The incidence of metabolic disorders have been sharply increased over the past 30 years due to unfavorable changes in environmental, social, economic, and behavioral factors. Metabolic disorders viz. diabetes, dyslipidemia, obesity are being regarded as major threats to human health and can rarely be cured. Though medical science has been successful in the eradication of many life threatening communicable diseases, metabolism related diseases have, paradoxically, nullified many a gains in public health. Studies indicate that unfavorable environment due to industrialization made human genetic background vulnerable that led to unprecedented rise in the prevalence of metabolism-related disorders. Several serious medical conditions have been linked to metabolic disorders such as high blood pressure, stroke and is also linked to higher rates of certain types of cancer.
Diabetes mellitus is one of the major causes of untimely illness and death in most countries and inflicts financial burden. It is defined as metabolic malfunctioning in which carbohydrate and lipid metabolisms are improperly regulated by insulin leading to characteristically elevated blood glucose levels. Persisting diabetic conditions might lead to damage of blood vessels, increased risk of coronary artery disease, myocardial infarction, atherosclerosis, claudication and stroke, blindness, nerve damage and in severe conditions may even lead to amputations (Saltiel, 2001; American Diabetes Association, 2006). Word Health Organization (WHO) has predicted that from 217 million diabetic people in 2005, worldwide diabetic population will increase to no less than 366 million by 2030. India has the highest occurrence of diabetes in the world with 32 million and is expected to increase to 78 million by 2030 (Wild et al., 2004). Asian Indians are genetically susceptible to insulin resistance and in conjunction with a sedentary lifestyle, higher consumption of fat and refined carbohydrates, are at risk of developing diabetes (Ramachandran et al., 1999).
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Diabetes care is a complex issue that demands many factors, beyond glycemic control, be addressed. Studies have shown that the long-term risk of complications can be minimized through best possible glycemic control. Diet management and physical activity is the most important step in the management and prevention of diabetes (American Diabetes Association, 2006b). In-take of complex carbohydrates and fibres, proteins (10-20% of diet), unsaturated fats is known to reduce post-parandial hyperglycemia. Physical activities help to decrease insulin resistance, consume excess glucose and fat and also increase GLUT4 at mRNA and protein level (Holmes and Dohm, 2004). In addition to insulin diabetes treatments are composed of several oral antihyperglycemic agents, including sulfonylureas, biguanides, thiazolidinediones, ï¡-glucosidase inhibitors, meglitinides and numerous combination treatments. A number of new drugs including dual peroxisome proliferator-activated receptor (PPAR) agonists, dipeptidyl peptidase IV (DPP-IV) inhibitors and glucagon-like peptide-1 (GLP-1) analogs have been introduced recently. The overall aim of all these treatments is to achieve a blood glucose level <7 mM at 2 h after lunch and HbA1c <7% and to avoid disabling hypoglycemia (Monnier and Colette, 2005).
Coast of diabetes related expenditure accounts for 5-10% of the total healthcare budget in many countries (Smyth and Heron, 2005). Overall, the budget allocated for diabetes care in India is too little to grant medical care for the patients and the burden of diabetes costs is born by the patients themselves. These costs can account for as much as 25% of the annual family income. In reality, efficacies of available antidiabetic drugs are still under debate due to either side effects or many patients who respond initially become insensitive to treatment over a period of time (16, UKPDS Group, 1995; Moller, 2001). For example, neither insulin injections nor oral antidiabetic drugs viz. acarbose, metformin and sulfonyl ureas can reinstate a normal euglycemia pattern, whether used alone or in combination, and whether administered as a regular or intensive regimen (Yki-JaÂ¨rvinen, 2001; American Diabetes Association 2002). Hence, newer and alternative approaches are necessary for better management of diabetes. Moreover, considering the fact that prevalence of diabetes is increasing in developing countries, with adequate treatment often not available, plant-derived active principles and plant-derivatives with purported hypoglycemic activity could be an alternative to existing treatments (Yeh et al., 2003; Bradley et al., 2007).
