Chitosan elicitation of Isatis tinctoria L. hairy root cultures for enhancing flavonoid productivity and gene expression and related antioxidant activity

Abstract

Elicitation for phytochemical enhancement via cost-effective elicitors can overcome the limitation of commercial application faced by plant cell and organ culture technology. Chitosan is a natural, low-cost, and nontoxic elicitor that can trigger plant defense responses with the concomitant enhancement in phytochemical biosynthesis. In this work, the elicitation of Isatis tinctoria L. hairy root cultures by chitosan was conducted to enhance the production of pharmacologically active flavonoids. In comparison with control (2.31 ± 0.29 mg/g DW), a 7.08-fold enhancement of total flavonoids (16.35 ± 0.88 mg/g DW) was achieved in 24 day-old I. tinctoria hairy root cultures elicited by 150 mg/L chitosan for 36 h. Interestingly, the multiple hydroxyl-substituted flavonoids (rutin, quercetin, isorhamnetin, and isoliquiritigenin) were noticed to increase significantly in chitosan-elicited I. tinctoria hairy root cultures. Moreover, the transcription of associated genes involved in flavonoid biosynthesis pathway was significantly up-regulated underlying chitosan elicitation, among which chalcone synthase and flavonoid 3'-hydroxylase might play an important role in flavonoid enhancement. Additionally, extracts from chitosan-elicited I. tinctoria hairy root cultures exhibited higher antioxidant activities with lower IC₅₀ values as compared with control. Overall, a cost-effective strategy via the simple chitosan elicitation is provided here to enhance the production of high-added value flavonoids in I. tinctoria hairy root cultures, which paves the way toward the successful commercialization of this in vitro culture system in the future.

Keywords: Antioxidant evaluation; Cost-effective elicitor; Molecular mechanism; Pharmacologically active flavonoids; Plant in vitro cultures.

Graphical abstract

Fig. 1

Effect of different concentrations of chitosan (50, 100, 150, 200, and 400 mg/L) with successive exposure time (0, 6, 12, 18, 24, 30, 36, 48, 60, 72, and 96 h) on total flavonoid yield in 24 day-old ITHRCs. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05).

Fig. 2

Representative LC–MS/MS total ion chromatograms of eight flavonoids in extracts form control and ITHRCs elicited by 150 mg/L chitosan for 36 h. RUT, rutin; NEO, neohesperidin; BUD, buddleoside; LIQ, liquiritigenin; QUE, quercetin; ISR, isorhamnetin; KAE, kaempferol; ISL, isoliquiritigenin.

Fig. 3

Yields of eight flavonoids in extracts form control and ITHRCs elicited by 150 mg/L chitosan for 36 h. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05). RUT, rutin; NEO, neohesperidin; BUD, buddleoside; LIQ, liquiritigenin; QUE, quercetin; ISR, isorhamnetin; KAE, kaempferol; ISL, isoliquiritigenin.

Fig. 4

Phenotype comparison of hairy root tissues from control and ITHRCs elicited by 150 mg/L chitosan for 36 h.

Fig. 5

Transcriptional profiles of flavonoid biosynthesis genes in ITHRCs elicited by 150 mg/L chitosan for different durations (6, 12, 24, 36, 48, 60, 72, and 96 h). PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3′H, flavonoid 3′-hydroxylase; FS, flavonol synthase. Mean ± SD values not sharing the same lowercase letters are significantly different (P < 0.05).

Fig. 6

Antioxidant activities of extracts from control and ITHRCs elicited by 150 mg/L chitosan for 36 h in DPPH radical scavenging assay (A) and β-carotene/linoleic acid bleaching test (B).