Modulatory effects of Terminalia arjuna against domoic acid induced toxicity in Caco-2 cell line

Abstract

Domoic acid is a potent marine algal toxin produced by diatomic genus of Pseudo-nitzschia causing amnesic shell fish poisoning. Domoic acid toxicosis mainly involves excitotoxic effects coupled with oxidative stress. The present study was aimed to evaluate the protective effects of hydro-alcoholic extract of Terminalia arjuna (TA) against domoic acid induced toxic effects in Caco-2 cell line. It was observed that the toxicity induced by domoic acid in Caco-2 cells was mediated by oxidative insult leading to morphological changes, DNA damage and apoptosis. In our study pre-treatment of the cells with TA (10, 20 and 30 μg/ml) showed significant protection against domoic acid induced morphological, oxidative and apoptotic damages in a dose dependent manner. The effect of phytocompounds present in TA viz., kaempferol and arjungenin showed significant protection against domoic acid induced toxicity in Caco-2 cell line. Hence, it could be inferred that the protective effect of TA extract against domoic acid induced toxicity could be due to the individual or synergistic effects of kaempferol and argungenin. However, further clinical studies are warranted to consider TA as a natural remedy to prevent amnesic shell fish poisoning.

Keywords: Apoptosis; Domoic acid; Morphological changes; Oxidative stress; Phytocompounds; Terminalia arjuna.

Fig. 1

LC–ESI–MS/MS chromatograms of some identified bioactive compounds from TA

Fig. 1

LC–ESI–MS/MS chromatograms of some identified bioactive compounds from TA

Fig. 2

Protective effect of TA against domoic acid induced cytotoxicity in Caco2 cells. a Response to pre-treatment of TA. Caco-2 cells were treated with hydro-alcoholic extract of Terminalia arjuna at different concentrations (10–50 µg/ml) for 24 h. b Cytotoxicity of domoic acid. Cytotoxicity of domoic acid was assessed in various doses of domoic acid i.e. 20–150 ng/ml and 75 ± 3 ng/ml was found to be the IC₅₀ value. c Effect of TA on domoic acid induced toxicity. Pre-treatment with TA (10–30 µg/ml) followed by domoic acid exposure (75 ng/ml) for 24 h. d Effect of phytocompounds present in TA against domoic acid induced cytotoxicity. Cell viability on pre-treatment of ursolic acid (UA) 250 ng/ml, kaempferol (K) 200 ng/ml, ellagic acid (EA) 300 ng/ml, arjunolic acid (AA) 150 ng/ml, arjungenin (AG) 200 ng/ml, followed by domoic acid (DA) exposure (75 ng/ml) for 24 h. Data are expressed as mean ± SEM of three independent experiments. In the TA treated groups Asterisk indicates statistically significance p < 0.05 vs control and Double asterisk indicates p < 0.05 vs domoic acid, whereas in phytocompounds treated groups Asterisk indicates significant difference p < 0.05 with respect to domoic acid; # indicates significant difference at p < 0.05 with respect to control

Fig. 3

Protective effect of TA pre-treatment on domoic acid induced structural alterations of Caco-2 cells. a Phase-contrast microscopy: (i) Control Caco-2 cells, (ii) Domoic acid (75 ng/ml) exposed cells for 24 h, (iii–v) Pre-treatment with TA (10 µg/ml, 20 µg/ml and 30 µg/ml) followed by domoic acid (75 ng/ml) exposure for 24 h. b Scanning electron microscopy: I and (i) Control Caco-2 cells showing normal morphology and normal cell size, II and (ii) Domoic acid exposed cells showing cell damage, III and (iii) Pre-treatment with TA (30 µg/ml) followed by domoic acid exposure showing protective effect. Images from I to III at 500× magnification and images from (i–iii) are at 3000× magnification. c Effect of pre-treatment with phytocompounds on domoic acid induced morphological changes in Caco-2 cells. (i): Control Caco-2 cells, (ii): Domoic acid (75 ng/ml) exposed cells for 24 h, (iii–vii): Pre-treatment with ursolic acid (UA) 250 ng/ml, kaempferol (K) 200 ng/ml, ellagic acid (EA) 300 ng/ml, arjunolic acid (AA)150 ng/ml, arjungenin (AG) 200 ng/ml respectively, followed by domoic acid (DA) exposure (75 ng/ml) for 24 h

