Effects and mechanisms of a concentrated goyasaponin fraction in 3t3 l1 cell line

CHAPTER 1 LITERATURE REVIEW AND OVERALL HYPOTHESES 1. Type 2 Diabetes and Obesity 1.1 Type 2 Diabetes Diabetes mellitus is characterized by abnormally high blood glucose levels (hyperglycemia) and caused by an altered secretory amount of insulin from pancreas as well as decreased effectiveness in insulin action [1, 2]. According to recent classification, Type 1 and Type 2 are two main types of this chronic disease [2, 3]. Type 1 diabetes, also known as juvenile-onset or insulin-dependent diabetes, is an autoimmune disease in which pancreatic islet cells are destroyed by antibodies produced by the body [2, 4]. Therefore, absolute deficiency in insulin production by the pancreas is always associated with Type 1 diabetes [3]. Type 2 diabetes, also known as adult-onset or non-insulin dependent diabetes, is the consequence of altered insulin secretion as well as resistance to insulin action [2, 4]. The prevalence of type 2 diabetes, which accounts for approximately 90 - 95% of all diagnosed cases of diabetes, is increasing worldwide and the global diabetic population is estimated to double from 151 million in 2000 to 300 million in 2025 [5, 6]. Meanwhile, annual medical expenditure associated with type 2 diabetes imposed a significant financial burden on society. In United States, type 2 diabetes accounts for more than $100 billion in healthcare costs annually [2] while in western European countries, 2-7% of total national health budgets has been spent on diabetes care [7]. Even more disturbing is the growing number of children and adolescents diagnosed with type 2 diabetes which was previously present in adults only [7, 8]. Additionally, as diabetes mellitus can lead to serious long-term 1    complications including heart disease, retinal damage, renal failure and stroke [9], it was ranked sixth as the leading cause of death in United States in 2004 [10]. In normal subjects, the plasma glucose levels are maintained with the aid of insulin [11]. Insulin is a multipotent hormone secreted from β-cell in pancreas and its most important effect is on glucose homeostasis [12]. First of all, insulin can stimulate the peripheral glucose uptake in muscle (around 60% of insulin-stimulated whole body glucose uptake), liver (around 30%) and adipose tissue (about 10%) [12]. At the same time, insulin also stores excess glucose as glycogen in liver and muscle [13, 14]. Insulin resistance, characterized as defects in insulin signaling in insulin-responsive tissues, occurs many years prior to diabetes onset and serves as a strong predictor for the type 2 diabetes development [15, 16]. Therefore, both normal insulin secretion and sensitivity to insulin action are of importance for maintenance of plasma glucose level. The developmental process of type 2 diabetes is a complex and multifactorial consequence as shown in Figure 1 proposed by Olefsky et al. in 1995 [16]. Several risk factors, including both genetic and acquired factors, make significant contributions to type 2 diabetes. In addition, further new insights into mechanism in the future would provide a better understanding of type 2 diabetes pathogenesis. 2    Figure 1. Pathology of type 2 diabetes concluded by Olefsky et al. in 1995 3    1.2 Obesity Obesity is another chronic disease associated with the excessive presence of body fat and it has become a leading public health problem in the world [17]. Similar to type 2 diabetes, increased prevalence of obesity among both adults and children has been observed in many countries throughout the world [18, 19]. According to National Centre for Health Statistics, it is reported that 61% of adults are overweight and 26% are obese in United States in 1999, as defined by body mass index (BMI) [20] . Globally, the prevalence of obesity has increased more than 75% since 1980 [17]. Obesity is a consequence of an imbalance in energy metabolism resulting from excessive food intake concurrent with decreased energy expenditure [17]. There are several explanations for this global increase in obesity rate. First of all, genetic factor makes dominant contribution to body weight as inheritability for obesity is estimated to be 50-90% [17]. Moreover, some acquired factors, including obesity-promoting changes in diet and sedentary lifestyle, also exacerbate this increased prevalence [17]. 1.3 Close Connection between Type 2 Diabetes and Obesity Obesity is closely associated with insulin resistance and is considered to be a leading risk factor for both type 2 diabetes and cardiovascular disease [21, 22]. Recent epidemiological studies have shown that BMI over 28 kg/m2 exponentially increase the risk of type 2 diabetes [23] . Various hypotheses have been proposed to explain the causal relationship between obesity and type 2 diabetes [24], one of the which accepted is the ‘lipotoxicity hypothesis’ as shown in Figure 2: in obese individuals, beta-cell dysfunction, 4    insulin resistance and impaired glucose tolerance develop in response to physiological dysfunction in muscle, liver and pancreas where excess lipids are deposited [24-26]. 1. Increased number of adipocytes (hyperplasia) Obesity 2. Enlarged size of adipocytes (hypertrophy) 1. Excess lipids cannot be stored in adipose tissue in the form of triglyceride Lipotoxicity 2. Excess lipids deposited insulin-sensitive tissues, such as muscle, liver and pancreas 3. Physiological dysfunction in muscle, liver and pancreas Insulin Resistance Beta-cell dysfunction Impaired glucose tolerance Type 2 Diabetes Figure 2. Proposed hypothesis of lipotoxicity 5    2. Adipose Tissue and 3T3-L1 Cell Line 2.1 Adipose Tissue Mature adipocytes are the main cellular component of adipose tissue. In addition, adipose tissue also contains other components, such as undifferentiated preadipocytes, immune cells (leukocytes, macrophages), nerve fibers, vascular stroma, lymph nodes and a matrix of collagen and reticular fibers [27]. There are essentially two types of adipose tissue in humans referred to as white adipose tissue (WAT) and brown adipose tissue (BAT) [27, 28]. BAT is found in fetuses and newborn infants and is practically absent in adults as its principal function is to burn fat to generate heat for newborns during the initial hours after birth [27, 28]. Uncoupling Protein-1 (UCP-1), exclusively expressed in brown adipocytes, plays a vital role in heat production as it uncouples electron transport from adenosine-5'-triphosphate (ATP) production allowing energy to dissipate as heat [27]. However, the predominant type of adipose tissue in humans is WAT [29]. Two types of WAT exist and are classified as visceral and subcutaneous [30]. Functional differences exist between these two types and it appears that individuals with visceral