosmetology. C. asiatica agitated shoot cultures were established to study the influence of ethephon, methyl jasmonate, L ‐phenylalanine (Eth 50 µM, MeJa 50 µM, L‐Phe 2.4 g/L of medium, respectively; seven variants of the supplementation) on the accumulation of secondary metabolites: the main centellosides (asiaticoside and madecassoside) and selected phenolic acids, and flavonoids in the biomass. Microshoots were harvested two and six days after the supplementation. Secondary metabolites were analyzed in methanolic extracts by UPLC‐MS/MS (centellosides) and by HPLC‐DAD (phenolics). In comparison with the reference cultures, the concentrations of individual secondary metabolites increased as follows: centellosides up to 5.6‐fold (asiaticoside), phenolic acids up to 122‐fold (p‐coumaric acid) and flavonoids up to 22.4‐fold (kaempherol). The highest production increase of individual compounds was observed for different variants of supplementation. Variant C (50 µM MeJa), the most optimal for centellosides and flavonoid accumulation, was selected for the experiment with bioreactors. Bioreactor Plantform?, compared to RITA^® system and agitated cultures, appeared to be the most advantageous for secondary metabolites production in C. asiatica shoot cultures. The phenolic acid, flavonoid, centelloside, and total secondary metabolite productivity in Plantform? system is 1.8‐fold, 1.7‐fold, 2.8‐fold, 2.1‐fold, respectively, higher than in MeJa elicitated agitated cultures, and 4.3‐fold, 7.3‐fold, 12.2‐fold, 7.2‐fold, respectively, higher than in control agitated cultures.     

Establishment of callus and cell suspension cultures of <Emphasis Type="Italic">Centella asiatica</Emphasis>

Methods for induction of callus and cell suspension cultures have been developed for the medicinally important herb Centella asiatica (L.) Urban. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis showed the presence of asiaticoside in the in vitro grown leaves, callus and cell suspension cultured cells.     

Conversion of α-amyrin into centellosides by plant cell cultures of Centella asiatica

Plant cell cultures of Centella asiatica produce small quantities of centellosides: madecassosid > asiaticosid > madecassic acid > asiatic acid. To obtain a more efficient production system of these bioactive triterpenoid compounds, we developed a process where the substrate, α-amyrin, was converted into centellosides by cell suspensions of C. asiatica. When α-amyrin in acetone was added at 0.01 mg/ml⁻¹ to the culture medium, together with the permeabilizing agent DMSO, after 7 days nearly 50% had penetrated the plant cells, of which almost 84% was transformed into centellosides. The system therefore efficiently converts α-amyrin into centellosides, thus opening a new possibility for the production of these compounds.     

Production of asiaticoside and madecassoside in <Emphasis Type="Italic">Centella asiatica in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

The localization was determined of the triterpenoids, asiaticoside and madecassoside, in different organs of glasshouse-grown plants and cultured material, including transformed roots, of two phenotypes of Centella asiatica (L.) Urban of Malaysian origin. Methanolic extracts of asiaticoside and madecassoside were prepared for gradient HPLC analysis. The two phenotypes of C. asiatica exhibited differences in terpenoid content that were tissue specific and varied between glasshouse-grown plants and tissue culture-derived material. Terpenoid content was highest in leaves, with asiaticoside (0.79 ± 0.03 and 1.15 ± 0.10 % of dry mass) and madecassoside [0.97 ± 0.06 and 1.65 ± 0.01 %(d.m.)] in the fringed (F) and smooth leaf (S) phenotypes, respectively. Roots of the F-phenotype contained the lowest content of asiaticoside [0.12 ± 0.01 %(d.m.)], whereas petioles of S-phenotype plants contained the lowest content of asiaticoside [0.16 ± 0.01 %(d.m.)] and madecassoside [0.18 ± 0.14 %(d.m.)]. Transformed roots were induced using Agrobacterium rhizogens and their growth was maximal on Murashige and Skoog basal medium supplemented with 60 g dm⁻³ sucrose. However, asiaticoside and madecassoside were undetectable in transformed roots and undifferentiated callus.     