Lipids are essential for energy homeostasis, reproductive and organ physiology, and various aspects of cellular biology. They are also linked to many pathological processes, such as obesity, diabetes, heart disease, and inflammation. Dyslipidemia is a broad term that refers to a number of lipid disorders. Most of the lipid disorders are related to diet and lifestyle, although familial disorders are important as well. The basic categories of dyslipidemia include: elevated low density lipoprotein cholesterol (LDLc), low levels of high density lipoprotein cholesterol (HDLc), excess lipoproteins or elevated triglycerides (TG) (Eaton, 2005). Recommendations of the Adult Treatment Panels (ATP) III under The National Cholesterol Education Program (NCEP) have provided guidelines for identification and treatment of lipid related disorders (ATP III, 2001).
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TG-rich VLDL released by liver delivers fatty acids to adipocytes for storage and to cardiac and skeletal muscle for energy consumption. Lipoprotein lipase (LPL) secreted by the adipocyte, muscle, and macrophage, plays an important role in VLDL fatty acid release, and its subsequent conversion to LDL. Cholesterol ester-rich LDL, on the other hand, delivers cholesterol to peripheral tissues and maintaining cell membrane integrity. In the reverse transport system, high density lipoprotein (HDL) transports excess cholesterol from extrahepatic cells, such as macrophages at the vessel wall, to liver, where it can be recycled or catabolized to bile acid (Russel, 1992).
Therapeutic intervention involves intervention at a macro-level and control of multiple risk factors using therapeutic lifestyle approaches by diet control and increased physical activity. it is clear that lifestyle therapies that combine diet and exercise interventions are efficacious, nonpharmacological strategies for the treatment of dyslipidemia. Lifestyle based treatments are particularly advantageous because diet and exercise elicit complementary effects on lipid proï¬les.
Physical inactivity substantially contributes to the risk factors of the dyslipidemia Physical activity and exercise reduces TG significantly in those with higher baseline levels and minimally for those with relatively normal levels (Durstine et al., 2002). Weight loss combined with favorable dietary changes enhances exercise-induced changes in the lipoprotein pattern, particularly in those with metabolic syndrome. A reduction in body weight of 5-10% reduces total cholesterol by up to 18%, TG up to 44%, and LDLc up to 22%, and increases HDLc by up to 27% (Wolf and Grundy, 1983; Eckel, 1999). Diet favorably alters the lipid/lipoprotein profile and may allow use of lower drug doses. Dietary change can bring a reduction of 20-30% reduction of LDL (ATP III, 2001). Saturated fatty acids decrease LDL receptor (LDLR) expression and increase LDLc levels. ATP III recommends reduced intake of SFA (~7% of total calories) with the remainder of total fats (25-35% of total calories) from polyunsaturated fatty acids and monounsaturated fatty acids and intake of less than 200 mg cholesterol per day.
A limited group of drugs are being used in the management of dyslipidemia depending up on patient's serum lipid profile, cardiovascular risk, and the liver and kidney functions (ATP III, 2001). Hypolipidemic agents include: 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (statins), nicotinic acid, ï¬bric acids, bile acid sequestrants, cholesterol absorption inhibitor, ezetimibe, Cholesteryl ester transfer protein inhibitor as well as combination therapies. These cholesterol-lowering agents were shown to decrease LDL-c levels up to 55%, increase HDL-c levels up to 35%, and decrease TG levels as much as 50%, depending on the drug and dose (4). Although these drugs produce desirable shifts in lipid levels within a short period of time, several safety concerns have surfaced regarding the long term use of these pharmacological agents (5-10). In particular, evidence suggests that the use of statins may result, although infrequently, in certain forms of myopathy, i.e., mild muscle aches to severe pain, restriction in mobility, as well as grossly elevated levels of creatine kinase (5). Additionally, liver toxicity, characterized by increases in hepatic transaminases, was also shown to result from prolonged use of statins at high doses (5, 7). Safety concerns regarding the use of bile acid sequestrants and ï¬bric acids were also reported (8 -10).