fat accumulation are more likely to develop metabolic and cardiovascular diseases [30]. In response to energy demands, WAT serves as a site for storing excess energy as triglyceride via lipogenesis and releasing energy in the form of free fatty acid/glycerol via lipolysis when there is a calories deficit [27, 29, 31]. In addition to its biological repertoire necessary for storing and releasing energy [32], WAT has been proved recently to be an essential endocrine organ which secretes a variety of bioactive proteins termed as adipocytokines, including adiponectin, leptin, resistin and visfatin, which have been proved to be actively involved in energy 6    regulation, lipid metabolism, insulin resistance, immunological response and vascular disease [32-34]. Moreover, WAT also expresses and secretes some other cytokines and chemokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 [35]. Besides these two roles of adipose tissue as mentioned above, Cao et al. in 2008 found that palmitoleate, a free fatty acid metabolite generated in and released from adipocytes, is a circulating factor that promotes the insulin sensitivity in liver and muscle [36]. This latest finding is a further step to demonstrate that lipid metabolism and glucose homeostasis are highly interconnected processes [37]. Therefore, due to its multifunctional characters, WAT has been focused by researchers as a possible central mediator of whole body insulin resistance in recent years [38]. 2.2 3T3-L1 is an established in vitro model of adipocyte biology The molecular and cellular events during the adipogenesis process have been studied on various cell culture models, including both preadipocyte cell lines and primary culture of adipose-derived stromal vascular precursor cells [32]. 3T3-L1 is a substrain of Swiss 3T3 murine cell line derived from disaggregated 17- to 19- mouse embryos [39]. It has been proved to be an established murine preadipocyte fibroblast which can be induced to adipocyte differentiation (adipogenesis) and completely convert into oil droplet-containing mature adipocytes [22]. Therefore, both differentiation process and adipocytokines secretion from mature adipocyte can be investigated under this cell line condition [22, 40]. 7    Progress has been made in understanding adipocyte differentiation process. First of all, these new findings provided the molecular and cellular basis of the adipose tissue growth of physiological and pathophysiological state. Additionally, they also provided means of developing strategies for both prevention and treatment of obesity [39]. Characterized by increased adipose tissue mass, obesity is determined by both enlarged size and increased number of adipocytes [41]. Therefore, several applicable anti-obesity mechanisms, including decreased preadipocyte proliferation, inhibition of adipocyte differentiation, reduced lipogenesis, increased lipolysis and enhanced free fatty acid oxidation were proposed by Wang et al. [42]. 2.3 Peroxisome Proliferator-Activated Receptor γ (PPARγ) Peroxisome proliferator-activated receptors (PPARs) constitute a subfamily of nuclear hormone receptors which regulate storage and catabolism of dietary fats [43-45]. There are three subunits of PPARs: α, δ and γ [46]. Among those PPAR subunits, the expression of PPARγ exhibits the predominant specificity in adipose tissue while smaller amounts are also present in skeletal muscle, liver, pancreatic β-cells, vascular endothelial cells and macrophages [46, 47]. The roles of PPARγ in both adipogenesis and differentiated adipocytes have been investigated extensively in vitro and in vivo. First of all, PPARγ plays a role as one key transcriptional factor in adipocyte differentiation [43, 46]. The expression of PPARγ is induced early in adipogenesis, subsequently induces the other transcriptional factors and promotes development of differentiation phenotype [48]. The observations that PPARγdeficient cells failed to differentiate to mature adipocytes demonstrated that PPARγ plays an 8    essential and irreplaceable role in the adipocyte differentiation [49, 50]. Secondly, PPARγ also play an important role in mature differentiated adipocytes. Tamori et al. found PPARγ functions at least in part by regulating relevant gene expressions to maintain the characteristics of mature adipocytes including free fatty acid (FFA) uptake and triglyceride accumulation [51]. Similarly, Way et al. also found that PPARγ activation with the potent PPARγ ligand GW1929 stimulated the expression of genes involved in lipogenesis and fatty acid metabolism in adipose tissue in Zucker diabetic fatty rat [52]. The therapeutic usage of PPARγ agonists is mainly focused on type 2 diabetes treatment. PPARγ has been identified as the receptor for thiazolidinediones (TZDs) and the antidiabetic effects of TZDs are mediated through PPARγ [4, 53, 54]. Therefore, adipose tissue, where PPARγ predominantly expressed, has become a main target tissue of TZDs. However, PPARγ agonists also bring in a paradox. On one hand, PPARγ agonists effectively reduce plasma glucose and ameliorate insulin resistance [55]. On the other hand, PPARγ agonists also promote adipocyte differentiation which potentially leads to obesity, a major risk factor for the development of type 2 diabetes. One of postulated explanations to this paradox is that PPARγ activation in rodents induced increased number of small adipocytes which typically demonstrate greater insulin sensitivity, more glucose uptake and lower rates of lipolysis when compared to large adipocytes [55-57]. However, further investigations are still in need to clarify this issue. 2.4 Adiponectin Adiponectin is predominantly secreted from mature adipocytes to influence insulin sensitivity by improving glucose and lipid metabolism [58, 59]. Adiponectin has been 9    regarded as a clinical marker of type 2 diabetes as high levels of adiponectin are associated with reduced risk of diabetes while reduced levels of adiponectin have been observed in type 2 diabetics and obese patients [60, 61]. Moreover, adiponectin also has become one of therapeutic targets for type 2 diabetes. First of all, adiponectin has been shown to suppress hepatic glucose production [62]. Moreover, via activating AMP-activated protein kinase (AMPK) pathway, adiponectin increases glucose uptake and stimulates fatty acid oxidation [63, 64]. 3. Antidiabetic Drugs: Thiazolidinediones (TZDs) and Metformin Besides the use of insulin in the treatment of diabetes, oral hypoglycemic agents such as sulphonylureas, thiazolidinediones (TZDs), α-glucosidase inhibitor, and metformin are also used to regulate plasma glucose level [65]. The principal modes of action of these antidiabetic agents are listed in Table 1 [4, 54, 66]. 