Chemicals
2,2-diphenyl-1-picrylhydrazyl (DPPH), 2’,7’-dichlorofluorescin diacetate (H2DCFDA), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS), monochlorobimane (MCB), Lipopolysaccharides (LPS) and primers from Sigma; DMEM and Fetal bovine serum (FBS) from HiMedia; FBS from Invitrogen; RPMI-1640, L-Glutamine, oligo-(dT) primer, M-MLV reverse transcriptase from Thermo Fisher Scientific and all other chemicals of AR grade were procured from SRL India Ltd and Merck India LTD.
Plant materials
T. portulacastrum L. plants were collected from fields in Kalyani, Dist. Nadia, West Bengal, India and were authenticated from the Department of Botany, University of Kalyani, Kalyani, Nadia (Voucher No. UD-101).
Preparation of TP extracts: Dried powder of different parts of T. portulacastrum such as leaves, stem and whole plant (100 g) was extracted with 500 ml petroleum ether for 24 h with constant shaking and filtered. This process was repeated twice. Ethyl acetate, acetone and ethanol solvent were used twice sequentially followed by petroleum ether. All the solvents were evaporated and dried. Further studies were carried out with ethanolic fractions.
Antioxidant capacity study
Antioxidant capacity of the different extracts of the TP was measured using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) (Maurya and Devasagayam 2010) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Maurya and Devasagayam 2010) radical scavenging assays, whereas metal reducing power was evaluated using ferric reducing power assay (FRPA) (Maurya and Devasagayam 2010) and molybdenum reduction assay (MRA) (Saxena et al. 2016).
Cell Lines:
Human hepatic cells (WRL 68) were cultured in Dulbecco’s modified Eagle’s Medium (DMEM) and RAW 264.7 cells in RPMI-1640 medium with L-Glutamine. For culturing both the cell line media were supplemented with 10% FBS (Thermo Fisher Scientific, 10500064) and 1% Pen-Strep (Thermo Fisher Scientific, 15140-122).
Evaluation of radioprotective property of T. portulacastrum extract
Radioprotective property of T. portulacastrum extract was evaluated using sub-cellular and cellular model systems. For sub-cellular assay, we have used murine mitochondrial membrane whereas for cellular assay we have employed human hepatic cells (WRL 68) as a model system.
Evaluation IR-induced lipid peroxidation
For evaluation of lipid peroxidation mouse mitochondrial membrane and human hepatic cells were used. Mitochondrial membrane fractions were isolated from the liver of male Swiss mice as described (Checker et al. 2010). Damage to the mitochondrial membrane fraction after radiation exposure was assessed in terms of lipid peroxidation (Maurya and Devasagayam 2010). Mitochondrial membrane fraction (a protein equivalent of 300 µg) was suspended in 300 µl of 10 mM potassium phosphate buffer, pH 7.4, and exposed to 50 Gy radiations in the absence and presence of different concentrations of TP extracts (pre-treated for 30 min at 37°C). After treatment, 900 µl TBA reagent (0.375% TBA, 0.25 M HCl, 15% trichloroacetic acid (TCA) and 6 mM Na2-EDTA) was added. The reaction mixture was incubated at 950C for 20 min, cooled to ambient temperature and centrifuged at 12,000 g for 5 min at 250C. Malondialdehyde (MDA) equivalents in the supernatant were estimated by measuring the fluorescence (as fluorescence provide more sensitivity) with excitation at 530 nm and emission at 590 nm using a microplate reader.
WRL 68 (2x106) cells were collected and treated with different T. portulacastrum extracts for 1 h at 370C for IR-induced cellular lipid peroxidation study inhuman hepatic cells. Subsequently cells were exposed to 8 Gy of IR. IR-induced cell membrane damage was assessed in terms of lipid peroxidation as described above [(Maurya and Devasagayam 2010).
MTT assay
To study the cytotoxicity and proliferation of cells after IR exposure, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-difenyl-tetrazolium bromide) was used. In brief, 15×103 WRL 68 cells were seeded in 96-well plate one day prior to addition of extract. Next day cells were treated with different concentration of the extracts and incubated for 1 h. These extract treated cells were exposed to 4 Gy of IR. After completion of incubation period, MTT assay was performed by adding 10 µl MTT solution (10 mg/ml) to each well. Formazan crystals formed inside the cells were dissolved by adding 100 µl of solubilizing buffer (0.01 N HCl in 10% SDS) and incubated for overnight at 37°C. The absorbance was measured at 570 nm using Synergy Bio-Tek (USA) microplate reader (Maurya et al. 2011).
Clonogenic assay
The clonogenic assay was used to evaluate the radioprotective efficiency of TP extract using WRL 68 cells. In brief, 2x103 exponentially growing cells were plated in a 6-well plate for overnight. Next day, cells were treated with different concentration of T. portulacastrum extracts 1h before 4 Gy IR-exposure. After irradiation, cells were cultured for 12 days at 37°C in CO2 incubator for the development of macroscopic colonies. The colonies were fixed with methanol, stained with 0.5% crystal violet and counted using a colony counter (Oxford Optronix, UK) (Jayakumar et al. 2015).