Clinical studies demonstrate that only 38% of patients receiving lipid-lowering therapy achieved NCEP-defined target levels (Breuer, 2001). Given the limitations of currently available lipid-lowering approaches, alternate therapy that is tolerable, safe, convenient, and removes excess lipid from the body is warranted. In view of these safety issues, the implementation of nonpharmacological therapies that beneï¬cially modulate lipid proï¬les without the risk of adverse affects would be highly advantageous. The Third Report of the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) recommends lifestyle therapies in place of drug therapies for patients who fall into an intermediate range of CHD risk (4).
Obesity is a grave, chronic disease in which excess body fat is accumulated such that health may be adversely affected. Obesity is defined as an excessively high amount of body fat in relation to lean body mass. Obesity is characterized by measuring body mass index (BMI) (Kopelman, 2000). BMI is expressed in mathematical formula in which a person's body weight in kilograms is divided by the square of his or her height in meters [wt/(ht) 2].
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The WHO has observed that the prevalence of obesity is rapidly rising to epidemic proportions around the world. Available data suggests that more than one billion adults population worldwide are overweight and at least 300 million of these are obese (Deital, 2003). Sedentary lifestyles, high-fat, energy-dense diets, and increased urbanization are the fundamental causes of this epidemic (James et al., 2001; Caballero, 2007). Obesity causes costly health problems, reduces life expectancy, and is associated with stigma and discrimination. Several other diseases have been linked to obesity (Table 3), including diabetes, heart diseases, high blood pressure, stroke and certain types of cancer (Aronne, 2002).
Management of obesity
Obesity is a chronic, stigmatized and costly disease. Because obesity can rarely be cured, treatment strategies are effective only as long as they are used. Many studies demonstrate that obese adults can lose about 0.5 kg per week by decreasing their daily intake to 500 to 1000 kcal below the caloric intake required for the maintenance of their current weight (Wadden and Foster, 2000). Over a period of six months, persons who combine caloric restriction and exercise may lose about 5 to 10 percent of body weight. It also helps long-term maintenance of a reduced weight (Blackburn, 1999; Smyth and Heron, 2005). In the later stages pharmacological intervention will become more prevalent. Present management of obesity essentially includes, exercise and diet modification, surgical intervention, treatment with approved drugs, plant extracts and plant derived compounds. Currently approved drugs for long-term treatment of obesity include sibutramine, which inhibits food intake, and orlistat, which blocks fat digestion (Yanovski and Yanovski, 2002). Drugs like diethylproprion, phendimetrazine and phentermine have been approved by the food and drug administration (FDA), USA for short-term use. Use of orlistat is associated with side effects such as oily spotting, faecal urgency and increased defecation and reduced absorption of fat-soluble vitamins, particularly vitamin E and ï¢ï€carotene (Yanovski and Yanovski, 2002). The principal side effects of sibutramine include dry mouth, insomnia and asthenia (Seagle et al., 1998). It also increases blood pressure and heart rate. The major side effects of ephedrine are an increase in heart rate and sense of palpitations, blurred vision, skin rash or nausea in some patients. Individuals taking chitosan need to take supplements of vitamin A, D and E (Yanovski and Yanovski, 2002).
Alternative therapies with desired biological effects are increasingly sought by patients with various chronic metabolic disorders. Alternative treatments have been most widely used in chronic diseases, which may be only partially alleviated by conventional treatment. Herbal formulations are the most commonly used alternative therapy for blood sugar control. Even in most advanced countries of Europe and America, 30-50% of patients with diabetes use some form of CAM therapy. Given this widespread use, clinical and research methods are needed to direct the safe use of CAM therapies that consider the evidence available, use objective measures of efficacy, are reproducible, and maintain tenets of individualized care.
