Green Synthesis of Antimicrobial Nanoparticles (Silver and Zinc Oxide) and Production of Commercially Important Secondary Metabolites from In Vitro-Derived Cultures of Linum usitatissimum L.

Abstract

2.1. ABSTRACT Linum usitatissimum L. is well-known for production of pharmacologically important secondary metabolites. Due to their tremendous beneficial effects on human health, these compounds are receiving greater attention throughout the World, especially in the treatment of various types of cancers. In present study, we have developed an efficient protocol for production of lignans like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG) and neolignans like dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) by exploiting in vitro callus cultures of flax. These cultures were established from stem and leaf explants, inoculated on Murashige and Skoog (MS) media supplemented with various concentrations of α-naphthalene acetic acid (NAA), thidiazuron (TDZ) and 6-benzyl adenine (BA). Results revealed that the leaf-derived calli (1.0 mg/l = 5.37 μM NAA) accumulated highest levels of biomass (DW; 15.7 g/l) with antioxidant activity of 91.51%, while highest production of total phenolics (111.09 mg/l) and flavonoids (45.02 mg/l) were observed in stem-derived calli (1.0 mg/l = 5.37 μM NAA). The high-performance liquid chromatography (HPLC) analysis revealed that the stem-derived calli (1.0 mg/l = 5.37 μM NAA) accumulated optimum concentrations of SDG (2.7 ± 0.021 mg/g DW), LDG (9.8 ± 0.062 mg/g DW) and DCG (13.8 ± 0.076 mg/g DW), while leaf-derived calli (1.0 mg/l = 5.37 μM NAA) showed optimum accumulation of GGCG (3.8 ± 0.022 mg/g DW) as compared to all other treatments. These results provided an evidence that the NAA differentially influence the production of lignans and neolignans in callus culture of flax. This study open new dimensions to devise strategies to enhance the production of these valuable metabolites. 3.1. ABSTRACT Flax lignans and neolignans are well-known for their incredible beneficial effects on human health, especially in the treatment of various types of cancers. The present study enhances the comprehension of biosynthesis of lignans, neolignans and other biochemical markers in in vitro callus and adventitious root cultures of flax. The results showed that the adventitious root culture was more efficient for production of lignans (at day-40) and neolignans (at day-30) than callus culture of flax. Reverse phase-high performance liquid chromatography (RP-HPLC) analysis showed that the accumulation of secoisolariciresinol diglucoside (5.5 mg/g DW) and dehydrodiconiferyl alcohol glucoside (21.6 mg/g DW) was 2-fold higher, while lariciresinol glucoside (11.9 mg/g DW) and guaiacylglycerol-βconiferyl alcohol ether glucoside (4.9 mg/g DW) was 1.5-fold higher in adventitious root culture than callus culture. Furthermore, the highest level of total phenolic production (119.01 mg/l) with antioxidant activity of 91.01% was found in adventitious root culture at day-40 while maximum level of total flavonoid production (45.51 mg/l) was observed in callus culture at day-30 of growth dynamics. These findings suggest that adventitious root culture can be scaled up to bioreactor for commercial production of these valuable metabolites. 4.1. ABSTRACT Lignans and neolignans are principal bioactive components of Linum usitatissimum L. (flax), having multiple pharmacological activities. In present study, we are reporting an authoritative abiotic elicitation strategy of photoperiod regimes along with UV-C radiations. Cell cultures were grown in different photoperiod regimes (24h-dark, 24h-light and 16L/8D h photoperiod) either alone or in combination with various doses (1.8-10.8 KJ/m2) of ultraviolet-C (UV-C) radiations. Secoisolariciresinol diglucoside (SDG), lariciresinol diglucoside (LDG), dehydrodiconiferyl alcohol glucoside (DCG), and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) were quantified by using reverse phase-high performance liquid chromatography (RP-HPLC). Results showed that the cultures exposed to UV-C radiations, accumulated higher levels of lignans, neolignans and other biochemical markers than cultures grown under different photoperiod regimes. 