Tinosporaside from Tinospora cordifolia Encourages Skeletal Muscle Glucose Transport through Both PI-3-Kinase- and AMPK-Dependent Mechanisms.

The stem of Tinospora cordifolia has been traditionally used in traditional Indian systems of medicine for blood sugar control, without the knowledge of the underlying mechanism and chemical constitution responsible for the observed anti-diabetic effect. In the present study, Tinosporaside, a diterpenoid isolated from the stem of T. cordifolia, was investigated for its effects on glucose utilization in skeletal muscle cells, which was followed by determining the anti-hyperglycemic efficacy in our diabetic db/db mice model. We found that tinosporaside augmented glucose uptake by increasing the translocation of GLUT4 to the plasma membrane in L6 myotubes, upon prolonged exposure for 16 h. Moreover, tinosporaside treatment significantly increased the phosphorylation of protein kinase B/AKT (Ser-473) and 5' AMP-activated protein kinase (AMPK, Thr-172). These effects were abolished in the presence of the wortmannin and compound C. Administration of tinosporaside to db/db mice improved glucose tolerance and peripheral insulin sensitivity associated with increased gene expression and phosphorylation of the markers of phosphoinositide 3-kinases (PI3Ks) and AMPK signaling in skeletal muscle tissue. The findings revealed that tinosporaside exerted its antidiabetic efficacy by enhancing the rate of glucose utilization in skeletal muscle, mediated by PI3K- and AMPK-dependent signaling mechanisms.

Gundelia tournefortii: Fractionation, Chemical Composition and GLUT4 Translocation Enhancement in Muscle Cell Line.

Type 2 diabetes (T2D) is a chronic metabolic disease, which could affect the daily life of patients and increase their risk of developing other diseases. Synthetic anti-diabetic drugs usually show severe side effects. In the last few decades, plant-derived drugs have been intensively studied, particularly because of a rapid development of the instruments used in analytical chemistry. We tested the efficacy of Gundelia tournefortii L. (GT) in increasing the translocation of glucose transporter-4 (GLUT4) to the myocyte plasma membrane (PM), as a main strategy to manage T2D. In this study, GT methanol extract was sub-fractionated into 10 samples using flash chromatography. The toxicity of the fractions on L6 muscle cells, stably expressing GLUTmyc, was evaluated using the MTT assay. The efficacy with which GLUT4 was attached to the L6 PM was evaluated at non-toxic concentrations. Fraction 6 was the most effective, as it stimulated GLUT4 translocation in the absence and presence of insulin, 3.5 and 5.2 times (at 250 µg/mL), respectively. Fraction 1 and 3 showed no significant effects on GLUT4 translocation, while other fractions increased GLUT4 translocation up to 2.0 times. Gas chromatography-mass spectrometry of silylated fractions revealed 98 distinct compounds. Among those compounds, 25 were considered anti-diabetic and glucose disposal agents. These findings suggest that GT methanol sub-fractions exert an anti-diabetic effect by modulating GLUT4 translocation in L6 muscle cells, and indicate the potential of GT extracts as novel therapeutic agents for T2D.

ß-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells.

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of ß-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERß-ERE luc expression system with greater response through ERß in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERß through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.

In Vitro Evaluation of Chemically Analyzed Hypericum Triquetrifolium Extract Efficacy in Apoptosis Induction and Cell Cycle Arrest of the HCT-116 Colon Cancer Cell Line.

Naturally derived drugs and plant-based products are attractive commodities that are being explored for cancer treatment. This in vitro study aimed to investigate the role of Hypericum triquetrifolium (50% ethanol: 50% water) extract (HTE) treatment on apoptosis, cell cycle modulation, and cell cycle arrest in human colon cancer cell line (HCT-116). HTE induced cell death via an apoptotic process, as assayed by an Annexin V-Cy3 assay. Exposing HCT-116 cells to 0.064, 0.125, 0.25, and 0.5 mg/mL of HTE for 24 h led to 50 ± 9%, 71.6 ± 8%, 85 ± 5%, and 96 ± 1.5% apoptotic cells, respectively. HCT-116 cells treated with 0.25 and 0.5 mg/mL HTE for 3 h resulted in 38.9 ± 1.5% and 57.2 ± 3% cleavage of caspase-3-specific substrate, respectively. RT-PCR analysis revealed that the HTE extract had no effect on mRNA levels of Apaf-1 and NOXA. Moreover, the addition of 0.125 mg/mL and 0.25 mg/mL HTE for 24 h was clearly shown to attenuate the cell cycle progression machinery in HCT-116 cells. GC/MS analysis of the extract identified 21 phytochemicals that are known as apoptosis inducers and cell cycle arrest agents. All the compounds detected are novel in H. triquetrifolium. These results suggest that HTE-induced apoptosis of human colon cells is mediated primarily through the caspase-dependent pathway. Thus, HTE appears to be a potent therapeutic agent for colon cancer treatment.

