To a small quantity of plant extract, 20 mL of distilled water was added and shaken in a graduated cylinder for 15 min lengthwise. Formation of foam indicated the presence of saponins.

The antioxidant activity of WETP was measured using the 2,2-diphenyl-2-picrylhydrazyl (DPPH) method. One millilitre of DPPH (0.01mM) was added to 3 mL of METP at various concentrations. The reaction mixture was then incubated in the dark at room temperature for 30 min. The absorbance was measured at 517 nm against a blank. The free radical-scavenging activity of the plant extract was determined by comparison with that of the methanol control. A blank was prepared without the addition of the extract. Ascorbic acid at various concentrations (10, 20, 30, 40, and 50 μg/ml) was used as the standard. A lower absorbance of the reaction mixture indicated a higher free radical scavenging activity. DPPH radical scavenging ability was calculated using the following equation:

Measurement of superoxide anion scavenging activity of different extracts was performed based on the method described by Nishimiki 16 with slight modifications. About 1 mL of nitroblue tetrazolium (NBT) solution (156 μM NBT in 100 mM phosphate buffer, pH 7.4), 1 mL NADH solution (468 μM in 100 mM phosphate buffer, pH 7.4) and 0.1 mL of sample solution of S. anacardium in water were mixed. The reaction was initiated by adding 100 μL phenazine methosulphate (PMS) solution (60 μM PMS in 100 mM phosphate buffer, pH 7.4) to the mixture. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against blank samples. The decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The % of inhibition was calculated by using following equation; % Inhibition= [(Absorbance control – Absorbance sample)/Absorbance control] x 100

The potential mutagenic effects of the plant extracts were investigated using a plate incorporation procedure 17. The assay was performed using the Salmonella typhimurium strains TA1535 and TA1538. Approximately 100 μl of the bacterial stock was incubated in 20 mL of Oxoid Nutrient broth for 16 h at 37°C on an orbital shaker. The overnight culture (0.1 mL) was added to 2 mL of top agar (containing traces of biotin and histidine) together with a 0.1 mL test solution (the plant extract, solvent control, or positive control) and 0.5 mL phosphate buffer (for exposure without metabolic activation). The top agar mixture was poured onto the surface of the agar plate and incubated for 48 h at 37°C. Following the incubation, the number of revertant colonies (mutants) was counted. All cultures were prepared in triplicate (except for the solvent control, where five replicates were performed) for each assay. This assay was repeated twice. The positive control (reference standard) used was sodium azide at a concentration of 5 μg/plate for the TA 1535 strain, and the positive control (Reference standard) used was nitrofluorine at a concentration of 5 μg/plate for the TA 1538 strain. Determination of mutagenic and antimutagenic potential of Semecarpus anacardium occurs via two different mechanisms [base pair and frame shift mutation] in Salmonella typhimurium strains.

Experimental groups

The Allium cepa assay is an efficient test for chemical screening and in situ monitoring of the genotoxicity of environmental contaminants. The test organism is usually a common onion of the Stuttgarter variety, which is mainly having the size 15-22 mm and weight of 2-4 g. Allium cepa chromosomal aberration test was carried out according to the methods described by Ragunathan et al. and Ping et al. 18, 16. Cell proliferation [mitotic induction or depression] and chromosomal abnormalities in the meristematic root cells of Allium cepa have been evaluated.

Experimental groups

Onions, as specified above, were kept for 4-5 days in different beakers filled with water for proper root growth. They were exposed to different concentrations (50,100,200 µg/mL) of Semecarpus anacardium in Petri dishes, and cyclophosphamide (40µg/mL) was used as the positive control. For detection of protective effect of Semecarpus anacardium, a few onions were exposed to cyclophosphamide in presence of the same concentration range of Semecarpus anacardium mentioned above at the same time. After 24 h (at least one mitotic cycle), chemically exposed roots of the different onions were collected in glass vials containing a fixative solution of methanol and glacial acetic acid (3:1) and stored overnight at 4°C.

