Bioassay-guided isolation and antioxidant properties of phytochemicals present in methanolic extract of Halosarcia indica from salt marsh ecosystems in Sri Lanka

Abstract

Halosarcia indica, belonging to the family Amaranthaceae, is a halophyte that exhibits unique adaptations to saline environments. Halophytes contain various bioactive chemicals, including plant polyphenols, which have garnered interest due to their antioxidant properties. Isolation of pure compounds responsible for the antioxidant activity is also very important in nutraceuticals, cosmetics, and pharmaceutical industries. The aim of the present study was to isolate and identify bioactive compounds from H. indica samples collected from Sri Lanka. Fresh plant materials were collected from salt marshes in Mannar district, Sri Lanka. Phytochemicals from finely powdered freeze-dried areal parts of H. indica were extracted using cold extraction and sequentially partitioned with methanol, dichloromethane (DCM), and hexane. The antioxidant activity of these fractions was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the Ferric Reducing Antioxidant Power (FRAP) assay, with butylated hydroxytoluene (BHT) serving as the positive control. The total phenolic content (TPC) was assessed using the Folin-Ciocalteu method, while the determination of total flavonoid content (TFC) was conducted through the aluminum chloride assay. Additionally, the total tannin content (TTC) was examined using the vanillin/HCl method. Finally, methanolic and DCM extractions were subjected to column chromatography, thin layer chromatography, and preparative thin layer chromatography techniques to isolate compounds responsible for antioxidant activity. The methanolic fraction exhibited the highest radical scavenging activity, with IC50 values of 69.78 ± 3.48 µg mL-1 in the DPPH assay and 81.38 ± 1.89 µg mL-1 in the ABTS assay, while the positive control BHT showed IC50 values of 20.91 ± 0.48 µg mL-1 and 22.93 ± 0.65 µg mL-1, respectively. In the FRAP assay, the methanolic fraction presented higher mean absorbance values of 0.598 ± 1.81 compared to that of the BHT 0.821 ± 1.82. Methanolic extracts of the H. indica had higher TPC (184.77 ± 3.21 mg GAE/g), TFC (190.69 ± 5.84 mg CE/g), and TTC (201.33 ± 4.21 mg GAE/g) values compared with DCM and hexane extracts. Methanolic extracts showed higher antioxidant activity followed by DCM and hexane extracts. Therefore, two pure compounds with antioxidant activities were isolated in methanolic (IC50 - 78.69 ± 1.24 µg mL-1) and DCM extracts (IC50 - 192.84 ± 1.65 µg mL-1) respectively. The study identified two antioxidant compounds from H. indica extracts, with methanolic extracts showing the highest activity. Further analysis will be done using spectroscopic methods. These two compounds will be identified using spectroscopic techniques, including 1H NMR, 13C NMR, HPLC, and mass spectrometry.