Molecular Epidemiology of Cutaneous Leishmaniasis from Azad Jammu and Kashmir, Pakistan and Plants Based Control

Abstract

Cutaneous leishmaniasis (CL) is a neglected tropical disease worldwide especially in the various rural areas of Pakistan including Azad Jammu and Kashmir (AJK). The leishmaniasis is prevalent in 102 countries/areas worldwide. Approximately, it effected 350 million people worldwide. Leishmania identification on species level is imperative to determine the clinical prognosis and to choose the most suitable therapeutic regimen. The chemotherapy has been used for Leishmania infection including amphotericin B, miltefosine, liposomal amphotericin, paromomycin, and pentavalent antimonials. However, several reports have been published regarding the limit of these drugs due to side effects, low efficacy, toxicity, increasing frequency of drug resistance, high cost, and the long period of treatments. The Leishmanial vaccines may not become available in the near future; the search for better drugs should be continued. To identify new treatments, natural products can provide an infinite source of chemical diversity. New drugs should be non toxic, cheap, more efficient, and easily available especially for the low-income population. Now a day, a special focus is on medicinal plants used against CL. The bioactive phytocompounds present in the plant derivatives can be a good source for discovering new antileishmanial medicines. In the current study, we conducted one active surveys in 2018 to investigate the molecular epidemiology of CL infection and Leishmania spp. identification in Azad Jammu and Kashmir. We have conducted another epidemological survey in continue study, to see trend in two years 2018 and 2019. The genetic diversity of two Leishmania species (Leishmania major and Leishmania tropica) were also determined in 2019 under studied population. The most prevalent species of (L. tropica) was cultured for evaluation of antileishmanial activity. For this purpose, we have conducted a survey for the leaves collection of medicinal important plants (Pyrus pashia (Rosacea), Malus pumila (Rosacea), Prunus persica (Rosacea), Pyrus communis (Rosacea), Prunus armeniaca (Rosacea), Ficus glomerata (Moraceae), and Diospyros lotus (Ebenacea) from AJK. We subjected the powder form of collected leaves to soxhelet apparatus for preparation of ethanolic crude extract. The ethanolic crude extract was subjected for phytochemical screening, cytotoxicity bioassays, and in-vitro antileishmanail activity against L. tropica. The most bioactive plant extract was subjected to column chromatography and thin layer chromatography for fraction isolation. All isolated fraction extracts were subjected for xvi antileishmanial activity of promastigotes and amastigotes of L. tropica and cytotoxicity. The most effective fraction was characterized by UV-Vis spectroscopy, FTIF, and GC-MS analysis. Subsequently, all identified compounds in feaction (F7) were analyzed for their bioactivity by in-silico molecular docking study. The current study revealed that more cases of CL in 2018 and 2019 were observed from Mirpur followed by Kotli and Muzaffarabad. The findings of 2018 and 2019 revealed that the highest monthly number of CL isolates in the summer were observed followed by spring, autumn, and winter respectively. Individual with age between 1-20 years of age were more likely infected with CL than others (21-40 and 41-60 years old) in both years. Most of the inhabitants were not getting any treatment nor used bed nets in 2018. Among all Leishmania spp. only one spp, (L. tropica) was observed in 2018 while three species (L. tropica, L. major, and L. infantum) were identified in 2019 survey. The inter-genetic diversity of L. tropica showed significant value (p=0.02) when comparing with L. tropica strains obtained from different distrcits of AJK. The L. major showed significant value (p=0.05) when comparing L. major starins with other L. major strains in 2019 survey. The leaves extract of Pyrus pashia (Rosacea), Malus pumila (Rosacea), Prunus persica (Rosacea), Pyrus communis (Rosacea), Prunus armeniaca (Rosacea), Ficus glomerata (Moraceae), and Diospyros lotus (Ebenacea) showed the presence of various phytochemical compounds (saponins, terpenoids, flavonoids, alkaloids phenols, tannins, and coumarins)by phytochemical analysis. Among all plant ethanolic extracts P. armeniaca showed highest Total Phenolic Content (279.62±5.40µgGAE/mgDW) and Total Flavonoid Content (205.70±2.41µgQA/mg DW), along with highest antioxidant activities (120.37±4.90µgAAE/mgDW) and Ferric Reducing Power values (278.71±1.03µgAAE/mgDW). All plant extracts showed cytotoxicity in the safety range >1000µg/ml except F. glomerata having LC50 values of 454.34µg/ml. The ethanolic leaves extract of P. armeniaca showed significant antileishmanial activity (IC50 16.18µg/ml) against promastigotes of L. tropica. Being the most effective crude extract, 142 fractions of P. armeniaca were isolated chromatography and then further 12 fractions were obtained by TLC. Out of all fraction extracts (F2-F12), fraction (F7) showed significantly the highest antipromastigotes activity with IC5011.48±0.82µg/ml compared with standard drug (amphoterecine B) used as positive control (i.e. 11.60±0.70. The amastigotes of L. tropica xvii showed IC50 21.03±0.98µg/ml compared with control 22.03±1.02µg/ml. The UV-Vis spectroscopic analysis of bioactive fraction (F7) revealed the presence of six peaks at 665.00, 607.00, 536.00, 505.50, 359.00, and 321.00nm with the absorption of 0.276, 0.085, 0.097, 0.109, 0.897, and 1.369 respectively. The FTIR spectra revealed the presence of alkane, aldehyde, carboxylic acid, thiols, alkynes, and carbonyls compounds from fraction F7. The GC-MS analysis of ethanolic fraction (F7) showed the presence of 9 compounds i.e (1) benzeneethanol, alpha, beta dimethyl, (2) carbazic acid, 3-(1 propylbutylidene)-, ethyl ester, (3) 1, 2-benzenedicarboxylic acid, diisooctyl ester, (4) benzeneethanamine a methyl, (5) 2aminononadecane, (6) 2-heptanamine-5-methyl, (7) cyclobutanol, (8) cyclopropyl carbine, and (9) nitric acid, nonyl ester. Subsequently, all (n=9) identified compounds were analyzed for their bioactivity by in-silico molecular docking study. Among all compounds, the 1, 2-benzenedicarboxylic acid, diisooctyl ester bound well to the PTR1 receptor. The fraction (F7) showed acceptable results with no cytotoxicity. It can be used for curing skin lesions of CL caused by L. tropica. However, in-vivo and in-vitro studies of the compound i.e. 1, 2-benzenedicarboxylic acid, diisooctyl ester required in the future. The antibacterial activity of 1, 2-benzenedicarboxylic acid, diisooctyl ester compound reported previously (Ajoke et al., 2014). Moreover, P. armeniaca is effortlessly accessible in different areas of Pakistan along with AJK, and inexpensive. Current findings can aid to constitute an effective alternative to side free drugs. The compound 1, 2- benzenedicarboxylic acid, diisooctyl ester is commercially available could be used in the treatment of skin lesions of CL caused by L. tropica