Extraction Of S. Khuzistanica Lab Report

Preparation of SKEO
The extraction of S. khuzistanica was performed by hydro distillation and clevenger apparatus for 5 hours. Once extraction was completed, the resulted essential oil was dehydrated using sodium sulfate (Merck). The essential oil was stored at temperature of 4ºC until using (12). In order to provide different concentrations of the extract, Dimethyl sulfoxide (DMSO) was used.
Gas chromatography/mass spectrometry analysis of essential oil
Isolation and measurement of the sample was done by coupled gas chromatography device (GC/MS) SHIMADZU 17 A with SHIMADZU mass spectrometry of QP5050A model and isolation of components was conducted in Fused Silica capillary column of the DBX-5 95% (polydimethylsiloxane) with a length of
The plates were incubated at 26±1°C for a week. In days 1, 3 and 5, the number of live promastigotes in each well was counted using hemacytometer cover glass and try pan blue staining. The dead parasites were blue and completely inactive and the live parasites were active and not dyed.
MTT assay
Antipromastigote effect of SKEO on promastigote stage of L. major by colorimetric cell viability MTT was assessed (13). Preparations plates were performed similar to those mentioned above and incubated at 25±1°C for 72h. After incubation, supernatants were thrown off by centrifuging plates in 2000 rpm for 5 min. Then 100 µl RPMI-PR- which included 10µl MTT (5mg/ml) was added to each well. The plates were incubated for 4h at 22°C in the dark. After that plates were centrifuged again for 5 min in 2000 RPM and supernatants discarded. One hundred µl DMSO was added to each well.
Positive control was cultured promastigotes in the complete medium with no drug. Also blank was the complete medium with no promastigotes and drugs. Absorbance was measured at 492nm by an ELISA reader. The 50% inhibitory concentration (IC50) was determined as antipromastigote activity using linear regression
The peritoneal macrophages were recovered, counted and the viability of cells percentage was determined (14). The protocol was approved by Committee on the Ethics of Animal Experiments, Lorestan University of Medical Sciences. Then the cells were transferred to glass coverslips inserted in 6-well plates at 5×105 cell/well and incubated at 37°C in 5% CO2 for 5 h. Afterward non- adherent cells were removed by washing with 37°C phosphate buffered saline (PBS) and RPMI1640 medium containing FBS 10%, 100 IU/ml penicillin and 100µg/ml streptomycin was added to each well and incubated overnight. No parasite was added to a group of plates that were considered for viability evaluation of macrophage during the study. The next day different concentrations of SKEO (0.1, 0.3, 0.6, 1.2, 2.5, 5, 10 and 20 µg/ml) and glucantime (0.2, 1, 5 and 25 μg/ml) were added into separate wells and incubated at 37°C in 5% CO2. The extract was not added to the number of wells considered as control group. Tests were performed as triplicate. On days 1, 3 and 5 after adding SKEO the coverslips were washed, fixed in methanol, and stained with Giemsa. The number of infected macrophages and amastigotes was determined by counting at least 200 macrophages in triplicate cultures. The results were expressed as the mean of infected macrophages and amastigotes per infected macrophage. IC50 values of the extract and MA were