Plant source were the main form of diabetes treatment before the introduction of insulin and oral hypoglycemic drugs. Herbal medicines are still the mainstay in the developing countries. More than 400 plants are known to have been used for the management of diabetes, and the studies have acknowledged the potential value of some of these plants (Marles and Farnsworth, 1995). Recent studies report that 30%-40% of patients with diabetes use some form of herbal therapy (Yeh et al., 2003; Garrow et al., 2006). Many herbs and herbal extracts are used as prescription drugs in the countries of European Union and the international market for medicinal plant is $ 80 billion (Wakdikar, 2004). In India, the herbal drug market is about $ one billion and the export of plant-based crude drugs is around $ 80 million. Herbal medicines also find market as nutraceuticals (health foods) whose current market is estimated at about $ 250 billion in USA and Europe (Kamboj, 2000). However, their safety and efficacy are yet to be evaluated by well designed in vitro as well as controlled clinical studies. Most of the studies related to the screening of plant products lack scientifically reliable methods and protocols. One reason being the non-availability of foolproof in vitro model to study the effect of plant product on rate limiting step of metabolic disorders. Non-standardized formulation of herbal products adds to the complexities that ultimately lead to difficulty in replication. Therefore, preparation of standardized medicinal herbs is mandatory in clinical evaluation and therapies.
For a sustained market for herbal medicine certain international specifications are to be met. It includes proper identification of medicinal plants, with botanical names with their common names in scientifically formulated medicines. Isolation and chemical characterization of acute ingredients including inorganic constituents, wherever possible is a basic requirement. Because herbal medicine that includes herbal extracts, powdered form of medicinal plants, minerals, organic matter, etc are being used by over 70% of world population, the WHO has defined use of herbal extracts. The guidelines of WHO state that in case the identification of an active principle is not possible, characterization of substance or mixture of substances (e.g., "chromatographic fingerprint") to ensure reproducible quality of the product (Akerele, 1992) is recommended because the herbal extracts may contain excipients in addition to the active ingredients. Multi-component botanical formulations can be standardized with techniques such as DNA fingerprinting, thin layer chromatography (TLC), liquid chromatography and mass spectroscopy in addition to approved manufacturing procedures (FDA, 2000). Although it is assumed that herbs used for metabolism related diseases are not to have the drawbacks of conventional drugs, undesirable herb-drug interactions should be anticipated for patients also receiving conventional medications. Pharmacological and clinical studies are to be done to ascertain their efficacy, safety and drug interactions. Such scientifically mandatory aspects will project herbal medicine appropriately and help in sustained global market .
A number of reviews and research findings highlight natural products and plants as potential antidiabetic drugs (Ivorra et al., 1989; Oubre et al., 1997; Imparl-Radosevich et al., 1998; Jarvill-Taylor et al., 2001; Anderson et al., 2004). There are several bioactive plant extracts that have been studied in vitro for hypoglycemic activity. A bioactive compound from Chinese plant Lithospermum erythrorhizon stimulates glucose uptake in 3T3-L1 adipocytes (Kamei et al., 2002). An extract from Lagerstroemia speciosa is documented to have insulin like glucose uptake stimulatory effect (Liu et al., 2001). With an objective to discover a non peptide, small active compound that exhibited insulin-mimetic activity, Salituro et al., (2001) screened over 50,000 samples of natural extracts for their ability to mimic insulin activity. They discovered a small non-peptidyl molecule demethylasterriquinone B1 structurally belong to quinones (L-783,281) from a fungal (Pseudomassaria) extract (Zhang et al., 1999; Qureshi et al., 2000). It is a paradox that despite the report of a number of plants with reported hypoglycemic activity, metformin is the only ethical drug approved for the treatment of hyperglycemia derived from a medicinal plant (Oubre et al., 1997).
Though a number of plants with some scale of hypoglycemic and hypolipidemic activity was reported, a significant amount of research as well as conventional usage suggests that fenugreek seeds (Trigonella foenum-graecum), bitter gourd fruit (Momordica charantia), and gurmar leaf (Gymnema sylvestre) are among the important plant products in terms of efficacy and safety.
