Bioassay-guided isolation of potent α-glucosidase inhibitory compounds from the fruit of Piper mullesua Buch–Ham ex D Don. and their in silico screening

Abstract Two bioactive compounds caffeic and sinapic acid were isolated from the fruit of the Piper mullesua Buch–Ham ex D Don using bioassay guided approach. These compounds were isolated from water fraction using column chromatography followed by semi preparative HPLC. These compounds showed very potent anti-diabetic and antioxidant activities. The molecular docking was carried out to predict the mode of interaction of the isolated compounds with α-glucosidase. The in vitro α-glucosidase inhibitory activity of caffeic and sinapic acid was determined, and their IC50 values were found 0.67 and 0.82 µg/ml, respectively. A QSAR equation was generated with an R 2 value of 84.81%, which is suitable enough for predicting the IC50 values of test molecules. The aforementioned finding confirms the isolated compounds show very significant anti-diabetic potential which is supported by the molecular docking and QSAR study. So, it has ample scope for drug development with further in vivo and clinical study. Graphical Abstract


Introduction
Bioactive compounds present in medicinal plants that have anti-diabetic potential are now a days mostly used to develop therapeutic anti-diabetic drugs. The epigallocatechin gallate, hyperin, rosmarinic acid are some of the natural compounds-based inhibitors of a-glucosidase reported previously (Yin et al. 2014;Chanu et al. 2018). The phenolic compounds are the most widely distributed antioxidants present in plants and these compounds reduce the risk of diabetes (Famuyiwa et al. 2019).
From the literature, it was found that the researcher carried out the studies on phytochemical and its biological properties on Piper species (Parra Amin et al. 2019; Li et al. 2020;Noshita et al. 2020). North-Eastern India has suitable habitat for the growth of the Piper species (Gajurel et al. 2001). Piper mullesua Buch-Ham ex D Don (Syn: Piper peepuloides Roxb.) also known as hill pepper, which grows under cover of dense vegetation bearing fruits having medicinal properties. The fruit of the plant is used to treat colds, coughs and control the sugar level as per traditional knowledge (Parmar et al. 1997). Accordingly, it was considered for evaluation of anti-diabetic properties and isolate a-glucosidase inhibitors. The aim of this study was to identify and isolate the active constituents of Piper mullesua Buch-Ham ex D Don through a bioactivity guided approach and evaluation of their anti-diabetic and antioxidant potential by different in vitro and in silico methods. Molecular docking and QSAR analysis were carried out to understand the interaction of bioactive molecules and a-glucosidase enzyme accordingly. The molecular docking and QSAR analysis for predict the a-glucosidase inhibitors has not been done for the plant species so far. This is the novelty of this study.