10    Table 1. Current therapeutic agents for type 2 diabetes Class Molecular Target(s) Sites of Action Main mode of action Adverse Effects Liver, muscle Decrease hepatic glucose output; Increase peripheral glucose uptake Gastrointestinal Disturbances; lactic acidosis PPARγ Fat, liver, muscle Increase insulin sensitivity Weight gain; Oedema; Anaemia SU receptor/ K+ ATP channel β-cell Increase insulin secretion Hypoglycaemia; Weight gain Intestine Decrease rate of intestinal carbohydrate digestion Gastrointestinal disturbances Fat, muscle, liver Decrease hepatic glucose output; Increase peripheral glucose uptake; Decrease lipolysis Hypoglycaemia; Weight Gain Biguanide (Metformin) Unknown TZDs (Pioglitazone, rosiglitazone) Sulfonylurea α-glucosidase inhibitor (acarbose) Insulin α-glucosidase Insulin receptor 11    TZDs and metformin are two important classes of drugs for the treatment of Type 2 diabetes but they counter insulin resistance via different cellular mechanisms [54]. Currently, two TZDs, pioglitazone and rosiglitazone remain on the US market while troglitazone was taken off because of liver toxicity [55]. TZDs function as PPARγ agonists. The main actions of TZDs include increasing systematic insulin sensitivity, increasing peripheral glucose uptake, reducing plasma fatty acid concentration and enhancing adiponectin secretion [15, 54]. Adiponectin influences insulin sensitivity by improving glucose and lipid metabolism and its decreased expression has been reported in models of obesity and diabetes [58, 59]. Moreover, TZDs also inhibited secretion of TNF-α, IL-6 and resistin which promoted muscle insulin resistance [67]. In addition, TZDs were proved to activate AMPK in rat liver and adipose tissue but it remains unclear whether this is a direct effect and/or mediated by PPARγ via increasing plasma level of adiponectin [68]. However, the safety of earlier generation TZD has been questioned as well as concerns over common side effects of newer generation of TZD such as weight gain, edema and heart failure [69]. Metformin, on the other hand, achieve hypoglycemic effects principally via suppressing hepatic glucose output [54]. It was found that metformin might mediate its insulin-sensitizing effect by directly activating AMPK pathway in rat liver and muscle [66]. In addition, a clinical study on type 2 diabetes patients demonstrated that metformin caused a significant increase in AMPK α2 activity in muscle after 10-week treatment [70]. These findings provide strong evidences that AMPK is the mediator, in part at least, of metabolic effects of metformin. However, unlike TZDs, metformin could remain weight stable [71], enhance lipolysis [53] and reduce triglyceride accumulation in adipocytes [72]. Moreover, it 12    was observed that metformin could not enhance secretion of adiponectin in both in vivo [73] and in vitro experiments [74]. 4. Momordica Charantia and Goyasaponins Although current therapeutic agents for type 2 diabetes are effective in controlling hyperglycemia, they also cause significant side effects as shown in Table 1. Some of the drugs such as sulphonylureas and TZDs frequently lead to weight gain which may further exacerbate the hyperglycemic conditions [23]. In view of these undesirable side effects as well as the increasing prevalence of type 2 diabetes, there is demands to search more efficacious agents with fewer side effects [38]. This fueled the search for alternative therapeutic substances [75] and led to a rising interest in dietary adjuncts and herbal products that exhibit hypoglycemic properties [76]. 4.1 Momordica Charantia The use of traditional functional foods, such as plants and herbal remedies to treat disease and symptoms has a long history of use in Asia and other developing countries [77, 78]. Momordica charantia is commonly known as bitter gourd or bitter melon [77]. It is a tropical climber belonging to the Cucurbitaceae family and has a unique bitter taste and is cultivated worldwide for its edible fruit [38]. Besides being consumed as a vegetable, M. charantia has been used to maintain health, prevent illnesses as well as manage chronic diseases, most notably diabetes, in the traditional medicinal systems of many cultures worldwide, including those of the Asian Indians, Chinese and South Americans [79]. Besides anti-diabetic properties, M. charantia has also been credited with antiviral, antitumor, 13    antileukemic, antibacterial, anthelmintic, antimutagenic, antimycobacterial, antioxidant, antiulcer, anti-inflammatory, hypocholesterolemic, hypotriglyceridemic, hypotensive, immunostimulant, and insecticidal properties [77]. However, it has been used extensively as a folk remedy for diabetes in Asia and is most widely studied with regards to its antidiabetic effects [78, 80, 81]. Previous studies on hypoglycemic activity of M. charantia have mainly focused on chemically-induced diabetic rodents, such as streptozotocin (STZ)-induced rats [82-84]. Momordica charantia extracts (methanol, ethanol and aqueous extracts, or fresh juice) were found to depress the level of plasma glucose [84, 85], stimulate glucose uptake into skeletal muscle cells after incubation with glucose [86], improve insulin sensitivity by ameliorating insulin signaling cascade in both skeletal muscle [87] and liver [88] of high-rat-red rats, enhance glucose transporter (GLUT4) protein content of plasma membrane in muscle tissue of rats [89], improve blood glucose tolerance [90], increase the number of pancreatic betacells [82], regenerate beta cells in islets of Langerhans in pancreas [91], reduce triglyceride content, decrease LDL-cholesterol and increase HDL-cholesterol [92, 93]. Clinical dietary trials using fruit juice of M. charantia have also shown similar serum hypoglycemic effects, such as decreased serum glucose concentration [94], improved glucose tolerance [94, 95], and a reduction of both fasting and postprandial serum glucose levels [92]. Therefore, the biochemical, pharmacological and histopathological profiles of M. charantia extracts in both in vivo and in vitro studies clearly indicate its potential antidiabetic activity and other beneficial effects against associated complications [96]. However, the active antidiabetic component of M. charantia has not been adequately identified, although a wide range of compounds have been isolated, notably steroidal-like saponins such as 14    cucurbitane-triterpene glycosides, oleanane-triterpene saponins [97] and proteins including insulin-like polypeptide-p and napin-like protein [98, 99]. 