The hexane and ethyl acetate were bought from Vetec (Rio de Janeiro, Brazil). Ultrapure water was treated in a Milli-DI^® Water Purification System (Millipore^® Bedford, MA, USA). Lactate dehydrogenase (LDH) was bought from Millipore Sigma. Folin-Ciocalteu, sodium carbonate, gallic acid (GA), aluminum chloride (AlCl₃), sodium nitrite, sodium hydroxide (NaOH), epicatechin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), low and normal melting agarose, disodium ethylenediaminetetraacetate dihydrate (Na₂EDTA), ribonuclease (RNase), Triton^™ X-100, NaOH, and Tris-base were all obtained from Sigma-Aldrich^® (St. Louis, USA). Fetal bovine serum (FBS), streptomycin-penicillin and trypsin-ethylenediaminetetraacetic acid (EDTA) solution were acquired from Biochrom KG (Berlin, Germany). Dimethyl sulfoxide (DMSO) was bought from VWR Chemicals (Solon, OH, USA). Phosphate-buffered saline (PBS) was bought from Lonza (Lutterworth, UK). SYBR™ Gold was supplied by Thermo Fisher Scientific (Madrid, Spain).

Neem leaf samples were harvested in the year 2015 and kindly provided by Embrapa Coastal Tablelands (Brazilian Agricultural Research Corporation), in Aracaju, Sergipe, Brazil. After this, neem samples were dried for 36 h at 45℃ in a kiln with hot air circulating and then milled up to particle size ranges from 0.5 mm to 1.0 mm.

The sequential PLE process was performed using 20 g of neem leaf in a stainless-steel extractor with an internal volume of about 100 mL at 25℃ and the flow rate was set at 1.0 mL/min for 60 min under 100 bar, for each solvent. The hexane extract (HE) was obtained in the first step. After this, the residual hexane was removed from the neem sample by using carbon dioxide (CO₂) under depressurization from 30 bar to 0 bar. The dried neem sample was used in a new extraction carried out with ethyl acetate at 25℃, a flow rate of 1.0 mL/min for 60 min under 100 bar, for obtaining the ethyl acetate extract (EAE). Thereby, obtaining two extracts from the same neem mass was possible using two distinct solvents (hexane and ethyl acetate). The hexane and ethyl acetate solvents were evaporated from their extracts in a hot-air circulation oven at 40°C [21]. All the extracts obtained were frozen at –20°C until analysis.

The TK6 cells used in this study originated from the European Collection of Cell Cultures (Cat. No. 95111735). TK6 cells were cultured in the atmosphere of 5% CO_2, 37°C in Roswell Park Memorial Institute (RPMI)-1640 medium (Merck, Darmstadt, Germany) supplemented with 2% L-glutamine, 10% FBS, and 1% penicillin/streptomycin.

TK6 cells were seeded with the density of approximately 25,000 cells per well in 96-well microplates (for analyzes of cell viability by the MTT and LDH) and 40,000 cells per well in 12-well microplates (for the cell cycle, apoptosis, and comet assays) and then incubated overnight. On the following day, cells were incubated for 48 h with neem extracts (dissolved in DMSO) at concentrations ranging from 2–25 µg/mL. All experiments for MTT and LDH assays were carried out using negative control (NC) prepared with 1% DMSO and positive control (PC) with 1% Triton^™ X-100, both prepared in the cell culture medium.

The cell viability of the neem extracts was assessed by MTT assay, according to Mosmann [25], 1983. After incubation for 48 h, the microplate was centrifuged (200 g) for 5 min. Sequentially, neem extracts were removed and 200 µL of MTT (0.5 mg/mL) were added to each well. The microplate was incubated at 37℃ for 4 h in the absence of light, and then centrifuged (200 g) for 5 min. Afterward, the supernatant was removed, and the formazan crystals produced were dissolved into 100 µL of DMSO. Abs was measured at 570 nm in a Cambrex ELx808 microplate reader equipped with KC4 Data Analysis Software (Biotek, Winooski, VT, USA) [26]. Cell viability (%) was calculated as follows: (Abs-sample/Abs NC) × 100.

Membrane integrity was detected by measuring LDH released from TK6 cells after neem extract treatment. LDH from dead cells that leaked was quantitatively assessed by cytotoxicity colorimetric kit (Millipore Sigma, Roche, Cat. No. 11644793001), according to the manufacturer’s procedure. Abs was measured in a Bio-Rad model 680 microplate reader at 490 nm using 655 nm background correction. The results were expressed as LDH release (%) and calculated according to Malhão et al. [27].

After treatment, TK6 cells were centrifuged (1,000 g) for 5 min. Then, the cells were resuspended in PBS and fixed in pre-chilled 70% ethanol at 4ºC overnight. Sequentially, the suspension cell was centrifuged for 5 min at 1,000 g. After PBS, the cell pellets were centrifuged (1,000 g) again for 5 min. The collected pellets were resuspended in 500 µL of staining solution of PI at 40 µg/mL and RNase (50 µg/mL), prepared in PBS (flow cytometry grade) and incubated for 30 min at 37°C. Cells (%) in each cell cycle phase [sub-G1 (apoptosis), G0/G1, S, and G2/M] were measured using a Guava^® easyCyte™ 8HT Flow Cytometer (Luminex, Austin, TX, USA), according to Valdiglesias et al. [28]. For this, a minimum of 10,000 events were acquired and evaluated using Guava™ Software version 3.1.1.

