Rutin-trihydrate (Fig.1), chloroquine diphosphate, DMSO, D-sorbitol, gentamycin, hypoxanthine, LPS (Escherichia coli 055:B5), DMEM, penicillin, streptomycin, Fetal Bovine Serum, MTT, Phosphate Buffered Saline, Triton X-100, potassium chloride, Thiobarbituricacid, Trichloroacetic acid, TMB substrate, were bought from Sigma-Aldrich, USA. Albumax II, RPMI- 1640, Fetal Bovine Serum, and fungizone were purchased from Gibco (Grand Island, United States) while (CM-H2DCFDA) chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate was procured from Invitrogen (Carlsbad, CA, United States). Mouse specific (IL-6, TNF-α, IFN-γ and IL-1β) ELISA Kits purchased from BD Biosciences.
2.2. In vitro Study
2.2.1. Maintainance of in vitro Plasmodium falciparum culture
The cultivation of P. falciparum sensitive and resistant (NF-54 and K1) strains was done in human red blood cells (B+) added with RPMI-1640 medium containing the supplements like hypoxanthine (370μM), HEPES (25 mM), NaHCO3 (0.2%), fungizone (25μg/mL ), gentamycin (40μg/mL) and Alumax II (0.5%) at 37°C and 5% CO2.Beside the change of growth medium every 24 hours, parasitaemia was routinely monitored through Giemsa stained thin smear of cultured parasite to achieve synchronized culture having initial stage parasite (ring stage).
2.2.2. Anti-plasmodial activity of rutin
For the in vitro study of the inhibition of P. falciparum growth, 200 µL of the ring stage P. falciparum culture having 1.2% parasitemia in 2% hematocrit was transferred to 96 well plate. The culture was then treated with rutin (0.1-100µM) and standard drugs (chloroquine and artesunate) and the culture without treatment served as negative control. After 48 hours of incubation at 37°C and 5% CO2, thin smear of culture was prepared from each well and stained with Giemsa stain to count parasitaemia, Determination of parasitemia was done based on the parasitized RBCs counted in around total 1000 erythrocytes while the percentage suppression of parasitemia was enumerated as [(A-B)/A] 100, where A is the mean percent parasitaemia in the negative control and B refers to the mean parasitaemia of the treatment group. The IC50 (Mean±SEM) was determined using non linear regression analysis from concentration-mediated growth inhibition data.
2.2.3. Isolation of Primary Macrophages
The previously described method (Bawankule et al. 2008) was followed to isolate the primary cells from the peritoneal cavity. Briefly, 1.0 mL of intraperitoneal injection of 3% protease peptone was given to eight-week-old Swiss albino female mice three days earlier than harvesting macrophage cells. Just before isolating cells, mice were subjected to ether anaesthesia and sacrificed by cervical displacement. The macrophage cells from the peritoneum were collected by lavage of the peritoneal cavity using chilled PBS (pH-7.4). Then, cells were washed and filtered through sterile gauze and viable cells were adjusted to the required density (0.5×106 -1×106 cells/mL) in a DMEM medium containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 µg/mL). After seeding, cells were incubated overnight at 37°C with 5% CO2.
2.2.4. The pro-inflammatory cytokine profile of rutin in primary macrophages
For the in vitro anti-inflammatory profile assessment of rutin, primary macrophages were seeded as described above. After 24 hours of seeding, cells were pre-treated with rutin at the concentration of 3, 10 and 30 μM along with dexamethasone (1 μM). The cells were then stimulated with LPS (1 µg/mL) and incubated for 24 hours at 37°C with 5% CO2. After incubation, supernatants from each well were harvested and stored at −80ºC immediately until analysis. Supernatants were tested for estimating the levels of the pro-inflammatory cytokines (IL-6, TNF-α and IL-1β) using mouse-specific Enzyme Immuno Assay (EIA) Kits according to the instructions of the manufacturer.
