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.     

高效液相法同时测定大鼠脑组织中积雪草苷和羟基积雪草苷浓度

目的:建立高效液相色谱(HPLC)同时测定脑组织中积雪草苷和羟基积雪草苷浓度的方法。方法:大鼠尾静脉注射积雪草苷、羟基积雪草苷和积雪草总苷,30 min后用生理盐水进行心脏灌注,去除脑组织中残留血液,取脑组织,经甲醇沉淀蛋白后,利用HPLC测定脑组织中积雪草苷和羟基积雪草苷浓度。结果:积雪草苷和羟基积雪草苷在0.5-12μg/mL线性关系良好,定量下限为0.5μg/mL,日内和日间精密度(RSD)均小于15%,准确度(RE)在-5.25%-9.16%,提取回收率在70.54%-102.61%。结论:该方法符合生物样品测定要求,可以同时测定大鼠脑组织中积雪草苷和羟基积雪草苷的浓度。     

Column-switching high-performance liquid chromatographic assay for determination of asiaticoside in rat plasma and bile with ultraviolet absorbance detection

A column-switching high-performance liquid chromatography (HPLC) method is described for the determination of asiaticoside in rat plasma and bile using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply deproteinated with acetonitrile prior to injection and bile was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of asiaticoside was accurate and repeatable, with a limit of quantification of 0.125 μg/ml in plasma and 1 μg/ml in bile. The calibration curves were linear in a concentration range of 0.125–2.5 μg/ml and 1–20 μg/ml for asiaticoside in rat plasma and bile, respectively. This method has been successfully applied to determine the level of asiaticoside in rat plasma and bile samples from pharmacokinetics and biliary excretion studies.     

A new ursane-type triterpenoid glycoside from Centella asiatica leaves modulates the production of nitric oxide and secretion of TNF-α in activated RAW 264.7 cells

One new ursane-type triterpenoid glycoside, asiaticoside G (1), five triterpenoids, asiaticoside (2), asiaticoside F (3), asiatic acid (4), quadranoside IV (5), and 2α,3β,6β-trihydroxyolean-12-en-28-oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl] ester (6), and four flavonoids, kaempferol (7), quercetin (8), astragalin (9), and isoquercetin (10) were isolated from the leaves of Centella asiatica. Their chemical structures were elucidated by mass, 1D- and 2D-nuclear magnetic resonance (NMR) spectroscopy. The structure of new compound 1 was determined to be 2α,3β,23,30-tetrahydroxyurs-12-en-28-oic acid 28-O-[α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl] ester. The anti-inflammatory activities of the isolated compounds were investigated on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Asiaticoside G (1) potently inhibited the production of nitric oxide and tumor necrosis factor-α with inhibition rates of 77.3% and 69.0%, respectively, at the concentration of 100 μM.     

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     

Evaluation of anxiolytic properties of Gotukola – (Centella asiatica) extracts and asiaticoside in rat behavioral models

The ayurvedic medicinal plant Gotukola (Centella asiatica) was evaluated for its anxiolytic properties. Specifically, this study assessed the effects of: Gotukola plant materials of different genotypic origin; hexane, ethyl acetate and methanol extracts of Gotukola; and asiaticoside, a triterpenic compound isolated from Gotukola. Various paradigms were used to assess the anxiolytic activity, including the elevated plus maze (EPM), open field, social interaction, locomotor activity, punished drinking (Vogel) and novel cage tests. The EPM test revealed that Gotukola, its methanol and ethyl acetate extracts as well as the pure asiaticoside, imparted anxiolytic activity. Furthermore, the asiaticoside did not affect locomotor activity, suggesting these compounds do not have sedative effects in rodents.     

Effect of asiaticoside on the healing of skin wounds in the carp Cirrhinus mrigala: An immunohistochemical investigation

In the present study effect of asiaticoside, on healing of skin wounds in Cirrhinus mrigala is reported. Skin wound, approx. 2 mm in diameter was excised using sterile disposable biopsy punch. Immediately after infliction of the wound, epidermis from wound edge starts migrating as thin sheet toward wound gap. Fronts of migrating epidermis gradually advance, and results in complete epithelialization of wound. Experiments were conducted for 30 days and fishes were divided into control, sham, vehicle control and asiaticoside treated groups. Immunohistochemical localization of proliferating cell nuclear antigen positive cells indicating cellular proliferation and caspase 3 positive cells reflecting apoptosis was carried out and their density at different post wound intervals in each fish group was analyzed. Significant increase in cellular proliferation as well as decrease in apoptosis in both epidermis and dermis in fish treated with asiaticoside compared to sham and vehicle control fish is observed at different intervals of wound repair. This suggests that in treated group healing of skin wounds in fish is enhanced than in sham and vehicle control groups. Asiaticoside treatment in healing of skin wounds would greatly be beneficial to fish farmers as it could protect fish from invasion of pathogens and check fish mortality.     

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.