Analysis of flavonoid content, antioxidant, antimicrobial and antibiofilm activity of in vitro hairy root extract of radish (Raphanus sativus L.)

Abstract

The production of biofilm inhibiting antimicrobial agents of plant origin is gaining significant importance in recent years. The inhibition of biofilm by natural agents is considered safe and provides greater scope for alternative antimicrobial strategies. The hairy root culture extracts established by infecting leaf explants with Agrobacterium rhizogenes R1000 and A4 strains in Raphanus raphanistrum L. subsp. sativus (L.) Domin (syn. R. sativus L.) have been studied in this regard. The total flavonoids and phenolics contents were estimated and their influence on the antioxidant and antibiofilm activities on the hairy root culture extracts were investigated. The R1000 strain produced 90.0% of hairy roots while the A4 strain produced 81.0% hairy roots in Murashige and Skoog (hormone-free ½ MS) medium containing 75 µM of acetosyringone within three days. The hormone-free ½ MS medium with 116.8 mM sucrose concentration produced highest biomass accumulation (14.11 g/L Fresh Weight (FW); 1.78 g/L Dry Weight (DW); Growth Index (GI) 7.06) after 30 days of culture compared to MS, B5 (Gamborg B5) and ½ B5 media. The integration of T-DNA of the Ri plasmid was confirmed by Polymerase Chain Reaction (PCR) analysis based on the amplification of rolB and rolC genes. The R1000 induced hairy roots extract (RHRE) produced high amount of phenolics (52.0 mg/g DW) and flavonoids (67.0 mg/g of DW). The quercetin content of RHRE (488.4 mg/g of DW) was determined by RP-HPLC and LC–MS/MS. The RHRE exhibited 92% inhibition against stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and in ferric reducing antioxidant power (FRAP) assay the absorbance was 3.62 at 593 nm. The RHRE also exhibited significant antimicrobial activity against Gram-negative and Gram-positive bacteria, particularly against MDR bacterium Pseudomonas aeruginosa, by inhibiting biofilm formation and EPS production in the bacterium. Our findings indicate the industrial applications of RHRE as potent antibiofilm agent probably due to the enriched quercetin content. The RHRE could also be used to prevent food contamination and spoilage in industrial environment. Factors affecting hairy root production have been optimized in Raphnus sativus (L.). Agrobacterium rhizogenes R1000 displayed better results compared to A4. Optimum flavonoid production obtained in ½-strength MS medium and 4% sucrose. R1000 hairy roots displayed higher antimicrobial, antibiofilm and antioxidant activity. The present study is a novel approach to improve quercetin content and can be used in food industry.