Ficus racemosa is an important medicinal plant of Assam with a wide range of ethnomedicinal values. Many researchers have pharmacologically investigated several parts of this plant. The present study investigated the heavy metal content, phytochemical and amylase and glucosidase inhibitory property of the plant along with in-silico docking and toxicity profile. Metallic content and trace elements play an important role in the physiology of plants and animals (Ozcan and Akbulut, 2008). Nevertheless, some heavy metals such as Cd, Cr, Pb, etc. are known to have toxic effects beyond certain limits. Several health complications related to lung, liver, kidney, and heart are reported from high exposure of heavy metals (Barbee and Prince, 1999; Prashanth et al., 2015). The present study found that the fruits of F. racemosa contain negligible amount of toxic elements as per the WHO permissible level (WHO, 1996). Several chemicals and active ingredients are reported from different parts of F. racemosa. GC-MS analysis in the present study revealed six major compounds from the fruits of F. racemosa. However, we did not find any literature regarding the biological reports of identified compounds. Lipinski’s rule of five is considered to be an important parameter that predicts the druglikeness of a compound. In the present in-silico druglikeness study, compounds C1, C3, and C4 were found to violate the rule in one or two parameters. LogP is a partition coefficient which determines the lipophilicity of a molecule, its absorption, distribution, and penetration in the body (Arnott and Planey, 2012). All the compounds identified from F. racemosa fruit extract were found to be lipophilic in nature while acarbose is strongly hydrophilic. Cell permeability of a drug candidate also depends on the topological polar surface area of the molecule. The increasing value of TPSA is associated with the non-permeability or non-bioavailability of the compounds (Palm et al., 1997). The upper limit of TPSA for a molecule to enter through cell membrane and brain is about 140Å² and 90Å², respectively (Pjouhesh and Lenz, 2005; Matsson and Kihlberg, 2017). The identified compounds from F. racemosa were found to have small TPSA value, and therefore highly permeable through the cell membrane.
The study of pharmacokinetics is an important prerequisite in the present-day drug discovery pipeline. The drug candidate needs to be easily absorbed and distributed throughout the body without any metabolism before reaching the target site. The lead compound needs to be less toxic and easy excretion from the body (Hodgson, 2019). The sum of the properties, as mentioned above, can be studied as ADMET profile. Computer-aided methods have been employed by many researchers to understand the ADMET profiles of compounds. By adopting the in-silico approaches, the cost and the time factor may be minimized compared to standard experimental approaches (Dimasi et al., 2003). The identified compounds from F. racemosa showed a moderate to high ADMET profile suggesting the possibility of a drug candidate. Because of its small size and lipophilic property, all the compounds are predicted to be easily absorbed by the human intestine, except C6. Organ toxicity such as hepatotoxicity, acute oral toxicity, and genotoxicity such as carcinogenicity, Ames mutagenesis also showed low to moderate levels of toxicological effects. The predicted LD50s were found to be 413.175 mg/kg, 1089.142 mg/kg, 1332.226 mg/kg, 967.882 mg/kg, 1438.699 mg/kg, and 1528.968 mg/kg for C1, C2, C3, C4, C5, and C6, respectively. Reference acarbose showed the lowest LD50 5874.473 mg/kg.
Amylase and glucosidase are two of the most important enzymes of chemotherapeutic drug targets in antidiabetic drug designing. By inhibiting these enzymes blood glucose level can be reduced significantly. Many research findings have revealed the α-amylase and α-glucosidase inhibitory properties of several medicinal plants (Kazeem et al., 2013; Somtimuang et al., 2018). In the present study methanolic crude fruit extracts of F. racemosa showed strong α-amylase and α-glucosidase enzyme inhibition. Earlier study by Chaware et al. (2020) reported several pharmacological properties of F. racemosa including the antidiabetic property of bark extracts. Flavonoids such as Kaempherol, Quercetin, Naringenin, and Baicalein isolated from stem bark of F. racemosa were found to have considerable antidiabetic property (Keshari et al., 2016). Baicalein is also reported to have glucose metabolism improving property in insulin-resistant HepG2 cells (Yang et al., 2019). Similarly, Deep et al. (2013) revealed the antidiabetic properties of several other plant species belonging to genus Ficus such as F. benghalensis, F. carica, F. glomerata, F. glumosa, and F. religiosa. Molecular docking is another aspect of drug designing approach which is a widely used, relatively fast, and economical computational tool that predicts the binding affinities between ligands and proteins. Using in-silico method virtual screening of a large number of chemicals can be screened to select the lead compounds of probable drug candidates (Huang and Zou, 2010). In-silico molecular docking has been used by many researchers to verify the effectiveness of phytochemicals (Ghaedi et al., 2020; Surriya et al., 2013; Swargiary et al., 2020). Ligand–enzyme binding and interaction studies have shown that the compounds from F. racemosa showed better binding affinity in both the enzymes than acarbose. The binding energy of C3 was found to be stronger in both the enzymes. Thus, the molecular docking interactions reflect the results of in-vitro enzyme assays indicating the α-amylase and α-glucosidase inhibitory property of Ficus racemosa. One of the most commonly used in-vitro methods of screening healthy cells to observe the growth, reproduction and morphological effects of chemical agents or plant extracts is the cytotoxicity test (Nath et al., 2020). Cytotoxicity is preferred as a pilot project test and an important indicator for toxicity evaluation of chemical agents as it is simple, fast, has a high sensitivity and can save animals sacrifices from toxicity assays (Li et al., 2015). In the present study, we used PBMC and splenocytes to test toxicity profiles of plants extract on healthy cells and result revealed limited toxicity with 5–10% cell death by apoptosis.
Pharmacophore modeling is a computationally effective and pragmatic technique for the discovery and optimisation of biologically active compounds (Wolber et al., 2008) and the study of intermolecular interactions in silico (Mortier et al., 2017). In current drug discovery field, pharmacophore features is most commonly used drug designing tool that led to establishment of more stable and targeted drugs with no or limited side effect in the host, as they are supposed to more precisely targeted (Bredel and Jacoby, 2004). The process of identifying a target, synthesizing bio-active compound with desired pharmacological action like minimal toxicity, high bioavailability, cost-effective synthesis, etc., and finally developing it to introduce in the market is a time-consuming, extremely complex and risky endeavour (Atanasov et al., 2015). To overcome these challenges, LigandScout, a standalone platform that address all the problems related to pharmacological action of drugs and it has been used in the present study to generate essential pharmacophore features of synthesized compounds (Wolber et al., 2008). The obtained pharmacophore features of aforesaid compounds revealed their potential role during intermolecular interaction with target proteins (Fig. 8a and b). For a set of molecules sharing a similar biological response, a ligand-based pharmacophore model can be derived by superposing them and determining the maximum number of overlapping chemical features (Wolber et al., 2008). The structure superimposition result showed that compound 3 possess most of the chemical descriptors common with other aligned compounds and hence bind more strongly with target proteins (Fig. 9 and Fig. 7). Further study is underway to isolate and characterize the bioactive compound of the plant and also to investigate the in vivo effect of isolated compounds of the plant.