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.     

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.     

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.     

In vitro production of adventitious roots containing asiaticoside from leaf tissues of Centella asiatica L.

Centella asiatica (Apiaceae) is an important medicinal herb used in a variety of herbal medicines worldwide. Although the whole plant contains important triterpenoids, a significant quantity of pharmacologically important phytochemicals collectively known as centellosides can be extracted from leaf tissues and not from other parts of this plant. Asiaticoside is one of the major centellosides and is used in holistic medicine for treating a variety of human ailments. Genotypes of C. asiatica of Indian origin accumulate significant quantities of asiaticoside in their roots, while genotypes from other continents contain insignificant quantities of this chemical. The main purpose of this study was to manipulate the leaf-derived callus of C. asiatica using a combination of plant growth regulators to generate a large quantity of adventitious roots. The presence of asiaticoside in callus and regenerated roots of C. asiatica was detected by thin layer chromatography as well as by high-performance liquid chromatography, and the accumulation of a significant quantity of asiaticoside was demonstrated by spectrophotometric analysis. The protocol developed for the regeneration of roots was simple, reproducible, and reliable for the possible commercial production of root biomass enriched for asiaticoside.     

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.     

Copper-Induced Modulation of Biomass Growth,Physiological Parameters,Bioactive Centellosides,and Expression of Biosynthetic Pathway Genes in an Important Medicinal Herb, <Emphasis Type="Italic">Centella asiatica</Emphasis>

Hydroponically established Centella asiatica plants were studied under different concentrations of copper (0, 0.32,1.6, and 3.2 µM) for their morphological, physiological, and biochemical characteristics. The plants grown in the medium without any exogenous supply of copper showed improved biomass accumulation with maximum fresh weight (FW?=?24.7 g plant^?1) and dry weight (DW?=?2.35 g plant^?1). The total chlorophyll content, leaf number, and the leaf area were higher (0.27 mg g^?1 DW, 20 and 100.54 cm²) in the plants grown under copper-deficient conditions. The toxic effects of increased levels of copper were evident by significant inhibition in growth and other morphological parameters. Copper treatment showed an increase in malondialdehyde (MDA) content and SOD activity. Bioactive phytochemical profiling using HPLC analysis revealed that higher levels of copper (1.6 and 3.2 µM) inhibit the accumulation of total centelloside content. The differential accumulation of centelloside content was further validated by the consistent pattern of expression of key pathway genes related to centelloside production in copper-treated C. asiatica plants. The study unveiled some of the important facts associated with the mechanism of copper tolerance in this important medicinal herb C. asiatica. The findings of the present study can be further used to provide better production of high value, in demand centellosides.     

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.     

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     

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 mug/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.     

Elicitation of 2,3-Dihydroxybenzoic Acid and Ajmalicine in a Catharanthus roseus Suspension Culture*

Addition of an elicitor preparation from cell walls of Phytophthora megasperma f. sp. glycinea (Pmg elicitor) to a newly established cell suspension culture of Catharanthus roseus induced extracellular free 2,3-dihydroxybenzoic acid, suggesting its role in pathogen defense. The same substance also accumulated intracellularly in a bound form. Treatment of the crude Pmg elicitor preparation with trypsin abolished elicitor activity, suggesting that the active fraction is proteinaceous. The cells became more sensitive to low but not to elevated elicitor concentrations when they were pretreated with 2,6-dichloroisonicotinic (DCIA) or 5-chlorosalicylic (5CSA) acid for about 1 day before addition of the elicitor. This indicates that the elicitor reception/transduction system becomes improved by these compounds known to be related to systemic acquired resistance against plant pathogens. The newly established cell line initially accumulated also the indole alkaloid ajmalicine, a process enhanced by Pmg elicitor. This potency was lost during subculturing for about 1 year and was also not restored by preincubation with DCIA or 5CSA. In contrast, elicitation of 2,3-dihydroxybenzoic acid synthesis was undiminished, suggesting that the Pmg elicitor perception system was still functioning and not the cause for the decline in elicited indole alkaloid production.     

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.     