4.2 Saponins Saponins are widely distributed in the plant kingdom and include a structurally diverse group of compounds [100]. They are amphiphilic in nature, consisting of triterpenoid, steroidal or steroid alkaloidal aglycones that are substituted with a varying number of sugar side chains [101-103]. M. charantia provides a rich source of triterpenoid saponins [104]. The triterpene aglycones share a similar basic structure where the 30 carbon atoms of the 6 linked isoprene units are arranged into 4- or 5- ring structures. The sites of glycone attachment may be one (monodesmosides), two (bisdesmosides) or three (tridesmosides) [103]. Triterpene saponins are typically bidesmosidic saponins, often with one sugar chain attached through an ether linkage at C-3 and one attached through an ester linkage at C28[103]. The unsubstituted, non-polar aglycones are classified as sapogenins. Triterpenes can be subdivided into around 20 groups depending on their molecular structures, including oleananes and cucurbitanes [104]. Saponins were initially a rather neglected area of research primarily because of great difficulties in their isolation and characterization [103]. With the advent of more sophisticated methods of isolation and structure elucidation through the last two decades, there has been great interest in these compounds [103]. Saponins occur as major constituents in active fractions isolated from many plants used in traditional medicine, and have been shown to possess a large variety of physiobiological activity, including anti-inflammatory, hemolytic, cholesterol lowering, and 15    anticancer properties [101-103, 105]. However, due to the structural complexity of saponins, only a few of these properties are common to all members of this diverse group [100]. 4.3 Goyasaponins Goyasaponin (in Japanese) means saponins extracted from bitter melon. Goyasaponin fraction, as elucidated by Murakami et al. [97], includes abundant types of cucurbitane-triterpene glycosides and oleanane-triterpene saponins, such as goyaglycoside group (a, b, c, d, e, f, g, h), goyasaponin group (I, II, III) and momordicoside group (A, C, F1, F2, I, K, L). The molecular formulae and structures of these compounds are shown in Figures 3 – 5. Over 40 different cucurbitane-type and oleanane-type triterpene saponins have been isolated from various parts of the of M. charantia plant [106] including the fruits[97, 106-108], seeds [109] and vines [110]. Recently, significant research interest has focused on isolation, identification and purification of cucurbitane-type triterpenoids from M. charantia. Utilizing mass spectrogram (MS) and nuclear magnetic resonance spectroscopy (NMR), there have been a number of reported new discoveries of cucurbitane-type triterpenoids isolated from M. charantia [106108, 110-112]. Moreover, it is noteworthy that one recent report documented that n-butanol soluble M. charantia goyasaponin fraction inhibited sucrose-loading serum glucose elevation in rats [81] which was an in vivo evidence demonstrating the hypoglycemic property of these compounds. However, mainly due to significantly low concentration of each identified single compounds as shown in Table 2 [97] and no commercial HPLC standards available on the market, research progress in this field is limited and much less is known about the bioactivity 16    of goyasaponins. In addition, there are very few reports identifying the possible mechanisms related to the anti-diabetic action at the cellular level of adipocyte, especially on regulation of adipocyte differentiation and adiponectin secretion. 17    Table 2. Yield percentage of each single goyasaponin compounds from fresh fruit [97] Compound Yield % from Fresh Fruit Compound Yield % from Fresh Fruit Compound Yield % from Fresh Fruit Goyaglycoside a 0.00008% Goyaglycoside h 0.00008% Goyasaponin I 0.00010% Goyaglycoside b 0.00005% Momordicoside A 0.0013% Goyasaponin II 0.00027% Goyaglycoside c 0.00007% Momordicoside C 0.00016% Goyasaponin III 0.00009% Goyaglycoside d 0.00008% Momordicoside F1 0.00011% Goyaglycoside e 0.00010% Momordicoside I 0.00006% Goyaglycoside f 0.00009% Momordicoside K 0.00003% Goyaglycoside g 0.00006% 18    OR1 R2 H GlcO Goyaglycoside a, b, c, d, e, g Goyaglycoside R1 R2 Molecular Formula a H OCH3 C37H60O9 b H OCH3; 3’-epimer C37H60O9 c CH3 OCH3 C38H62O9 d CH3 OCH3; 3’-epimer C38H62O9 H C42H68O13 OCH3; 3’-epimer C43H70O14 β-Dglucopyranosyl β-Dglucopyranosyl e g OH O OH OGlc OH H H O GlcO Glc - Glc - O Goyaglycoside f (C42H68O13) Goyaglycoside h (C42H70O15) Figure 3. Molecular formulae and structures of goyaglycosides a – h 19    C O O CH 3 OH COOH O O OH O OH CHO H OH O OH H O H OH O O CH3 OH O H O CH 3 O OH OH O H OH OH H OH OH OH OH Goyasaponin I (C65H102O31) C O O CH 3 COOH O O OH H OH O OH O OH OH H OH OH O O OH CHO CH 3 O OH H OH OH O H OH O O CH3 OH O H O H O H OH OH OH OH Goyasaponin II (C70H110O35) COOH O COOH O O C H3 C O H O OH O O OH H OH O OH OH OH H OH Goyasaponin III (C49H76O19) Figure 4. Molecular formulae and structures of goyasaponins I, II, III 20    OH OH R1 OH OH H H R 2O Glc - Glc - O Momordicoside A (C42H72O15) Momordicoside R1 C CH3 D H R2 βgentiobiosyl βgentiobiosyl Molecular Formula C42H72O14 C42H70O13 OR 1 O 2 R O Momordicoside R1 R2 F1 CH3 β-D-glucopyranosyl Molecular Formula C37H60O8 F2 H β-D-allopyranosyl C36H58O8 G CH3 β-D-allopyranosyl C37H60O8 I H β-D-glucopyranosyl C36H58O8 Figure 5. Molecular formulae and structures of selected momordicosides 21    OR OHC HO OGlc Momordicoside R K CH3 Molecular Formula C37H60O9 L H C36H58O9 Figure 5 (Continued). Molecular formulae and structures of selected momordicosides 22    5. Overall Hypotheses, Objectives and Implications of This Study 5.1 Overall Hypotheses A concentrated goyasaponin fraction (CGF) from M. Charantia fruit was obtained using an extraction and concentration method. It is hypothesized that this fruit CGF shows a potential as a PPARγ agonist and plays a similar role as TZDs which can influence preadipocyte proliferation, process of adipocyte differentiation and adiponectin secretion of differentiated adipocytes by modulating cell signaling. In addition, neither the saponin contents in M. Charantia seed nor bioactivity has been investigated thoroughly. It is proposed that seed CGF shows similar effects as fruit CGF on this in vitro model. These findings may be important to elucidate the effects and relevant mechanisms of M. Charantia on anti-obesity and type 2 diabetes prevention and/or management. 5.2 Overall Objectives M. charantia is widely used as a traditional functional food for the treatment of type 2 diabetes while adipose tissue is one of the targets for anti-diabetic drugs. Based on proven hypoglycemic effect of goyasaponin fraction on rats [81], the overall objective of this study is to investigate whether a concentrated saponin fraction (CGF) extracted from both M. charantia fruit and seed show a potential as PPARγ agonist by evaluating their effects and relevant mechanisms of in the 3T3-L1 murine cell line. (1) To optimize methods of extracting and concentrating saponin fractions from both M. Charantia fruit and seed and to obtain lyophilized CGF in powder for further in vitro 23    experiments. (2) To investigate the effect of CGF extracted from Momordica charantia fruit and seed in 3T3-L1 cell model related to diabetes and obesity. (3) To elucidate related mechanisms of these observed activities. 