Enhanced Production of Bryonolic Acid in Trichosanthes cucumerina L. (Thai Cultivar) Cell Cultures by Elicitors and Their Biological Activities

Abstract

Bryonolic acid is a triterpenoid compound found in cucurbitaceous roots. Due to its biological activities, this compound gets more attention to improve production. Herein, we carried out efficient ways with high bryonolic acid productions from Trichosanthes cucumerina L., a Thai medicinal plant utilizing plant cell cultures. The results showed that calli (24.65 ± 1.97 mg/g dry weight) and cell suspensions (15.69 ± 0.78 mg/g dry weight) exhibited the highest bryonolic acid productions compared with natural roots (approximately 2 mg/g dry weight). In the presence of three elicitors (methyl jasmonate, yeast extract, and chitosan), cell suspensions treated with 1 mg/mL of chitosan for eight days led to higher bryonolic acid contents (23.56 ± 1.68 mg/g dry weight). Interestingly, cell culture and root extracts with high bryonolic acid contents resulted in significantly higher percent cell viabilities than those observed under control (1% v/v DMSO) treatment in Saos-2 and MCF-7 cells. The present study indicated that T. cucumerina L. cell cultures are alternative and efficient to produce the biologically important secondary metabolite.

Keywords: Trichosanthes cucumerina L.; bryonolic acid; callus; cell suspension; elicitors.

Figure 1

The effect of TC explants from leaves (a,f), cotyledons (b,g), hypocotyls (c,h), epicotyls (d,i), and roots (e,j) on MS media supplemented with 1 mg/L IBA and 1 mg/L BA after subculturing for one week (a–e) and three weeks (f–j).

Figure 2

Profile of growth according to fresh and dry weight (g in 30 mL) of TC cell suspensions at the indicated cultured times (week). Data are presented as the mean ± standard deviation from three cell lines. The asterisk indicates significance (* p-value < 0.05 by t-test from Microsoft Excel 2016).

Figure 3

Effects of elicitors on bryonolic acid production in TC cell suspension cultures. (A) Methyl jasmonate (50, 100, and 200 µM), (B) yeast extract (0.1, 0.5, and 2% w/v), and (C) chitosan (1, 50, and 100 mg/mL). Elicitors were added to four-week cell suspensions, which were observed after treatment for 2, 4, 6, and 8 days compared with controls (70% ethanol, sterile water, or 1 N acetic acid, respectively). Data are presented as the mean from three independent cell lines, and error bars show the standard deviations. The asterisk indicates significance (* p-value < 0.05, ** p-value < 0.01 compared with control group by t-test from Microsoft Excel 2016).

Figure 4

Effect of two concentrations (50 and 100 µg/mL) of MeOH extracts (TC1: root, TC2: leaf, TC3: stem, TC4: loofah, TC5: cell suspension and TC6: callus) and bryonolic acid (BA) compared with 1% v/v DMSO in the (A) MCF-7 and (B) Saos-2 cell lines. DMEM and cisplatin were used as negative and positive controls, respectively. The bar graph and error bars represent the mean and standard deviation from the experiments, respectively. The asterisk indicates significance (* p-value < 0.05 compared with the 1% v/v DMSO group).

Figure 5

Wound healing assay in Balb/c 3T3 cells treated with two concentrations (50 and 100 µg/mL) of MeOH extracts (TC1: root, TC5: cell suspension and TC6: callus) and bryonolic acid (BA) compared with 1% v/v DMSO treatment. The bar graph and error bars represent the mean and standard deviation of the length (µm) from the experiments.