Bioassay Guided Fractionation and in vitro Anti-plasmodial Activity of Ficus deltoidea and Ficus benjamina

Background: Malaria is infectious vector born disease affecting 212 million people belonging to 97 countries globally in the year 2016. Although this number has reduced progressively from last one decade but recent failure of currently available antimalarial drug therapy has accentuated the urgent need to explore different novel approaches in Anti-plasmodial drug discovery. Objective: The aim of the present study was to evaluate the Anti-plasmodial activity of traditional medicinal plants Ficus deltoidea and Ficus benjamina. Materials and Methods: Crude petroleum ether and hydro alcoholic extract of both the plant species were evaluated for Anti-plasmodial activity by schizont maturation inhibition assay using 3D7 plasmodium strains. Results: It was observed that petroleum ether extract of F. benjamina leaves showed most promising inhibitory effect on the growth of schizonts with IC50 14.5 μg/ml. Bio-assay guided fractionation of petroleum ether extract of F. benjamina led to the hexane and chloroform fraction with high Anti-plasmodial activity (IC50 4.0 μg/ml and IC50 7.8 μg/ml respectively). Further, phytochemical investigation of F. benjamina indicated the presence of various valuable phytochemicals belonging to class of steroids, terpenoids and phytosterols. Conclusion: This study has revealed the Anti-plasmodial activity of F. deltoidea and F. benjamina for the first time. Significant Anti-plasmodial activity and preliminary phytochemical studies of F. benjamina indicates its rich chemical diversity which make this plant a good candidate for isolating new molecule that could serve as new lead in Anti-plasmodial drug discovery.


INTRODUCTION
Malaria is still considering a grievous parasitic disease caused by Plasmodium falciparum and other species of plasmodium, killing 429000 of the total 212 million infected people globally in the year 2016. 1 Although this morbidity and mortality rate is gradually reducing every year but still P. falciparum is regularly gaining attention due to its high adaptive character, fast sexual reproduction and rapid development of resistance against most of the currently available antimalarial drugs. However, World Health Organization (WHO), succeed somewhere in controlling the resistance by prescribing combination of various synthetic and semi synthetic drug molecules in the form of Artemisinin combination therapy (ACT). 2 But recent failure reports of ACTs, rapid re-emergence of malaria in certain ACT treated malarial patient and decreasing efficacy of standard antimalarial drug chloroquine (CQ) convinced the global chemists to focus on exploring some other new strategies in Anti-plasmodial drug discovery. 3,4 Various approaches such as development of new scaffolds, structural modification of existing molecules, better understanding of parasite life cycle and its mechanism of drug action, synthesizing hybridized drug molecules, designing of new ACT and plant based antimalarial drug discovery can be adopted to fulfill the need of timely requirement. Among the several strategies followed for the development of new drugs for the malaria, isolation and identification of novel biomolecules from plant sources is gaining much more importance than other approaches. 5,6 Traditional medicinal plants gives us most effective antimalarial drugs in the form of quinine and artemesinin isolated from Cinchona calisya and Artemisia annua respectively. 7,8 Similarly, large number of such plants like Azadirachta indica, Tinospora cordifolia, Carica papaya with acclaimed antimalarial activity used in the traditional medicine were extensively evaluated scientifically and have now become a part of the modern world health care system. 9,10,11 However, still there are large numbers of plants which are used traditionally to cure malarial fever and not yet explored and reported scientifically. Hence, nature is considered as an ever-evolving source of medicinally important plant secondary metabolites. Ficus is one of the largest genus in the Moraceae family which comprises of approximate 800 species globally and approximate 115 species are distributed in India. 12 Several members of this genus were employed as one of the economical source of medicine because of its rich chemical diver-leum ether and 80% ethanol for 24 h by hot percolation method using soxhlet apparatus. Both the crude extracts were concentrated under reduced pressure at 45°C and freeze dried to produce powder form of extract. The residues obtained were weighed accurately and stored at 4°C. The crude plant extracts were fractionated into different fractions based on their Anti-plasmodial activity. The petroleum ether extract of FBL was suspended in water and this aqueous solution was extracted successively with hexane, chloroform and butanol by partition extraction method Figure 2.