5.3 Implications of This Study There are two implications of this study. First of all, because adipose tissue is one of the target tissues for antidiabetic agents, assessments of both fruit CGF and seed CGF effects on 3T3-L1 preadipocyte proliferation, adipocyte differentiation and adiponectin secretion can shed more light on the underlying mechanisms of the bioactive compounds responsible for the extensively investigated antidiabetic properties of M. charantia. Secondly, the discovery of any enhanced bioactivity of M. charantia goyasaponins from fruit or seed will constitute a basis for determination of any potential for its development into a nutraceutical product. 24    CHAPTER 2 CONCENTRATION OF GOYASAPONIN FRACTIONS 1. Objectives (1) Sample preparation of CGF from both M. Charantia fruit and seed for further in vitro experiments. (2) Component analysis of CGF by HPLC-MS based on molecular weight confirmation. 2. Materials and Methods 2.1 Preparation of Concentrated Goyasaponin Fraction from M. charantia Fruit M. charantia fruit was purchased from a local supermarket, washed, separated from the seeds and aerial fibers, cut into small pieces and lyophilized. Dried fruit pieces were powdered and stored at -15℃ until extraction. Lyophilized samples were refluxed in methanol for 4 h, filtered (No.1 Whatman paper, Maidstone, England) and vacuum evaporated. The residue was dissolved in deionized water and centrifuged for 5 minutes at 500 x g. The supernatant was then applied equally to five Amberlite XAD4 columns (Sigma, St. Louis, MO) with a bed volume of 140 cm3 each at a flow rate of 3 bed volume / hour (BV/H) followed by 1 L water wash at a rate of 5 BV/H. The sample was eluted by ethanol (500 mL) in each column at a rate of 3 BV/H, and concentrated by vacuum evaporation. The residue was dissolved in water and lyophilized and is herein referred to as the fruit concentrated goyasaponin fraction (Fruit CGF) as shown in Figure 6. 25    Lyophilized M. charantia fruit powder Reflux with methanol, Discard residue. Methanolic extract Removal of methanol under vacuum and dissolved in deionized water Aqueous extract Solid phase extraction using Amberlite® XAD-4; eluted with ethanol Ethanolic extract Removal of ethanol and dissolved in deionized water Concentrated aqueous extract Lyophilization Fruit CGF in powder form Figure 6. Schematic representation of methodology used in preparation of concentrated goyasaponin fraction from M. charantia fruit. 26    2.2 Preparation of Concentrated Goyasaponin Fraction from M. charantia Seed Sun-dried M. charantia seeds were purchased from a local herbal store (Ban Lee Huat Seed Pte Ltd., Singapore). Seeds (4kg) were grounded and stored at -15°C. Each time, ground M. charantia seeds were weighed (35 g), refluxed in 500mL methanol for 4h. The mixture was filtered through filter paper twice (Whatman No.1) and vacuum evaporated. Concentrated methanolic extracts from all batches of refluxed extracts were combined before liquid-liquid extraction to minimize variations between batches. The concentrated methanol extract (70ml) was first partitioned between 140mL ethyl acetate (EtOAc) (Tedia, Fairfield, USA) and 70mL deionized water. The EtOAc phase was discarded while the aqueous phase was further partitioned between 125mL n-butanol (Sigma, St. Louis, MO) and 75mL deionized water. The aqueous phase was discarded while the n-butanol phase, containing the saponins, was vacuum evaporated. The residue was dissolved in 400ml deionized water and centrifuged for 5 minutes at 500 x g. The supernatant was then applied equally to one Amberlite XAD4 column with a bed volume of 140 cm3 at a flow rate of 3 bed volume / hour (BV/H) followed by 1 L water wash at a rate of 5 BV/H. The sample was eluted by ethanol (500 mL) in each column at a rate of 3 BV/H, and concentrated by vacuum evaporation. The residue was dissolved in water and lyophilized and is herein referred to as the seed concentrated goyasaponin fraction (Seed CGF) as shown in Figure 7. 27    Ground M. charantia seeds Reflux with methanol, Methanolic extract Removal of methanol under vacuum Concentrated methanolic extract Partition between ethyl acetate and deionized water Aqueous phase Ethyl acetate phase (Discarded) Partition between n-butanol and deionized water n-butanol phase Aqueous phase (Discarded) Removal of n-butanol and dissolved in deionized water Aqueous extract Solid phase extraction using Amberlite® XAD-4; eluted with ethanol Ethanolic extract Removal of ethanol and dissolved in deionized water Concentrated aqueous extract Lyophilization Concentrated Seed Goyasaponin Fraction in powder form Figure 7. Schematic representation of methodology used in preparation of concentrated goyasaponin fraction from M. charantia seeds 28    2.3 Identification of Goyasaponin Compounds in CGF from both fruit and seed by High Performance Liquid Chromatography-Mass Spectrum (HPLC-MS) A Waters Symmetry C18 column (3.9 × 150 mm, 5 µm) was used for the separation of fruit CGF. Fruit CGF powder was dissolved in methanol and syringe filtered (0.45 µm). Solvent A consisted of 0.05 % (v/v) acetic acid in water; solvent B was 0.05 % (v/v) acetic acid in acetonitrile and the column temperature was held constant at 30 °C. The flow-rate was 1 mL/min and the elution program was as follows: Time: 0 25 min (95-0% A, 5-100% B), 25 - 30 min (0% A, 100% B), 30 - 35 min (0-95% A, 100 5% B). The detection wavelength was 208 nm and injection volume was 20 µL. A Phenomenex Synergi column (RP100A, 2.00 × 50 mm, 2 µm, CA, USA) was used for the separation of seed CGF. Seed CGF powder was dissolved in methanol and syringe filtered (0.45 µm). Solvent A consisted of 0.05 % (v/v) acetic acid in water; solvent B was 0.05 % (v/v) acetic acid in acetonitrile and the column temperature was held constant at 30 °C. The flow-rate was 0.5 mL/min and the elution program was as follows: Time: 0 - 35 min (95-0% A, 5-100% B), 35 - 40 min (0% A, 100% B), 40 - 45 min (0-95% A, 100 - 5% B). The detection wavelength was 208 nm and injection volume was 20 µL. Detection and molecular weight confirmation of the goyasaponin compounds were established by a LC - MS (Finnigan LCQ quadrupole ion trap) system in both positive and negative mode. The MS conditions were set to an ion spray voltage of 4.5 kV with a capillary temperature of 300 °C and capillary voltage of 31 eV, 1 mL/min (Fruit CGF) or 0.5 mL/min (Seed CGF) was delivered to ESI-MS and the remaining diverted to waste. The scan mass spectra were in the m/z range of 100–2000. 