The ZEA, dichloro-dihydro-fluorescein diacetate (DCFH-DA), rhodamine 123, caspase-3 kit, and 0.22 μm of syringe filters were obtained from Sigma–Aldrich (Bengaluru, India). Acetonitrile, methanol, dimethyl sulfoxide (DMSO), and analytical grade water were obtained from Merck (Bengaluru, India). Dulbecco’s modified Eagle’s medium (DMEM), citrate buffer solution (CBS), an antibiotic solution of penicillin and streptomycin, fetal bovine serum (FBS), and Dulbecco’s phosphate buffered saline (D-PBS) were obtained from HiMedia (Mumbai, India). Live/dead cell assay detection kit was obtained from Invitrogen Detection Technologies (Bengaluru, India), and plastic ware for cell culture was obtained from Nunc (Bengaluru, India).

The seeds of J. curcas was collected from Tamil Nadu region, India, and washed thoroughly with distilled water, and dried for 2 weeks at room temperature. The husk was separated from the seeds and crushed into a fine powder at a particle size of 500 μm with a micro hammer mill. A quantity of 450 g fine powder was blended with hot distilled water containing 450 g of ZnCl₂ for 1 h and dried out at 60 ± 5°C in a hot-air oven. The leftover material was filled in a steel container with a tight lid and repacked with another concentric steel container. The linear empty space of the container was filled with sand in the form of layer-by-layer up to outer container brim. The arrangement for carbonization was made under traces oxygen between the voids of material and reaction was executed at 800 ± 5°C for 60 min in a furnace. Following, reaction mixture was cooled and excess ZnCl₂ was subjected to leaching out by immersing in a hot solution of 1 M HCl at 80 ± 5°C for 24 h in the oven. The material was washed with hot water till the chloride was detached (tested by the silver nitrate method) and dried out at 105 ± 5°C in a hot-air oven and filtered through 250–500 μm size filter. The obtained material was designated as AC of J. curcas (ACJC) and stored in a desiccator at room temperature under dry place for a further purpose.

The physicochemical characteristics of the prepared ACJC were investigated using various analytical techniques including X-ray diffraction (XRD), Raman spectroscopy, Scanning electron microscope–energy dispersive X-ray (SEM–EDX), and Brunauer–Emmett–Teller (BET). The XRD analysis was carried out to understand the structural characteristics of the ACJC using X’Pert PRO X-ray Diffractometer (PANalytical, Spectris Technologies Pvt. Ltd., India) with Cu Kα radiation source (λ = 1.541 Å) in the 2𝜃 range from 10° to 70° with a scanning rate 3°/min. The average crystalline size of the prepared sample was calculated using Scherrer equation (Patterson, 1939). The Raman spectrum of ACJC was recorded using Renishaw’s in Via Raman microscope (Renishaw India, Bengaluru) with spectrometer attachment. Green laser with 514 nm wavelength set in a low power mode (0.5% of maximum power) was used to excite the sample. The surface micro-morphology and chemical configuration of ACJC were assessed using SEM (Quanta 200, ICON Analytical Equipment Pvt. Ltd., United States) attached EDX detector. The precise surface area of ACJC was determined using BET surface area analyzer (Micromeritics TriStar II, United States).

The experiments were undertaken individually for six times (n = 6) and results were expressed in mean ± SD. The statistical analysis was executed by one-way ANOVA following Dunnett’s test and p-value < 0.05 was stated as statistically significant (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).

Since last decade, researchers around worldwide have been highly focused on to develop low-cost AC from the abundant and cheaply accessible natural resources. The plant sources are a highly suitable for synthesis of AC due to its high cellulose and hemicellulose content, and the cellulose and hemicellulose have exhibited a high potential sorption competence toward a variety of toxic compounds (Correa et al., 2017; Ooi et al., 2017). Henceforth, in the present study, cellulose- and hemicellulose-rich seed shells of J. curcas were used as a raw material to synthesize of AC by ZnCl₂ chemical activation method.