The next day, roots were placed in a clear Petri dish containing a mixture of 2% aceto orcein and 1N HCl in a 5:1 ratio. Next, the Petri dish was heated gently for 2-5 sec in a yellow flame because it emits less heat and was kept for 1-1.5 h. Subsequently, a small drop of 45% acetic acid was placed on a clear microscopic glass slide, and the root tip (approximately 1-2 mm) was placed on the glass slide with acetic acid. It was then kept for a few seconds to wash off excess stain. A cover slip was also placed above it in such a way that no air bubbles were formed, and the root tips were squashed slightly with a gentle tap of the thumb or matchstick. Finally, the open interface of the coverslip and slide was sealed with paraffin or DPX so that the cells would not dry. Five slides were prepared and analysed for each treatment group to determine the mitotic index and various chromosomal aberrations.

The antigenotoxic activity of Semecarpus anacardium against cyclophosphamide-induced genotoxicity in mice bone marrow erythrocytes was evaluated.

Experimental groups

Micronucleus test procedure

Micronucleus assay was performed according to OECD guidelines (TG-474) 20. Thirty animals were assigned to this assay, with six animals per group. One group of animals (Group 1) was used as a control and was administered distilled water. The second group (Group 2) was treated with cyclophosphamide (40 mg/kg body weight) i.p. for two consecutive days, and the last three groups (Group 3, Group 4, and Group 5) were given Semecarpus anacardium 50 mg/kg, 100 mg/kg, and 200 mg/kg body weight orally along with cyclophosphamide. Semecarpus anacardium leaf extract was dissolved in water and administered in divided doses before and 4 h after cyclophosphamide injection. After 48 h of treatment, the animals were sacrificed, and the femur bone was collected. The proximal ends of the bone were opened and the bone marrow was flushed into a centrifuge tube with chilled phosphate-buffered saline (pH-7.4).

The bone marrow suspension was centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and the pellets were re-suspended with 0.2 mL of the same phosphate buffer saline. A drop of the cell suspension was placed on a clear slide and smeared. The smear was air-dried and fixed with methanol for 10 minutes. After fixation, slides were stained with Giemsa solution (1x) for 15 min and covered with dibutyl phthalate Polystyrene Xylene (DPX) to make the slides permanent. 1000 polychromatic erythrocytes (PCEs) were observed at 1000x magnification with oil immersion for each treatment group.

Chromosomal aberration test was performed according to previously published methods 24. The same grouping of animals (Group 1 to Group 5) and treatments were maintained in this method, as mentioned in section 2.5.1, with six animals per group. Colchicine (metaphase arresting agent) was administered to animals at 4 mg/kg, i. p., 3 h before sacrifice. After that, the animals were sacrificed, and both femur bones were dissected, followed by opening of the proximal end of the bones with a small incision. The bone marrow was flushed with 1.5 mL of 2.2% freshly prepared chilled sodium citrate solution in a centrifuge tube and centrifuged at 3000 rpm for 10 min. After centrifugation, the supernatant was removed and 1.5 mL of 0.075 M potassium chloride solution was added to the centrifuge tubes, and the cells were re-suspended and incubated for 30 min at 37°C. After incubation, the cell suspensions were centrifuged, and the supernatant was replaced with freshly prepared cold fixative methanol and glacial acetic acid (3:1) and kept for 10 min. The suspensions were then centrifuged for 10 min at the same rpm (3000 rpm). Afterwards, the supernatants were discarded by leaving one or two drops of fixative with cell pellets. Cells were smeared by dropping the cell-fixative suspension in clear glass slides and air drying for 10 min.

Air-dried slides were then stained with 1x Giemsa stain for 15 min and washed gently in water to remove excess stain. Finally, the slides were dried and mounted with DPX. At least five slides were made for each treatment group, and 100 well-separated metaphase chromosomes were analyzed for any type of aberration.

Statistical analysis

Statistical analysis was performed using one-way ANOVA followed by Dunnett’s comparison test, and all results are presented as mean ± standard error of the mean (SEM). GraphPad Prism software version 5 was used for the statistical analysis.