29    3. Results The yield of lyophilized CGF powder (1663.62 mg) from fresh fruit (4.915 kg) is approximately 0.03385% while the yield of lyophilized CGF powder (3336 mg) from sun-dried seed (4 kg) is approximately 0.0834%. A variety of goyasaponin compounds were identified in the both fruit CGF and seed CGF on the basis of molecular fragment match to reported molecular weights in the literature [97]. The suspected compounds contained in the Fruit CGF are listed in Table 3. For a given retention time, up to three compounds in Fruit CGF were identified based on identical molecular weight. Compounds momordicilin, momordicoside A, goyaglycoside g, momordicoside E were identified individually without overlapping molecular weights. Similarly, suspected compounds in Seed CGF were listed in Table 4. 30    Table 3. Molecular weight confirmation of isolated goyasaponin compounds in fruit CGF by LC-MS No. Retention Time 1 15.8 2 17.59 Identification 5 18.54 -18.62 20.23 -20.31 21.58 6 21.88 7 22.84 8 24.17 goyaglycoside c /goyaglycoside d goyaglycoside a /goyaglycoside b /momordicoside K momordicoside F2 /momordicoside I goyaglycoside c /goyaglycoside d momordicoside E goyaglycoside a /goyaglycoside b /momordicoside K goyaglycoside c /goyaglycoside d momordicilin 9 34.87 momordicoside A 3 4 [M+MeOH]- [M+H]+ [M+CH3COOH]- [M+Na]+ 693.5 650.2 619.1 721.4 720.2 707.4 721.4 571.5 847.6 Table 4. Molecular weight confirmation of isolated goyasaponin compounds in seed CGF by LC-MS No. Identification [M+MeOH]- 1 Retention Time 1.11 Momordicoside D 815.44 2 6.18 Momordicoside A 3 8.74 Momordicoside A 4 9.79 Momordicoside D 5 23.96 Momordicin 6 25.54 Momordicoside C 831.50 7 31.54 Momordicoside C 831.54 8 32.69 Goyaglycoside e/f 9 42.54 Momordicoside C [M+H]+ [M+K]+ 874.93 817.03 815.68 478.10 782.44 831.73 31    [ M+CH3COOH ]- 4. Discussion In the first stage of project, methods for extraction and concentration of goyasaponin fractions from both M. Charantia fruit and seed were investigated and optimized. From HPLC-MS results, a variety of goyasaponin compounds were identified in both fruit CGF (Table 3) and seed CGF (Table 4) on the basis of molecular fragment matched to reported molecular weights in the literature. In addition, lyophilized CGF from both fruit and seed were obtained for further in vitro experiments. Further investigation could be focused on isolation and purification of single goyasaponin compounds from both fruit and Seed. First of all, a successive ethyl acetate and n-butanol liquid-liquid extraction could be used to remove impurities and concentrating the saponin fraction in methanolic extract. This two-step chromatography separation method, on a laboratory scale, would offer an equally efficient isolation as compared to multiple-steps chromatography that is commonly used. Secondly, the cucurbitane-type triterpene glycosides could be separated into its individual components by normal-phase silica gel followed by HPLC manual collection. This method can be scaled-up with ease. However, further studies need to be done to optimize the parameters and to reduce the cycle time of isolation. When one isolated component was obtained, it could be identified using common spectroscopy methods, such as LC-MS, according to previously reported molecular fragment. Further investigations are still in need to obtain the configuration of the side chains and the whole structure to confirm its identity, such as nuclear magnetic resonance (NMR) spectroscopy which could aid in identifying the structures of the aglycone as well as the structures and linkage of glycone moieties. Besides, other chemical identification and confirmation studies could be employed, such 32    as enzymatic, acid or basic hydrolysis to confirm the structures of further isolated goyasaponin compounds. 33    CHAPTER 3 EFFECTS OF FRUIT CGF ON 3T3-L1 CELL LINE 1. Hypothesis CGF from M. Charantia fruit influences preadipocyte proliferation; affects adipocyte differentiation and influences secretion of adiponectin in the 3T3-L1 murine cell model. 2. Objectives First of all, effect of fruit CGF on preadipocytes viability is determined by MTT assay in order to demonstrate the bioactivity of CGF. The mechanisms of this altered preadipocyte proliferation after CGF treatment would be investigated by further LDH assay, apoptosis assay and cell cycle analysis. Secondly, effect of fruit CGF on adipocyte differentiation could be displayed via both Oil-Red-O staining and triglyceride content test on adipocytes. The mechanism of the fruit CGF treatment in the whole adipogenesis would be demonstrated by PPARγ expression of adipocytes via western blot assay. More importantly, the effect of fruit CGF on adiponectin secretion from differentiated adipocytes would also be tested. 3. Materials and Methods 3.1 Cell Culture and Adipocyte Differentiation 34    3T3-L1 preadipocytes were purchased from American Type Culture Collection (Manassas, VA). Preadipocytes were incubated in Dulbecco's modified Eagle medium (DMEM) (Sigma, Germany) supplemented with 10% fetal bovine serum (FBS) (Biowest, Miami, Florida), and 1% antibiotic-antimycotic (100 U/mL penicillin, 100 µg/mL streptomycin sulfate, 0.25 µg /mL amphotericin B) (Gibco, Invitrogen, Canada) at 37℃ in a humidified 5% CO2 incubator (Sanyo, Tokyo, Japan). Cells were maintained at a cell concentration not exceeding 6 x 104 cells/mL and were subcultured by total medium replacement using 0.25% (w/v) trypsin - 0.53 mM EDTA (Gibco, Canada) every 2 - 3 days. The viable cell number was assessed in quadruplicate by trypan blue exclusion dye using a hemocytometer. Preadipocytes were seeded in 24-well plates at a density of 5 x 104 cells/well for 48 h. Adipogenesis (Day 0) was initiated with medium supplemented with 0.5mM 1Isobutyl-3-methylxanthine (IBMX) (Sigma) and 1 μM dexamethasone (DEX) (Sigma). The culture medium was replaced 48 h later (Day 2) with DMEM containing 10 µg/mL insulin (Sigma) for 48 h replaced thereafter with DMEM every 48 h. . On Day 8, adipogenesis was complete and adipocytes had acquired intracellular lipid droplets [113]. 3.2 Cell Viability (MTT Assay) Preadipocyte cell viability was assessed by the MTT (3-(4,5-Dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) viability assay. 3T3-L1 preadipocytes were seeded in 96-well plates (2500 cells/well) and allowed to attach overnight. After 24 h, culture medium with CGF (200-400 µg/mL) was added to each well and the cells were incubated for 24, 48 and 72 h and untreated cells acted as control. After each period, 35    medium and test compounds were replaced with 10 µL MTT solution (5 mg/mL MTT in PBS) and 90 µL DMEM to each well. After 4 h, 100 µL solubilization solution (10% SDS in 0.01M HCl) was added to each well and incubated overnight as described by Popovich and Kitts [114]. The plates were measured by a spectrophotometric microplate reader (Thermo Multiskan Spectrum, Helsinki, Finland) at 570 nm with a reference wavelength of 650 nm. Absorbance (A) of each treated group was equal to A570nm – A650nm. The preadipocyte viability percentage was expressed by A CGF / A untreated control x 100% and the 50% Inhibitory Concentration (IC50) values were calculated by linear regression. 