The crystalline phase of synthesized ACJC was identified from the XRD result (Figure 1). The diffraction patterns agree well with the standard JCPDS file (41–1487), which confirmed the formation of crystalline structures of the graphitic carbon. The obtained diffraction peaks are indexed to the corresponding peaks for (002), (100), and (101) crystal planes of graphitic carbon. The presence of a sharp peak around 26° has confirmed the crystalline structure of ACJC which portrays the superior alignment of disordered graphitic carbon layers to form the crystalline turbostratic structure (Li et al., 2007). The average crystallite size of ACJC was calculated from the FWHM value of the highly intense peak corresponding to the (002) crystal plane. It is found that the ACJC has an average crystallite size of 1.07 nm. The peak splitting observed in the X-ray diffraction peaks are due to the transition of carbon from a highly symmetric phase to a lower one. It also shows the presence of microporous wall structure of ACJC. The XRD results confirmed that ACJC has a better crystallographic structure as well-organized aromatic carbon that is more stable than amorphous-like carbon and these results were further evaluated by Raman spectroscopic analysis.

Figure 2 shows the Raman spectrum recorded by exciting the sample using a green laser with 514 nm excitation wavelength. The Raman spectrum of ACJC elucidates the chemical structure and crystalline nature of the prepared sample. The 514 nm selectively excites the π state of sp² hybridized carbon molecules. The obtained Raman peaks around 1303 and 1589 cm^-1 are indexed to the D band and G band of the graphitic carbon particles, respectively. The G band represents the peak corresponding to vibrational modes arises due to the stretching of bonds associated with the pairs of sp² hybridized carbon atoms present either in carbon molecular chain or in aromatic ring. The E2g symmetry modes around 1589 cm^-1 in the G band confirm the presence of highly crystalline and ordered nature of graphitic carbon in ACJC. Compared to the G band of graphite around 1575 cm^-1, the higher value of peak at 1589 cm^-1 for the G band of ACJC sample is attributed to the C–C bond bending in the sample due to the porous nature of the ACJC. The D band observed around 1303 cm^-1 corresponds to the A1g mode called as lattice breathing mode. The presence A1g breathing mode reveals the structural disorder in the sample. Moreover, the peak corresponding to the D3 band absent in the ACJC, which confirms that the amorphous phase of carbon between graphitic layers is absent in the ACJC sample. Hence, it confirms that the ACJC sample is highly crystalline nature (Lazzarini et al., 2016).

The SEM analysis was undertaken to determine the surface morphology of the synthesized ACJC and micrographs were recorded at different magnifications (500× and 1000×) as shown in Figure 3A. The obtained images revealed the honeycomb void structure analogous to the turbostratic morphology of the carbon particles, and this result is in perfect agreement with the structural data obtained from XRD analysis. The ZnCl₂ chemical activation method has established new microporous as well as widened existing microporous by increasing the volume and surface area of the microporous, and these pores were formed because of ZnCl₂ evaporation during carbonization. These large volumes of microporous developed by ZnCl₂ were helpful in engulfing the contaminants. The volume and surface area of microporous depends on the structure of precursor applied for the carbonization purpose.

The elemental analysis of ACJC was carried out using EDX technique and results suggest that the seed shells of J. curcas contain a good amount of carbon. The carbonization of seed shells of J. curcas by ZnCl₂ chemical activation has increased the carbon content and indicated successful carbonization of seed shells of J. curcas results in the formation of ACJC. Figure 3B elucidated the presence of carbon (C) and oxygen (O) in ACJC and wt. % of C and O were 93.36 and 6.64, respectively. A small amount of oxygen is present in the ACJC due to the oxidation of carbon during the heat treatment process.

The adsorption capacity of AC depends on volume, size, and specific surface area of the pore (Dizbay-Onat et al., 2017). The specific surface of ACJC was assessed from adsorption experiments on the basis of the BET method, and the micropore volume (V) was evaluated by calculating the adsorbate volume. The t-curve was analyzed to measure the thickness of the adsorbed film (Senniappan et al., 2016). The BET analysis was helpful in establishing the relationship between surface area and material properties of samples and the effect of activation temperature, activator, impregnation ratio in the formation of pore formation (Ab Ghani et al., 2017). In the present study, ACJC with the greater surface area was obtained by ZnCl₂ chemical activation at a higher temperature (Liu et al., 2016). The porous structure of ACJC is due to the “swelling effect” caused by the electrolytic action of ZnCl₂ which breaks the lateral bonds in the cellulose molecules and increases the pore density in the molecular structure of seed shells of J. curcas. Therefore, microporosity and surface area of ACJC has increased (Liu et al., 2016). The S_BET, micropore area, and average pore diameter of ACJC were observed as 822.78 (m²/g), 255.36 (m²/g), and 8.5980 (Å), respectively (Figures 4A,B). In support of our result, IUPAC has also concluded from S_BET results that the occurrence of type I isotherm with high pressure at horizontal plateau could result in the development of narrow pore size scattered microporous material. The Fractal dimension, which is a measure of the roughness of a surface of ACJC, was estimated by applying the Frenkel–Halsey–Hill (FHH) equation to N₂ adsorption isotherms. The results showed that the change in Fractal dimension was insignificant and the complete development of pores on the surface of ACJC was due to the activation of ZnCl₂ (Carrott and Carrott, 2007).