In Vivo and In Vitro Antidiabetic Efficacy of Aqueous and Methanolic Extracts of Orthosiphon Stamineus Benth.

Orthosiphon stamineus is a popular folk herb used to treat diabetes and some other disorders. Previous studies have shown that O. stamineus extracts were able to balance blood glucose levels in diabetic rat animal models. However, the antidiabetic mechanism of O. stamineus is not fully known. This study was carried out to test the chemical composition, cytotoxicity, and antidiabetic activity of O. stamineus (aerial) methanol and water extracts. GC/MS phytochemical analysis of O. stamineus methanol and water extracts revealed 52 and 41 compounds, respectively. Ten active compounds are strong antidiabetic candidates. Oral treatment of diabetic mice with O. stamineus extracts for 3 weeks resulted significant reductions in blood glucose levels from 359 ± 7 mg/dL in diabetic non-treated mice to 164 ± 2 mg/dL and 174 ± 3 mg/dL in water- and methanol-based-extract-treated mice, respectively. The efficacy of O. stamineus extracts in augmenting glucose transporter-4 (GLUT4) translocation to the plasma membrane (PM) was tested in a rat muscle cell line stably expressing myc-tagged GLUT4 (L6-GLUT4myc) using enzyme-linked immunosorbent assay. The methanol extract was more efficient in enhancing GLUT4 translocation to the PM. It increased GLUT4 translocation at 250 µg/mL to 279 ± 15% and 351 ± 20% in the absence and presence of insulin, respectively. The same concentration of water extract enhanced GLUT4 translocation to 142 ± 2.5% and 165 ± 5% in the absence and presence of insulin, respectively. The methanol and water extracts were safe up to 250 µg/mL as measured with a Methylthiazol Tetrazolium (MTT) cytotoxic assay. The extracts exhibited antioxidant activity as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. O. stamineus methanol extract reached the maximal inhibition of 77 ± 10% at 500 µg/mL, and O. stamineus water extract led to 59 ± 3% inhibition at the same concentration. These findings indicate that O. stamineus possesses antidiabetic activity in part by scavenging the oxidants and enhancing GLUT4 translocation to the PM in skeletal muscle.

Drug Discovery of Plausible Lead Natural Compounds That Target the Insulin Signaling Pathway: Bioinformatics Approaches.

The growing smooth talk in the field of natural compounds is due to the ancient and current interest in herbal medicine and their potentially positive effects on health. Dozens of antidiabetic natural compounds were reported and tested in vivo, in silico, and in vitro. The role of these natural compounds, their actions on the insulin signaling pathway, and the stimulation of the glucose transporter-4 (GLUT4) insulin-responsive translocation to the plasma membrane (PM) are all crucial in the treatment of diabetes and insulin resistance. In this review, we collected and summarized a group of available in vivo and in vitro studies which targeted isolated phytochemicals with possible antidiabetic activity. Moreover, the in silico docking of natural compounds with some of the insulin signaling cascade key proteins is also summarized based on the current literature. In this review, hundreds of recent studies on pure natural compounds that alleviate type II diabetes mellitus (type II DM) were revised. We focused on natural compounds that could potentially regulate blood glucose and stimulate GLUT4 translocation through the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. On attempt to point out potential new natural antidiabetic compounds, this review also focuses on natural ingredients that were shown to interact with proteins in the insulin signaling pathway in silico, regardless of their in vitro/in vivo antidiabetic activity. We invite interested researchers to test these compounds as potential novel type II DM drugs and explore their therapeutic mechanisms.

In Vitro and In Silico Evaluation for the Inhibitory Action of O. basilicum Methanol Extract on α-Glucosidase and α-Amylase.