Results and discussions
The bioactive molecules were isolated using a bioassay-guided approach. Initially, the fruit of the plant was extracted in methanol and the a-glucosidase assay was carried out in methanol extract and the IC 50 value was found to be 4.58 ± 0.23 mg/ml which is very promising for further study. The methanol extract was fractionated then in ethyl acetate, n-butanol, and water. The IC 50 value was found 10.219 ± 0.37 and 7.897 ± 0.18 mg/ml for n-butanol and water extract respectively. The ethyl acetate part is inactive in the a-glucosidase assay. The four sub-fractionated parts from the water fraction i.e., WF-1, WF-2, WF-3, and WF-4 were also subjected for this inhibitory analysis. The IC 50 value was found 5.89 ± 0.08, 2.106 ± 0.54, 0.704 ± 0.0.16, and 2.89 ± 0.35 mg/ml in WF-1, WF-2, WF-3, and WF-4, respectively. The caffeic acid and sinapic acid which was isolated from the WF-3 subfraction using semi-preparative HPLC ( Figure 1). The HPLC chromatogram of the compounds is shown in Figure S1 (Supplementary materials). The presence of the compounds was further confirmed through HRMS spectral analysis having molecular ion peak at m/z 180.1135 and 224.2052 for caffeic and sinapic acid, respectively, given in faction WF-3 ( Figure S2) (Supplementary materials). It is supported by reported information (Jeong et al. 2011; Van et al. 2016). Table 1 shows the different IC 50 values of the prepared extract and subfractions along with the isolated compounds.
Further, the isolated compounds were confirmed by their 1 H and 13 C-NMR spectra. The 1 H NMR spectra clearly suggest the structure of isolated compounds which was given in Figure S3 (A-B) validated by 13 C-NMR spectra in Supplementary materials Figure S4  (A-B). The NMR data are reported in section 2 in supplementary materials. The reported information is supported by previous data (Jeong et al. 2011; Van et al. 2016).
The calibration curves of both the compounds in Figure S5 showed good linear regressions. Table S1 gave the linear range and content of the isolated compounds.  The sinapic acid and caffeic acid content were found to be 0.0601 mg/L and 0.0239 mg/L, respectively. It was reported that the caffeic acid derivative like rosmarinic acid has shown significant results in in-vivo assay (Chanu et al. 2018). So, it has ample scope for further in vivo study.
To study the interaction of caffeic and sinapic acid with a-glucosidase, molecular docking studies were carried using the CDOCKER module of Biovia Discovery Studio 2019 (DS). The structure of a-glucosidase was retrieved from RCSB-Protein Data Bank, where it was complexed with standard inhibitor acarbose. The site where acarbose was bound to a-glucosidase was chosen as the active site for screening the binding efficacy of caffeic and sinapic acid to a-glucosidase. The structure of caffeic, sinapic acid, and standard inhibitor (positive control) were minimized using the full energy minimization module of DS. Molecular CDocker interaction energies of caffeic and sinapic acid with a-glucosidase (PDB ID: 5NN8) are À25.9692 kcal/mol and À28.4307 kcal/mol respectively (Table S2). The caffeic acid forms four conventional Hbonds with the residues ASP282, MET519, LEU677, and ARG600 and one Pi-Pi T shaped interaction with TRP481, while sinapic acid also forms four conventional Hbonds with the residues ASP282, ASP518, ARG600, and LEU677 and one Pi-Pi T shaped interaction with TRP481. Upon comparing the interactions of these compounds with that of the standard inhibitor acarbose, it was found that the CDocker interaction energy is À26.7226 kcal/mol. It forms four conventional H-bonds with ASP282, ASP404, ARG600, ASP616, and HIS674. The generated QSAR model shows a significant coefficient of determination R 2 ¼ 84.81%. Using this model, pIC 50 of caffeic acid, sinapic acid and acarbose was found to be 5.87325, 5.88645, and 6.03058, respectively. Therefore, it has been observed that both the compounds show significant inhibition of the enzyme and good agreement with experimental information. The 2D and 3D interactions from the docking results are given in Supplementary materials Figures 6S-7S. The generated QSAR plot is shown in Figure 8S, which is significantly capable of predicting the pIC 50 of the test set molecules.
The plant based natural antioxidant compounds can protect b-cells from reactive oxygen species (ROS) which can prevent diabetes. Therefore, an alternative for the management of obesity and diabetes are mainly medicinal plants which help to maintain low blood glucose as well as boost body antioxidant system and insulin regulation (Patel et al. 2012).
The antioxidant activity of the extracts was analyzed through three different methods, that is, DPPH, ABTS, and FRAP (Table S3). The WF-3 has the highest scavenging activity confirmed by all three assays. The isolated compounds, that is, caffeic and sinapic acid from WF-3 may possibly be the active antioxidant components as these have the lowest IC 50 values. The predominant DPPH scavenging activity was found which may be due to presence of the both isolated caffeic and sinapic acid. The isolated molecules may interact with DPPH scavengers to be produced a more stable intermediate or product than other scavengers produced in ABTS and FRAP assay (Chelleng et al. 2021).

Conclusion
The two potential anti-diabetic and antioxidant agents, that is, caffeic and sinapic acid were isolated from the fruit of Piper mullesua Buch-Ham ex D Don by bioassay guided approach. The IC 50 value clearly revealed that the responsible compounds show very significant a-glucosidase enzyme inhibitory properties. Molecular docking and QSAR studies of the isolated compounds revealed the possible interaction with a-glucosidase enzyme inhibitors. Molecular docking studies of the isolated compounds result in a good agreement with the in-vitro analysis data that suggests that both caffeic and sinapic acid inhibits the enzyme significantly. So, there is ample scope of in-vivo and clinical study for developing pharmaceutical products for health benefit.