Materials
quercetin (Alfa Aesar, Heysham, England) were purchased from Merck KGaA (Darmstadt, Germany). Hyaluronic sodium salt (HA-Na, Mw = 280 × 103) was provided by Contipro (Dolní Dobroucˇ, Czech Republic).
Quercetin nano phytosome and particle size analysis
Thin layer hydration method with different concentrations of Quercetin, PC and cholesterol were used to prepare the Phytosomes. Quercetin and cholesterol were melted in methanol and dichloromethane, respectively in a round bottom flask and the solvent were evaporated using rotary evaporator (Heidolph, Germany), kept an overnight at 45°C up to creating thin dry film in the bottom of the flask. The film was hydrated with distilled water in a rotary at 45°C, sonicated at 45°C and homogenized with 20,000 rpm to decrease phytosomes size. The particle size analyzer (Wing SALD 2101, Japan) were used to measure the particle size and particle size distribution and stated by the volume median diameter (VMD)(Pourhajibagher et al. 2021).
Determination of encapsulation efficiency (EE)
An appropriate amount of dispersion was transferred in Millipore Amicon® Ultra filtration tube, centrifuged (Sigma-3k-30, Germany) for 5 min at 5000 rpm and then the supernatant was collected and amount of free Quercetin was spectrophotometrically determined at 210 nm. The encapsulation efficiency has been determined according to the following equation(Cesarone et al. 2019):
$$EE\left(\%\right)=\frac{Amount of the entrapped quercetin in the noisomes}{Total amounts of the quercetin}\times 100$$
Parasites
Leishmania major (MRHO/IR/75/ER) standard strain was collected from Leishmaniasis Research Center, School of Public Health, Tehran University of Medical Sciences, Iran and was cultured in RPMI-1640 medium (Sigma-Aldrich, Bornem, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (Gibco from Thermo Fisher Scientific, Merelbeke, Belgium) and 1% penicillin/ streptomycin (Gibco 31331, Breda, The Netherlands) at 37°C, 5% CO 2. P. falciparum (Pf 3D7 strain) were obtained from Pasteur Institute of Iran and cultured in a modified medium contained of 16.20 g/L RPMI 1640 (Sigma, Munich, Germany), 11.11 mM glucose, 0.20% sodium bicarbonate (Sigma, Munich, Germany), 50 g/mL gentamicin (Gibco,Waltham, MA, USA), 45 g/mL hypoxanthine (Sigma, Munich, Germany), 25 mM HEPES, 0.50% Albumax I (Gibco, Waltham, MA, USA), and incubated at 37 C in an atmosphere of 5% O2, 5% CO2, and 90% N2 (Raeisi et al. 2020, Elmi et al. 2021).
In vitro anti-leishmanial assay
The promastigotes density of L. major was 1×105 parasites/mL and all experiments were carried out in 96-well plates in triplicate. Quercetin loaded phytosome and quercetin (100 µL) in concentrations of 100, 200 and 400 µg/ml was added to culture medium and 100 µL of L. major promastigote in culture medium were added (total volume 200 µL). The viability of the promastigotes was measured using trypan blue as a vital staining. To calculate the IC50, quercetin were used at concentrations ranging from 2.5 to 100 µg/ml and the IC50 values were assessed from dose response growth inhibition curves by Microsoft excel software. Phosphate-buffered saline (PBS) and glucantime were used as negative and positive controls, respectively in each set of experiment(Esboei et al. 2018).
In vivo anti-leishmanial assay
Thirty BALB/c females at aged of 4–5 weeks and weighed 30–40 g were purchased from pasture Institute of Tehran (Tehran, Iran) and kept in standard conditions (light and temperature) and divided into 6 groups including 3 treatments group (100, 200 and 400 µg/ml), negative, positive and non-treated groups. L. major promastigotes (1×106) in stationary phase were intradermally injected in the tail base of each mouse. After 21 days, nodules and ulcers were appeared and the treatments were applied two times per a day at the ulcer site and 150 µg/ml of treatments were used in each time using a cotton applicator for 4 weeks. The diameters of the lesions were measured using digital caliper and the mice were weighted by a digital scale weekly. All parts of the in vivo experiments were performed according to the ethically standards of animal and was approved by the Institutional Ethics Committee of Research of Islamic Azad University, Tonekabon branches(Esboei et al. 2018).
In Vitro Anti-Plasmodial Assay
100 µl of quercetin in concentrations of 100, 200, 400 µg/ml and Chloroquine (Sigma-Aldrich, New Delhi, India) and PBS as positive and negative groups, respectively were added to round-bottom 96-well. 100 µl of the parasites in culture medium were added. During incubation time, 100 L of SYBR green I lysis buffer (Tris (20 mM, pH 7.5), saponin (0.008%, w/v), Triton X-100 (0.08%, v/v) and EDTA (5 mM)) were added to the each well, lightly shaked and then incubated in dark at 37 C for 1 h. After incubation period, fluorescence intensity was then measured with a victor fluorescence multi-well plate reader () at 485 and 530 nm, respectively. The IC50 values were calculated using the IC Estimator-version 1.2 software (http://www.antimalarial-icestimator.net/MethodIntro.htm) (Free Software Foundation, Boston, MA, USA)(Elmi et al. 2021).
Cytotoxicity Assay
To assess the cytotoxicity effects of the mentioned drugs, Non-cancer (WI38) and cancer (J774) mammalian cell lines were cultured in DMEM medium supplemented with FCS 10% and CO2 5%. Diluted cells (100µL) at density of 1 × 104 cells/mL were added into each well of 96-well plates and incubated for 24 h at 37 ºC. After incubation time, 100 µL of quercetin loaded phytosome and quercetin at concentrations of 400 µg/mL was added to each well and incubated for 72h. Then, the medium was removed and 100 L of MTT was added to each well and incubated for 45 minutes. The MTT were discarded, 100 L of DMSO was added and the Absorbance was measured on a spectrophotometer at 570 nm. All experiments were done in triplicate(Zafari et al. 2017).
Hemolytic Test
Five mL from human blood was collected and washed three times with Phosphate buffered saline (PBS). The quercetin loaded phytosome and quercetin alone at concentration of 400 µg/mL were prepared in microplate and triton X100 and PBS (Sigma-Aldrich, St. Louis, USA) were set as positive and negative controls, respectively. 100 µL Red blood cells (RBCs) suspension (2%) was added to each well and incubated for 2 h at 37°C. after incubation time, The microplate was centrifuged for 10 min at 1200 g and the absorbance of the supernatant was determined at 540 nm. The percentage of hemolysis for each sample was calculated by the following formula(Elmi et al. 2021):
Hemolysis % =\(\frac{Plasma hemoglobin concentration}{Total blood hemoglobin }\)
Statistical analysis
Data were analyzed using SPSS v. 11.5 and the mean values less than 0.05 was accepted as statistical significant level. In this study mean values were analyzed by Two-way ANOVA and student's t-test.