Reagents
Marketed preparations of cyclophosphamide (injections, Sigma–Aldrich), cyclosporine (ampoules, Biocon Ltd., India), ketoconazole (tablets, Albatross Pharmaceuticals, India), ampoxin (injections, Unichem Laboratories, India), and cisplatin (MP Biomedicals ) were used in the study. Fetal bovine serum (FBS), trypsin EDTA solution, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), antibiotic and antimycotic preparations, trypan blue 0.4%, and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from HiMedia Laboratories Pvt. Ltd., India. 4, 6 Diamino-2 phenylindole (DAPI), 2, 7-dichlorofluoresceindiacetate (DCFH-da) dyes were purchased from sigma.
Cell line and animals
The A549 human lung cancer cell line was obtained from the National Centre for Cell Science (NCCS), Pune, Maharashtra, and was maintained at 37 °C in RPMI-1640 medium supplemented with 10% FBS under 5 % CO2 and humidified condition in a CO2 incubator [19, 20]. Six-week-old C57bl6 mice were obtained from Mahaveera Enterprises, Hyderabad, India (Registration no: 1656/PO/bt/S/12/CPCSEA). Animals were of both sexes. The average weights of animals were between 25 and 30 g. The protocol for the study was presented in the Institutional Animal Ethics Committee (IAEC) meeting, and was approved on 20/10/2018. An approval certificate was issued by the committee with Protocol number: PERD/IAEC/2018/014.
Collection of plant material
Uraria picta (Jacq.) DC was collected in the month of October 2018 from Gandhi Bazar, Ahmedabad, Gujarat. The plant was authenticated by a taxonomist. A voucher specimen was submitted to the department (Voucher specimen number: BVPPERD/PP/1118/809). Plant material was shade dried, powdered, and stored in an airtight container.
Preparation of methanolic extract of Uraria picta (Jacq.) DC
Powdered aerial plant parts of Uraria picta were weighed (300 g) and suspended in a conical flask containing methanol (2 L). The suspension was kept undisturbed for 3 days with occasional shaking. The procedure was repeated twice with the remaining marc to achieve the complete extraction of phytoconstituents. Total filtrate obtained was combined and kept in a rotavapor apparatus at 50 °C to evaporate methanol. Methanolic extract of Uraria picta (MEUP) after solvent evaporation was combined, weighed and its percent yield was calculated [21].
Preliminary phytochemical screening of extract
The preliminary phytochemical analysis of the MEUP was carried out to confirm the presence of different classes of the phytoconstituents in the extract including tannins, flavonoids, alkaloids, saponins, glycosides, and polyphenols [22].
Estimation of total phenolic content
Estimation of total phenolics in MEUP was carried out. Extraction of powdered plant material (500 mg) was done in 50% aqueous methanol. Filtrate was collected after extraction and the volume of the filtrate was made up to 50 ml with methanol. For estimation, 10 ml of double-distilled water and 1.5 ml of the Folin–Coicalteu reagent was added to 0.1 ml of the extract; and the solution was incubated for 5 min. To this, 4 ml of 20% sodium carbonate was added and the volume of the solution was made up to 25 ml with double-distilled water, mixed thoroughly, and incubated at room temperature for 30 min. The absorption of the mixture was recorded at 765 nm. Gallic acid (100 μg/ml) was used as standard. Total phenolic content in MEUP was expressed as gallic acid equivalent (GAE) [23].
Estimation of total flavonoid content
Estimation of total flavonoid in MEUP was done by adding 125 μL of MEUP solution in methanol to 75 μL of 5% NaNO2 solution. The resulting mixture was allowed to stand for 6 min, followed by the addition of 150 μL of aluminum trichloride (10%) and further incubation for 5 min. The final volume of solution was adjusted to 2.5 ml with distilled water after the addition of 750 μL of NaOH (1 M), incubated for 15 min, and allowed to acquire a pink appearance. Absorbance was measured at 510 nm. Catechin was used as standard. The total flavonoid content in MEUP was expressed as catechin equivalent (CE) [24].