Diabetes mellitus is a metabolic disease that predominates, nowadays. It causes hyperglycemia and consequently major health complications. Type II diabetes is the most common form and is a result of insulin resistance in the target tissues. To treat this disease, several mechanisms have been proposed. The most direct route is via inhibiting the intestinal enzymes, e.g., α-glucosidase and α-amylase, responsible for intestinal polysaccharide digestion that therefore would reduce the absorption of monosugars through the intestinal walls. In this study, we shed the light on this route by testing the inhibitory effect of Ocimum basilicum extract on the enzymes α-glucosidase and α-amylase in vitro and in silico. Experimental procedures were performed to test the effect of the O. basilicum methanol extract from aerial parts followed by the in silico docking. 500 µg/mL of the extract led to 70.2% ± 8.6 and 25.4% ± 3.3 inhibition on α-glucosidase and α-amylase activity, respectively. Similarly, the effect of caffeic acid, a major extract ingredient, was also tested, and it caused 42.7% ± 3.0 and 47.1% ± 4.0 inhibition for α-amylase and α-glucosidase, respectively. Docking experiments were performed to predict the phytochemicals responsible for this robust inhibitory activity in the O. basilicum extracts. Several compounds have shown variable levels of inhibition, e.g., caffeic acid, pyroglutamic acid, and uvasol. The results indicated that O. basilicum can be a potent antidiabetic drug.

Anticancer activity and phytochemical composition of wild Gundelia tournefortii.

Artichoke-like wild thistles are often used in Palestinian cuisine. One of the most commercially recognized species of these wild edible thistles is Gundelia tournefortii, a common plant in the Mediterranean region. G. tournefortii, or 'Akoob' in Arabic, remains uncultivated, harvested wild by local populations and considered highly valuable due to its reputed health benefits. The present study aimed to investigate the anticancer effects of G. tournefortii on the human colon carcinoma HCT-116 cell line. Methanol and hexane extracts were identified to exert considerable antitumor activity against the HCT-116 cancer cell line, while the aqueous extract was inactive. The phytochemical profiles of the methanol and hexane extracts were investigated using gas chromatography-mass spectrometry. A total of 6 of the 27 natural compounds identified, including sitosterol, stigmasterol, lupeol, gitoxigenin, α-amyrin and artemisinin, have been previously validated as being active against cancerous cells. Therefore, the presence of these phytochemicals in G. tournefortii is of importance in its role in preventing and treating cancer.

In Vitro Evaluation of the Potential Use of Propolis as a Multitarget Therapeutic Product: Physicochemical Properties, Chemical Composition, and Immunomodulatory, Antibacterial, and Anticancer Properties.

Propolis is a resin that honeybees produce by mixing saliva and beeswax with exudate gathered from botanical sources. The present in vitro study investigated the potential use of propolis as a multitarget therapeutic product and the physicochemical properties, chemical composition, and immunomodulatory, antioxidant, antibacterial, and anticancer properties of a propolis extract from the northern Morocco region (PNM). Pinocembrin, chrysin, and quercetin were the main phenolic compounds of PNM as measured in HPLC. The PNM showed significant inhibitory effects against all tested Gram-positive and Gram-negative strains and showed high antioxidant activities by scavenging free radicals with IC50 (DPPH = 0.02, ABTS = 0.04, and FRAP = 0.04 mg/ml). In addition, PNM induced a dose-dependent cytostatic effect in MCF-7, HCT, and THP-1 cell lines at noncytotoxic concentrations with IC50 values of 479.22, 108.88, and 50.54 µg/ml, respectively. The production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) was decreased in a dose-dependent manner in LPS-stimulated human peripheral blood mononuclear cells (PBMNCs), whereas the production of the anti-inflammatory interleukin-10 (IL-10) was increased in a dose-dependent manner reaching 15-fold compared to the levels measured in untreated PBMNCs. Overall, the results showed that the traditionally known multitarget therapeutic properties of the PNM seem to be mediated, at least in part, through cytostatic, antibacterial, and immunomodulatory effects.

Gundelia tournefortii Antidiabetic Efficacy: Chemical Composition and GLUT4 Translocation.

In the present in vitro study, we tested the chemical composition, cytotoxicity and antidiabetic activity of two distinct extracts of wild Artichoke-like vegetable, Gundelia tournefortii: methanol and hexane. GC/MS phytochemical analysis of G. tournefortii methanol and hexane extracts revealed 39 compounds reported here for the first time in G. tournefortii out of the 45 detected compounds. Only Stigmasterol was present in both extracts. The efficacy of G. tournefortii extracts in enhancing glucose transporter 4 (GLUT4) translocation to the plasma membrane (PM) was tested in L6 muscle cells stably expressing myc-tagged GLUT4 (L6-GLUT4myc) using cell-ELISA test. Results obtained here indicate that methanol and hexane extracts were safe up to 250 µg/ml as measured with MTT and the LDH leakage assays. The methanol extract was the most efficient in GLUT4 translocation enhancement. It increased GLUT4 translocation at 63 µg/ml 1.5- and 2-fold relative to the control in the absence and presence of insulin, respectively. These findings indicate that G. tournefortii possesses antidiabetic activity in part by enhancing GLUT4 translocation to the PM in skeletal muscle.

