Patients and Methods: This study was a cross-sectional study that included adult men and women aged 20-60 years old. The subjects had no chronic or metabolic disease. This research was conducted from April to November, 2020, in North Sumatra Province, Indonesia. The parameters studied were demographics, daily food intake, anthropometry and a history of obesity in adolescence, and for the participants' fathers and mothers. The statistical test used was the chi-squared test/Fisher test.

Results: This study included 136 research subjects, 60 male and 76 female; based on the results of the study, 47.8% were found to be obese, but food intake showed a low intake (96.2%). There was a significant relationship between a history of obesity in adolescence and incidences of obesity (≥30 kg/m2) in the mother and father, with significance values of p=0.01, p=0.004, and p=0.001, respectively.

METHODS: Crude extract obtained from the dried leaves using 80% methanolic solution was further partitioned using different polarity solvents. The resultant extracts were investigated for their α-glucosidase inhibitory potential followed by metabolites profiling using the gas chromatography tandem with mass spectrometry (GC-MS).

RESULTS: Multivariate data analysis was developed by correlating the bioactivity, and GC-MS data generated a suitable partial least square (PLS) model resulting in 11 bioactive compounds, namely, palmitic acid, phytol, hexadecanoic acid (methyl ester), 1-monopalmitin, stigmast-5-ene, pentadecanoic acid, heptadecanoic acid, 1-linolenoylglycerol, glycerol monostearate, alpha-tocospiro B, and stigmasterol. In-silico study via molecular docking was carried out using the crystal structure Saccharomyces cerevisiae isomaltase (PDB code: 3A4A). Interactions between the inhibitors and the protein were predicted involving residues, namely LYS156, THR310, PRO312, LEU313, GLU411, and ASN415 with hydrogen bond, while PHE314 and ARG315 with hydrophobic bonding.

AIM OF THE STUDY: In the present study, we investigated the effects of TCS against insulin resistance in muscle cells through integrating in vitro experiment and identifying its active biomarker using metabolomics and in molecular docking validation.

MATERIALS AND METHODS: We used centrifugal partition chromatography (CPC) to isolate 33 fractions from methanolic extract of TCS, and then used UHPLC-Orbitrap-HRMS to identify the detectable metabolites in each fraction. We assessed the insulin sensitization activity of each fraction using enzyme-linked immunosorbent assay (ELISA), and then used confocal immunocytochemistry microscopy to measure the translocation of glucose transporter 4 (GLUT4) to the cell membrane. The identified active metabolites were further simulated for its molecular docking interaction using Autodock Tools.

RESULTS: The polar fractions of TCS significantly increased insulin sensitivity, as measured by the inhibition of phosphorylated insulin receptor substrate-1 (pIRS1) at serine-312 residue (ser312) also the increasing number of translocated GLUT4 and glycogen content. We identified 58 metabolites of TCS, including glycosides, flavonoids, alkaloids, coumarins, and nucleotides groups. The metabolomics and molecular docking simulations showed the presence of minor metabolites consisting of tinoscorside D, higenamine, and tinoscorside A as the active compounds.

METHODS: Liquid-liquid partition chromatography was used to separate methanolic extract to get hexane, ethyl acetate, butanol and residual aqueous fractions. The total antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging and ferric reducing antioxidant power assay (FRAP). The antidiabetic activity of methanol extract and its consequent fractions were examined by α-glucosidase inhibitory bioassay. The chemical profiling was carried out by gas chromatography coupled with quadrupole time-of-flight mass spectrometry (GC Q-TOF MS).

METHODS: The aqueous ethanolic leaf extracts of C. caudatus were characterized by NMR and LC-MS/MS. The total phenolic content and α-glucosidase inhibitory activity were evaluated by the Folin-Ciocalteu method and α-glucosidase inhibitory assay, respectively. The statistical significance of the results was evaluated using one-way ANOVA with Duncan's post hoc test, and correlation among the different activities was performed by Pearson's correlation test. NMR spectroscopy along with multivariate data analysis was used to identify the metabolites correlated with total phenolic content and α-glucosidase inhibitory activity of the C. caudatus leaf extracts.

RESULTS: It was found that the α-glucosidase inhibitory activity and total phenolic content of the optimized ethanol:water (80:20) leaf extract of the plant increased significantly as the plant matured, reaching a maximum at the 10th week. The IC50 value for α-glucosidase inhibitory activity (39.18 μg mL- 1) at the 10th week showed greater potency than the positive standard, quercetin (110.50 μg mL- 1). Through an 1H NMR-based metabolomics approach, the 10-week-old samples were shown to be correlated with a high total phenolic content and α-glucosidase inhibitory activity. From the partial least squares biplot, rutin and flavonoid glycosides, consisting of quercetin 3-O-arabinofuranoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and quercetin 3-O-xyloside, were identified as the major bioactive metabolites. The metabolites were identified by NMR spectroscopy (J-resolve, HSQC and HMBC experiments) and further supported by dereplication via LC-MS/MS.