3.3 Cell Toxicity Test by LDH Assay LDH was measured as previously described [114] with minor modifications. Pyruvate and NADH was purchased from Sigma. Tris, EDTA buffer was purchased from National University of Singapore Medical Institute (NUMI). Preadipocytes were seeded in 24-well plates at a density of 5 x 104 cells/well and allowed to attach overnight. CGF extract in culture medium was added at a concentration of 308 µg/mL, equivalent to the IC50 value determined from a 72-hour MTT assay, described above. Untreated preadipocytes acted as control groups and all cells were incubated at 37℃ in a 5% CO2 humidified incubator for 24, 48, and 72 h. Cell-free supernatant was obtained by centrifugation at 400g for 10 minutes. Two milliliters of Tris-EDTA-NADH buffer (pH 7.4, 50 mmol/L Tris buffer, 5 mmol/L EDTA, 150 µmol/L NADH) and 50 µL cell-free supernatant were mixed in a 24-well plate and incubated at 37℃ for 10 minutes. Prewarmed (37℃) pyruvate solution (200 µL) was added to each well and the reaction 36    velocity was measured in the first three minutes by a spectrophotometric micro-plate reader at 340 nm at 37℃. 3.4 Apoptosis Assay and Cell Cycle Analysis Preadipocytes were seeded in 24-well plates at the density of 5 x 104 cells/well. After 24 h, CGF powder was added to each well at a concentration of 200, 300, and 400 µg/mL with the untreated cells acting as a control. After treatment for 24, 48 and 72 h, adherent and non-adherent cells were harvested by trypsinization and centrifugation at 400g for 10 minutes. The pellet was washed twice in PBS and centrifuged to remove the PBS. The pellet was vortexed vigorously while 1 mL of ice-cold 70% ethanol was added slowly and allowed to fix overnight at 4 ℃. Ethanol was removed by centrifugation at 300g for 10 minutes and 1 mL PBS containing Propidium iodide (PI) (50 µg/mL, Sigma) and RNase A (100 µg /mL, AppliChem, Germany) was added and incubated for 1 hour. Cell cycle was analyzed using a FACS Vantage flow cytometer (Becton-Dickinson, Mountain View, CA). The percentage of each cell cycle phase after acquiring 10,000 cells was analyzed using WinMDI 2.8 software (Joseph Trotter, Scripps, CA). 3.5 Oil-Red-O Staining Preadipocytes were seeded on quadriPERM 4-well plates (Greiner Bio-One, Germany) and adipocyte differentiation was induced as described above. Three CGF concentrations (100, 200 and 300 µg/mL) were tested and untreated cells acted as controls. On Day 8, 10% formalin in PBS was added to replace the cultured medium and allowed to incubate for 5 minutes at room temperature, followed by fresh formalin for 1 37    hour. Each well was washed by 60% isopropanol and allowed to dry, incubated with 60% Oil Red O solution for 10 minutes and washed (5 times) with deionized water. Cell morphology was assessed using a CX 31 biological microscope equipped with a C-5060 digital camera (Olympus, Nagano, Japan). 3.6 Cell Lysate Preparation and Protein Concentration Determination Mammalian Cell Lysis / Extraction Reagent (CelLytic-M) and adipocyte lysate was prepared according to the manufacturer’s instructions (Sigma). Briefly, cells were washed with PBS and 150 µL cell lysis reagent was added to each well and allowed to mix on a rotary stage for 15 minutes. The cell lysate was collected and centrifuged at 15000g for 15 minutes to pellet the cellular debris. The supernatant was transferred to a chilled test tube and stored at -80℃. The protein concentration of the cell lysate was determined with Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol and were measured by a spectrophotometric micro-plate reader at 595nm. 3.7 Triglyceride Content Test Preadipocytes were seeded in 24-well plates and adipocyte differentiation was induced as described above. Three concentrations of CGF (100, 200 and 300 µg/mL) were tested and incubated for 8 days. On Day 8, adipocytes were collected and lysed as described above. Triglyceride content was determined by a commercial triglyceride kit (Wako Pure Chemical, Osaka, Japan). Protein concentration of lysate was determined as 38    described above. The results were expressed as triglyceride (mg) / protein (mg) in each group. 3.8 Expression of PPARγ by Western Blot Rabbit polyclonal primary antibody of PPARγ, adiponectin, Beta Actin and goat polyclonal secondary antibody to rabbit IgG - H&L horse radish peroxidase (HRP) were purchased from Abcam (Cambridge, UK). The same cellular protein (9 µg per lane) as used in triglyceride content test was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% polyacrylamide separating gel (Mini-PROTEAN Tetra Cell, Bio-Rad, Hercules, CA). The protein was transferred to a nitrocellulose membrane (ClearPAGE, C.B.S. Scientific, Del Mar, CA) by semi-dry transfer (C.B.S. Scientific). The membranes were blocked for 1 hour in 5% blotting grade blocker (Bio-Rad) in PBST (0.05% Tween 20 in PBS, pH 7.4). Membranes were incubated with diluted primary antibody (PPAR -γ (1:300), Beta Actin (1:2000)) at room temperature overnight and with the secondary antibody (1:2000) in PBST for 1 hour. Between each step, membranes were washed 3 times for 5 minutes each. The antibody/protein complexes were visualized by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Pierce, Rockford, IL). Fluorchem FC2 Imaging System (Alpha Innotech, San Leandro, CA) was used to detect the chemiluminescent signals of target protein bands. The expression of proteins was quantified densitometrically using the software GelPro 32 (Media Cybernetics, Bethesda, Md) and expressed relative to reference bands of Beta Actin. 39    3.9 Secretion of Adiponectin from Differentiated Adipocytes by Western Blot Rabbit polyclonal primary antibody of adiponectin was purchased from Abcam. On Day 8, differentiated adipocytes were washed with serum-free medium twice. Four concentrations of CGF (50, 100, 200 and 300 µg/mL) in medium were replaced and incubated for 12h and 24h respectively. Untreated cells act as negative control while 10 µM Troglitazone (0.02% dimethyl sulfoxide (DMSO) in medium, v/v) (sigma) acts as positive control. Medium of 10 µL was taken for detection of released adiponectin by SDS-PAGE and Western Blot methods as mentioned above with diluted primary antibody of Adiponectin at 1:2000. The number of viable adipocytes was measured by Trypan Blue exclusion. Adiponectin expression was quantified using the software GelPro 32 and normalized to viable cell number. 