Methanolic extract of coconut (Cocos nucifera L.) haustorium mitigates pro-oxidant-mediated apoptotic cell death via Nrf-2 pathway and lipopolysaccharide-induced cytokine release in cells

2.1 Reagents used in the experiment and cell lines

Normal colon epithelial cells from rats (IEC-6) and murine macrophages (Raw 264.7) were procured from the cell culture collection center of the National Centre for Cell Science-DBT, Pune. The cells were maintained under standard conditions using complete growth media (RPMI-1640) with 10% FBS, 5% CO₂ at 37 °C.

2.2 Collection, extract preparation, and characterization of coconut haustorium

The sprouted coconuts (12–13 month old West coast tall variety) were collected from Kozhikode and de-husked, dried in an incubator at 50 °C and then powdered. The powder was extracted with 100% methanol and dried under vacuum concentration. The methanolic extract of coconut haustorium (CHE) was prepared by Soxhlet extraction (Borosil, Mumbai, India). The dried coconut haustorium powder was extracted using methanol (100%) for 6 h continuously. Total phenol contents (Ahmed and Tavaszi-Sarosi, 2019) and flavonoid contents (Wang et al., 2020) were determined by standard procedures. The phytochemical composition was analyzed by using LC/MS analysis (Malayil et al., 2020).

2.3 Detection of biologically safe concentration of CHE and cytoprotective effect

The nontoxic concentration of haustorium extract of Coconut (CHE) and cytotoxic activity of H₂O₂ and malondialdehyde (MDA) was determined by MTT assay (Nawaz et al., 2021). The cytoprotective effect was studied according to the protocols described by (Malayil et al., 2020). The percentage of cell death was calculated by comparing it with a control cell. The percentage inhibition of cell death with respect to the free radical control group was determined as follows; $$\%Inhibitionofcelldeath=\frac{\mathit{Cell}deathincontrol-Celldeathintreatment}{\mathit{Cell}deathinControl}\times 100$$

2.4 Analysis of intracellular antioxidants and indicators of oxidative insults

The intra-cellular reduced glutathione (GSH) level in µmoles/mg protein was determined by the reaction of Ellman’s reagent (DTNB) (Nair et al., 2016). The activity of antioxidant enzyme catalase (CAT) was determined as the degradation of hydrogen peroxide per unit time as previously described (Illam et al., 2017). The lipid peroxidation indicator, thiobarbituric acid reactive substances (TBARS) and level of conjugated dienes (CD) were spectrophotometrically quantified according to the methods described by Narayanankutty et al. (2017). All these antioxidant parameters and oxidative stress status were finally expressed as nanomoles or IU per milligram of protein (Malayil et al., 2020).

2.5 Gene expression analysis by qPCR

The cells were harvested and cDNA was synthesized using CellAmp™ kits (Takara Bio, India) (Job et al., 2021a). The thermal cycling and sequences of primers used in the study and their sequences are given as supplementary material 1. The expression of each gene in different treatments was calculated by the 2^-ΔΔCT method against the internal standard as beta-actin (Rao et al., 2013).

2.6 Inhibitory effect of CHE on Lipopolysaccharide-induced cytokine production from Raw 264.7 cells

The murine macrophage cell line Raw 264.7 was initially exposed to different doses of CHE (10, 20, and 40 µg/mL) for 24 h and later stimulated to secrete pro-inflammatory cytokines by the treatment with lipopolysaccharide (1 mg/mL). The levels of proinflammatory cytokines being released by the macrophages (IL-1β, IL-6, and TNF-α) were estimated using PeproTech ELISA kits (Rocky Hill, USA) according to the instruction manual. The quantity of nitric oxide in the media was quantified as described previously (House et al., 2020).

2.7 Statistical analysis

The data was presented in the manuscript as mean ± SD for different concentrations, each carried out in triplicate assays. Levels of significance and statistical operations were performed using one-way analysis of variance (ANOVA) followed by Tukey Kramer post hoc test (Graph pad Prism 7.0 version, La Jolla, USA).

