OBJECTIVE: The objective of this study was to investigate the effects of four different polyols, namely, ethylene glycol, erythritol, xylitol and sorbitol on the acid-denatured states of CGB lectin.
METHODS: CGB lectin was subjected to acid denaturation at pH 2.5 and pH 1.5, both in the absence and presence of 30% (w/v) polyols, i.e. ethylene glycol, erythritol, xylitol and sorbitol. Thermal denaturation of the acid-denatured states was also studied in the absence and presence of these polyols. Different spectroscopic probes such as tryptophan fluorescence, ANS fluorescence and far-UV CD spectral signal were used to monitor structural changes in the acid-denatured states of CGB lectin in the presence of polyols.
RESULTS: Presence of erythritol, xylitol and sorbitol in the incubation mixture was found to stabilize the lectin at both pH 2.5 and pH 1.5, as evident from the burial of the hydrophobic clusters and decreased polarity around Trp residues. These polyols also stabilized the acid-denatured states of CGB lectin against thermal denaturation by shifting the thermal transition curves towards higher temperatures. Exposure of the acid-denatured states of CGB lectin, obtained at pH 2.5 and pH 1.5 to 61°C and 51°C, respectively, induced formation of non-native β-structures, compared to that present at 25°C, and this phenomenon was significantly suppressed in the presence of these polyols. Based on the spectral data, both sorbitol and erythritol appeared to exude better stabilizing effect. On the other hand, ethylene glycol was shown to destabilize the aciddenatured states of CGB lectin.
CONCLUSION: Thermal stabilization of the lectin was noticed in the presence of erythritol, xylitol and sorbitol at both pH 2.5 and pH 1.5. These polyols also stabilize the secondary and tertiary structures of the acid-denatured CGB lectin at 25°C. Ethylene glycol was proved to be a destabilizer of the acid-denatured CGB lectin.
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.
CONCLUSION: For high phytomedicinal quality, the 10th week is recommended as the best time to harvest C. caudatus leaves with respect to its glucose lowering potential.