UV-C mediated accumulation of pharmacologically significant phytochemicals under light regimes in in vitro culture of Fagonia indica (L.)

Abstract

Fagonia indica (L.) is an important medicinal plant with multitude of therapeutic potentials. Such application has been attributed to the presence of various pharmacological important phytochemicals. However, the inadequate biosynthesis of such metabolites in intact plants has hampered scalable production. Thus, herein, we have established an in vitro based elicitation strategy to enhance such metabolites in callus culture of F. indica. Cultures were exposed to various doses of UV radiation (UV-C) and grown in different photoperiod regimes and their impact was evaluated on biomass accumulation, biosynthesis of phytochemicals along antioxidant expression. Cultures grown under photoperiod (16L/8D h) after exposure to UV-C (5.4 kJ/m²) accumulated optimal biomass (438.3 g/L FW; 16.4 g/L DW), phenolics contents (TPC: 11.8 μgGAE/mg) and flavonoids contents (TFC: 4.05 μgQE/mg). Similarly, HPLC quantification revealed that total production (6.967 μg/mg DW) of phytochemicals wherein kaempferol (1.377 μg/mg DW), apigenin (1.057 μg/mg DW), myricetin (1.022 μg/mg DW) and isorhamnetin (1.022 μg/mg DW) were recorded highly accumulated compounds in cultures at UV-C (5.4 kJ/m²) dose than other UV-C radiations and light regimes.. The antioxidants activities examined as DPPH (92.8%), FRAP (182.3 µM TEAC) and ABTS (489.1 µM TEAC) were also recorded highly expressed by cultures under photoperiod after treatment with UV-C dose 5.4 kJ/m². Moreover, same cultures also expressed maximum % inhibition towards phospholipase A2 (sPLA2: 35.8%), lipoxygenase (15-LOX: 43.3%) and cyclooxygenases (COX-1: 55.3% and COX-2: 39.9%) with 1.0-, 1.3-, 1.3- and 2.8-fold increased levels as compared with control, respectively. Hence, findings suggest that light and UV can synergistically improve the metabolism of F. indica and could be used to produce such valuable metabolites on commercial scale.

Figure 1

(a) Accumulation of Biomass in callus cultures of Fagonia indica under photoperiod (16L/8D h) after eliciting with various treatments (0–10.8 kJ/m²) of UV-C radiations. (b) Accumulation of biomass in callus cultures of Fagonia indica under contineous light (24 h) after eliciting with various treatments of UV-C radiations. (c) Accumulation of biomass in callus cultures of Fagonia indica under complete dark (24 h) after eliciting with various treatments (0–10.8 kJ/m²) of UV-C radiations. Values represented as mean ± SE of three replicates. Different (black for FW, red for DW) letters represent significant differences between the various experimental conditions (p < 0.05).

Figure 2

Combinatorial effects of UV-C treatments (0–10.8 kJ/m²) on morphology of callu cultures of F. indica grown for 30 days under light regimes; (a) P1–P2 stands for UV-C treated cultures in photoperiod (16L/8D h), (b) C1–C7 represents UV-C elicited cultures in cont. light (24 h) and (c) D1–D2 shows UV-C mediated cultures in complete dark (24 h).

Figure 3

(a) Determination of Phenolic Contents in callus cultures of Fagonia indica elicited with various treatments (0–10.8 kJ/m²) UV-C radiations and grown in Photoperiod (16L/8D h). (b) Determination of Phenolic Contents in callus cultures of Fagonia indica elicited with various treatments (0–10.8 kJ/m²) of UV-C radiationsand grown in contineous light (24 h). (c) Determination of phenolic contents in callus cultures of Fagonia indica elicited with various treatments (0–10.8 kJ/m²) of UV-C radiationsand grown in complete darkness (24 h). Values represented as mean ± SE of three replicates. Different (black for TPC, red for TPP) letters represent significant differences between the various experimental conditions (p < 0.05).

Figure 4

(a) Determination of flavonoids contents in callus cultures of Fagonia indica elicited with different doses (0–10.8 kJ/m²) of UV-C radiations and photoperiod (16L/8D h). (b) Determination of flavonoids contents in callus cultures of Fagonia indica elicited with different doses (0–10.8 kJ/m²) of UV-C radiations and contineous light (24 h). (c) Determination of flavonoids contents in callus cultures of Fagonia indica elicited with different doses (0–10.8 kJ/m²) of UV-C radiations under complete dark (24 h). Values represented as mean ± SE of three replicates. Different (black for TFC, red for TFP) letters represent significant differences between the various experimental conditions (p < 0.05).

Figure 5

Productivity of 11 different phenolic compounds quantified via HPLC, in callus cultures of Fagonia indica after elicitation with UV-C radiations (0–10.8 kJ/m²) and maintained in different light regimes; cont. light (24 h), dark (24 h) and photoperiod (PHP, 16L/8D h). Values represented as mean ± SE of three replicates. Different (black for UV + PHP, red for UV + cont light, green for UV + dark) letters represent significant differences between the various experimental conditions (p < 0.05).

Figure 6

Free radical scavenging activity of UV-C radiations (0–10.8 kJ/m²) mediated callus cultures of Fagonia indica grown in different light regimes [cont. light (24 h), dark (24 h) and photoperiod (PHP, 16L/8D h)]. Values represented as mean ± SE of three replicates. Different (black for UV + PHP, red for UV + cont light, green for UV + dark) letters represent significant differences between the various experimental conditions (p < 0.05).

Figure 7

(a) Anti-inflammatory activities of callus cultures of Fagonia indica elicited with UV-C radiations (0–10.8 kJ/m²) and grown in photoperiod (16L/8D h) using sPLA2, 15-LOX, COX-1 and COX-2 cell free in vitro assays. The activities of extracts were expressed as % inhibition. (b) Anti-inflammatory activities of callus cultures of Fagonia indica elicited with UV-C radiations (0–10.8 kJ/m²) and grown in cont. light (24 h) using sPLA2, 15-LOX, COX-1 and COX-2 cell free in vitro assays. The activities of extracts were expressed as % inhibition. (c) Anti-inflammatory activities of callus cultures of Fagonia indica elicited with UV-C radiations (0–10.8 kJ/m²) and grown in complete dark (24 h) using sPLA2, 15-LOX, COX-1 and COX-2 cell free in vitro assays. The activities of extracts were expressed as % inhibition. Employed under the same experimental conditions as sample extracts, reference inhibitors were used as positive controls: Ibuprofen (10 µM) was used as positive control for COX-1 and COX-2 activity leading to enzyme inhibition of 31.4 ± 0.8% and 29.8 ± 1.2%, respectively; Thioetheramide-PC (5 µM) was used as sPLA2 inhibitor, resulting in an inhibition of 43.7 ± 0.8%; Nordihydroguaiaretic acid (100 µM) was used as 15-LOX inhibitor, leading to an inhibition of 30.6 ± 0.7%. The same volume of extraction solvent was used as blank. Values represented as mean ± SE of three replicates. Different letters represent significant differences between the various experimental conditions (p < 0.05).

Figure 8

Graphical represenation of synergistic effects of UV-C treatments (0–10.8 kJ/m²) and light regimes (cont. light (24 h) and complete dark (24 h) on callus cultures of Fagonia indica.