Sleman Kadan

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 μg/mL, whereas all other tested extracts exhibited cytotoxic effects at concentrations higher than 500 μg/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.

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.

Anticancer Activity of Anise (Pimpinella anisum L.) Seed Extract

Cancer incidence is much lower in India than in western countries. The reason is not fully understood, but the high spice consumption could be one of the contributing factors. Anise is one of the plants grows in India and people from our region believe that anise seeds are helpful in cancer prevention and treatment. In this study, anticancer activity of eth- anol extract of anise (Pimpinella anisum L.) seed was investigated. MTT and LDH assays revealed that ethanolic extract have cytotoxic activity on human prostate cancer cell line (PC-3) at concentrations found safe to normal cells (rat skeletal muscle cell line (L6)). Treatment with anise seeds extract caused anti proliferative and apoptotic effects, with IC50 value of 400 µg/mL to cancer cells. Thus, anise could be one of the foods that attribute to cancer prevention and treatment. It could also be a natural source of novel anticancer compounds with anti proliferative and/or apoptotic properties and it is worth to work on for isolation and identification of novel anticancer drug candidates.

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.

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.

Herbal-Derived Anti-obesity Compounds and Their Action Mechanisms

Recent researches demonstrated the potential of phytochemicals to prevent and to counteract obesity. Multiple combinations of phytochemicals can result in a synergistic activity that increases their bioavailability and action on multiple molecular targets, offering advantages over chemical treatments. Herbal-derived compounds have potential for controlling appetite, inhibiting pancreatic lipase activity, stimulating thermogenesis and lipid metabolism, promoting lipolysis and adipogenesis, and inducing apoptosis in adipocytes. Appetite reduction and inhibiting pancreatic lipase activity represent the first line in the regulation of body weight. Stimulating lipolysis and thermogenesis represents an essential target for the development of anti-obesity drugs. Furthermore, targeting adipocyte life cycle using various dietary bioactives that affects different stages of adipocyte life cycle represents also an important target for the development of new anti-obesity drugs. In this regard, different stages of adipocyte development that are targeted by anti-obesity drugs can include preadipocytes, maturing preadipocytes, and mature adipocytes. Various herbal-derived active compounds, such as genistein, daidzein, cyanidin, proanthocyanidin, xanthohumol, apigenin and luteolin, kaempferol, myricetin, quercetin, epigallocatechin gallate, conjugated linoleic acid, docosahexaenoic acid, quercetin, resveratrol, and ajoene, affect adipocytes during specific stages of development, resulting in either the induction of apoptosis or inhibition of adipogenesis. Although numerous molecular targets that can be used for both treatment and prevention of obesity have been identified, targeted single cellular receptor or pathway has resulted in limited success. Therefore, targeting several cellular pathways simultaneously with multiple phytochemicals to achieve synergistic effects might be an appropriate approach in the management of obesity.

Prevention and Treatment of Obesity-Related Diseases by Diet and Medicinal Plants

Obesity increases the risk for many pathological processes including type 2 diabetes, hypertension, hyperlipidemia, cardiovascular diseases, and certain types of cancer. Thus, obesity mitigation strategies should take into account these secondary pathologies in addition to promoting weight loss. Progress in biological and particularly in biochemical studies with respect to adipocytes in recent years has gradually clarified that the dysregulation of adipose tissue expansion accompanied by hyperplasia (increase in the total number) and hypertrophy (increase in the size) of adipocytes causes obesity. Adipocytes are well recognized as endocrine secretory cells as well as fat storage cells, which produce biologically active molecules such as hormones, cytokines, and other factors. These molecules, collectively called adipocytokines (recently often called adipokine), are involved in regulating adipocyte functions and metabolism through a network of endocrine, paracrine, and autocrine signals and thus modulate adipocyte biology. Adipocytokines include tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), plasminogen activator inhibitor-1 (PAI-1), angiotensinogen, tissue factor, transforming growth factor-β (TGF-β), leptin, adiponectin, resistin, and certain chemokines such as monocyte chemoattractant protein-1 (MCP-1). Most adipocytokines are increasingly produced with increasing adiposity and are closely associated with obesity-related pathologies (e.g., metabolic syndrome, diabetes, and cardiovascular diseases). In this chapter, we will focus on prevention and treatment of obesity-related diseases by diets and medicinal plants.

Novel anti diabetic phytochemicals: Isolation, protein target and mechanism of action