Stimulation of asiaticoside accumulation in the whole plant cultures of <Emphasis Type="Italic">Centella asiatica</Emphasis> (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, CdCl₂, CuCl₂ 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     

An optimized biotechnological system for the production of centellosides based on elicitation and bioconversion of Centella asiatica cell cultures

Centella asiatica is a herbaceous plant of Asian traditional medicine. Besides wound healing, this plant is recommended for the treatment or care of various skin conditions such as dry skin, leprosy, varicose ulcers, eczema, and/or psoriasis. Triterpene saponins, known as centellosides, are the main metabolites associated with these beneficial effects. Considering the interest in these high value active compounds, there is a need to develop biosustainable and economically viable processes to produce them. Previous work using C. asiatica plant cell culture technology demonstrated the efficient conversion of amyrin derivatives into centellosides, opening a new way to access these biomolecules. The current study was aimed at increasing the production of centellosides in C. asiatica plant cell cultures. Herein, we report the application of a new elicitor, coronatine, combined with the addition of amyrin‐enriched resins as potential sustainable precursors in the centelloside pathway, for a positive synergistic effect on centelloside production. Our results show that coronatine is a powerful elicitor for increasing centelloside production and that treatments with sustainable natural sources of amyrins enhance centelloside yields. This process can be scaled up to an orbitally shaken CellBag, thereby increasing the capacity of the system for producing biomass and centellosides.     

Enhancement of centelloside production from cultured plants of Centella asiatica by combination of thidiazuron and methyl jasmonate

In order to produce centellosides from whole plant cultures of Centella asiatica (L.) Urban, we evaluated the synergistic effects of thidiazuron (TDZ) and methyl jasmonate (MJ) on whole plant growth and centelloside production. After 4 weeks of treatment with 0.025 mg/L of TDZ coupled with 0.1 mM MJ, the production of madecassoside and asiaticoside from whole plant cultures was estimated to be 2.40- and 2.44-fold, respectively, above that of MJ elicitation alone. When whole plants were treated with a growth regulator and an elicitor, the growth of whole plants, as compared to the controls, did not differ. Additionally, total phytosyterol content in the leaves of whole plants co-treated with MJ and TDZ was 1.08-fold greater than those of MJ alone. These results demonstrate that combined treatments not only stimulate the accumulation of centellosides in the leaves but also inhibit the reduction of phytosterol levels caused by MJ elicitation.     

Isolation and characterization of squalene synthase cDNA fromcentella asiatica (L) urban

We have cloned and characterized a gene for squalene synthase (SQS) fromCentella asiatica (L) Urban, a species that produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Its full-length cDNA clone was isolated by RACE PCR. The sequence ofpSQS contains an open reading frame of 1248 nucleotides, which code for 416 amino acids with a molecular mass of 47.3 kDa. Southern analysis revealed that one copy might exist in the C.asiatica genome. We also determined that 0.1 mM methyl jasmonate was sufficient to up-regulate those levels ofCaSQS mRNA.     

Centella asiatica (L.) urban from Nepal: Quali-quantitative analysis of samples from several sites,and selection of high terpene containing populations for cultivation

Centella asiatica (L.) Urban is widely used in traditional medicine in many countries and in the formulation of drugs and cosmetics, and is therefore suitable as a trade item for the development of medicinal plants for the population of Nepal. The aim of this work was to select plant populations of C. asiatica with high contents of secondary metabolites growing in various localities in Nepal, and to enhance knowledge of the cultivation of this plant. Quali-quantitative analysis of bioactive triterpenes (asiaticoside and asiatic acid) and phenol derivatives (flavonoids and caffeoyl esters) was performed by HPLC-DAD-ELSD. The highest quantities of triterpenes and phenols were found in samples from the Gorkha and Chitwan districts. Regarding cultivated plants, soil fertilisation is critical, since over-rich soils affect secondary metabolite content. Plants growing in sand-rich soils produce more terpenes. This work provides indications on how to select high-terpene producing germplasm and recommendations for plant cultivation.     

Estimation of flavonoid and centelloside accumulation in leaves of Centella asiatica L. Urban by multiparametric fluorescence measurements