In vitro evaluation of anti-diabetic activity and cytotoxicity of chemically analysed Ocimum basilicum extracts.

The aim of this study was to evaluate the role of glucose transporter-4 (GLUT4) in the anti-diabetic effects of methanol, hexane and dichloromethane extracts of the aerial parts of Ocimum basilicum (OB) and to analyze their phytochemical composition. Phytochemical analysis of the three extracts by GC/MS using the silylation derivatization technique revealed 53 compounds, 17 of them were found for the first time in OB. Cytotoxic and anti-diabetic properties of the extracts were evaluated using L6-GLUT4myc muscle cells stably expressing myc epitope at the exofacial loop (GLUT4). No cytotoxic effects were observed in treated cells up to 0.25 mg/ml extract as measured with MTT and LDH-leakage assays. GLUT4 translocation to the plasma membrane was elevated by 3.5 and 7 folds (-/+ insulin) after treatment with OB extracts for 20 h. Our findings suggest that the observed anti-diabetic properties of OB extracts are possibly mediated in part through one or more of the 17 new identified compound.

In Vitro Evaluations of Cytotoxicity of Eight Antidiabetic Medicinal Plants and Their Effect on GLUT4 Translocation.

Despite the enormous achievements in conventional medicine, herbal-based medicines are still a common practice for the treatment of diabetes. Trigonella foenum-graecum, Atriplex halimus, Olea europaea, Urtica dioica, Allium sativum, Allium cepa, Nigella sativa, and Cinnamomum cassia are strongly recommended in the Greco-Arab and Islamic medicine for the treatment and prevention of diabetes. Cytotoxicity (MTT and LDH assays) of the plant extracts was assessed using cells from the liver hepatocellular carcinoma cell line (HepG2) and cells from the rat L6 muscle cell line. The effects of the plant extracts (50% ethanol in water) on glucose transporter-4 (GLUT4) translocation to the plasma membrane was tested in an ELISA test on L6-GLUT4myc cells. Results obtained indicate that Cinnamomon cassia is cytotoxic at concentrations higher than 100 µ g/mL, whereas all other tested extracts exhibited cytotoxic effects at concentrations higher than 500 µ g/mL. Exposing L6-GLUT4myc muscle cell to extracts from Trigonella foenum-graecum, Urtica dioica, Atriplex halimus, and Cinnamomum verum led to a significant gain in GLUT4 on their plasma membranes at noncytotoxic concentrations as measured with MTT assay and the LDH leakage assay. These findings indicate that the observed anti-diabetic properties of these plants are mediated, at least partially, through regulating GLUT4 translocation.

Indexing molecules for their hERG liability.

The human Ether-a-go-go-Related-Gene (hERG) potassium (K(+)) channel is liable to drug-inducing blockage that prolongs the QT interval of the cardiac action potential, triggers arrhythmia and possibly causes sudden cardiac death. Early prediction of drug liability to hERG K(+) channel is therefore highly important and preferably obligatory at earlier stages of any drug discovery process. In vitro assessment of drug binding affinity to hERG K(+) channel involves substantial expenses, time, and labor; and therefore computational models for predicting liabilities of drug candidates for hERG toxicity is of much importance. In the present study, we apply the Iterative Stochastic Elimination (ISE) algorithm to construct a large number of rule-based models (filters) and exploit their combination for developing the concept of hERG Toxicity Index (ETI). ETI estimates the molecular risk to be a blocker of hERG potassium channel. The area under the curve (AUC) of the attained model is 0.94. The averaged ETI of hERG binders, drugs from CMC, clinical-MDDR, endogenous molecules, ACD and ZINC, were found to be 9.17, 2.53, 3.3, -1.98, -2.49 and -3.86 respectively. Applying the proposed hERG Toxicity Index Model on external test set composed of more than 1300 hERG blockers picked from chEMBL shows excellent performance (Matthews Correlation Coefficient of 0.89). The proposed strategy could be implemented for the evaluation of chemicals in the hit/lead optimization stages of the drug discovery process, improve the selection of drug candidates as well as the development of safe pharmaceutical products.