The half-maximal inhibitory concentration, also known as the IC50 value, is denoted in µg/mL, and it is said that the lower the IC50 value, the greater the free radical scavenging by the antioxidant compound. The IC50 value of ascorbic acid was 7.283 µg/mL, and the IC50 value of leaf extract of Semecarpus anacardium leaf extract was 37.239 µg/mL. The DPPH-scavenging activity of the leaf extract of Semecarpus anacardium and the reference standard ascorbic acid are presented in Table 1.

The half-maximal inhibitory concentration, also known as the IC50 value, is denoted in µg/mL, and it is said that the lower the IC50 value, the greater the free radical scavenging by the antioxidant compound. The IC50 value of ascorbic acid was 11.432 µg/mL and IC50 Value of Semecarpus anacardium leaf extract was 34.191 µg/mL (Table 2).

The AMES test was carried out to evaluate the genotoxic and antigenotoxic potential of Semecarpus anacardium leaf extract. The assay was performed without metabolic activation which was carried out by addition of the S9 mix, and the number of reverted colonies was counted. Based on these results, it can be concluded that Semecarpus anacardium leaf extract at a dose of 10 mg/plate is non-mutagenic. It was also noted that in the presence of mutagens, the number of revertant (histidine +) colonies of Salmonella was not reduced, suggesting that the Semecarpus anacardium leaf extract does not have any antimutagenic properties. The TA 1535 strain was used for checking the base pair mutation, and the TA 1538 strain was used for observing frameshift mutations. The results are presented in Table 3 and Figure 1.

Using this assay, the mitotic depressant and genotoxic/antigenotoxic activity of Semecarpus anacardium leaf extract were evaluated. The mitotic index (M.I) was calculated using the following formula: Mitotic index (M. I) = [(number of dividing cells/total number of cells) × 100].

The leaf extract of Semecarpus anacardium considerably reduced the number of dividing cells in a dose-dependent manner. There was a significant reduction in the mitotic index from 2.6 ± 0.830 to 2.2 ± 0.372 when the dose of leaf extract was increased from 50 µg/mL to 200 µg/mL showing that it has mitotic depressant activity in a dose dependent manner (Table 4 and Figure 2).

Allium cepa cells were subjected to chromosomal analysis to study the effect of Semecarpus anacardium leaf extract on cyclophosphamide-induced aberrations. A significant reduction in the chromosomal aberration percentage was visible as they were reduced from 1.3 ± 3.56 to 0.9 ± 2.53, 0.5 ± 2.67 and 0.3 ± 2.15 in Group 3, Group 4, and Group 5 in a dose depended manner (Table 5 and Figure 3).

Micronucleus assay was performed and the number of micro nucleated polychromatic erythrocytes (MNPCEs) were counted for the determination of antigenotoxic (protective) effect of Semecarpus anacardium leaf extract on mice bone marrow erythrocytes. The leaf extract significantly reduced the number of MNPCEs (P < 0.05) in a dose-dependent manner when administered simultaneously with cyclophosphamide. The MNPCE count in the positive control cyclophosphamide (40 mg/kg) group was 29 ± 1.623. However, when 50, 100, and 200 mg/kg Semecarpus anacardium leaf extract was administered along with cyclophosphamide in Gr-3, 4, and 5, a significant reduction in count to 16.66 ± 1.441, 9.33 ± 1.247, and 5 ± 0.816, respectively, was observed in a dose-dependent manner. The maximum percentage reduction in the micronucleus polychromatic erythrocyte cell count was observed in Group 5 (82.75%) (Table 6 and Figure 4).

The same treatment plan was followed in the micronucleus assay. Metaphase arrested cells of about hundred in number were observed in each group, for chromosomal abnormalities and the observations were made. The aqueous extract of Semecarpus anacardium reduced the number of chromosomal abnormalities induced by cyclophosphamide in mouse bone marrow cells. The total aberration % was calculated using the following formula:

Total aberration % = (Total no of aberrations / 500) × 100

The total aberration % was found to be 25.69 ± 0.32% in cyclophosphamide-treated group 2, which was reduced significantly to 16.33 ± 1.57, 9.23 ± 1.64%, and 6.12 ± 1.09 in groups 3, 4, and 5, respectively, in a dose-dependent manner when the leaf extract was administered along with cyclophosphamide (Table 7 and Figure 5).