Statement of the Problem: The associations of serum zinc, copper and Zn/Cu ratio with clinical/biochemical characteristics in both types of diabetic patients were explored in this study. Methodology & Theoretical Orientation: Serum levels of zinc and copper were measured by atomic absorption spectrophotometer in 26 type 1 and 80 type 2 diabetic patients, along with 205 age/gender-matched healthy controls. Findings: Significantly decreased levels of Zn and Zn/Cu ratio were observed in both types of diabetic patients compared to controls, more clearly in type 1 (p<0.001). Positive and significant correlations between Zn and age (r=0.460, p<0.001), Zn and BMI (r=0.344, p<0.001), Zn/Cu and total cholesterol (r=0.207, p=0.033), and Zn and the number of coronary risk factors (Spearman’s r=0.311, p=0.001) were found. No significant relationship between Zn, Cu and Zn/Cu ratios with other biochemical parameters including HbA1c and in terms of family history, smoking, medications, micro vascular and macrovascular complications was found in both types of diabetic patients. Conclusion & Significance: Alterations of trace elements have been reported to have effects on the pathogenesis and progression of diabetes mellitus. Although decreased Zn/Cu ratio was found in our diabetic patients, no significant correlations were observed with clinical/biochemical characteristics except BMI, cholesterol, total cholesterol, and the number of coronary risk factors in our patients. Further studies may be the final arbiter of this issue.G drugs have been proposed as a monoor combined therapy for type 2 diabetes mellitus for the outstanding features, but natural GLP-1 is hardly used in clinic due to its short half-life, while short-acting analogs/receptor agonists have poor compliance in patients for frequent dosing. This study aims to introduce a novel long-acting GLP-1 receptor agonist, which is an antibody fusion protein by linking the human GLP-1 derivative to a humanized GLP-1R antibody via a peptide linker, and to evaluate its anti-diabetic effects and duration. Glutazumab is characterized by receptor binding and reporter gene assay, and its specificity was investigated through addition of exendin (9-39) and Ab1 which were the cognate receptor antagonist and antibody respectively. To evaluate the anti-diabetic effects, glutazumab was studied in diabetic KKAy mice by single dose and repeated doses. The blood glucose, food/water intake, body weight and gastric emptying was measured in the single dose study, while blood glucose, GSP, HbA1c, insulin and lipid were determined in the repeated-dose study. The oral glucose tolerance and hyperglycemic clamp test were performed to assess the β-cell function. In all the experiments, dulaglutide served as a control. Glutazumab significantly binds and activates GLP-1R, while the natural receptor antagonist exendin (9-39) showed no inhibition except in the presence of the antibody Ab1. Single injection of Glutazumab remarkably decreased blood glucose for 3~6 days in normal ICR mice and diabetic KKAy mice. Repeated injections of glutazumab also evidently reduced non-fasting and fasting blood glucose fluctuation, decreased GSP and HbA1c levels, improved impaired oral glucose tolerance and β-cell function and ameliorated the dyslipidemia in diabetic KKAy mice. These results demonstrated that glutazumab is a novel long-acting GLP-1 receptor agonist with excellent anti-diabetic effect in KKAy mice, and suggested that it may be a potential treatment for type 2 diabetes.

Teucrium polium extracts stimulate GLUT4 translocation to the plasma membrane in L6 muscle cells

Teucrium polium L. (TP) is recommended by herbal and integrative practitioners for the treatment of diabetes. However, its mechanism of action in stimulating GLUT4 activity and translocation to the plasma membrane is still unknown. This in vitro study examined the chemical composition, cytotoxicity, and antidiabetic activity of three TP distinct extracts: water/ethanol (WTP), methanol (MTP), and hexane (HTP). The compositions of the TP extracts were determined by GC/MS. MTT and LDH assays were used to assess the toxicity of the extracts. The efficacies of the TP extracts in enhancing glucose transporter-4 (GLUT4) translocation to plasma membrane (PM) were tested in L6 muscle cells, stably expressing myctagged GLUT4 using the cell-ELISA test. GC/MS phytochemical analysis of MTP and HTP extracts revealed 10 compounds in each extract, and only palmitic acid was communal in these two extracts. WTP, MTP, and HTP extracts were safe up to 63, 63, and 250 μg/ml, respectively. The HTP extract was the most efficient in GLUT4 translocation enhancement, while the least efficient was the WTP extract. In addition, the HTP extract increased the GLUT4 translocation at 32 μg/ml by 2and 3-fold relative to the control in the absence and presence of insulin, respectively. A similar result was obtained with the MTP extract at 63 μg/ml. In contrast, WTP extract in the presence of insulin had no effect and in the absence of insulin had slightly enhanced GLUT4 translocation. These findings indicate that TP antidiabetic activity is mediated in part by enhancing GLUT4 translocation to the PM in skeletal muscle.

Introduction to Medicinal Plant Safety and Efficacy

The utilization of herb-based products to prevent/treat diseases is a therapeutic modality that has stood the test of long history of use and has played important roles in the development of conventional medicine. Indeed, many of the currently utilized pharmacological classes of drugs include a phytochemical prototype. Aspirin, atropine, codeine, ephedrine, digoxin, L-dopa, morphine, quinine, reserpine, taxol, and tubocurarine are a few examples of drugs, which were originally discovered through the study of medicinal plants and traditional knowledge of indigenous people. Black seeds, garlic, ginseng, ginger, ginkgo, olive oil, pomegranate, milk thistle, and St. John’s wort are a few examples of medicinal plants, which are gaining popularity among modern physicians and researcher, and this trend is likely to continue partly due to high cost involved in the development of patentable synthetic drugs. There is growing evidence to show that pharmacological effects of medicinal plants are potentiated through synergistic mechanisms and/or side effect-neutralizing combinations.