Fig. 3

Protective effect of TA pre-treatment on domoic acid induced structural alterations of Caco-2 cells. a Phase-contrast microscopy: (i) Control Caco-2 cells, (ii) Domoic acid (75 ng/ml) exposed cells for 24 h, (iii–v) Pre-treatment with TA (10 µg/ml, 20 µg/ml and 30 µg/ml) followed by domoic acid (75 ng/ml) exposure for 24 h. b Scanning electron microscopy: I and (i) Control Caco-2 cells showing normal morphology and normal cell size, II and (ii) Domoic acid exposed cells showing cell damage, III and (iii) Pre-treatment with TA (30 µg/ml) followed by domoic acid exposure showing protective effect. Images from I to III at 500× magnification and images from (i–iii) are at 3000× magnification. c Effect of pre-treatment with phytocompounds on domoic acid induced morphological changes in Caco-2 cells. (i): Control Caco-2 cells, (ii): Domoic acid (75 ng/ml) exposed cells for 24 h, (iii–vii): Pre-treatment with ursolic acid (UA) 250 ng/ml, kaempferol (K) 200 ng/ml, ellagic acid (EA) 300 ng/ml, arjunolic acid (AA)150 ng/ml, arjungenin (AG) 200 ng/ml respectively, followed by domoic acid (DA) exposure (75 ng/ml) for 24 h

Fig. 4

Protective effect of TA pre-treatment on domoic acid induced apoptosis. a Effect of TA on domoic acid induced loss of mitochondrial membrane potential. Pre-treatment with TA (10 µg/ml, 20 µg/ml and 30 µg/ml, respectively) followed by domoic acid (75 ng/ml) exposure for 24 h. The value of MMP was expressed as relative fluorescence intensity compared to control. *Statistically significant differences, *p < 0.05 vs control and **p < 0.05 vs domoic acid. b Effect of TA on domoic acid induced nuclear damage using DAPI staining. (i) Control Caco-2 cells, (ii) Domoic acid (75 ng/ml) treated cells exposed for 24 h, (iii–v) Pre-treatment of TA (10, 20, and 30 µg/ml, respectively) followed by domoic acid (75 ng/ml) exposure for 24 h. Arrow marks in the image represent cells undergoing apoptosis

Fig. 5

Effect of pre-treatment of TA and phytocompounds on domoic acid induced oxidative stress. a, b Reactive oxygen species (ROS). c, d Nitrite production (NO). Pre-treatment of TA (10, 20, and 30 µg/ml, respectively) and phytocompounds (ursolic acid (UA) 250 ng/ml, kaempferol (K) 200 ng/ml, ellagic acid (EA) 300 ng/ml, arjunolic acid (AA) 150 ng/ml, arjungenin (AG) 200 ng/ml,) followed by domoic acid (75 ng/ml) exposure for 24 h. The values of ROS were expressed as relative fluorescence intensity and NO was expressed as relative absorbance compared to domoic acid treatment. In TA treated groups *indicates statistically significance p < 0.05 vs control and ** indicates p < 0.05 vs domoic acid, whereas in phytocompounds treated groups * indicates significant difference p < 0.05 with respect to domoic acid; # indicates significant difference at p < 0.05 with respect to control

Fig. 6

Protective effect of TA on domoic acid induced modulation of glutathione reductase and catalase. 1. Control 2. Domoic acid (75 ng/ml) 3–5. TA-10, 20 and 30 µg/ml, respectively. GAPDH - Glyceraldehyde 3-phosphate dehydrogenase, GR-glutathione reductase, CAT-catalase. Relative band intensities are shown in separate bar diagrams for GR and CAT