The contact time involved between ACJC (adsorbent) and ZEA (adsorbate) is one of the key factors that considerably influence the adsorption process (El-Sayed et al., 2014). The effect of contact time on the adsorption of ZEA by ACJC was investigated with a different initial concentration of ZEA (25, 50, 75, and 100 μg/mL) and 1 mg/mL of ACJC at pH 7. Based on the kinetic investigations, the plot of ZEA adsorption by ACJC (μg/mg) versus contact time (min) was presented in Figure 5A. The results showed the rapid adsorption of ZEA by ACJC for initial 15 min of contact time. Following the contact time, adsorption of ZEA was slow down until equilibrium was obtained and the time taken to obtain adsorption equilibrium time was 120 min. A quantity of 21.27 ± 1.26, 21.56 ± 0.48, 22.21 ± 0.64, and 23.02 ± 0.82 μg of ZEA was adsorbed by 1 mg of ACJC at equilibrium time for the different initial concentration of 25, 50, 75, and 100 μg/mL of ZEA, respectively. The reason behind the adsorption of ZEA by ACJC may be the involvement of strong attraction forces between the positive sites of cationic ZEA and anionic sites of ACJC (Lemke et al., 1998). The study has established ZEA adsorption capability of ACJC over short period of time and it could be effective for decontamination of ZEA.

The effect of different concentration of ACJC (0.5–4 mg/mL) on the adsorption of ZEA (100 μg/mL) was studied at pH 7. The concentration of ZEA adsorption was enhanced with an increase in the dose of ACJC. The kinetic model of ZEA adsorption (μg/mL) versus concentration of ACJC (mg/mL) for a period of adsorption equilibrium time (120 min) was depicted in Figure 5B. A quantity of 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mg/mL of ACJC has adsorbed 12.26 ± 0.20, 23.02 ± 0.82, 34.61 ± 0.52, 46.29 ± 0.95, 53.72 ± 0.67, 66.58 ± 0.86, 71.15 ± 1.46, and 86.92 ± 1.39 μg of ZEA with initial concentration of 100 μg/mL of ZEA, respectively. The quantity of ZEA adsorbed was increased with increasing the dose of ACJC and results showed that it was in the mode of dose-dependent. The reason for the increase in adsorption of ZEA was due to the availability of more number of surface adsorption sites resulting from the increased concentration of ACJC (Sayğılı and Güzel, 2016). The study concluded that the concentration of ACJC was a determinant factor for adsorption of ZEA.

The effect of pH on adsorption of ZEA by ACJC was studied by varying the pH (3, 4, 5, 6, 7, 8, and 9) of media with different concentration of ZEA (25, 50, 75, and 100 μg/mL) and 1 mg/mL of ACJC. The kinetic model of ZEA adsorption by ACJC (μg/mg) versus pH of media at adsorption equilibrium time (120 min) was showed in the Figure 5C. The results showed that adsorption of ZEA was noticeably not altered with a change in pH of media. Our result was in accordance with the previous report of Avantaggiato et al. (2014). The pH of media alters the surface charge of adsorbent and the degree of ionization of adsorbate, and it can alter the shift in kinetics and equilibrium of the adsorption process, and it is highly significant during the electrostatic interactions. Generally, the charge of the adsorbate depends on its pK_a value and the adsorbate exists mainly in protonated form at pH < pK_a and in deprotonated form at pH > pK_a. Therefore, the effect of pH on adsorption of ZEA by ACJC was dependents on the degree of ionization molecules. However, ZEA has pK_a value of 7.6 with diphenolic compound structure and some amount of phenolate anion present in water at pH 7 and the degree of ionization of ZEA was negligible. Therefore, the effect of pH on ZEA adsorption was negligible (Avantaggiato et al., 2014).

Desorption studies of different concentration of ACJC were undertaken with changing the pH of the media. The ZEA adsorption study was carried out at pH 7 with 100 μg/mL of ZEA and different concentration (0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 mg/mL) of ACJC for a period of adsorption equilibrium time and desorption studies were executed for period 30 min in the media of pH 3. The amount of ZEA desorbed from adsorbed amount of ZEA was observed as 1.59 ± 0.02, 2.77 ± 0.14, 4.08 ± 0.31, 5.38 ± 0.24, 7.06 ± 0.71, 9.24 ± 0.59, 10.79 ± 0.64, and 12.85 ± 0.72 μg/mL for 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 mg/mL of ACJC, respectively (Figure 5A). The results showed that low amount of ZEA was desorbed due to change in the pH of media and suggested that adsorption of ZEA by ACJC was stable with a change in pH of media.

