Phytochemical characterization and evaluation of antioxidant, antimicrobial, antibiofilm and anticancer activities of ethyl acetate seed extract of Hydnocarpus laurifolia (Dennst) Sleummer

Abstract

Various functional groups were observed in the FTIR analysis of Hydnocarpus laurifolia seeds ethyl acetate extract such as O-H, N-H, C-H, -CH₂, O=C=O, C=O, C=O-NH, and CH_3, etc. Eleven bioactive compounds were detected via GC-MS and the predominant compounds include (1S)-2-cyclopentene-1-tridecanoicacid (chaulmoogric acid) (80.59%); 2-cyclopentene-1-undecanoic acid (hydnocarpic acid) (6.76%); cyclobutylamine (5.28%); methyl thioacetate (ethanethioic acid) (4.84%); lignoceric acid (2.21%). The TPC and TFC values were 0.110 ± 0.04 GAE g^-1 and 0.175 ± 0.05 g CE g^-1 respectively. Ethyl acetate extract showed strong DDPH free radical scavenging activity with IC₅₀ value 10.64 ± 0.48 µg ml^-1 and antioxidant activity index 3.759. The ethyl acetate extract also exhibited potential ABTS radical scavenging efficacy with a very low IC₅₀ value, i.e., 07.81 ± 0.48 µg ml^-1. P. aeruginosa was the most sensitive bacteria to the extract with 33.16 ± 0.88 mm inhibition zone and MIC: 3.12 mg ml^-1, MBC: 6.25 mg ml^-1. P. aeruginosa biofilm was inhibited by ethyl acetate extract 99.22% at MIC concentration. The LM images displayed a decrease in the number of biofilm cells and FE-SEM micrographs showcased the extensive decrease as well as disintegration in biofilm. Additionally, ethyl acetate extract was found selectively cytotoxic to the K562 cancer cells having an IC₅₀ of 25.41 μg ml^-1 and barely cytotoxic to normal PBMCs having an IC₅₀ of 482.54 μg ml^-1, and the selectivity index value was 18.99. Data validate scientifically the traditional use of H. laurifolia seeds in folk medicines and confirmed that it can be used in modern phytomedicines as an antioxidant, antimicrobial, antibiofilm, and anticancer agent and is toxicologically safe.

Keywords: Antibiofilm activity; Anticancer activity; Antimicrobial activity; Antioxidant activity; FTIR; GC–MS; Hydnocarpus laurifolia.

Fig. 1

FTIR spectrum analysis of H. laurifolia seeds ethyl acetate extract

Fig. 2

GC–MS chromatogram of H. laurifolia seeds ethyl acetate extract

Fig. 3

DPPH radical scavenging activity of H. laurifolia seeds ethyl acetate extract

Fig. 4

ABTS radical scavenging activity of H. laurifolia seeds ethyl acetate extract

Fig. 5

Growth curves of P. aeruginosa on the exposure of H. laurifolia seeds ethyl acetate extract. a 1.56 mg ml⁻¹, b 3.12 mg ml⁻¹, c 6.25 mg ml⁻¹, and d non-treated control

Fig. 6

Antibiofilm activity of H. laurifolia seeds ethyl acetate extract against P. aeruginosa biofilm by ring assay

Fig. 7

Percentage inhibition of P. aeruginosa biofilm by H. laurifolia seeds ethyl acetate extract

Fig. 8

Light microscopy (100 X) of P. aeruginosa biofilm treated with a non-treated control, b 0.78 mg ml⁻¹, c 1.56 mg ml⁻¹, d 3.12 mg ml⁻¹ concentration of H. laurifolia seeds ethyl acetate extract

Fig. 9

FE-SEM micrographs of P. aeruginosa biofilm treated with, a non-treated control, b 0.78 mg ml⁻¹, c 1.56 mg ml⁻¹, d 3.12 mg ml⁻¹ concentration of H. laurifolia seeds ethyl acetate extract

Fig. 10

Cytotoxicity of different concentrations, a Non-treated control, b 12.5 µg ml⁻¹, c 25 µg ml⁻¹, d 50 µg ml⁻¹, e 100 µg ml⁻¹, and f 200 µg ml⁻¹ of H. laurifolia seeds ethyl acetate extract on K562 cells

Fig. 11

Percent cell inhibition and viability of PBMCs by H. laurifolia seeds ethyl acetate extract via MTT bioassay