Antileishmanial Activity of Cassia fistula L., Morus nigra L. and Ziziphus jujuba Mill., Plant Extracts

Aim: To investigate the different antileishmanial activities of extracts of Cassia Fistula L., Morus Nigra L. and Ziziphus Jujuba Mill. Methodology: In this method, three (03) plants having concentrations between 500 – 2000 μg/mL were subjected to KWH23 strains of L. tropica in which Standard drug was Amphotericin B and have negative control for 24 – 48 hours. To check the in-vivo studies, plant extract was tested on BALB/c mice (Iqbal et al., 2016). Results: It showed that inhibition (mean) of KWH23 strains at 500, 1000, 1500, 2000 μg/mL after 48 hours were 92.1 ± 0.02, 95.00 ± 0.05, 97.09 ± 0.07 and 98.05 ± 0.05 % respectively. It decreased the lesion size (mean) from 0.8 ± 0.1 mm to 0.40 ± 0.2 mm having significance value p < 0.01 after 8th week, and cure at 200 mg/Kg against intracellular amastigotes in BALB/c mice was 90.00% (95% Cl = 80.05 – 97.00). Conclusion: The result shows that Ziziphus jujuba Mill. leaves possess significant antileishmanial activity. Original Research Article Iqbal et al.; JPRI, 33(46B): 466-471, 2021; Article no.JPRI.75329 467


INTRODUCTION
Leishmaniasis is an infectious disease, which is prevalent all over the world. It belongs to genus Leishmania. It affects millions of people annually around the globe [1]. Existing remedies have high cost, drug resistance, toxicity and high dose regimen. Natural entities exhibit safety and efficacy in terms of drug potential against infectious diseases [2].
Ziziphus jujuba Mill. is commonly known as "Ber" belong to family Rhamnaceae. It is native to china and is cultivated in Australia, southern and eastern Asia and Europe. As traditional medicine, different parts of the plant, especially the fruits, leaves, and stem bark are used for thousands of years. In this plant, various pharmacological activities observed including antifungal, antioxidant, anti-inflammatory, anticancer, immunostimulating, hepatoprotective, antiobesity and antiulcer due to presence of flavonoids, phenolics, triterpenic acids etc [3].
Cassia fistula L. locally known as "Amaltas" (family: Caesalpinioidease) is cultivated in Asia, China, West Indies and South Africa. Its fruit is used to treat fever, leprosy, abdominal pain and various skin diseases, contain aspartic acid, Fe and Mn, Glutamic acid, lysine, Isoflavone, biochanin A, Kaempherol, tannic matter etc [4].
Morus Nigra L. is locally termed as "Shahtoot" (family: Moraceae), which is cultivated in Africa, Asia, America and Europe. Different parts of plant including root, fruits, bark and twigs contain flavonoids, triterpenes and saponins exhibit pharmacological effects like antioxidant, anticancer, antibacterial [5] and antileishmanial [1].
In this work, Anti-leishmanial effects (in-vitro and in-vivo) of Leaves of Ziziphus jujuba Mill., Fruit of Morus Nigra L. and Fruit of Cassia Fistula L. were checked against L. tropica parasites.

Plant Material Collection and Crude Extract Preparation
Leaves

Antileishmanial Activity (In-vitro)
In this study, KWH23 strains of L. tropica were used for antileishmanial analysis of MNF, ZJL and CFF where as method was adopted from

Anti-leishmanial Activity (In-vivo)
In this process, BALB/c mice (Male) having weight of 20-32 gm and aged of 6-8 weeks were used which were supplied by National Institute of Health, Pakistan. This method was adopted from Iqbal et al., 2017a.

Statistical Analysis
In-vivo experimental work having IC50 value, percentage cure rate and mean lesion size (mm) was determined by Non-linear regression and was measured by Graph Pad Prism 6 software, whereas level of significance was p < 0.05. Percentage inhibition of cell was Analysed in triplicate and measurements of data was analyzed in Mean±SD.

Antileishmanial (In-vivo) Test
In the albino mice that are infected with 0.02 mL KWH23 strains (1.5  10 6 promastigotes/mL) effective in-vivo anti-leishmanial results of ZJL, MNF, and CFF after 36 -120 days has been shown (

DISCUSSION
In current work, ZJL at different concentrations showed inhibition of KWH23 strains (p<0.0)1 after 48 hours which is an agreement with previously work reported [1].
Secondary metabolites such as triterpenic acid, saponins, and phenolics are confirmed in ZJL as per previous reportings by Yoshikawa et al. [6][7]. Antileishmanial activity in ZJL is due to the presence of these secondary metabolites. This antileishmanial activity in ZJL is more than other plants (CFF and MNF) as studied in this work. Mean lesion size was decreased after 8 th week and cure after 120 days in BALB/c mice when ZJL extract was administered at regular interval of time. In this case, significant inhibition of Metabolic pathways of KWH23 strains are due to administration of ZJL extract [1].
It is first time to report the antileishmanial activities of ZJL, CFF and MNF against KWH23 strains which showed significant results.

CONCLUSION
The result highlighted that due to presence of phenol, flavonoids, and triterpenes in Ziziphus jujuba Mill. It showed remarkable antileishmanial effects against L. tropica and this effective part of the plant can be a significant source for the latest anti-leishmanial agents.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for