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Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica.

ABSTRACT: Centella asiatica (Apiaceae) is a tropical/subtropical medicinal plant, which contains a variety of triterpenoids, including madecassoside, asiaticoside, madecassic acid, and asiatic acid. In this study, we tested the efficiency of hairy root (HR) induction in C. asiatica from leaf and petiole explants. Leaves and petioles collected from C. asiatica plants were suspended in agro-stock for 30 min and co-cultured with Agrobacterium rhizogenes for 3 days to induce HR formation. The transformation efficiency of leaf and petiole explants was approximately 27% and 12%, respectively. A total of 36 HR lines were identified by PCR-based amplification of rol genes, and eight of these lines were selected for further analysis. Among all eight HR lines, the petiole-derived lines HP4 and HP2 displayed the highest growth index (37.8) and the highest triterpenoids concentration (46.57 mg∙g^-1), respectively. Although triterpenoid concentration was >2-fold higher in leaves than in petioles of C. asiatica plants, the accumulation of triterpenoids in petiole-derived HR cultures was 1.4-fold higher than that in leaf-derived HR cultures. Additionally, in both leaf- and petiole-derived HR cultures, terpenoid production was higher in HRs than in adventitious roots. These results demonstrate that the triterpenoid content in the explant does not affect the triterpenoid content in the resultant HRs. The HR culture of C. asiatica could be scaled up to enable the mass production of triterpenoids in bioreactors for the pharmaceutical and cosmetic industries.

SUBMITTER: Baek S

PROVIDER: S-EPMC8781555 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Centella asiatica (L.) Urban extracts are widely used as food, drugs and cosmetics, and the main active compounds are glycosides (madecassoside and asiaticoside) and aglycones (madecassic acid and asiatic acid). Green extraction is an interesting concept that can produce safe and high-quality extracts that use less solvent, time and energy with the environmental friendly. This study investigated the optimum conditions for extracting a triterpenoid glycoside-enriched C. asiatica extract using microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). Central composite design and response surface methodology (RSM) were used for the experimental design and data analysis. Four-month-old C. asiatica tetraploid plants were selected as the elite raw material containing high amount of triterpenoid glycosides for the extraction experiments, and the triterpenoid content was determined by a validated HPLC method. The results demonstrated that the RSM models and equations were reliable and could predict the optimal conditions to enhance C. asiatica extract yield, glycoside and aglycone amounts. The percent of ethanol was the major factor that had a significant effect on C. asiatica yield and glycoside and aglycone content during MAE and UAE. The maximum triterpenoids content in extract; 7.332 ± 0.386% w/w madecassoside and 4.560 ± 0.153% w/w asiaticoside 0.357 ± 0.013% w/w madecassic acid and 0.209 ± 0.025% w/w asiatic acid were obtained by MAE with 80% ethanol at 100 watts for 7.5 min, whereas the optimal conditions for highest total triterpenoids extraction from dry plant was UAE with 80% ethanol, temperature 48 °C, 50 min enhanced 2.262 ± 0.046% w/w madecassoside, 1.325 ± 0.062% w/w asiaticoside, 0.082 ± 0.009% w/w madecassic acid and 0.052 ± 0.007% w/w asiatic acid as secondary outcome. Moreover, it was found that MAE and UAE consumed energy 59 and 54%, respectively, lower than that of the conventional method, maceration, in term of kilowatt-hour per gram of total triterpenoids. These optimized green conditions could be recommended for C. asiatica extraction for triterpenoid glycoside-enriched extracts production for the pharmaceutical or cosmeceutical industries and triterpenoids quantitative analysis in raw materials.

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Development of Hairy Root Cultures for Biomass and Triterpenoid Production in Centella asiatica.

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