Inb500

For cultivation process Hamid et al. (2012) method was used with some modifications. From the samples leishmanial parasite was cultured in the RPMI 1640 (Sigma, USA) culture media. The quantity of 0.3 g/30 mL of RPMI 1640 media was dissolved in (10 g/1000 mL) distilled water and added to 20 separate small screwed caped tubes. To avoid any bacterial and fungal contamination, the antibiotic Kanamycin was mixed. To the test tubes 1 mL of that sample was added in which amastigote was observed. At 25°C the test tubes were placed in incubator (Memmert type Inb 500, Germany). After 11 days of incubation period, promastigote of Leishmania tropica culture was observed with Giemsa staining and seen under Olympus Microscope at different magnifications 10x, 40x, and 100x [18 ].

The culture medium RPMI-64 (Gibco, USA) was prepared for the purpose of the culturing of the Leishmania parasite from the samples. We dissolved the 0.3 g/30 mL of medium RPMI-64 in distilled water (10.43 g/1000 mL of distilled water) and distributed it amongst the 10 vials of bijou bottle, each having 3 mL of the dissolved media with supplemented 10% fetal bovine serum. The antibiotic consisting of penicillin G and kanamycin were mixed in cultural media to avoid bacterial contamination. The bijou bottles were placed in ice jar and were taken to the field where we collect the samples from the identified suspected person. The skin scrapings were directly mixed from the lesion to each bottle in ice jar and transported to the Department Lab of Kohat University of Science and Technology, Kohat. Then the medium was incubated at 26°C in incubator (Memmert Type INB500, Germany) and was examined at the different life stages of Leishmania tropica and was identified under 100x magnification of microscope for every 24 hours till up to 10 days.

The total phenol content was determined by the Folin–Ciocâlteu method with minor modifications [71 (link)]. Thus, a quantity of 0.5 mL was taken from each extract prepared as described above, over which 1.25 mL of Folin–Ciocâlteu reagent (Sigma-Aldrich Chemie GmbH, Munich, Germany) was added, diluted 1:10 with distilled water. The samples were incubated for 5 min at room temperature. After incubation, 1 mL of Na₂CO₃ (Geyer GmbH, Renningen, Germany; 60 g/L aqueous solution) was added. The samples were incubated for 30 min at 50 °C, using the thermostat (INB500, Memmert GmbH, Schwabach, Germany). The absorbance was read at 750 nm using a UV–VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany). As a control, we used 70% ethanol (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The calibration curve was obtained with gallic acid (2.5–250 μg/mL). Results were expressed in mg GAE per kg dry matter (d.m.). All determinations were made in triplicate.