Fenugreek and its therapeutic applications
The genus Trigonella is one of the six genera of the subfamily or tribe Trifoliae of the family fabaceae (Papilionaceae) within the order Leguminoceae. According to Hutchinson (1964), Trigonella contain 70 species. Fenugreek (Trigonella foenum-graecum) is one of the oldest medicinal plants of Mediterranean origin and cultivated worldwide. Cultivated area of fenugreek in India accounts for 50,600 ha with a production of 64,220 tons and an export of ~20,000 tons (DSAD, 2007). The seeds and leaves are used to prepare extracts or powders for medicinal purpose. Several uses of fenugreek are known including the treatment of indigestion, weakness, edema of the legs and baldness. In India, fenugreek is generally used as a condiment and lactation stimulant. A number of clinical as well as animal studies have highlighted hypoglycemic and hypolipidemic properties of fenugreek seeds (Basch et al., 2003).
Hypoglycemic effects of fenugreek
Studies on antidiabetic actions of fenugreek seeds were initiated 70 years ago (Moissides, 1939). Later, Fourier (1948) observed that the consumption of coarsely ground fenugreek seeds improved severe diabetes in human subjects. This property was confirmed in chemically induced diabetic animals (Ribes et al., 1984; Khosla et al., 1995). Aqueous extract of fenugreek leaves and seeds administered both orally and intraperitoneally (i.p.) possess hypoglycemic effect in normoglycemic and diabetic rodents (Abdel-Barry et al., 1997; Zia et al., 2001; Vats et al., 2002). Alcoholic or methanol extracts of fenugreek seeds are also shown to have significant antihyperglycemic activity (Ali et al., 1995; Vats et al., 2004). It is also shown that fractions which contain fibres, saponins and proteins decreased hyperglycemia in both normal and diabetic dogs (Ribes et al., 1984; Valette et al., 1984). Studies in obese rats indicated that hypoglycemic effect of soluble fibre extracted from fenugreek seeds is mediated by inhibition of intestinal glucose uptake (Srichamroen et al., 2009).
Fenugreek seeds were found to diminish hyperglycemia, urinary sugar excretion and serum cholesterol in normal and diabetic subjects (Sharma, 1986a; Sharma et al., 1990). Despite a significant reduction in blood glucose, in some studies no significant change was observed in insulin level following fenugreek seeds administration to type 2 diabetics (Madar et al., 1988), rats (Madar, 1984), or dogs (Ribes et al., 1984). Fenugreek seeds have been reported to suppress clinical symptoms of diabetes, polyuria, polydypsia, weakness and weight loss (Sharma, 1986b). It has also been demonstrated that the hypoglycemic properties of fenugreek is not destroyed by cooking or roasting (Sharma, 1986a; Khosla et al., 1995). Raghuram et al., (1994) showed that fenugreek seeds powder when given in the diet for 15 days to diabetic patients prior to glucose load improved glucose tolerance. Most importantly, ingestion of an experimental diet containing 25Â g fenugreek seeds powder shown to have no renal or hepatic toxicity in diabetic patients (Sharma et al., 1996a). Fenugreek seeds intake increases molar insulin binding sites suggesting that antidiabetic activity is mediated at the insulin receptor (IR) level (Raghuram et al., 1994; Kannappan and Anuradha, 2009).
The whole grain of fenugreek seeds per 100 g of edible portion contains 369 calories, 7.8% moisture, 28.2 g protein, 5.9 g fat, 54.5 g total carbohydrate, 8 g fiber, 3.6 g ash (Al-Habori and Raman, 2002). The biologic and pharmacological actions of fenugreek seeds and leaves are attributed to a number of constituents, such as steroids (steroid sapogenin), N-compounds (trigonelline), flavanoids (quercetin, vitexin, isorhamnetin) glycosides, polyphenolic substances (scopoletin, coumarin) volatile constituents, amino acids (4-hydroxy isoleucine) etc. In early reports hypoglycemic effect of fenugreek seeds was attributed to its major alkaloid trigonelline (Mishkinsky, 1967; Shani et al., 1974). A free amino acid, 4-hydroxyisoleucine, isolated from fenugreek seeds is shown to stimulate insulin secretion in vivo and in vitro (Sauvaire et al., 1998). Other postulated hypoglycemic constituents of fenugreek are coumarin (Shani et al., 1974), scopoletin and fenugreekine (Ghosal et al., 1974), fibre content (Sharma, 1986). However, no substantial or in vitro studies clinical studies were performed to prove the efficacy of these compounds categorically. It is also to be noted that, with the exception of guanidine, many of the compounds isolated from plants are small molecules such as alkaloids, flavanoids, glycosides, steroids, aminoacids or minerals that are not suitable for pharmaceutical development drugs for the management of metabolic disorders (Oubre et al., 1997; Day, 1998). Moreover, medicinal plant extracts used for treating hyperglycemia may contain number of components which together contribute to over-all effectiveness. Therefore isolating individual component from such extracts may not be effective.