2.2.5. Determination of cell viability
The effect of rutin on the viability of primary macrophages was studied using MTT assay. In short, primary macrophages (0.5 × 106 cells /well) were seeded in 96 well plate and incubated in CO2 incubator at 37°C with 5% CO2 for 24 hours. After incubation, 20 μL MTT (5 mg/mL in PBS) was added into each well and incubated for another 4 hours. Following incubation, media containing MTT was replaced with DMSO (100μL) to solubilize the formazan crystals formed. The absorbance was recorded at 550nm and viability of the cells has been represented in terms of percentage (%) of survival.
2.2.6. Quantification of Reactive Oxygen Species (ROS) generation
IntracellularROS level was determined as previously described (Becker et al., 2003). Briefly, RAW 264.7 cells were cultured using DMEM media containing 10% FBS. For the measurement of intracellularROS, 1ᵡ106 cells/well was seeded in a 6 well plate and incubated for 24 hours at 5% CO2 and 37˚C. After incubation, cells were pretreated with rutin at the concentrations of 3, 10 and 30 μM along with dexamethasone (1 μM). After 30 minutes, cells were stimulated with LPS (1μg/mL) for intracellular ROS generation and again incubated for 24 hours. After 24 hours incubation, cells were incubated with fluorescent dye CM-H2DCFDA (20μM) in culture medium and were further incubated in CO2 incubator for 20 minutes. Following incubation, the cells were washed twice with PBS and also harvested in PBS after trypsinization. The fluorescence was measured using a flow cytometer (BD Biosciences) operational with a 488 nm argon laser as light source. Using FACS Diva software version 7.1 (BD Biosciences) percentage and mean fluorescence intensity of CMH2DCFDA positive cells were calculated. Similarly, intracellular ROS level was also measured using spectro fluorometer (Spectramax i3x, Molecular Device) at 485 nm of excitation and 520 nm emission wavelength. Data was expressed as fluorescence unit which depicted the ROS level in rutin treated cells as compared to LPS-induced cells.
2.3. In vivo study
2.3.1. Animals and ethical approval
The protocol (CIMAP/IAEC/2020-23/01) followed for the anti-malarial experiment was approved by the Institutional Animal Ethics Committee (IAEC) along with the approval of the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), Government of India (Registration No: 400/01/AB/CPCSEA).
2.3.2. Mice and parasite infection
To evaluate the suppressive anti-malarial activity of rutin, adult Swiss albino male mice which were bred in-house and aged 6-8 weeks (20 ± 2 g) were chosen as experimental host. Random grouping of animals with 6 mice each in 5 groups was followed by seven days acclimatization in the experimental facility, kept under the standard environmental conditions of 23 ± 2°C, 12 hour light/dark cycle with the supply of food and water on an ad libitum basis. Chloroquine-sensitive rodent malaria strain Plasmodium berghei K-173 was maintained at institute by a continuous passage in mice and the blood-stage parasites were preserved in liquid nitrogen.
2.3.3. Anti-malarial study of rutin in P. berghei infected mouse model
According to the method of Knight and Peters and few modifications in an already published report (Mohanty et al. 2013), each experimental mice was inoculated intraperitoneally with 0.2 mL suspension of infected blood carrying approximately 1×106 P. berghei K-173 parasitized red blood cells diluted in sterile ACD. After an hour of infection, mice were administered with an oral dosage of rutin (25, 50, 100 mg/kg/day) prepared in 0.7% CMC while mice receiving vehicle (0.7% CMC) alone were considered as vehicle-treated group. The standard drug chloroquine at the dose of 10 mg/kg/day was orally given and considered as the positive control. The same dose regime was repeated once daily for four days.
