Chemicals and reagents
The analytical reagent grade (EMPARTA ACS grade) solvents like petroleum ether (40–60 °C), toluene, chloroform, ethyl acetate, methanol, ethanol and analytical TLC aluminium plates were purchased from Merck Millipore, Burlington, Massachusetts, USA. The different analytical grade (AR) chemicals like sodium bicarbonate, HEPES buffer, D-glucose, D-sorbitol, formic acid, phosphate buffer solution 10X (PBS), Giemsa stain, sodium pyruvate, dimethyl sulfoxide (DMSO) and plastic wares used in the biological activity screening were purchased from HiMedia Pvt. Ltd., Mumbai, India. Molecular biology grade Fetal bovine serum (FBS), gentamycin, amphotericin-B, penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), RPMI-1640 (Roswell Park Memorial Institute-1640), DMEM (Dulbecco’s Modified Eagle’s medium) were purchased from Gibco-BRL, Life Technologies Inc., Gaithersburg, MD 20884–9980, USA and Sigma Aldrich, St. Louis, MO, USA. The marker compound and standard drug, namely, quercetin (HPLC grade ≥ 95%) and chloroquine phosphate (Certified Reference Material grade) were purchased from Sigma-Aldrich, St. Louis, MO, USA.
Plant materials
The information regarding the use of different plants as herbal remedies for the treatment of fever associated with malaria was collected by interviewing local practitioners in the form of questionnaires and from local books.
Herbariums of the two selected plants (Voucher specimen no. DU/DRS/HRB/NG/2018–19/FS-01 and DU/DRS/HRB/NG/2018–19/FS-05) were prepared according to standard procedure and sent to Botanical Survey of India, Shillong for identification and authentication. Then the plant parts used in the preparation the traditional herbal remedy were collected and processed according to the guideline of Good Agricultural and Collection Practice of WHO [30]. The processed plant materials were coarsely powdered with a mechanical grinder and stored in airtight containers.
Extraction of the plant materials
The extraction of the powdered plant materials was carried out using cold maceration technique using petroleum ether (40–60 °C), chloroform, ethyl acetate, methanol and hydro-alcoholic (1:1) solvent system. Besides, one sample from each plant were prepared as per the traditional practices. In the cold maceration, about 500 g plant material was taken with 3 l of solvent and kept for 72 h with occasional shaking [31]. After that, the solvents were removed under reduced pressure at low temperature using a rotary evaporator (IKA Rotary Evaporator RV 8 V). In the case of the hydro-alcoholic extract, the water part was removed by lyophilisation of the sample using laboratory freeze dryer (IIC Industrial Corporation). The extracts were preserved in glass sample bottles and kept in − 20 °C for further use. The extracts were prepared at ratio of 1:6 during cold maceration process. The yields of the extracts of C. maxima were found to be 0.262%, 0.562%, 2.882%, 1.151%, 8.305% and 0.167% w/w with petroleum ether (40–60 °C), chloroform, ethyl acetate, methanol, hydro-alcoholic and traditional solvent system respectively. Similarly, in case of A. nilagirica, the yields of the extracts were found to be 0.757%, 1.627%, 1.891%, 2.062%, 1.770% and 0.215% w/w with petroleum ether (40–60 °C), chloroform, ethyl acetate, methanol, hydro-alcoholic and traditional solvent system respectively.
In vitro antimalarial screening of the extracts
Preparation of standard and test samples
For this study, 1 mg/ml stock solutions of the extracts were prepared by using incomplete RPMI-1640 media containing 0.5% DMSO. Chloroquine phosphate was used as standard drug in this study and prepared 100 μg/ml stock using the same protocol as used for extracts for further use.
In vitro culture of malaria parasites
The chloroquine sensitive (3D7) and chloroquine resistant (RKL-9) strains of the malaria parasite P. falciparum were obtained from the Parasite Bank of National Institute of Malaria Research (Indian Council of Medical Research), New Delhi. The strains of P. falciparum were maintained in fresh A+ erythrocytes suspended in RPMI-1640 medium supplemented with 25 mM HEPES, 1% D-glucose, 0.23% sodium bicarbonate, gentamycin (40 mg/ml), amphotericin-B (0.25 mg/ml) and 10% heat-inactivated AB+ serum at 37 °C and 5% CO2 environment [32, 33]. After every 24 h, the used medium was replaced with fresh medium supplemented with 10% heat-inactivated AB+ serum and the parasitemia level was maintained below 2%.