Phytochemical screening
All the crude extracts and fractions were filtered and examined for the presence of major phytoconstituents as per standard protocol of preliminary phytochemical analysis. 30 In vitro cultivation of Plasmodium parasites In vitro blood stage culture of CQ-sensitive strain (3D7) of P. falciparum was used to test the Anti-plasmodial activity of different plant extracts and its fractions. The culture of erythrocytic stage of malaria parasite was maintained at Malaria Parasite Bank, National Institute of Malaria Research, New Delhi, India using modified method of Trager and Jensen. 31 Isolated 3D7 strain of P. falciparum was cultivated in human AB +ve red blood cells using RPMI 1640 medium supplemented with AB Rh+ serum (10 %), 5 % sodium bicarbonate and 40 μg/ml of gentamycin sulphate. The culture was incubated at 37°C under a gas mixture of 2% O 2 , 5% CO 2 , and 93% N 2 in CO 2 incubator. Initial parasitemia was maintained between 0.5% -1.0%. The growth of the parasite was daily observed by microscopic examination of thin blood smear treated with Geimsa stain and when the parasitaemia is above 3% in the initial culture it should be subcultured by replacing the old media with fresh RBCs and complete media. Percentage parasitaemia is calculated by counting the infected erythrocytes in a total of 10,000 erythrocytes.  18,19 Ethnomedical studies revealed the significance of F. benjamina leaves (FBL) and F. deltoidea leaves (FDL) in the management of respiratory disorders, 20 burn injury, 21 diabetes, 22 cancer, 23 and inflammation. 24 The presence of wide range of chemical compounds such as flavnoids, coumarins phytosterols and phenolics are also considered as an important factor to accept the ethnopharmacological claims and modern pharmacological studies. 25,26,27,28,29 However, there is no scientific data yet reported in literature which specifies the Anti-plasmodial activity of FD and FB. With the above prospective of searching traditional medicines for the better Anti-plasmodial molecules, the various extracts of FDL, FBL and fractions of most active crude extract are evaluated for In vitro Anti-plasmodial activity by schizont maturation inhibition assay.  Table 2. Specimens of the same were preserved in the herbarium section of the Khalsa College of Pharmacy, Amritsar, and Punjab for further reference.

Preparation of plant extracts and fractions
Procured leaves were cleaned properly and shade dried in open air for 6-7 days and then, pulverized in mixture grinder to get a coarse powder in dry form. 500 g of dried powder was successively extracted with petro-

Schizont maturation inhibition assay
Stock solution (1000 µg/ml) of test extracts and fractions were prepared by dissolving 1 mg of lyophilized crude extracts and its fractions in 100 µl of DMSO and 900 µl of incomplete media (without Plasmodium strains). The 100 µl of this stock solution was placed into the 96 well plate previously inoculated with 100 µl of Plasmodium cultured blood mixture media in two-fold serial dilution to obtain drug concentrations of 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2.0 and 0.9 µg/ml. Two initial concentrations of 500 µg/ml and 250 µg/ml were not considered for the Anti-plasmodial drug evaluation because of its possibility of high toxicity to RBCs as well as to follow the WHO protocol of Anti-plasmodial drug screening. 32 Prepared well plates were incubated in the controlled gas mixture at 37 °C for 24-30 h for schizont maturation. After 24 h of incubation, a thick smear was prepared from the control well to observe maturity of schizonts and if more than 10% schizonts (with more   than three nuclei) were seen in the control wells, the experiment was considered valid for study Figure 3. The thin smear of each well was then prepared and after Giemsa staining the schizonts was counted out of a total 200 asexual parasites. IC 50 values, indicates the concentration of the test sample where 50% of schizont maturation is inhibited as compared with parasite development in positive control. Negative control is maintained with fresh red blood cells, positive control is maintained with parasitized blood cell culture without any treatment.

RESULT AND DISCUSSION
In the present study, the crude extracts of FDL and FBL were evaluated for Anti-plasmodial activity by schizont maturation inhibition assay Table 3. According to WHO guidelines, Anti-plasmodial activity was classified as follows: highly active/good at IC 50 < 5 µg/ml, promising at 5-15 µg/ml, moderate at 15-50 µg/ml and inactive at >50 µg/ml. 33 Based on this classification, the crude hydro-alcoholic extract ( Table 4. The preliminary phytochemical analysis confirms the presence of steroids, terpenoids, alkaloids and phytosterols in FBPE and its bio-fractions except butanol fraction (FBF b ).
The results obtained from this study revealed the rich chemical diversity of FBL which could be responsible for the Anti-plasmodial activity of FBPE and its bio-fractions. Literature also implicated several members of steroids, terpenoids and phytosterols have showed high significant inhibitory effect on the growth of Plasmodium parasite. 34,35 It could be considered that the Anti-plasmodial activity of FBL and its bio-fractions can be ascribed to the phytochemical constituents present in them, which further justified the use of FBL for the treatment of malaria in the tradi-

CONCLUSION
The present study demonstrates the successful application of traditional plant based drugs in the treatment of malaria. In vitro Antiplasmodial drug screening of F. deltoidea and F. benjamina leaves justifies its traditional usefulness in modern medicine. The crude petroleum ether extract of F. benjamina was found to be more active of two allied species of genus Ficus. Moreover, its hexane and chloroform fractions can be considered as an important lead in the discovery of new antiplasmoidal drug molecule which could only be possible by isolating pure active constituents. Thus, a further study on the isolation of Anti-plasmodial molecules from the active fractions of F. benjamina is in process and will be reported in near future.