Evaluation Of The Cellular Redox Status
To study the mechanism of the T. portulacastrum extracts for radioprotection, cellular redox study was carried out. For this cellular ROS and intracellular thiol (GSH) levels were monitored using H2DCFDA and monochlorobimane (MCB) fluorescence dye respectively.
Measurement of cellular ROS level
2’,7’-dichlorofluorescin diacetate (H2DCFDA) is a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. 4×106 WRL-68 cells were incubated with 10 µM H2DCFDA in serum-free medium by incubating at 37°C for 45 min. After incubation, cells were washed to remove the excess dye and suspended in the phosphate-buffered saline (PBS, pH = 7.4). For studying inhibition of radiation-induced ROS formation, cells were exposed to IR in presence of different concentrations of the T. portulacastrum extracts and incubated at 370C for 1 h. The fluorescence intensity of the oxidized probe was read using a microplate reader (excitation/emission wavelength, 485/ 520 nm) (Maurya and Devasagayam 2010).
Intracellular GSH
Monochlorobimane (MCB) is a fluorescence dye which has a high affinity for GSH. In this study, MCB was used to measure cellular thiol (GSH) levels. WRL-68 cells were incubated with the different concentrations of T. portulacastrum extracts for 1 h at 37°C. Monochlorobimane (40 µM, 30 min at 37°C) was used to measure the level of the GSH in these cells. Fluorescence emission (excitation/emission wavelength, 380/460) from cellular sulfhydryl-reacted monochlorobimane was measured using a microplate reader (Checker et al. 2010).
Evaluation Of Anti-inflammatory Property Of Tp Extract
The anti-inflammatory effects of T. portulacastrum extract in LPS-stimulated RAW 264.7 macrophages were evaluated by nitric oxide (NO) assays and quantitative real-time reverse transcription-polymerase chain reaction analysis of expression of inflammatory genes. For studying the cytotoxicity in RAW 264.7 activated with LPS and treated with the T. portulacastrum extract, MTT assay was performed as described previously. GSH concentration was measured through Ellman's reagent and calculated from standard curve using pure GSH (Moron et al. 1979).
Nitric oxide (NO) assay
RAW 264.7 cells (0.5×105 cells/well) were seeded into 96 well plates for 24 h. Next day, cells were pre-incubated with different concentrations of T. portulacastrum extracts (0, 31.25, 62.5, 125 µg/ml) for 1 h and further stimulated with 500 ng/ml of LPS. The culture supernatants were collected 24 h after the LPS stimulation, and the concentrations of NO were measured using Griess reagent. 100 µl of culture supernatant was mixed with 100 µl of Griess reagent (sulfanilamide 1%, 2% phosphoric acid and 0.1% NEDD in water) and the mixture was incubated at room temperature for 10 min before measuring the absorbance at 550 nm. In all experiments, fresh culture medium was used as the blank and sodium nitrite was used as the standard (Kacem et al. 2015).
Semi-quantitative PCR: For semi-quantitative PCR, RAW 264.7 cells were pretreated with T. portulacastrum extract for 1 h followed by LPS treatment. Total cellular RNA was extracted using Trizol following manufacturer’s protocol. cDNA was synthesized from RNA using oligo-(dT) primer by M-MLV reverse transcriptase (Thermo Fisher, 28025-013). Specific primers for TNF-α, Nrf- 2 and iNOS were used for PCR reactions and then run on 1.5% Agarose gel. GAPDH primers were used for normalization of mRNA quantity respectively (Ahuja et al. 2016). The following primers were used for semi-quantitative PCR: iNOS (forward 5ʹ-TTCTTCCAGCTCAAGAGCCAGAAA-3ʹ; reverse 5ʹ-GGGATTGCATTTCGCTGTCT-3ʹ), Nrf2 (forward 5ʹ-CCCGAATTACAGTGTCTTAATACCG-3ʹ; reverse 5ʹ- AGGTGGGATTTGAGTCTAAGGA-3ʹ), TNF-α (forward 5ʹ-ATGGCCTCCCTCTCATCAGTTC-3ʹ; reverse 5ʹ-GGGAGTAGACAAGGTACAACCC-3ʹ), GAPDH (forward 5ʹ-TGATGACATCAAGAAGGTGGTGAAG-3ʹ; reverse 5ʹ-TCCTTGGAGGCCATGTGGGCCAT-3ʹ).
Statistical analysis
All experiments with T. portulacastrum extracts were performed in triplicate and mean ± standard error (SE) of each triplicate result was considered for statistical analysis. Analysis of results was performed using the Statistical Package for Social Science, version 23 (SPSS, Chicago, Illinois) software. Significant differences were assessed through the one-way analysis of variance (ANOVA), followed by the Tukey test for individual differences. A value of P < 0.05 was used to evaluate statistical significance.