MTT assay
The MTT assay is based on the principle that the mitochondrial dehydrogenase enzyme of live cells reduces the yellow MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) dye into purple formazan crystals [25]. MTT assay was done to assess the cytotoxicity of MEUP against A549 cancer cells. A549 human lung cancer cells were maintained at 37 °C in RPMI 1640 medium supplemented with 10% FBS under 5% CO2 and 95% air in a CO2 incubator. Cells were seeded in a 96 well plate (10,000cells/well) and incubated for 24 h. After the incubation period, media was discarded and cells were treated with different concentrations of MEUP (37.5 μg/ml, 75 μg/ml, 150 μg/ml, 300 μg/ml, 450 μg/ml, 600 μg/ml and 750 μg/ml) and cisplatin (4.5 μg/ml, 9.0 μg/ml, 13.5 μg/ml, 18.0 μg/ml) followed by incubation for another 24 h. The assay was carried out in triplicate. After 24 h, treatment media was discarded and MTT (5 mg/ml) was added in each well and incubated for 4 h. After incubation, MTT was discarded from the plate. Formazan crystals were solubilized by adding 100 μl of DMSO in each well. A microplate reader (BioTek, USA) was used to record the absorbance at 570 nm. The absorbance of untreated control well was taken as 100% and relative percent cell viability was calculated taking absorbance obtained from untreated control well as 100%. % cell viability was calculated by the formula:
Percent cell viability = (Absorbance of test × 100)/Absorbance of control [26]
IC50 values of cisplatin and MEUP were calculated by a four-parameter logistics model using Sigma Plot 12.0 (Systat Softwares Inc).
Trypan blue exclusion assay
The trypan blue exclusion assay is based on the principle that dead cells take the trypan blue dye due to their compromised cell membrane permeability [27]. The assay was carried out to determine the cytotoxicity caused by MEUP to A549 cancer cells. A549 cancer cells were seeded in 6 well plates (0.5 million/well) and kept for 24 h at 37 °C and 5% CO2 in a CO2 incubator. After 24 h, media was discarded and cells were treated with different concentrations of MEUP (300 μg/ml, 450 μg/ml, and 600 μg/ml). Cisplatin was taken as the positive control (IC50 concentration). After treatment, detached cells in media were taken in a tube while the live cells were harvested using trypsin and added in the same tube. A total of 100 μl of cells were taken and mixed with 100 μl of 0.4% trypan blue dye (1:1) and kept undisturbed for 4 min. A drop of the mixture was taken in the Neubauer chamber and observed under the microscope and cell counting was done. Dead cells were identified by their ability to take blue color while the live cells did not take any color. Percent cell death was calculated by the formula [28]:
$$ \mathrm{Percent}\ \mathrm{cell}\ \mathrm{death}=\mathrm{Dead}\ \mathrm{cell}\mathrm{s}/\mathrm{Total}\ \mathrm{cell}\mathrm{s}\ \mathrm{X}\ 100 $$
Evaluation of apoptosis (DAPI staining)
4, 6 Diamino-2 phenylindole (DAPI) is a dye that binds to DNA strands robustly. The binding of DAPI with DNA strand gives fluorescence that can be detected under the fluorescence microscope [29]. For this, cells were seeded in a 12 wells plate (1 million cells/ well) and kept in a CO2 incubator under a humid condition for 24 h at 37 °C and 5% CO2. After incubation, media was discarded and cells were washed with PBS, followed by the treatment with cisplatin (IC50 concentration) and different concentrations of MEUP (300 μg/ml, 450 μg/ml, and 600 μg/ml) for 24 h. After treatment, 1 ml of methanol was added in each well for 20 min for cell fixation. Cells were then washed with PBS followed by the addition of DAPI (300 nM) to each well and incubated for 30 min. After incubation, dye was decanted from each well and cells were washed with PBS (1–2 ml) twice. Observations were made under a green fluorescence microscope after adding PBS [30, 31]. For quantitative estimation of effects of increasing concentration of MEUP on nuclear morphology, numbers of apoptotic cells/100 cells were measured [32, 33].
Evaluation of antioxidant effect: determination of intracellular ROS
DCFH-da assay was carried out to assess the ability of MEUP to reduce the intracellular reactive oxygen species. Cells were seeded in a 12-well plate (1 million/well) and incubated for 24 h in a CO2 incubator at 37 °C and 5% CO2. After the incubation period, media was discarded from the wells and fresh media containing cisplatin (IC50 concentration) and different concentrations of MEUP (300 μg/ml, 450 μg/ml, and 600 μg/ml) were added and incubated for another 24 h at 37 °C in a CO2 incubator under 5% CO2 and humidified conditions. H2O2 (1 mM) was taken as the positive control. DCFH-da was prepared in reduced serum in the final concentration of 1 μM. Cells were washed with phosphate buffer saline (PBS) and loaded with 200 μl of dye and incubated for 30 min in dark. After the incubation period, cell washing was done with PBS twice and further loaded with PBS. Observations were made under the fluorescence microscope. Fluorescence intensity was quantified using the software ImageJ [34, 35].
