Improving Flavonoid Accumulation of Bioreactor-Cultured Adventitious Roots in Oplopanax elatus Using Yeast Extract

Abstract

Oplopanax elatus is an endangered medicinal plant, and adventitious root (AR) culture is an effective way to obtain its raw materials. Yeast extract (YE) is a lower-price elicitor and can efficiently promote metabolite synthesis. In this study, the bioreactor-cultured O. elatus ARs were treated with YE in a suspension culture system to investigate the elicitation effect of YE on flavonoid accumulation, serving for further industrial production. Among YE concentrations (25-250 mg/L), 100 mg/L YE was the most suitable for increasing the flavonoid accumulation. The ARs with various ages (35-, 40-, and 45-day-old) responded differently to YE stimulation, where the highest flavonoid accumulation was found when 35-day-old ARs were treated with 100 mg/L YE. After YE treatment, the flavonoid content increased, peaked at 4 days, and then decreased. By comparison, the flavonoid content and antioxidant activities in the YE group were obviously higher than those in the control. Subsequently, the flavonoids of ARs were extracted by flash extraction, where the optimized extraction process was: 63% ethanol, 69 s of extraction time, and a 57 mL/g liquid-material ratio. The findings provide a reference for the further industrial production of flavonoid-enriched O. elatus ARs, and the cultured ARs have potential application for the future production of products.

Keywords: Oplopanax elatus; elicitation duration; elicitation time; elicitor concentration; flash extraction; yeast extract.

Figure 1

Effect of yeast extract (YE) concentration on biomass (a) and flavonoid content (b) and productivity (c) of Oplopanax elatus adventitious roots (ARs). The 45-day-old fed-batch bioreactor-cultured ARs were transferred to flasks and treated with YE for 8 days. Data represent the mean value ± standard deviation (n = 3). The different letters within the same column indicate significant difference by Duncan’s multiple range test at p < 0.05.

Figure 2

Effect of yeast extract (YE) on biomass (a) and flavonoid content (b) and productivity (c) of Oplopanax elatus adventitious roots (AR) at different ages. The 35-, 40, and 45-day-old fed-batch cultured O. elatus ARs were transferred to flasks and treated with 100 mg/L YE (the control group was added with equal amount of the medium) for 8 days in the YE group. Data represent the mean value ± standard deviation (n = 3). The different letters within the same color column indicate significant difference by Duncan’s multiple range test at p < 0.05. ** indicates significant difference between the groups of YE and control by Student’s t-test at p < 0.01.

Figure 3

Effect of yeast extract (YE) treatment duration on biomass (a) and flavonoid content (b) and productivity (c) of Oplopanax elatus adventitious roots (ARs). The 35-day-old fed-batch bioreactor-cultured ARs were transferred to flasks and treated with 100 mg/L YE (the control group was added with equal amount of the medium) in the YE group. Data represent the mean value ± standard deviation (n = 3). The different letters within the same line indicate significant difference by Duncan’s multiple range test at p < 0.05. ** indicates significant difference between the groups of YE and control at each time point by Student’s t-test at p < 0.01.

Figure 4

Comparison of antioxidant ability of extracts from YE-untreated (C-ARE) and -treated (YE-ARE) adventitious roots (ARs) of Oplopanax elatus from the fed-batch bioreactor culture. (a) DPPH scavenging rate. (b) ABTS⁺ scavenging rate. (c) Fe³⁺ chelating rate. The ARs in YE group were treated with 100 mg/L YE for 4 days on day 35 of fed-batch bioreactor culture. The ARs in the control group were cultured for 49 days in fed-batch bioreactor culture system. DPPH = 2,2-diphenyl-1-picrylhydrazyl; ABTS = 2′2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). EC₅₀ = concentration for 50% of maximal effect. Data represent the mean value ± standard deviation (n = 3).

Figure 5

Effect of extraction solvent (a), solvent (ethanol) concentration (b), extraction time (c), and liquid−material ratio (d) on flavonoid yield of Oplopanax elatus adventitious roots from fed-batch bioreactor culture. Data represent the mean value ± standard deviation (n = 3). The different letters within the same column indicate significant difference by Duncan’s multiple range test at p < 0.05.

Figure 6

Interaction effect of two factors on flavonoid yield of extract from Oplopanax elatus adventitious roots (ARs). (a) interaction between ethanol concentration and extraction time. (b) interaction between ethanol concentration and liquid−material ratio. (c) interaction between extraction time and liquid−material ratio. The ARs were harvested from bioreactors after 4 days of yeast extract (100 mg/L) treatment on day 35 of fed-batch bioreactor culture.

Figure 7

Experiment process.

Figure 8

Chemical structure (a) and high-performance liquid chromatography profiles of rutin, quercetin, and kaempferide standards (b) and adventitious root sample of Oplopanax elatus (c). (1) Rutin. (2) Quercetin. (3) Kaempferide.