3.6 KJ/m2 dose of UV-C radiations resulted in 1.86-fold (7.1 mg/g DW) increase in accumulation of SDG, 2.25-fold (21.6 mg/g DW) in LDG, and 1.33-fold (9.2 mg/g DW) in GCGG in cell cultures grown under UV + photoperiod than their respective controls. While cell cultures grown under UV + dark showed 1.36-fold (60.0 mg/g DW) increase in accumulation of DCG in response to 1.8 KJ/m2 dose of UV-C radiations. Additionally, 3.6 KJ/m2 dose of UV-C radiations also resulted in 2.82-fold (195.65 mg/l) increase in total phenolic production, 2.94-fold (98.9 mg/l) in total flavonoid production and 1.04-fold (95%) in antioxidant activity of cell cultures grown under UV + photoperiod. These findings open new dimensions for reliable, consistent and enhanced production of biologically active lignans and neolignans at commercially feasible levels. 5.1. ABSTRACT Green synthesis of silver nanoparticles (AgNPs) by using plants is an emerging class of Nanobiotechnology. It revolutionizes all domains of medical sciences, by synthesizing the chemical-free AgNPs for various biomedical applications. In current report, AgNPs were successfully synthesized by using whole plant extract (WPE) and Thidiazuron-induced callus extract (CE) of L. usitatissimum. The phytochemical analysis revealed that the total phenolic and flavonoid content were higher in CE than WPE. Ultra violet-visible spectroscopy (UV-Vis) of synthesized AgNPs showed characteristic surface plasmon band in the range of 410-426 nm. Bioreduction of CE-mediated AgNPs was completed in shorter time than that of WPE-mediated AgNPs. Scanning electron microscopy (SEM) showed that both types of synthesized AgNPs were spherical in shape but CE-mediated AgNPs were smaller in size (19-24 nm) and more scattered in distribution than WPEmediated AgNPs (49-54 nm). X-ray diffraction (XRD) analysis confirmed crystalline nature (face-centered cubic) of both types of AgNPs. Fourier-transform infrared spectroscopy (FTIR) revealed that the polyphenols and flavonoids were mainly responsible for reduction and capping of synthesized AgNPs. Energy dispersive X-ray analysis (EDX) further confirmed the successful synthesis of AgNPs. Moreover, the synthesized AgNPs were found stable over months with no change in the surface plasmon bands. More importantly, CE-mediated AgNPs displayed significantly higher bactericidal activity against multiple drug resistant human pathogens than WPE-mediated AgNPs. The present work highlighted the potent role of TDZ in in vitro-derived cultures for enhanced biosynthesis of chemical-free AgNPs, which can be used as nanomedicines in many biomedical applications. 6.1. ABSTRACT The use of plants and plant-derived materials for biosynthesis of zinc oxide nanoparticles (ZnONPs) is developing into a lucrative field of green nanotechnology and gaining more importance owing to its simplicity, rapidity, and eco-friendliness. In present study, a novel and efficient green approach has been developed for biosynthesis of ZnONPs by exploiting the in vitro platform of plants. Two different in vitro cultures extracts i.e.; callus extract (CE) and adventitious root extract (RE) of flax were used as a source of reducing and stabilizing agents. Phytochemical analysis revealed that the RE was rich in phytochemical reducing agents as compared to CE. UV-visible spectroscopy showed that the bioreduction of RE-mediated ZnONPs completed in shorter time than CE-mediated ZnONPs. Scanning electron microscopy showed that CE-mediated ZnONPs were spherical with weak agglomeration but the RE-mediated ZnONPs were hexagonal in shape with uniform distribution of particles. X-ray diffraction analysis showed that the both type of ZnONPs exhibited the same crystalline nature (wurtzite hexagonal) but vary in their sizes. REmediated ZnONPs were smaller in size (34.97 nm) than CE-mediated ZnONPs (61.44 nm). Fourier-transform infrared spectroscopy revealed that the polyphenols (lignans), carboxylic acids and aromatic compounds were mainly involved in reduction and capping of both type of ZnONPs. Moreover, the RE-mediated ZnONPs showed more potent antibacterial and antileishmanial activity against multidrug resistant bacterial strains and parasite of Leishmania major than CE-mediated ZnONPs. The present work highlighted the potent role of in vitro cultures of flax in enhanced biosynthesis, antibacterial and antileishmanial activities of ZnONPs.