Antioxidant, Antimicrobial, and Bioactive Potential of Two New Haloarchaeal Strains Isolated from Odiel Salterns (Southwest Spain)

Abstract

The need to survive in extreme environments has furnished haloarchaea with a series of components specially adapted to work in such conditions. The possible application of these molecules in the pharmaceutical and industrial fields has received increasing attention; however, many potential bioactivities of haloarchaea are still poorly explored. In this paper, we describe the isolation and identification of two new haloarchaeal strains from the saltern ponds located in the marshlands of the Odiel River, in the southwest of Spain, as well as the in vitro assessment of their antioxidant, antimicrobial, and bioactive properties. The acetone extract obtained from the new isolated Haloarcula strain exhibited the highest antioxidant activity, while the acetone extracts from both isolated strains demonstrated a strong antimicrobial activity, especially against other halophilic microorganisms. Moreover, these extracts showed a remarkable ability to inhibit the enzyme cyclooxygenase-2 and to activate the melanogenic enzyme tyrosinase, indicating their potential against chronic inflammation and skin pigmentation disorders. Finally, the aqueous protein-rich extracts obtained from both haloarchaea exhibited an important inhibitory effect on the activity of the acetylcholinesterase enzyme, involved in the hydrolysis of cholinergic neurotransmitters and related to several neurological diseases.

Keywords: anti-inflammatory; antimicrobial; antioxidant; bioactive substances; haloarchaea.

Figure 1

Molecular Phylogenetic Analysis by Maximum Likelihood Method. The trees represent a comparison among the 16S rRNA sequences from the new strains isolated, H. hispanica HM1 (A) and H. salinarum HM2 (B), and a series of reference archaeal sequences. Multiple alignments were generated by MUSCLE (MUltiple Sequence Comparison by Log-Expectation) and the trees were constructed with MEGA 7. The numbers at nodes indicate the bootstrap values calculated for 1000 replicates. The name and the NCBI access number are indicated for all the reference sequences. Black triangles indicate the new strains identified.

Figure 2

Antioxidant activity of aqueous and acetone extracts (1 mg mL⁻¹) obtained from the haloarchaeal strains H. hispanica HM1 and H. salinarum HM2, determined by DPPH (1,1-Diphenyl-2-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assays, or FRP (Ferrous ion reduction potential) and CCA (Copper chelating activity). For each assay, the data were submitted to one-way variance analysis (ANOVA). Bars are followed by different superscript letters (a, b or c), which denote groups with significant differences according to the Duncan’s Multiple Range Test (p < 0.05) (A). The half maximal inhibitory or effective concentration (IC₅₀ or EC₅₀) were calculated when the activity was higher than 65% (B). These values were normalized to the content of proteins and carotenoids in the extracts, being the protein concentration of the aqueous extract 47 and 30 µg per mg DW and the carotenoid content of 10 and 12 µg per mg DW, respectively, for H. hispanica HM1 and H. salinarum HM2.

Figure 3

In vitro activity of haloarchaeal extracts on enzymes related to diabetic, neurodegenerative skin pigmentation, and inflammatory diseases. Percentages of inhibition of the enzymes: α-amylase, α-glucosidase, acetylcholinesterase (AChE), tyrosinase (TYRO) and cyclooxygenase 2 (COX-2) by the all extracts obtained in different solvents (hexane, dichloromethane, ethyl acetate, chloroform, aqueous buffer and acetone) from H. hispanica HM1 (A) and H. salinarum HM2 (B) strains are represented. A known inhibitor for each enzyme was included as control. For each assay, the data were submitted to one-way variance analysis (ANOVA). Bars are followed by different superscript letters (a–f), which denote groups with significant differences according to the Duncan’s Multiple Range Test (p < 0.05).

Figure 4

Antimicrobial activity of acetone extracts. Antimicrobial activity was indicated by zones of inhibition of growth or halos. Taking into account the diameter of the halo, the susceptibility of the different microorganisms to the extracts was classified in four reference groups: (A) 0.5–1 cm (+) in Bacillus cereus; (B) 1–2 cm (++) in Staphylococcus aureus; (C) 2–4 cm (+++) in Dunaliella bardawil and (D) 4–6 cm (++++) in Dunaliella salina.

Figure 5

Minimal Inhibitory Concentration plate assay. Dilutions of the active extracts were assayed against one of the most susceptible microorganisms of each group. Halophilic microorganisms, represented by Dunaliella salina and Halogeometricum, showed to be more sensitive to the extracts.