2.3.4. Parasitaemia and survival determination
From 4th day post-infection, thin blood smears by tail snip were prepared on every alternate day until day 28th. After staining with Giemsa stain, percent parasitemia was examined by calculating total parasitized and normal RBCs over three optical fields with at least 300 RBCs per field. Determination of parasitemia was done based on the parasitized RBCs counted in around total 1000 erythrocytes, the percentage suppression of parasitemia was enumerated as [(A-B)/A] 100, where A is the mean percent parasitaemia in the negative control (vehicle-treated group) and B refers to the mean parasitaemia of the treatment group. Additionally, for assessing the mean survival time and percent survival, all the treated and non treated groups were followed up to 28 days to record and assess the mortality of individual mice in each group.
2.3.5. Quantification of Hemoglobin and glucose
On the peak day of parasitaemia (8th day), haemoglobin and blood glucose level were assessed in P. berghei infected mice. For estimating haemoglobin, the standard Drabkin’s cyanmethemoglobin method was followed as per the manufacturer’s guidelines. Glucose estimation was done using glucometer (Dr. Morepen, GlucoOne) according to the procedure described by (Mohanty et al. 2013).
2.3.6. Quantification of pro-inflammatory cytokines
Another set of experiment was performed to assess the role of rutin in modulating the profile of inflammatory mediators in the P. berghei infected mice. On the peak day (8th day), mice were bled through retro-orbital plexus and collected blood was processed to obtain serum. The animals were then sacrificed for the collection of the whole brain. The pro-inflammatory cytokines (IL-6, TNF-α and IFN-γ,) were then quantified from serum and brain homogenate by using the ELISA kits.
2.3.7. Quantification of malondialdehyde (MDA) content
The amount of MDA in liver, brain and spleen tissues was determined by following the method of Okhawa et.al. (1979) with the modifications described by Oakes & Van Der Kraak. (2003) . The quantification of LPO was done by using the standard curve of MDA and calculated as nM MDA / mL of tissue homogenate.
2.4. Combination study of chloroquine and rutin
2.4.1. Anti-plasmodial profile of chloroquine and rutin combination
The modified fixed ratio method was performed for the study of interaction between chloroquine and rutin (Fivelman et al., 2004). Firstly, the IC50 i.e.50% inhibitory concentration against the chloroquine-resistant strain K1 of P. falciparum was determined for chloroquine and rutin as described in section 2.2.2. The 5 times higher of the respective IC50 concentrations of chloroquine and rutin were diluted two-fold with culture medium to initial concentrations, then mixed volume by volume at 4:1 (1.016μM: 20.5μM), 3:2 (0.777μM: 41μM), 2:3 (0.518μM: 61.5μM), 1:4 (0.259μM: 82μM). All these ratios were further diluted two-fold. Then, IC50 values were determined for different combinations (chloroquine and rutin), the individual IC50 concentrations and the IC50 values of the combination were used for the calculation of fractional inhibitory concentrations (FIC) and ƩFIC using the following formula:
ƩFIC= FIC value of chloroquine + FIC value of rutin
2.4.2. Anti-malarial combination study of chloroquine and rutin in P. berghei infected mouse model
Another experiment was performed to evaluate the antimalarial potential of rutin in combination with chloroquine. The experiment was carried out as described in section 2.3.3 and mice were treated with an oral dose of rutin (25 mg/kg), CQ (2.5 mg/kg) and a combination of rutin (25mg/kg) and CQ (2.5 mg/kg) along with CQ (10 mg/kg) as the positive control. All the doses were prepared in 0.7% CMC (vehicle) and the group that received only CMC was called as vehicle-treated group. The parasitaemia,haemoglobin and survival were determined to evaluate the potential of rutin and chloroquine combination in P. berghei infected mice.
2.5. Statistical Analysis
The results in the present study were expressed in termsof Mean± SEM (in vitro; n=3, in vivo; n=6) and analysis was done using Graph Pad Prism 5. The comparison between vehicle-treated and rutin-treated groups was made using one-way analysis of variance (ANOVA) along with Turkey′s multiple comparison tests. The P-value P<0.05 was taken as statistically significant.