With an objective to assess the hypoglycemic and hypolipidemic effects of fenugreek seeds, we have employed innovative and contemporary in vivo and in vitro mechanism based studies. In this regard, novel preparations of extracts of fenugreek seeds with hypoglycemic and hypolipidemic activity was established. Aqueous, dialyzed fenugreek seeds extract (FSE) with hypoglycemic activity was documented with chromatographic fingerprinting. With an objective to elucidate the hypoglycemic effects of the extract, a cell line of stably expressing myc and eGFP tagged glucose transporter 4 (GLUT4) in CHO cells expressing human insulin receptor (HIRc) denoted as CHO-HIRc-myc-GLUT4eGFP cells was developed. This cell model was utilized to establish a novel real time, visual, cell-based GLUT4 translocation assay based on GLUT4 associated GFP fluorescence using cooled charge-coupled device camera attached to a fluorescent microscope. This method with the aid of video imaging provides foolproof visual evidence useful for assessing GLUT4 translocation modulators. Further studies revealed that extract stimulated glucose uptake in CHO-HIRc-mycGLUT4eGFP cells in a dose-dependent manner. This effect was mediated by GLUT4 translocation to the plasma membrane (PM) from the intracellular space. FSE mediated GLUT4 translocation and glucose uptake was inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor and bisindolylmaleimide 1 (BIS-1), a protein kinase C (PKC) specific inhibitor suggesting the involvement of insulin signaling pathway. In vitro phosphorylation analysis revealed that, like insulin, FSE also induced tyrosine phosphorylation of a number of proteins including insulin receptor (IR), insulin receptor substrate 1 (IRS-1), and p85 subunit of PI3-K, in both 3T3-L1 adipocytes and human hepatoma cells, HepG2 with one striking difference being that FSE induced PI3-K downstream signals involves only PKC and had no effect on protein kinase B (Akt) activation. These results indicate that hypoglycemic effect of fenugreek seeds, at least in part, is mediated through activating insulin signaling pathway in adipocytes and liver. Additionally, extract is not a general activator of tyrosine kinases and also had no effect on fat accumulation in differentiating 3T3-L1 cells.
Fenugreek seeds extract exhibited hypoglycaemic activity in diabetic mice irrespective of the stain of mice or chemical agent used to induce diabetes. Hypoglycemic effectivenees was achieved because of significant enhancement in liver GK and HK activities in par with that of insulin. Chronic administration of extract in diabetic mice reduced hyperglycemia and this effect was further sustained for ten days. This extract was capable of improving intraperitoneal glucose tolerance in normal glucose loaded mice and was accompanied with the reduction in serum insulin concentration. These results are indicative of an extrapancreatic mode of action of FSE. However, FSE exhibited only marginal hypoglycemic activity when administered orally. It is concluded that this novel FSE preparation corrects metabolic alterations associated with diabetes by exhibiting insulin-like properties and has a potential for clinical applications.
