Phytochemical profiling of salak (salacca zalacca) fruit flesh extracts through metabolomics approach and investigations of antidiabetic and antioxidant potential

Abstract

Fruits of salak (Salaaca zalacca) is traditionally used and commercialized as an antidiabetic agent. However, the scientific evidence to proof this usage is quiet lacking. Therefore, this research was aimed at evaluating the antidiabetic activity of S. zalacca fruit using proton-nuclear magnetic resonance (1H-NMR) spectroscopy-based metabolomics, investigating its mechanism of action, profiling the identified antidiabetic agents using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) based metabolomics, and developing a validated regression model using Fourier transform infrared spectroscopy (FTIR) based fingerprinting. Firstly, the flesh of S. zalacca fruit was subjected to two different drying methods namely; oven and freeze-drying and then extracted using three different methods i.e., Soxhlet, sonication and maceration. For metabolomics study, the fruit was extracted by different ratios of ethanol and water (0, 20, 40, 60, 80, 100%, v/v). The extracts obtained were subjected to different in vitro assays including α-glucosidase inhibitory activity, DPPH and FRAP. The E60 extract exhibited the highest α-glucosidase inhibitory activity and therefore was chosen for further in vivo antidiabetic analysis using 1H-NMR based metabolomics approach. LC-MS and GC-MS based metabolomics approaches were used in profiling the bioactive metabolites present in the extract. A validated calibration model was developed using FT-IR based fingerprinting. Results obtained showed that the freeze-dried samples extracted with sonication exhibited the highest bioactivity compared to other extracts. The E60 extract contained the highest amount of TFC and TPC and hence displayed highest inhibitory activity against 1, 1-diphenyl-2 picrylhydrazyl (DPPH) radical, ferric reducing and α-glucosidase. Several α-glucosidase inhibitors such as stearic acid, palmitic acid, β-sitosterol, myo-inositol, and ethyl 4-[5-methyl-2-oxo-1',2',5',6',7',7'a-hexahydro-1H-spiro[indole-3,3'-pyrrolizine]-2'-ylamido]benzoate were identified through LC-MS and GC-MS based metabolomics. The docking results predicted the interactions of all the metabolites identified to be in non-competitive mode involving majorly hydrophobic interactions with the protein residues. Some of the residues involved are PRO456, AGR467, LYS406 HID295, ASN259 LEU313, LYS125, ARG315, PHE303 VAL266, ILE262, ARG263 TYR158, HIE280 ARG315 etc. The validated regression model based on the FTIR based fingerprinting was successfully developed for the first time through this study in predicting the α-glucosidase inhibitory activity activities of the new set of extracts of S. zalacca. In vivo toxicological study was performed using 15 male Sprague Dawley rats were administered different concentrations for continuous 15 days and there were no abnormal activity and mortality observed throughout the study period. Thus, further antidiabetic investigation was conducted on this extract using obese-diabetic induced rats (OBDC). Results obtained indicated that administration of the E60 at 400 mg/kg bw for 6 weeks significantly (P < 0.05) decreased the blood glucose level and also normalized the blood lipid profile of rats. However, based on the data obtained from the metabolomics, the metabolite profiles of the urine of tested rats could not be fully normalized by the E60 extract. Lastly, histopathology of kidney and pancreas of both normal and diabetic treated rats showed normal and improved conditions when compared to diabetic control group. Findings of our study further support the traditional claims of S. zalacca fruits in management of diabetes.