The adsorption isotherms were deducted by plotting the quantity of ZEA adsorbed per unit of mass of ACJC (q_e) against the quantity of ZEA present in the external phase (C_e) under an equilibrium condition. The obtained data were analyzed by trial version of OriginPro 8.5 software program to fit the isotherm models of Langmuir and Freundlich by the equations (2) and (5), respectively (El-Sayed et al., 2014). The Langmuir model delivered well appropriate correlations for isotherm adsorption plots. The quantity of ZEA adsorbed per unit mass of ACJC was increased gradually by increasing the dose of ACJC. The isotherms presented an exponential graph and a typical Langmuir shape (Figure 6A). The isotherm provided a good fit line to experimental adsorption data with small variance and correlation coefficient (R²) of 0.9989. The separation factor or equilibrium parameter (R_L) of Langmuir isotherm model was obtained from Eq. 3 to evaluate the favorability of ZEA adsorption by ACJC. The R_L values were observed in between 0.0116 and 0.0451 and it suggested that adsorption of ZEA by ACJC was favorable (El-Sayed et al., 2014). The energy of adsorption (b) was observed as a 0.8460 mL/mg and the maximum ZEA adsorption capacity of ACJC was 23.14 μg/mg. The Freundlich isotherm model was constructed with ln q_e versus ln C_e following equation (5) (Figure 6B). The obtained correlation coefficient of 0.7049 suggested a high degree variance and model was not fitted (El-Sayed et al., 2014). The study concluded that adsorption of ZEA by ACJC was well fitted with Langmuir isotherm model rather than Freundlich, and these results also demonstrated the monolayer sorption of ZEA by homogeneous sites of ACJC (El-Sayed et al., 2014).

The results of the study concluded that ACJC as an efficient decontaminating agent for ZEA. Further investigation should be undertaken to propose a detailed binding mechanism of ZEA with ACJC. Also, regeneration studies should need to carry in detail to recover the adsorbent. So that it will enhance the economic feasibility of the decontamination process. Furthermore, a variety of mycotoxins will be encountered in food and feed matrices, and therefore, the competitive adsorption potential of ACJC should need to be assessed with multi-component mycotoxins.

In the therapeutic field, AC has found a diversity of application and used as an antidote to trap the swallowed chemicals, toxins, and overdose drugs in the gut and block their entry into the bloodstream. Therefore, the present study was lastly focused on to evaluate the adsorption feature of ACJC as an antidote against ZEA-induced cytotoxicity under in vitro studies. Many studies have proved the cytotoxicity of ZEA and reported that ZEA induces the toxicity by damaging the cellular membrane, generating intracellular ROS molecules, depleting the mitochondrial membrane potential (ΔΨ_M), and the apoptosis process (Venkataramana et al., 2014). The antidote action of ACJC against ZEA-induced toxicity was checked by assessing the cell membrane damage, generation of ROS molecules, mitochondrial membrane potential, and apoptosis.

The live/dead cell assay is a dual staining technique comprising of calcein AM and ethidium homodimer. The calcein AM is cell-permeant dye and in the live cells, calcein AM converted to a green-fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterases, and therefore, live cells appear in green under GFP filter, while ethidium homodimer crosses the damaged cell membrane and binds to the nucleus of the cell and emits bright red fluorescence in dead cells under RFP filter (Haugland et al., 1994). In present study, 8.69 ± 3.30% of live cells (green fluorescent cells) were noticed in ZEA treated cells and the percentage of dead cells (red fluorescent cells) were significantly (p < 0.05) high in ZEA treated cells compared to control cells and the results evidenced that ZEA-induced the cell death by damaging the cellular membrane (Figures 7A,B). On the other hand, cells exposed with ACJC were presented 95.46 ± 2.39% (p < 0.05) of live cells and results showed the non-toxicity of ACJC. The cells exposed together with ACJC and ZEA were exhibited 90.28 ± 5.44% (p < 0.05) of live cells and results indicated that ACJC has decreased the cytotoxicity of ZEA by lowering the availability of ZEA in media through adsorption.