Antimalarial activity screening & determination of IC50 values
The antimalarial screening of the extracts was carried out against both 3D7 and RKL-9 strain of P. falciparum by Giemsa staining light microscopy method. For antimalarial testing, initially the asynchronous P. falciparum parasites were synchronized to obtain only the ring stage parasitized cells by treating with 5% D-sorbitol [34]. The initial ring stage parasitemia was maintained at 0.5% in 4% haematocrit using complete medium and fresh A+ erythrocytes before using in the screening. For screening, test and standard drugs were taken in nine different concentrations by two fold serial dilutions for both the 3D7 and RKL-9 strains in 96 well plates. The concentration ranges for test drug and standard drug were 50 to 0.19 μg/ml and 5 to 0.019 μg/ml respectively. All the treatments were performed in triplicates. Then parasitized blood was added to the wells of 96-well plate containing 100 μl of test and standard samples to carry out the assay. The plates were incubated at 37 °C in an environment of 5% CO2 for 36–40 h in a CO2 incubator. After the incubation period, blood smears were prepared in glass slide from each well and fixed by treating with methanol. The slides were stained with 10% Giemsa stain prepared in 1% phosphate buffer solution (PBS). After that number of schizonts (3 or more merozoites containing) per 100 asexual parasites were counted under a light microscope (Leica DM1000) at 1000X (oil emersion) magnification [35]. The percentage inhibition for each concentration was calculated by the following equation:
$$ \% Inhibition=1-\frac{no. of\ schizonts\ in\ test}{no. of\ schizonts\ in\ negative\ control}\times 100 $$
Finally, the IC50 values were calculated by plotting nonlinear regression curve between log dose vs percentage (%) inhibition using GraphPad Prism (GraphPad Prism v.7 San Diego, California, USA). Based on the obtained IC50 values, the extracts were categorized in to active (< 10 μg/ml), intermediate (10–25 μg/ml) or inactive (> 25 μg/ml) categories [36].
In vitro cytotoxicity study
The most active extract obtained for each plant from the in vitro antimalarial study were further taken for in vitro cytotoxicity study using MTT assay [37]. The study was carried out against normal murine macrophage RAW264.7 cell line. Approximately, 1 × 104 cells/ml were cultured in DMEM (Dulbecco’s Modified Eagle’s medium) media supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10% FBS (Fetal Bovine Serum), penicillin (100 units/ml), streptomycin (10 μg/ml) and allowed to incubate at 37 °C in a humidified 5% CO2 environment. After 80% cell confluency, the cells were treated with different concentrations (50, 100, 200 and 500 μg/ml) of the selected test extracts and incubated for 24 h. Cells without any treatment were considered as control. After 24 h incubation, 0.5 mg/ml MTT was added to each well of the plates and incubated for a further 5 h. After the completion of the incubation, formed formazone complexes were dissolved properly in MTT solvent and the absorbance was taken at 570 nm using a microplate reader (Multiskan™ FC Microplate Photometer). The experiment was performed in triplicates and the percentage of cell viability was calculated for each concentration and compared to control cells without any treatment. Finally, CC50 (cytotoxic concentration) were determined for the test extracts.
Phytochemical and qualitative fingerprint analysis
The various biological activities shown by plant extracts are mainly due to the presence of the different secondary metabolites like alkaloids, flavonoids, glycosides, terpenoids, saponins, steroids, tannins etc. [38]. The active extract from each plant was analysed by chemical reagents to detect the presence of those secondary metabolites using standard procedures as described in the earlier study [39].
The qualitative fingerprint analysis of an extract under the controlled environment is one of the quality control parameters for herbal products [40, 41]. In this study, the qualitative fingerprints of the active extracts were developed using HPTLC densitometry analysis under control environment of temperature and humidity. The study was carried out by using Camag TLC Scanner 4, semiautomatic sample spotter Linomat 5, UV visualization cabinet (deuterium, tungsten and mercury lamp), 100 μl Hamilton dosage syringe, TLC aluminium plate pre-coated with silica gel 60 F254 (10 cm × 10 cm) and glass twin trough chamber (10 cm × 10 cm).
Initially, stock solutions of the marker compound (quercetin for A. nilagirica)/semi-purified fraction for C. maxima (1 mg/ml) and active extracts (10 mg/ml) were prepared in their respective solvents by sonicating for 15 min. Then the samples were centrifuged at 2000 rpm for 5 min and the supernatants were transferred to the sample vials by filtration for further use.
The experiment was carried out at a temperature of 25 ± 2 °C, relative humidity 55%. In the TLC plate (10 cm × 10 cm), 2 μL of the marker compound or semi-purified fraction in duplicate and 4 μL of the active extracts in quadruplicate were applied as a band of 8 mm × 1 mm in size at a distance of 8 mm from the bottom. After applying the sample, the plates were developed using 10 ml mobile phase composed of toluene: ethyl acetate: methanol: formic acid at a ratio of 3:5:1:0.5 using the glass twin trough chamber (10 cm × 10 cm). The plates were developed up to a distance of 7 cm and air-dried at room temperature. Then plates were visualized under UV cabinet at 254 nm and 366 nm. After that, the plates were scanned at 254 nm in absorbance mode and at 366 nm in fluorescence mode using the Camag TLC scanner 4 linked with VisionCAT 2.5 software. During scanning, the slit dimension was kept at 5 × 0.2 mm and the scanning speed was employed at 20 mm/s [42].