University of Khartoum

In vitro Glycogen Phosphorylase Inhibition Coupled with LC/MS Profiling and Antioxidant Activity of Nauclea latifolia Smith Fruits

In vitro Glycogen Phosphorylase Inhibition Coupled with LC/MS Profiling and Antioxidant Activity of Nauclea latifolia Smith Fruits

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Title: In vitro Glycogen Phosphorylase Inhibition Coupled with LC/MS Profiling and Antioxidant Activity of Nauclea latifolia Smith Fruits
Author: Abd Algaffar, Shereen Omer Elfarouk
Abstract: Background: The long-term effects of the current clinically used anti-diabetic drugs are still unsatisfactory with various adverse effects which limit their usage. Considering its central role in glycogen metabolism, glycogen phosphorylase (GP); an allosteric protein with at least six different ligand binding sites that modulate its enzymatic activity; has recently emerged as an important therapeutic target to discover potent antidiabetic drugs for type 2 diabetes (T2D). Meanwhile, recent evidence indicates the intricate association of the inflammatory damage characterizes diabetes and the pathophysiological events mediated through pancreatic islets reactive oxygen species (ROS) in the loss of β-cell function. Traditional medicine in Sudan have been used for centuries to treat diseases including diabetes, hence there are already vast literatures available for data mining and among them Nauclea latifolia fruit (Karmadoda) standing very prominently. Therefore, during the last few years, considerable attention has been given to scientifically validate the bioactivity of some antidiabetic traditional plants. The aim of this study is to validate the traditional use of N. latifolia fruit as antidiabetic agent and identify its chemical components, molecular target, and mechanism of action in comparison with the intact fruits. Methods: The plant including its fruits was taxonomically identified as N.latifolia Smith and herbarium specimens were deposited. Both the intact and processed fruits were extracted with 70% ethanol, concentrated, and fractionated with petroleum ether, chloroform and ethyl acetate respectively. The crude extract and all its respective fractions were screened for (GP) inhibition using GPa. Caffeine and quercetin were used as standards. The Antioxidant activity was performed according to standard methods using DPPH (2,2 Diphenylpicrylhydrazyl) and ABTS (2,2’- Azinobis( 3-benzothiazoline- 6 sulfonic acid). Scavenging capacity of the compounds was compared with that of Ascorbic acid and Trolox. The quantification of the flavonoids contents was executed according to Aluminum chloride colorimetric assay method and total flavonoid content was expressed as quercetin equivalent per 100g dry weight of sample. The total phenolic content was determined using Folin-Ciocalteu reagent and the results were expressed as gallic acid equivalents per 100g dry plant matter. LC-PDAESIMS analysis were performed on apparatus with an electrospray source assisted by the DataAnalysis® software. The online SciFinder Nauclea genus database “Nauclea DB” was used to predict from all NPs of Nauclea genus. SpectrusX processor, C+HNMR Predictors and DB software (ACD/Labs, 2014 Release) were used to predict the chemical shifts of these natural products. Molecular Operating Environment (MOE. v. 2008.10, Chemical Computing Group, Canada) and Discovery Studio Visualizer Softwares were used for the visualization of the docking results and generating the images. Results: Systematic screening of N.latifolia Smith intact fruit and its traditionally processed fruit (Karmadoda) revealed a remarkable GP inhibition for the intact fruit (95.57%) in comparison with the processed fruit. The DPPH and ABTS radical scavenging activity revealed that the intact fruit ethyl acetate fraction exhibited the most prominent antioxidant activity (89.50±.0..0%DPPH RSA± SD) which far exceeding its respective processed counterpart (83.57±0.02 %DPPH RSA± SD). Statistical analysis indicated that processing of fruits had a direct negative significant impact (P< 0.05) on both the antidiabetic and antioxidant activities. Quantification of both the total phenolics and total flavonoid contents revealed higher polyphenolic contents in the intact fruits compared to processed ones. Planar chromatographic analysis and hyphenated LC/MS techniques resulted in the identification of five β-carboline alkaloids, a flavonoid glycoside and chlorogenic acid in the intact fruit, while the processed fruit exhibited different chemical profiles characterised by the presence of only three β-carboline alkaloids besides the flavonoid glycoside in addition to a bis (2-ethylhexyl) phthalate which is considered as an artefact. Eventually, among the allosteric molecular targets analysed computationally, the catalytic site proved to be the most appropriate to accommodate both the phenolic and alkaloids identified in the fruit as ligands with the flavonoids luteolin 3'- methyl ether 7-glucuronide-4'-rhamnoside and chlorogenic acid scoring -28.39 and -21.42 Kcal/mol, respectively, among the phenolic compounds, as well as the 10- hydroxy strictosamide and strictosidine scoring - 28.40 and -23.07 Kcal /mol, respectively. Conclusions: The intact fruit is superior in terms of its antidiabetic and antioxidant capacity than the processed fruit. The catalytic site has been identified as the main N. latifolia molecular target for GP antidiabetic activity
URI: http://khartoumspace.uofk.edu/123456789/27035


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