Recently, we have shown the relevance of nitrogen (N), phosphorus (P) and potassium (K) supply levels for resource partitioning between primary and secondary metabolism, and the concentration of centellosides in Centella asiatica L. Urban leaves. So far, no efforts have been made to investigate the effects of mineral supply on flavonoid concentrations in this species. Here, we aimed to examine the accumulation of centellosides in C. asiatica leaves in vivo by means of non-destructive fluorescence measurements using products of the secondary metabolism, particularly the epidermal flavonols and anthocyanins, as reference. For this purpose we conducted three discrete experiments in a greenhouse having N, P and K levels as experimental factors. Our results reveal that flavonoid and anthocyanin accumulation is affected by N, P and K fertigation in the same way as the centelloside accumulation. More precisely, limitations in plant growth were accompanied by higher flavonoid and anthocyanin concentrations, confirming the proposed trade-off between the plant's primary and secondary metabolism. The fluorescence-based flavonol (FLAV) and anthocyanin (ANTH_RG) indices correlated fairly with flavonoid and especially with anthocyanin concentrations. Moreover, centellosides were positively correlated with the FLAV and ANTH_RG indices, and with the BFRR_UV index, which is considered as universal ‘stress-indicator’. Thus, here we indicate for the first time, that the fluorescence-based indices FLAV, ANTH_RG as well as BFRR_UV enable the monitoring of flavonoid and centelloside concentrations in leaves of C. asiatica. Our results support and highlight the significant potential for further development and application of fluorescence-based sensors in ecophysiological research as well as in the production of medicinal plants.     

Asiaticoside production from centella (Centella asiatica L. Urban) cell culture

Petiole explants of centella plants (Centella asiatica L. Urban) were cultured on Murashige and Skoog (MS) solid medium containing 20 g/L sucrose, supplemented with 1.0 mg/L benzylaminopurine and 1.0 mg/L naphthaleneacetic acid for callus production. To establish a cell suspension culture, 2 g of fresh callus was cultured in 50 mL of the same medium but without solid agent at a 100 rpm agitation speed. Every 2 g of culture was subcultured in fresh MS liquid medium for maintenance. After 24 days of culture at a 120 rpm agitation speed, the centella cell biomass reached a maximum of 9.03 g/50 mL on the same MS medium with 30 g/L sucrose and a 3 g inoculum size. A high performance liquid chromatography analysis showed that asiaticoside content in 24-day old suspension cultured cells (45.35 mg/g dry weight) was significantly higher (4.5 fold) than that of in planta leaves (10.55 mg/g dry weight).     

Therapeutic properties and pharmacological activities of asiaticoside and madecassoside: A review

Centella asiatica is an ethnomedicinal herbaceous species that grows abundantly in tropical and sub-tropical regions of China, India, South-Eastern Asia and Africa. It is a popular nutraceutical that is employed in various forms of clinical and cosmetic treatments. C. asiatica extracts are reported widely in Ayurvedic and Chinese traditional medicine to boost memory, prevent cognitive deficits and improve brain functions. The major bioactive constituents of C. asiatica are the pentacyclic triterpenoid glycosides, asiaticoside and madecassoside, and their corresponding aglycones, asiatic acid and madecassic acid. Asiaticoside and madecassoside have been identified as the marker compounds of C. asiatica in the Chinese Pharmacopoeia and these triterpene compounds offer a wide range of pharmacological properties, including neuroprotective, cardioprotective, hepatoprotective, wound healing, anti-inflammatory, anti-oxidant, anti-allergic, anti-depressant, anxiolytic, antifibrotic, antibacterial, anti-arthritic, anti-tumour and immunomodulatory activities. Asiaticoside and madecassoside are also used extensively in treating skin abnormalities, burn injuries, ischaemia, ulcers, asthma, lupus, psoriasis and scleroderma. Besides medicinal applications, these phytocompounds are considered cosmetically beneficial for their role in anti-ageing, skin hydration, collagen synthesis, UV protection and curing scars. Existing reports and experimental studies on these compounds between 2005 and 2022 have been selectively reviewed in this article to provide a comprehensive overview of the numerous therapeutic advantages of asiaticoside and madecassoside and their potential roles in the medical future.     

Investigation of HIV-1 Viral Protein R Inhibitory Activities of Twelve Thai Medicinal Plants and Their Commercially Available Major Constituents

Viral protein R (Vpr) is an accessory protein in Human immunodeficiency virus-1 (HIV-1) and has been suggested as an attractive target for HIV disease treatment. Investigations of the ethanolic extracts of twelve Thai herbs revealed that the extracts of the Punica granatum fruits, the Centella asiatica aerials, the Citrus hystrix fruit peels, the Caesalpinia sappan heartwoods, the Piper betel leaves, the Alpinia galangal rhizomes, the Senna tora seeds, the Zingiber cassumunar rhizomes, the Rhinacanthus nasutus leaves, and the Plumbago indica roots exhibited the anti-Vpr activity in HeLa cells harboring the TREx plasmid encoding full-length Vpr (TREx-HeLa-Vpr cells). Moreover, the investigation of the selected main constituents in Punica granatum, Centella asiatica, A. galangal, and Caesalpinia sappan indicated that punicalagin, asiaticoside, ellagic acid, madecassic acid, madecassoside, zingerone, brazilin, and asiatic acid possessed anti-Vpr activities at the 10 μM concentration. Among the tested extracts and compounds, the extracts from Centella asiatica and Citrus hystrix and the compounds, punicalagin and asiaticoside, showed the most potent anti-Vpr activities without any cytotoxicity, respectively.     