The antidote action of ACJC on ZEA-induced toxicity in neuro-2a cells was also assessed by determining the intracellular ROS molecules by DCFH-DA probe. The DCFH-DA is a regularly used quantitative method to determine the oxidative species. The DCFH-DA is deacetylated to the non-fluorescent compound by cellular esterases and further oxidize to 2′,7′-dichlorofluorescein (DCF) fluorescent molecules by intracellular ROS (Kumar et al., 2016; Sellamani et al., 2016). In the present study, the percentage of intracellular ROS (267.24 ± 19.46%) was high in ZEA treated cells compared to control (p < 0.05). Another hand, percentage of intracellular ROS molecules was not much affected in ACJC treated cells (110.23 ± 8.06) and these results were in line with live/dead cells assay and confirmed the non-toxicity of ACJC (Figures 8A,B). The results suggested that ZEA induces the cytotoxicity by oxidative stress through the generation of intracellular ROS molecules (Venkataramana et al., 2014). When cells exposed to a combination of ZEA and ACJC, the percentage of intracellular ROS molecules (118.37 ± 10.79%) was relatively less and not affected (p < 0.05). The reason is that ACJC has lowered the availability of ZEA in media by adsorption, and therefore, the level of intracellular ROS molecules was not affected by ZEA.

The antidote action of ACJC on ZEA-induced toxicity in neuro-2a cells was assessed by determining ΔΨ_M using probe rhodamine 123. The rhodamine 123 is cell permeable, cationic probe, and emits green fluorescence by the sequestered action of metabolically active mitochondria and measured as an indicator for ΔΨ_M of the cell. The depletion in ΔΨ_M of cell ceases the synthesis of adenosine triphosphate (ATP) molecules and activates the apoptosis process (Scaduto and Grotyohann, 1999). In the present study, ΔΨ_M of cells was significantly (p < 0.05) depleted to 32.53 ± 6.17% on exposure of ZEA (Figures 9A,B) and another hand, ΔΨ_M was noticeably not affected by the treatment of ACJC (96.01 ± 1.85) compared to control (p < 0.05). Also, cells treated together with ZEA and ACJC was noticeably (p < 0.05) not exhibited depletion in ΔΨ_M (92.90 ± 3.66). The results concluded that ACJC has adsorbed the ZEA and lower availability of ZEA in media and therefore, ZEA has failed to deplete the levels of ΔΨ_M of the cell.

The apoptosis is the common way to induce the death of the cells by a variety of toxic compounds (Venkataramana et al., 2014). In the present study, antidote action of ACJC on ZEA-induced toxicity was determined by measuring the levels of caspase 3 using caspase-3 detection kit following the instructions of the manufacturer. The caspase 3 is an associate of the cysteine-aspartic acid protease family and its successive activation plays a principal role in the accomplishment of cellular apoptosis. The caspase 3 hydrolysis the acetyl Asp-Glu-Val-Asp 7-amido-4-methyl coumarin (Ac-DEVD-AMC) and releases the fluorescent AMC. The concentration of AMC released is directly proportional to the level of caspase 3 and apoptosis (Riss et al., 2016). The concentration of the AMC released was determined from the calibration curve of AMC standards. In the present study, the level of caspase 3 was elevated to 186.53 ± 12.66% on the treatment of ZEA compared to control (p < 0.05) and it concluded that ZEA induces the death of cells by apoptosis (Figure 10). While cells exposed with ACJC have noticeably (p < 0.05) not presented the elevated levels of caspase-3 (102.77 ± 3.31). The cells exposed to a combination of ZEA and ACJC was also noticeably (p < 0.05) not exhibited the elevated levels of caspase 3 (109.38 ± 5.93) and this is due to lower availability of ZEA in media by means of adsorption of ZEA by ACJC.

All the above studies collectively concluded that ACJC has established as an antidote against ZEA-induced cytotoxicity. Furthermore, in vivo studies are required to evaluate the application of ACJC as an antidote against ZEA-induced toxicity. However, in support of our study Abdel-Wahhab et al. (2015) has assessed the antidote action of AC on DON-induced cytotoxicity and genotoxicity in rat and concluded that AC could be better antidotes to treat DON-induced toxicity. The JECFA also evaluated the toxicity of AC prepared by the ZnCl₂ chemical method and recommended as food and feed additive to decontaminate a variety of toxic compounds (JECFA, 2010). Thus, the study concluded ACJC could be used as decontaminating and antidote against the ZEA-induced toxicity and the comprehensive conclusion of the study was presented in graphical design in Figure 11.