Polyphenolic characteristics, antimicrobial and antioxidant activity of Zingiber neesanum (Graham) Ramamoorthy rhizomes and identification of volatile metabolites by GC-MS analysis.

Rhizomes of Zingiber neesanum (Graham) Ramamoorthy, endemic to Western Ghats and subject to few studies, were analysed for bioactivity and phytochemical composition. High total tannin (TT) content in isopropanol extract [55.261 ± 6.623 mg TAE.100 g-1 DW] contributed to antimicrobial activity against gram negative Enterococcus fecalis (21.7 ± 0.6 cm) while high total flavonoid (TF) content in ethyl acetate extract [681.94 ± 33.87 mg CE.100 g-1 DW] accounted for the antifungal activity against Mucor rouxii (9.7 ± 0.6). Isopropanol extracts also showed high anti-oxidant activity as determined by DPPH and FRAP assays. Major bioactive phytochemical constituents in Z. neesanum rhizome identified by GC-MS analysis included 2-Methyl-7-nonadecene (13.99%; antimicrobial), Actinomycin C2 (8.57%; antineoplastic) and Deoxyspergualin (12.55%; immunosuppressive).

Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability.

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50-80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.