Regeneration of Centella asiatica plants from non-embryogenic cell lines and evaluation of antibacterial and antifungal properties of regenerated calli and plants

Background

The threatened plant Centella asiatica L. is traditionallyused for a number of remedies. In vitro plant propagation and enhanced metabolite production of active metabolites through biotechnological approaches has gained attention in recent years.

Results

Present study reveals that 6-benzyladenine (BA) either alone or in combination with 1-naphthalene acetic acid (NAA) supplemented in Murashige and Skoog (MS) medium at different concentrations produced good quality callus from leaf explants of C. asiatica. The calli produced on different plant growth regulators at different concentrations were mostly embryogenic and green. Highest shoot regeneration efficiency; 10 shoots per callus explant, from non-embryogenic callus was observed on 4.42 μM BA with 5.37 μM NAA. Best rooting response was observed at 5.37 and 10.74 μM NAA with 20 average number of roots per explant. Calli and regenerated plants extracts inhibited bacterial growth with mean zone of inhibition 9-13 mm diameter when tested against six bacterial strains using agar well diffusion method. Agar tube dilution method for antifungal assay showed 3.2-76% growth inhibition of Mucor species, Aspergillus fumigatus and Fusarium moliniformes.

Conclusions

The present investigation reveals that non-embryogenic callus can be turned into embryos and plantlets if cultured on appropriate medium. Furthermore, callus from leaf explant of C. asiatica can be a good source for production of antimicrobial compounds through bioreactor.     

Determination and quantification of asiaticoside in endophytic fungus from <Emphasis Type="Italic">Centella asiatica</Emphasis> (L.) Urban

Centella asiatica (L.) Urban is a highly considered medicinal plant owing to its secondary metabolites asiaticoside, madecassoside, asiatic acid, and madecassic acid. The asiaticoside, one of the most important constituents of the plant, is a triterpenoid saponin having memory enhancement property. Given its medicinal properties, we isolated and characterized endophytic fungi from this plant with the aim to screen these microorganisms for asiaticoside production. In total, we isolated 13 endophytic fungi from the leaves of the plant, out of which one of the isolates produced asiaticoside. This asiaticoside producing isolate was identified as Colletotrichum gloeosporioides by internal transcribed spacer-based rDNA sequencing. The presence of asiaticoside in ethyl acetate extract of C. gloeosporioides was confirmed by LC–MS. The production of asiaticoside measured in relation to incubation time and subculture generation revealed presence of 62.29?±?3.36 µg/100 mL of asiaticoside by C. gloeosporioides on the 15th day in first subculture generation followed by a decrease in subsequent generations. A similar trend was also shown by yield and growth curve of C. gloeosporioides. The asiaticoside production and yield were found to be positively correlated. This paper reported the production of asiaticoside by an endophytic fungus C. gloeosporioides for the first time. The present findings definitely provide an impetus to the production of asiaticoside by utilizing the endophytic source.

Graphical Abstract

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Chemical compound studied in this article: Asiaticoside (PubChemCID: 108062)

     

Effect of growth regulators on asiaticoside production in whole plant cultures of<Emphasis Type="Italic">Centella asiatica</Emphasis> (L) urban

We investigated the effects of growth regulators on whole-plant cultures derived from nodes ofCentella asiatica. A B5 liquid medium including 0.01 mg L^-1 2,4-D resulted in decreased growth and asiaticoside production. Among the cytokinins tested (TDZ, BA, zeatin, and kinetin), TDZ was the best supplement for the promotion of asiaticoside biosynthesis. To directly estimate this effect, we measured asiaticoside content in the leaf, the main organ for synthesis. The addition of TDZ did not affect asiaticoside accumulation. Nevertheless, our results suggest that treatment with exogenous TDZ may enhance the production of asiaticoside in cultures simply through an increase in biomass.