Plant products and plants in the management of dyslipidemia
Sitostanol/sterol esters, available as regular and low-fat food supplements, inhibit intestinal absorption of dietary and biliary cholesterol and are most effective in persons with high cholesterol absorption and low cholesterol synthesis (Lichtenstein et al., 2001). Fish oils rich in omega-3 polyunsaturated fatty acids have been proven to be beneficial in reducing TG levels, blood pressure, and CHD and are relatively well tolerated. However, a therapeutic dosage regimen is not defined yet. Soluble fiber from plant source appears to affect both hepatic cholesterol and lipoprotein metabolism, thus increasing bile acid loss. The result is a decrease in hepatic cholesterol concentrations and upregulation of LDLR. A meta-analysis of randomized controlled trials demonstrated that 2-10 g/day of soluble fiber was associated with small but significant decreases in TG and LDLc with no changes in HDLc levels (Brown et al., 1999).
A number of plant extracts and plant products have been evaluated for hypolipidemic activity in animal models of dyslipidemia. Studies concerned with bitter melon in cholesterol fed hamsters have shown that it contains some components that could ameliorate lipid disorders (Senanayake et al., 2004). Coriander seeds when incorporated into high fat diet and added cholesterol had a significant hypolipidemic action (Dhanapakiam et al., 2008). Flavonoid rich extract obtained from seeds of Eugenia jambolana is also shown to induce hypolipidemic activity in STZ induced diabetic rats (Sharma et al., 2008). Hypolipidemic action of this plant extract was found to be through up regulation of PPAR-ï§. Garlic (Allium sativum), soyprotein and guggulipid (Commifora mukul) are commonly used natural products for the management of dyslipidemia. Garlic is indicated by the German Commission E for use in the support of dietary measures for treating hyperlipidemia (Kannar et al., 2001). Mechanism based studies have shown that constituents of garlic inhibit HMG-CoA reductase. A meta-analysis of 38 controlled clinical trials has shown relation between soy protein consumption and serum lipid concentrations in humans (Anderson et al., 1995). Soy protein is believed to upregulate LDLR, resulting in LDLc lowering.
In addition to fenugreek, only limited numbers of plants have been investigated for the application in the management of obesity. Randomized, controlled trials have shown that intake of vegetables 400 g/day and fruit 300 g/day contribute to weight reduction in obese patients (Svendsen et al., 2007). Green tea polyphenol, epigallocatechin gallate can act directly to inhibit differentiation of preadipocytes and to induce apoptosis of mature adipocytes is proposed as an important adjunct in the treatment of obesity (Li et al., 2005). Extract of Panax ginseng berry is shown to decrease body weight of ob/ob mice (Attle et al., 2002). Moderate use of grape juice and white wine are also known to decrease body fat, waist circumference, blood pressure, blood glucose, insulin, TG and cholesterol (Flechtner-Mors et al., 2004). Based on the in vitro studies using 3T3-L1 cells, Garcinia extract is proposed to be useful in preventing obesity (Hasegawa, 2001).
Hypolipidemic effects of fenugreek
A significant number of clinical as well as animal experiments have shown that fenugreek leaves and seeds possess hypolipidemic effects. Fenugreek seeds extract are reported to lower triglycerides (TG) and total cholesterol in a dose dependent manner in streptozotocin (STZ)-induced diabetic rats (Annida et al., 2004; Xue et al., 2007). Soluble dietary fibre fraction of fenugreek seeds decreases TG cholesterol and low density lipoprotein cholesterol (LDLc) in type 2 diabetic rats (Hannan et al., 2003). Combined treatment of sodium orthovanadate and fenugreek seeds can ameliorate altered lipid metabolism in alloxan (AXN)-diabetic rats (Yadav et al., 2004). Ethanol extract from fenugreek seeds contains hypolipidemic components which appear to be saponins. Steroid saponins from fenugreek seeds are reported to decrease total plasma cholesterol without any change in TG in normal and diabetic rats (Petit et al., 1995). Fenugreek seeds treatment selectively reduces the LDLc and very low density lipoprotein cholesterol (VLDLc) in AXN-diabetic rats with no toxicological effects.
