Production of Flavonoids in Pegaga (Centella Asiatica L. Urban) Cell Suspension Culture

Centella asiatica L. Urban (Umbelliferea), locally known as ‘Pegaga’ was an herbal plant that had been used in traditional medicine in Asia for many centuries. Its medicinal values are attributed to the presence of flavonoid compounds. Since there was still no information available on the flavonoid production in cultured tissues, studied have been carried out in evaluating the distribution of the flavonoid content particularly kaempherol, quercertin, luteolin and apigenin in intact plants of the four accessions of C. asiatica collected several locations in Malaysia as well as in callus and cell suspension cultures. The total flavonoid content has been determined by using spectrophotometry methods. The flavonoid compounds present in the cell cultures have been analyzed with thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) techniques. Results obtained from the studies revealed that the four accessions of C. asiatica which showed distinctive differentiation in morphological characteristic were differed in the biochemical constituents especially in their flavonoid contents. The flavonoid constituents were detected in the range of 1.93 ± 0.094 to 8.99 ± 0.346 mg/g dry weight in the whole plant tissues. Flavonoids were also successfully detected in callus (0.98 ± 0.097 to 2.46 ± 0.021 mg/g dry weight) and cell suspension culture (0.67 ± 0.056 to 0.89 ± 0.044 mg/g dry weight). The flavonoid content found in cultured tissues were lower that that produced in the intact plant tissues. In the TLC analysis for the leaf tissues of intact plant, the possible of flavonoid compound might presented were kaempherol, naringerin, luteolin, (+)-catechin and rutin. Similar results were also been observed in callus, cell suspension and cell suspension media in the TLC analysis. Further investigation using HPLC showed that leaf tissues of intact plant accession UPM03 has rutin, luteolin, quercertin and kaempherol while others accession only have rutin or/and kaempherol. An additional flavonoid compound called apigenin was detected in callus and cell suspension. As a result, culture conditions such as different cell size aggregations, pH values, light, inoculum sizes as well as interaction between plant growth regulators have been assessed to optimize and enhance the selected flavonoid production in the chosen cell suspension cultures. Cell suspension UPM03 in aggregation size of 250-500 μm with the highest cell growth rate (0.69 g fresh weight/day) was suitable to produce flavonoid in 100 mL shake flask of C. asiatica suspension cells. The cells grow better and produce more flavonoid (3.66 ± 0.13 mg/g dry weight) under the initial pH of 5.7 with the presence of light when supplemented with 3 mg/L 2,4-D and 1 mg/L kinetin. In addition, the responses of cell to selected elicitors and precursors have also been investigated. The biosynthesis of flavonoid was elicited by the addition of 1mg/L of both yeast extract (7.32 ± 0.17 mg/g dry weight) and salicylic acid (7.11 ± 0.16 mg/g dry weight). Elicition with 400 μM methyl jasmonate have increased flavonoid content to 7.82 ± 0.2 mg/g dry weight. However, the addition of 1mg/L casein hydrolysate (4.91 ± 0.15 mg/g dry weight) and chitosan (4.29 ± 0.13 mg/g dry weight) have no significant effect if compared to the control culture (5.08 ± 0.22 mg/g dry weight). The usage of 60 mg/L phenylalanine and 40 mg/L tyrosine have resulted in 14-fold and 3-fold increase in production of flavonoid, respectively. Further study on the synergistic effect of combination of precursor (60 mg/L phenylalnine) and elicitor (400 μM) showed that the cell suspension UPM03 not only produce the highest flavonoid content in cell (67.07 ± 6.47 mg/g DW) but also in the media (7.73 ± 0.12 mg/g). Thus, it was proven that the synergistic bioprocess was a very useful strategy to enhance the flavonoid production particularly luteolin in in vitro cultures of C. asiatica.

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