Preparation of drugs for in vivo administration
Animal dosing was done according to their respective body weights. MEUP was dissolved in 10% polyethylene glycol (PEG) 400. Cisplatin, cyclosporine, and cyclophosphamide were dissolved in normal saline. Ketoconazole was dissolved in water.
Immunosuppression
Immunosuppression of C57BL6 mice was done as per the protocol described by Jivrajani et.al [36]. Cyclosporine and ketoconazole were administered at a dose of 30 mg/kg intraperitoneally and 10 mg/kg orally, respectively for 7 days. WBC count was taken on day 0 and day 7 to confirm the immunosuppression. Blood sampling was done in isoflurane-anesthetized animals from the retroorbital sinus and collected in 1.5-ml heparinized microcentrifuge tubes. WBC and lymphocyte counts were determined using an automated hematology analyzer (VetScan HM-5; Abaxis Inc., Union City, CA, USA). Cyclophosphamide was administered at a dose of 60 mg/kg subcutaneously on days 1 and 3 before injecting tumor cells.
Preparation of tumor cell line
Semi-confluent A549 human lung cancer cells were detached by 0.25% trypsin. Cells were collected in a 15 ml tube and centrifuged at 200×g for 5 min at 4 °C. Supernatant media was decanted and the settled cells were washed with culture media by centrifugation at 200×g for 5 min at 4 °C. The settled cells were finally suspended in a known volume of culture media. Cell counting was done using Neubauer’s chamber. The viability of cells was determined by trypan blue exclusion assay. Collected cells were kept on ice and injected in animals immediately [36].
Cancer cell implantation
Immunocompromised C57BL6 mice (males, 4–6 weeks old) were selected for the study. The average weight of animals was between 25 and 30 grams. Prior to the injection of cancer cells, hairs from the shoulder blade of each animal were removed by waxing. Cells were injected in a volume of 0.1 ml/mice containing about 5 million cells. Tumor volume was measured by a Vernier caliper on every alternate day. Treatment was started after the tumor volume reached between 80 –100 mm3 [36].
In vivo antitumor activity
Tumor bearing mice having tumor volume between 80 and 100 mm3 were randomized and divided into 4 groups (n = 6/group) comprising of disease control (DC), positive control (PC), test group 1 (T1), and test group 2 (T2). Positive control (PC) received cisplatin (2 mg/kg) intraperitoneally (i.p.) twice a week as per the standard protocol [20, 37,38,39]. The disease control group received 0.2 ml of PBS orally. Test group 1 received a lower dose of MEUP (200 mg/kg) and test group 2 received a higher dose of MEUP (400 mg/kg) orally and once a day for 20 days. Tumor volume was measured every 4 days by the formula:
Volume (mm3) = (A) × (B2)/2, where A was the largest diameter (mm) and B the smallest (mm). Treatment was continued for 20 days. Animals were sacrificed at the end of the study by CO2 asphyxiation; tumors were excised and weighed [36].
Change in body weight and histopathological analysis
Body weights of animals were taken on every alternate day to observe any severe fluctuations in their respective body weights. At the end of the study, vital organs (lung, kidney, spleen, liver, and heart) were excised and histopathology was done to check any signs of toxicity to vital organs. For histopathology, tissue specimens were fixed in 10% neutral buffered formalin and kept at 4 °C for 24 h. Specimens of kidneys, lungs, heart, liver, and spleen were cut into 3–4-μm thickness. Staining was done with hematoxylin and eosin. The sections were then analyzed under a microscope for any morphological changes [40].
Statistical analysis
Graph pad prism 8 and sigma plot 12.0 software were used to carry out the statistical analysis of the data. The IC50 value was analyzed by a four-parameter logistic model using the SigmaPlot 12.0 (Systat Software Inc.). One-way ANOVA and Student’s t tests were employed for multiple comparisons of groups. A P value < 0.05 was considered significant while the data was expressed as mean ± SD.