Interaction between abscisic acid and nitric oxide in PB90‐induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures

Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor‐induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up‐regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90‐induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor‐induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up‐regulation of Str and Tdc. ABA‐induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO‐induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90‐induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90‐induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:994–1001, 2013     

Effects of Gamma Stress and Carbon Dioxide on Eight Bioactive Flavonoids and Photosynthetic Efficiency in <Emphasis Type="Italic">Centella asiatica</Emphasis>

Increased atmospheric CO₂ and gamma irradiation have a significant impact on the plant photosynthetic apparatus and organic compound production. In this study, we evaluated the effect of elevated CO₂ on the photosynthetic efficiency and production of defensive secondary metabolites (flavonoids) induced by gamma irradiation as a physical elicitor in Centella asiatica. Irradiated and non-irradiated 10-week-old plants of C. asiatica were exposed to 400 and 800 μmol mol^?1 of atmospheric CO₂ in growth chambers for 2 h every day until six weeks. A CO₂-enriched atmosphere initially improved the photosynthetic efficiency and ameliorated the detrimental impact of gamma irradiation on the photosynthetic apparatus, increasing carbon allocation into the flavonoid pathway. Elevated CO₂ combined with gamma irradiation resulted in the highest concentration of flavonoids in C. asiatica tissues compared with the other treatments. There was an enhancement in rutin (2.49 fold), naringin (2.15 fold), fisetin (4.07 fold), and morin (4.62 fold) with rising CO₂ concentrations from 400 to 800 μmol mol^?1 in the irradiated plants. With increasing CO₂ concentration, the compensation point and the respiration declined, whereas the apparent quantum yield and the maximum net photosynthesis (A _max) rate increased. The efficiency of photosystem II (PSII) was improved in the irradiated plants grown under high concentrations of CO₂. The total carbohydrate concentration reached the maximum value at the highest level of CO₂, followed by gamma irradiation combined with the highest level of CO₂. Irradiated plants of C. asiatica grown under elevated CO₂ could be superior to non-irradiated plants due to increased carbon availability both for the flavonoid biosynthesis and for the photosynthetic pathway.     

Zum Einfluß des bakteriellen Phytotoxins Coronatin auf pflanzliche Zellsuspensionskulturen

Effect of the Bacterial Phytotoxin Coronatine on Plant Cell Cultures The influence of the bacterial phytotoxin coronatine on cell cultures of Lycopersicon peruvianum and Chenopodium album was studied. It was detectable that the cell culture L. peruvianum more related to the host plant of the phytopathogenic bacteria reacts more sensitive to coronatine. Concentrations of 3.5 ng coronatme/ml culture liquid mduce reductions in growth and the TTC-activity. In the case of the other used cell culture the critical amount of coronatine was higher than 7 ng/ml culture liquid. In further experiments the concentrations of free amino acids and proteins within the cell were followed. After 6–7 days of culture a slightly recovery of the cultures treated with coronatine was detectable.     

Effects of Environmental and Nutritional Factors on Production of the Polyketide Phytotoxin Coronatine by Pseudomonas syringae pv. Glycinea

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH₄Cl (1 g), MgSO₄ · 7H₂O (0.2 g), KH₂PO₄ (4.1 g), K₂HPO₄ · 3H₂O (3.6 g), and FeCl₃ (2 μM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH₄Cl, phosphate, Mg, and SO₄), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO₃ or micromolar levels of FeCl₃ significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18°C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of P. syringae tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.     

Preparation of mixed alginate elicitors with high activity for the efficient production of 5′-phosphodiesterase by Catharanthus roseus cells

When various autoclaved microbial cells suspensions (exogenous elicitors) were added to Catharanthus roseus cell cultures, its growth was inhibited but 5′-phosphodiesterase (PDase) production was stimulated. The greatest effect was with autoclaved Alteromonas macleodii: the dry cell concentration decreased from 13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen species) minimized the cell growth inhibition but enhanced PDase production (0.474 U/ml) about 20 times higher than the control (no addition). The method for the preparation of mixed alginate elicitors with high activities containing exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and trans-4,5-dihydroxy-2-cyclopenten-1-one was developed. The mixed alginate elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and the productivity was increased 120-fold compared to the control without cell growth inhibition.