To evaluate the DNA damage, the alkaline comet assay was performed according to Costa et al. [29], with slight modifications. TK6 cells treated with neem extracts for 48 h were washed twice with prechilled PBS. Sequentially, TK6 cells were centrifuged at 1,000 g for 5 min. Cell pellets obtained were resuspended in 200 µL of 0.6% low melting point agarose (LMA). Then, the cells were dropped onto a frosted slide previously coated with 1% normal melting point agarose (2 replicates in each slide). The slides were placed on ice for 5 min and then immersed in a lysis solution [100 mmol/L EDTA, 2.5 mol/L sodium chloride, 10 mmol/L Tris-base, 10 mol/L NaOH (pH 10), and 1% Triton^™ X-100] at 4℃ for 1 h, in the absence of light. Sequentially, slides were placed on a horizontal tank and immersed in a freshly made alkaline electrophoresis solution (0.3 mol/L NaOH and 1 mmol/L Na₂EDTA) for 40 min in the absence of light. Electrophoresis was performed for 20 min at 1.15 V/cm. Then, the slides were washed for 10 min with prechilled PBS at 4℃, 10 min with prechilled ultrapure water at 4℃, fixed with 70% and 96% ethanol for 15 min (for each solvent) at room temperature, and dried overnight. Before analysis, slides were stained with SYBR™ Gold for 30 min. After that, the slides were visualized at a magnification of 250× using a Nikon Eclipse E400 microscope equipped with an epi-fluorescence attachment and analyzed by using Comet Assay IV software (Perceptive Instruments, Suffolk, UK). At least 100 cells were scored and DNA in the comet tail (TDNA) was used as the DNA damage parameter (%) [30].

Results are shown as a mean ± standard error of the mean (SEM). Statistical analysis was performed with GraphPad Prism 5.0 (La Jolla, California, USA). A unidirectional variance analysis (ANOVA), followed by a post hoc Tukey’s test, was carried out to verify the differences between the control and exposed cells. The in vitro cytotoxicity at half-maximal inhibitory concentration (IC₅₀) value was obtained through nonlinear regression of the dose-response curve fit. The significant difference was concluded at a P < 0.05.

In general, the results of this present study demonstrated that flavonoids and other phenolic compounds were present in and EAE extracts (Figure 1). In the HE, the TPC-HE and TFC-HE were respectively 3.20 mg/g of extract ±1.20 mg/g of extract and 14.61 mg/g of extract ± 0.60 mg/g of extract. A new extraction using ethyl acetate, in the same sample extracted by hexane, allowed a significantly higher amount of TFC-EAE (65.50 mg/g of extract ± 1.17 mg/g of extract) and TPC-EAE (52.08 mg/g of extract ± 0.88 mg/g of extract).

This study used MTT and LDH assays to identify cytotoxicity in TK6 cells. According to the results of the MTT assay (Figure 2), HE and EAE significantly decreased TK6 cell viability at concentrations between 15 µg/mL and 25 µg/mL; this decrease in viability is more pronounced after exposure to EAE. The EAE at 25 µg/mL was more significantly cytotoxic to TK6 cells than HE at 25 µg/mL. However, EAE at 25 µg/mL showed a similar result to PC. This result is indicative of a high level of cell damage and toxicity caused by EAE at 25 µg/mL.

IC₅₀ values were calculated based on these results and were 18.88 µg/mL ± 0.35 µg/mL for HE, and 16.62 µg/mL ± 0.05 µg/mL for EAE. Three concentrations of each extract were chosen for the other experiments based on the results obtained in the cell viability experiments (IC₅₀).

LDH level was performed to quantitatively assess by measuring the activity of LDH released from the damaged or dead TK6 cells after exposure to concentrations from 10 µg/mL to 20 µg/mL of HE and EAE for 48 h. As shown in Figure 3, the EAE 20 µg/mL caused a more significant LDH leakage to TK6 cells than HE 20 µg/mL. LDH leakage increased significantly from 30.51% ± 1.82% to 62.31% ± 6.41% after exposure to EAE 15 µg/mL and EAE 20 µg/mL, respectively.

The possible effect of HE and EAE on the induction of cell apoptosis was investigated by evaluation of subregion G0/G1 of the cell cycle distribution, indicating fragmentation of DNA in late apoptotic stages [31, 32]. In all tested concentrations, the cells in the sub-G0/G1 (%) region increased after treatment with HE and EAE (Figure 4A and B).

The induction of apoptosis was higher after exposure to EAE when compared to HE; the treatment with the same concentration (15.0 µg/mL) of HE and EAE led to 2.52% and 11.29% of cells in the subregion G0/G1, respectively. In addition, EAE induced apoptosis in a dose-dependent which was not seen after treatment with HE.

The results in Figure 5A and B show that both extracts, HE and EAE, were able to alter the TK6 cell cycle after a treatment of 48 h, with an arrest in the G0/G1 phase. The cells (%) in G0/G1 phase increased from 72% (NC) to 83% after treatment with 15 µg/mL of either HE or EAE. In consequence, a reduction in the S and G2/M phases was observed in all tested conditions.

The results in Figure 6A and B exhibit the DNA damage in TK6 cells after a 48-h treatment with various concentrations of EAE and HE.

DNA damage increased significantly in TK6 cells after treatment with HE and EAE, compared to the NC in all tested concentrations. The induced DNA strand breaks were similar for both HE and EAE.