3.1 Total phenolic and flavonoid content as well as phenolic composition

The total phenol content of CHE was 44.32 ± 2.29 mg gallic acid equivalent (GAE) and the flavonoids content in the methanol extract of coconut haustorium was 7.48 ± 0.42 mg quercetin equivalent (QE). Further, the chemical characterization of the extract using LC-MS identified the presence of various simple phenolic acids and flavonoid compounds (Supplementary material 2).

3.2 Determination of cytotoxicity and biologically safe concentrations

The cytotoxicity of CHE was negligible up to a concentration of 50 µg/mL in both IEC-6 as well as macrophages (Raw 264.67) cells. The cell death was found to be<2% at the dose below 40 µg/mL and hence, the 10, 20, and 40 µg/mL were chosen for cytoprotective study. The cytotoxicity analysis by MTT determined the half-maximal cell death values for malondialdehyde as 352.18 ± 9.4 µM and hydrogen peroxide as 448.10 ± 6.8 µM. Thus, the concentrations of MDA and hydrogen peroxide were set as 450 and 350 µM (Fig. 1).

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Fig 1

The cytotoxicity of hydrogen peroxide and malondialdehyde (a) methanolic extract of Coconut haustorium (b) determined using MTT assay for 48-hour period.

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3.3 Cytoprotective effect of coconut haustorium

Treatment with H₂O₂-induced significant cell death (cell viability 49.1 ± 1.4%) in immortalized intestinal cells (IEC-6), at a concentration of 350 µM compared to the untreated cells. Further, pre-treatment with the CHE at the respective doses 10, 20, and 40 µg/mL protected the cell death induced by H₂O₂; the cell viability at these doses was estimated to be 55.8, 68.4, and 86.9% (Fig. 2a).

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Fig 2

Protective effect of the CHE against the toxicity induced by hydrogen peroxide (a) and malondialdehyde (b) in immortalized intestinal cells (IEC-6).

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Similarly, malondialdehyde (450 µM) treatment reduced the cell viability of IEC-6 cells to 48.8 ± 1.3%. The dose-dependent protective effect of CHE was observed in these cells upon pre-treatment with doses 10, 20, and 40 µg/mL. The cell viability during the respective treatment doses were 59.2, 70.4, and 91.5% (Fig. 2b).

3.4 Effect on cellular (IEC-6) antioxidant defence

3.5 Changes in the expression pattern by qPCR

The treatment of IEC-6 cells with H₂O₂ (350 µM) or MDA (450 µM)-induced a significant elevation in the RNA expression of apaf-1, caspase 3, and caspase 7 genes (p < 0.001). Compared to the MDA models, the apoptosis induction in terms of fold change in these genes were more prominent in the hydrogen peroxide alone treated groups. However, pre-treatment with the different doses of CHE dose-dependently reduced the gene expression pattern of genes such as caspase-3, caspase-7 and apaf-1 (p < 0.01). On contrary to the expression of apoptotic genes, the Nrf-2 and heme oxygenase-1 expression was lowered in the H₂O₂ or MDA-treated cells. However, treatment with CHE has significantly induced the expression of these two genes (p < 0.001) and the effect was found to be dose-dependentFigure 3.

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Fig 3

The effect of pre-treatment with CHE on the expression of apoptotic genes and antioxidant genes in cells treated with hydrogen peroxide (a) and malondialdehyde (b).

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3.6 Anti-inflammatory activity in LPS-stimulated macrophages

Lipopolysaccharide was used to stimulate Raw 264.7 cells to induce the secretion of cytokines and nitric oxide radicals. In LPS alone treated macrophages, the release of cytokines like interleukins (IL-1β, IL-6) and TNF-α were significantly elevated (p < 0.001). Concomitantly, there observed a significant elevation in nitric oxide radicals in these cells. Pre-treatment with the polyphenol-rich CHE for 24 h at doses 20 and 40 µg/mL significantly reduced the secretion of IL-1β, IL-6 (p < 0.01) as well as TNF-α (p < 0.05). Further, the nitric oxide radicals were significantly reduced by the pre-treatment with CHE dose 20 and 40 µg/mL (Table 2).