Bioactive Flavonoids from the Stem Bark of Cordia dichotoma Forst.: Identification, Docking and ADMET Studies

Cordia dichotoma Forst. (F. Boraginaceae) has been traditionally used for the management of a variety of human ailments. In our earlier work, the antidiabetic activity of methanolic bark extract of C. dichotoma (MECD) has been reported. In this paper, two flavonoid molecules were isolated (by column chromatography) and identified (by IR, NMR and Mass spectroscopy/spectrometry) from the MECD with an aim to investigate their antidiabetic effectiveness. Molecular docking and ADMET studies were carried out using AutoDock Vina software and Swiss ADME online tool, respectively. The isolated flavonoids were identified as 3,5,7,3’,4’-tetrahydroxy-4-methoxyflavanone-3-O-L-rhamnopyranoside and 5,7,3’-trihydroxy-4-methoxyflavanone-7-O-L-rhamnopyranoside (quercitrin). Docking and ADMET studies revealed the promising binding affinity of flavonoid molecules for human lysosomal alpha-glucosidase and human pancreatic alpha-amylase with acceptable ADMET properties. Based on computational studies, our study reports the antidiabetic potential of the isolated flavonoids with predictive pharmacokinetics profile.


Results and Discussion
The phytochemical analysis revealed the presence of flavonoids, alkaloids, glycosides, saponins, steroids, carbohydrates and proteins in the methanolic bark extract of C. dichotoma Forst (MECD).

Compound 1 (MECD-1)
Subfraction 20-78 was purified by column chromatography on silica using methanol:ethyl acetoacetate to yield the pure compound 1 (120 mg). The isolated compound 1 was obtained as pale yellow amorphous powder with a melting range of 163-165 o C. The structure of the isolated compound 1, represented in Figure 1, was elucidated by UV, IR, 1HNMR, 13CNMR and Mass spectroscopic/spectrometric analyses.

Molecular docking
Molecular docking is used to understand the drug-receptor interaction, binding affinity and binding orientation of bioactive molecules into the target protein molecule.
The objective behind docking study is to predict a particular biological activity based on the binding orientation/ affinity of small molecule ligands to the appropriate target active site [9]. In the docking study, the binding affinity was predicted in terms of the interaction energy (kcal/mole). Results of docking (binding) energies are given in Table 1.
Both the compounds exhibited very good binding affinity against both alpha-glucosidase and alpha-amylase enzyme. The compound 1 (MECD-1) exhibited more binding affinity against alpha-amylase compared to the alpha-glucosidase. On the hand, the compound 2 (MECD-2) showed better affinity against alpha-glucosidase than alpha-amylase. No much variation in binding energies between these two enzymes were observed. Overall, both the isolated flavonoids exhibited significant inhibitory potential of human glucosidase and amylase enzymes. Post-docking visualization of protein-ligand complexes revealed that the compounds interacted with active site residues of the protein molecules through the formation of predominantly hydrogen bonding interactions (Figure 3 & Figure 4).  Ser524 residues, whereas the compound 2 interacted with Asp616, His674 and Leu678 residues through H-bonds (Figure 4a, 3b). The 3D diagrams revealed the binding conformation and binding poses of the compounds at the catalytic site of alpha-amylase (Figure 4c, 3d) were observed. diabetes [13]. The traditional usefulness about the antidiabetic potential of C. dichotoma is mentioned in literature [2][3][4]. In our earlier studies, the antidiabetic activity of the methanolic bark extract of C. dichotoma has already been reported [4]. Moreover, literature suggest that the flavonoids content demonstrates antidiabetic efficacy of many plants [14][15][16][17][18]. Our docking study validates the antidiabetic claim about C. dichotoma reported in traditional medicines as well as in recent literature. Although the isolated phytocompounds are already established bioactive flavonoids with many scientific reports from past literature, their antidiabetic potential determined by in silico (molecular docking) methods with alpha-glucosidase and alpha amylase inhibitory activities has been reported for the first time. Our study may thus provide an avenue for further investigation with these bioflavonoids for their development as potent antidibaetic molecules with alpha-glucosidase and alpha-amylase inhibitory agents for the treatment of type 2 diabetes mellitus.

ADMET
Results of predicted ADMET (absorption, distribution, metabolism, excretion and toxicity) data showed that both the isolated compounds possess good solubility profile, which is in favour of their oral bioavailability. There is a prediction of poor intestinal absorption, while the compounds were predicted to be non-inhibitors of the cytochromess (CYP450) [19]. Poor intestinal absorption might be due to their limited oil/water partition coefficient (logP) values (-1.64 and -1.84). CYP450 enzymes are largely involved in drug metabolism. Non-inhibition of CYP450 enzymes suggests that compounds do not suppress the metabolic fucntion of the enzymes. Inhibition can lead to increased bioavailability of compounds that normally undergo extensive first-pass elimination or to decreased elimination of compounds dependent on metabolism for systemic clearance. Compounds did not exhibit the property of blood brain barrier (BBB) penetration. It substantiates that the compounds are devoid of producing CNS toxicities.
Furthermore, quercitrin (compound 2) was predicted to be a substrate to permeability of glycoprotein (p-gp), whereas the other flavonoid molecule (compound 1) did not show such property. Glycoprotein is responsible for the efflux of drug molecules out of the target cells [20]. A good drug candidate should not only have sufficient efficacy against the therapeutic target, but also show appropriate ADMET properties at a therapeutic dose. It is therefore inevitable to evalute the ADMET profile of drug-like molecules to avoid the failute of candidate drugs at the clinical stage of drug development [21]. Forst. was submitted at the herbarium of the department for future reference.

Preparation of methanolic bark extract
The shade-dried barks of C. dichotoma were pulverized to a coarse powder and defatted using petroleum ether by cold maceration method [2] to remove fat, latex and non-polar compounds of high molecular weights. The defatted plant residues were then macerated succesively with methanol to obtain the desired extract [3,4].

Phytochemical analysis
Chemical tests for the screening and detection of phytochemical constituents of the MECD were carried out using the standard procedures [22,23].

Isolation of phytocompounds
The MECD was subjected to column chromatographic separation using silica gel

Identification of isolated compounds
Ultraviolet (UV)-visible spectra were recorded on Shimadzu UV-1700 UV-visible spectrophotometer and the wave lengths of maximum absorption (max, nm) were reported. Infrared (IR) spectra were obtained on a Bruker Alpha Fourier Transform (FT-IR) spectrometer using KBR disc and reported in terms of frequency of absorption (υmax, cm -1 ) . 1 H and 13 C Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker Avance II 400 FT-NMR spectrometer at 400 and 100 MHz, respectively using tetramethylsilane (TMS) as an internal standard (δ 0.00 ppm) and CDCl3 as a solvent.
Mass spectra were obtained on a LC-MS Water 4000 ZQ instrument using electrospray
Prior to docking, The docking was performed in the AutoDock Vina software [26] in accordance with the standard procedure. The protein crystal structure was prepared prio to the docking process. Hydrogen atoms were added to the protein structure, and all ionizable residues were set at their default protonation at pH 7.

Conclusions
This study reports two bioactive flavonoids, viz., Predictive ADMET study demonstrates acceptable pharmacokinetics of the isolated compounds. In silico study needs to be further validated by in vitro and in vivo experimental assays in order to confirm the antidiabetic effectiveness for the flavonoids reported herein. Our study may thus provide an avenue for further investigation with these bioflavonoids for their development as potent antidibaetic molecules with