In a double blind placebo controlled study, it has been reported that hydroalcoholic extract of fenugreek seeds decreases serum TG and increases high density lipoprotein cholesterol (HDLc) in type 2 diabetic patients (Gupta et al., 2001). In a short term study, fenugreek seeds also decrease total cholesterol in diabetic patients (Sharma and Raghuram, 1990; Sharma et al., 1990). Ingestion of an experimental diet containing 25 g fenugreek seeds powder for 24 weeks resulted in a significant reduction of TG, total cholesterol, LDLc and VLDLc in type 2 diabetic patients (Sharma et al., 1996b). The consumption of germinated fenugreek seeds powder is shown to reduce total cholesterol and LDLc in human subjects (Sowmya and Rajyalakshmi, 1999). Presence of saponins is proposed to be essential for hypolipidemic activity of fenugreek seeds (Ribes et al., 1987; Sauvaire et al., 1991). In contradiction, incorporation of defatted or saponin depleted fenugreek seeds in the experimental diet of hyperlipidemic subjects also significantly reduced serum total cholesterol, LDLc and TG levels, with no change in HDLc (Sharma et al., 1991; Prasanna, 2000).
Although fenugreek has a long history in folk medicine, there is scarcity of data regarding their efficacy related to obesity studies. Fenugreek seeds extract is shown to reduce the body weight gain induced by a high-fat diet in obese mice (Handa et al., 2005). The extract decreases plasma TG gain induced by oil administration. Oral supplements of fenugreek seeds have the ability to alleviate obesity-associated pathologies in Zucker obese rats, an animal model of obesity (Raju and Bird, 2006). Reduction in plasma levels of TG and total cholesterol in association with reduction in epididymal adipose weight is proposed to be due to the presence of soluble fibre or 4-hydroxyisoleucine content of fenugreek seeds (Handa et al., 2005; Srichamroen et al., 2008).
Since protein or protein derived products of legumes are shown to have hypolipidemic properties, precipitable protein from fenugreek seeds was extracted and a novel thermostable extract of fenugreek seeds (TEFS) was developed. Chromatographic finger printing was established and hypolipidemic effect of this extract was investigated in vitro and in vivo. Hypolipidemic effect of TEFS was evaluated in vitro by employing differentiating and differentiated 3T3-L1 cells, and HepG2 cells cultured in normal or sterol enriched conditions. Hypolipidemic effect was studied by Oil Red O staining, quantifying decrease in accumulation of fat or by western blot analysis of adipogenic and lipogenic factors. At molecular level, TEFS inhibited accumulation of fat in differentiating and differentiated 3T3-L1 cells via decreased expression of adipogenic factors such as peroxisome proliferator activated receptor-ï§ (PPAR-ï§), sterol regulatory element binding protein-1 (SREBP-1) and CAAT element binding proteins-ï¡ (C/EBP-ï¡). This effect can be compared to that of mediated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or cerulenin, known pharmacological inhibitors of differentiation. Following TEFS treatment, cellular triglycerides (TG) and cholesterol levels decreased significantly in HepG2 cells because of reduced expression of SREBP-1, at mRNA as well as protein level. Under sterol enriched condition, TEFS upregulated low density lipoprotein receptor (LDLR) expression resulting in enhanced LDL uptake. Treating fat supplement fed C57BL6/J mice with TEFS for 15 days caused decrease in serum TG, LDL-cholesterol (LDLc) and body weight in a dose and time dependent manner. Results indicate that hypolipidemic effect of TEFS is due to inhibition of fat accumulation and upregulation of LDLR.
Though we were successful in elucidating the mechanism of action of defined fenugreek product, were not successful in isolating its active principle (s) individually using close to foolproof visual assays. For example sensitive GLUT4 translocation assay was used to screen fractions obtained from gel filtration and ion exchange column and HPLC column. Biological activity was spread across various fractions and hence specific activity was never found increasing. Present study is a cumulative effort towards understanding the molecular mechanisms and related signaling pathways that contribute to hypoglycemic and hypolipidemic effects of fenugreek seeds. Taken together, the studies mechanistically and scientifically highlight the fact that fenugreek seeds and its derived products have potential application in the management of glucose and lipids related metabolic disorders. As an offshoot of this work, in vitro models and methods developed in this study could be used for screening natural products suitable for the development of new hypoglycemic or hypolipidemic drugs.