Screening for potential GSK-3 inhibitors from medicinal plants in Sabah

Norhaniza Johansah (2015) Screening for potential GSK-3 inhibitors from medicinal plants in Sabah. Masters thesis, Universiti Malaysia Sabah.

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Abstract

Glycogen synthase kinase-3 (GSK-3), a serine/threonine protein kinase, is ubiquitously expressed in the mammalian cells as GSK-3α and GSK-3β. The aberrant activity of GSK-3 has been implicated in various human diseases, such as type II diabetes mellitus, cancer, and neurodegenerative diseases. Metabolites capable of inhibiting GSK-3 are important to suppress overexpression of GSK-3, thus, potentially useful as therapeutic approach for these diseases. A total of 112 plant samples collected from various places in Sabah were extracted using 99.9% (v/v) methanol and assigned codes based on plant parts the samples were prepared from. Prior to GSK-3β yeast screening assay using genetically-engineered H10075 yeast strain, the extracts were concentrated under vacuum and redissolved to a stock concentration of 100 mg/ml. Based on the screening data, eight plant samples (TWU002L, G021W, DL2-1W, GE01W, S1M2L, S1M2St, S1M2R and S3M4Fr (first sampling)) exhibited GSK-3β inhibitory activities, while, 13 plant samples (S3M4Fr (second sampling), S3M4St, JT27L, TWU004Rh, K001B, GS010B, S12M14Fr, S12M14St, S27M1Fr, KB50Fr, BN002L, BN003L and BK09St) showed toxic activities. Consistent inhibitory activities were detected in three out of eight potential plant samples; GE01W (Gleichenia truncata), S1M2L (Turnera ulmifolia) and S3M4Fr (Diospyros sp.), and these were further partitioned into hexane (HE), ethyl acetate (EAE), chloroform (CE), chloroform-methanol (C-ME), butanol (BE) and aqueous extracts (AE). Of these partitioned extracts, GE01W.CE, GE01W.C-ME, GE01W.EAE, S1M2L.CE and S3M4Fr.HE displayed GSK-3β inhibitory activities. From these, GE01W.CE, S1M2L.CE and S3M4Fr.HE were selected to be fractionated via column chromatography (CC). GE01W.CE was subjected to solvent precipitation, followed by a series of column chromatographic separations using chloroform:methanol mixtures with increasing polarities as the mobile phases to yield four active fractions (GE01W.CE.Prep.FrA.F1, GE01W.CE.Prep.FrA.F8, GE01W.CE.Prep.FrA.F9 and GE01W.CE.Prep.FrA.F11). Out of these, fraction GE01W.CE.Prep.FrA.F8 with the most significant inhibitory activity, was further analyzed via HPLC and LC-MS/MS. S1M2L.CE and S3M4Fr.HE were chromatographically-eluted with chloroform:methanol (19:1) and hexane:dichloromethane (3:7), respectively. LC-MS/MS analysis of GE01W.CE.Prep.FrA.F8 revealed the presence of two main phytocomponents; pcoumarylhexose (pCAH) and p-coumaric acid methyl ester (pCAME). pCAME exhibited non-selective inhibitory activity at 0.4, 1.0 and 2.0 mg/disk. However, at 0.1 and 0.2 mg/disk, pCAME showed potential GSK-3β inhibitory activity. Phytochemical screening of GE01W demonstrated the presence of flavonoids, coumarins, saponins, tannins, phenolics, terpenoids, sugar and polysaccharides, and traces of alkaloids. In GSK-3β enzymatic assay using Kinase-Glo® Luminescent Assay kit, different concentrations of pCAME in the reaction mixtures resulted in a significant decrease of Vmax (V’max values decreased from about 1.8 to 0.5) without large changes in the apparent Km values (K’m values ranging from 1.6 to 3.0 μM), suggesting inhibition of GSK-3β by pCAME through ATP non-competitive binding mode. pCAME recorded an IC50 value of 18.88 μM. As conclusions, pCAME identified in G. truncata, is a potential novel non ATP-competitive inhibitor of GSK-3β. pCAME and G. truncata extracts may therapeutically useful for treatment of various diseases, such as type II diabetes mellitus and Alzheimer’s disease.

Item Type:	Thesis (Masters)
Keyword:	Glycogen synthase kinase-3, p-coumaric acid methyl ester, Inhibition, Type II diabetes, Alzheimer's disease, Flavonoids, Coumarins, Saponins
Subjects:	Q Science > QK Botany > QK1-989 Botany > QK710-899 Plant physiology
Department:	FACULTY > Faculty of Science and Natural Resources
Depositing User:	DG MASNIAH AHMAD -
Date Deposited:	11 Apr 2025 11:16
Last Modified:	11 Apr 2025 11:16
URI:	https://eprints.ums.edu.my/id/eprint/43459

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