Characterization of multicomponent antioxidants from Haloferax alexandrinus GUSF-1 (KF796625)

Abstract

The present study was aimed to exploit the haloarchaeon Haloferax alexandrinus GUSF-1 (KF796625) for the presence of biomolecules possessing antioxidant activity. The culture produced a bright orange pigment when grown aerobically in nutrient rich medium with 25% crude solar salt. Biomolecules from cell-free supernatant and from the cells of the culture were individually extracted through the assistance of solvents of different polarities, such as ethanol, methanol and hexane, and monitored for scavenging of stable free radicals. Each of the extracts showed varying capacities to scavenge DPPH^•(20, 31, and 80% DPPH^• RSA; 160.19, 248.29 and 640.76 AAE µg g^-1 of cells) at 1 mg mL^-1. The extracellular ethanolic extract was polysaccharide in nature, equivalent to 47 µg mL^-1 of glucose when assayed with the phenol-sulfuric acid method. The Fourier Transform-Infra Red spectroscopy confirmed the characteristic glycosidic peaks between 2000 and 1000 cm^-1. Similarly, the glycerol diether moiety separated from hydroxylated methanolysates through thin-layer chromatography scavenged free radicals (10.47% DPPH^• RSA; 80.03 AAE µg g^-1 of cells). Further, the hexanolic extract exhibited spectral characteristics of red carotenoids and resolved into distinct compounds when separated by thin-layer chromatography using different developing systems. All separated compounds were positive for the DPPH^• reaction (13-30% DPPH^• RSA; 100-240 AAE µg g^-1). Chemical profiling of the hexanolic extract using the high resolution-liquid chromatography-mass spectroscopy-diode array detector analysis confirmed the presence of different carbon length isoprenoids; C₃₀: tetrahydrosqualene, C₄₀: 3-hydroxyechinenone, astaxanthin, canthaxanthin, lycopene, phytofluene, phytoene and C₅₀: bisanhydrobacterioruberin, monoanhydrobacterioruberin, bacterioruberin and haloxanthin. Thus, we conclude that the synergistic actions of all these components contribute to the antioxidant activity of the culture and that the antioxidant activity of the exopolysaccharide, glycerol dither moiety, tetrahydrosqualene, haloxanthin and 3-hydroxyechinenone is recorded as the first report for Haloferax alexandrinus GUSF-1 (KF796625). Therefore, recommended for use in microbial industrial biotechnology.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-020-02584-9.

Keywords: Carotenoids; DPPH•; Exopolysaccharides; Glycerol diether moiety; HR-LC/MS–DAD; Haloarchaea.

Fig. 1

Characterization of ethanolic extract from Haloferax alexandrinus GUSF-1 (KF796625). a UV spectrum showing a single peak at 193 nm, b DPPH^• decolorization of ethanolic extract monitored by the spectrophotometer; which shows that both the peaks of DPPH^• (325 and 517 nm) are abolished by the ethanolic extract, indicating the free radical scavenging capacity, c FTIR spectrum revealing the characteristic glycosidic peaks between 2000 and 1000 cm⁻¹

Fig. 2

Characterization of hydroxylated methanolysate from cells of Haloferax alexandrinus GUSF-1 (KF796625): The thin-layer chromatogram was resolved in petroleum ether (40–60 °C): diethyl ether (85:15,v/v) and developed with a iodine vapor, b 10% dodecaphosphomolybdic acid in absolute ethanol and heated at 150 °C, c decolorization of hexanolic DPPH^• solution by compound at R_f 0.2 and d DPPH^• spot assay

Fig. 3

FTIR spectrum of glycerol diether moiety extracted from hydroxylated methanolysate from cells of Haloferax alexandrinus GUSF-1 (KF796625)

Fig. 4

Decolorization of DPPH^• by hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625). a Bright orange hexanolic extract, b UV–Vis absorption spectrum of hexanolic extract with peaks in the UV and visible region and c the deep purple color of DPPH^• (left) is decolorized by the hexanolic extract rendering it straw yellow (right), an indication of its potent free radical scavenging capacity

Fig. 5

FTIR spectrum of hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625)

Fig. 6

Thin-layer chromatograms of hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625) developed in a acetone:petroleum ether (20:80; v/v), b methanol:chloroform (7:93; v/v), and c acetone:hexane (50:50; v/v) and viewed as (1) visible spots and (2) after spraying with DPPH^•. The hexanolic extract was also resolved in d n-heptane: benzene (90:10, v/v) and viewed as (1) visible spots, (2) after spraying with DPPH^• and (3) exposed to iodine vapor

Fig. 7

Chemical profile of hexanolic extract from cells of Haloferax alexandrinus GUSF-1 (KF796625). a HR-LC/MS–DAD chromatogram of hexanolic extract revealing a total of 12 peaks labeled according to the increasing retention times (R_t). Identification of the separated compounds was achieved by comparing the m/z values obtained with the Agilent Mass Hunter Qualitative Analysis Software version (B.05.00), Japanese carotenoid database, Pubchem, and reported literature. Diode Array Detector response at b 320, c 447 and d 490 nm