3.10 Statistical Analysis One-way ANOVA (SPSS 12.0) was used to detect the significant differences (p < 0.05) using the Duncan Post hoc multiple comparisons of observed means. Each experiment was performed in triplicate and the results were expressed as mean ± standard deviation. 4. Results 4.1 Dose-Response Relationship Fruit CGF treatment caused a dose and time dependent reduction in preadipocyte cell viability as assessed by MTT viability assay. Figure 8 shows that at each time period the cell viability significantly (p < 0.05) deceased with increasing concentration 40    with one exception at concentrations between 250 – 300 µg/mL after 24 h exposure. IC50 values decreased as exposure times increased, the IC50 values were calculated by linear regression and determined to be 401.67 ± 4.04 µg/mL for 24 h, 313.67 ± 10.41 µg/mL for 48 h and 308.33 ± 11.59 µg/mL for 72 h respectively. 41    Figure 8. The effect of fruit CGF on cell viability in 3T3-L1 preadipocytes. Cells were treated with CGF at concentrations of 200, 250, 300, 350, 400 µg/mL for 24h (a), 48h (b) and 72h (c) respectively. Panel (d) shows the relative effect at both time and concentration. MTT assays had six replicates for each treatment. Values are expressed as means ± standard deviation from three independent experiments. Means with different letters (a, b, c, d, e) are significantly different at each time period (p < 0.05). 42    4.2 Increased LDH Release Figure 9 shows the effect of a fruit CGF treatment on lactate dehydrogenase (LDH) release, a marker of membrane integrity, from preadipocytes treated for 24 – 72 h at the 72 h IC50 concentration of 308.33 ± 11.59 µg/mL. Fruit CGF significantly (p < 0.05) increased the release of LDH after 48 and 72 h of treatment. It is noteworthy that all time periods showed more LDH release when compared to the untreated controls. Figure 9. The effect of fruit CGF on preadipocyte LDH activity. Cells were treated with CGF for 24h, 48h and 72h respectively at concentrations of 308 µg/mL, the IC50 concentration determined from a 72-hour MTT assay with five replicates. Values are means ± standard deviation from three independent experiments. Means with different letters (a and b) are significantly different (p < 0.05). 43    4.3 Disturbed Cell Cycle The effect of fruit CGF on 3T3-L1 preadipocyte cell cycle events were measured by flow cytometry. Representative DNA cell cycle histograms are shown in Figure 10 and the proportion of the cell population in each phase is listed in Table 5. Although the percentage of cells in the sub-G1 category was determined to be significantly increased compared to the control cells, the absolute increase in cell percentages were less than 1% of total cell DNA counts. Therefore, the actual influence of CGF on inducing apoptosis in 3T3-L1 preadipocytes was minor. In contrast, CGF perturbed the cell cycle and increased cells in the G2/M cell cycle phase beginning at concentration of 300 µg/mL at 48 and 72 h and showed an increase of 35 and 28 %, respectively. A percent change increase of 17% at 24 h, 51% at 48 h and 82 % at 72 h were observed at a concentration of 400 µg/mL. Concurrently, a significant (p < 0.05) decrease in G1 phase cells were observed at concentrations greater than 300 µg/mL at all time periods when compared to the respective control populations. The percentage decrease for a concentration 300 µg/mL CGF was 8.1% at 24 h, 12.1% at 48 h and 6% at 72 h. Moreover, at a concentration of 400 µg/mL the percentage decrease was calculated to be 14.5, 21, and 22 % at 24, 48 and 72 h, respectively. 44    Figure 10. DNA cell cycle histograms of preadipocytes. Cells were treated with fruit CGF at concentrations of 200, 300, 400 µg/mL for 24h, 48h and 72h respectively. Untreated cells acted as control groups. Cells were fixed in 70% ethanol and stained with PI as described in Materials and Methods. DNA histograms shown are representative of an assay repeated in three independent experiments with similar results. 45    Table 5. DNA cell cycle results of preadipocytes treated with fruit CGF from 24 – 72 hours. Three replicates were performed for each treatment and repeated for three separated times. Values are expressed as means ± standard deviation. An asterisk (*) denotes a significant difference from control. sub-G1 (%) G1 (%) 24h 48h 72h 24h 48h 72h control 0.39 ± 0.05 0.29 ± 0.07 0.33 ± 0.17 64.82 ± 4.31 79.09 ± 1.19 78.28 ± 1.59 200 µg/mL 0.65 ± 0.08* 0.39 ± 0.04 0.57 ± 0.16 63.16 ± 6.18 75.50 ± 1.41 75.17 ± 3.59 300 µg/mL 0.60 ± 0.05* 0.47 ± 0.04* 0.44 ± 0.09 59.57 ± 3.93* 69.46 ± 1.39* 73.60 ± 4.54* 400 µg/mL 0.82 ± 0.25* 0.78 ± 0.11* 0.79 ± 0.10* 55.45 ± 3.73* 62.58 ± 2.36* 61.25 ± 5.64* S (%) G2/M (%) 24h 48h 72h 24h 48h 72h control 12.28 ± 1.35 6.42 ± 0.57 5.98 ± 0.84 20.75 ± 2.34 13.76 ± 1.07 13.20 ± 0.49 200 µg/mL 12.67 ± 2.46 7.38 ± 1.17 5.72 ± 1.70 20.97 ± 2.07 15.49 ± 0.41 14.74 ± 1.26 300 µg/mL 13.44 ± 0.92 9.59 ± 1.67* 6.42 ± 2.05 23.62 ± 2.14 18.55 ± 0.99* 16.86 ± 1.11* 400 µg/mL 16.01 ± 0.71* 13.57 ± 1.30* 11.02 ± 3.74* 24.35 ± 2.20* 20.71 ± 1.45* 24.06 ± 1.63* 46    4.4 Suppressed differentiation from preadipocytes to adipocytes The fruit CGF was added to adipogenesis medium at concentrations of 100, 200 and 300 µg/mL during the adipogenesis process to determine the influence on lipid accumulation during the differentiation of preadipocytes to adipocytes. Figure 11 shows representative images of Oil-Red-O stained cells after CGF treatment for 8 days and the adipogenesis was clearly observed as suppressed by CGF in a dose-dependent manner. Moreover, CGF also significantly decreased the intracellular triglyceride content in adipocytes dose-dependently (Figure 12(A)). It is noteworthy that all concentrations tested significantly (p < 0.05) reduced intracellular triglyceride accumulation compared to untreated control adipocytes. Moreover, the adipogenesis-related protein expression level of PPARγ was assessed by western blot. Fruit CGF at three concentrations of 100, 200 and 300 µg/mL were added with each medium change over the course of adipogenesis with untreated cells acting as control groups. As shown in Figure 12 (B), a key transcription factor of adipocyte differentiation, expression level of PPARγ was down-regulated dose dependently which was in accordance with the morphological data as well as decreased intracellular triglyceride content. 47    Figure 11. Oil-Red-O stained adipocyte on Day 8. Fruit CGF extract at three concentrations of 100 (b), 200 (c) and 300 µg/mL (d) were added with each medium change during the entire adipogenesis process. The untreated cells acted as control groups (a). Intracellular lipid droplets were stained with 60% Oil-Red-O solution and morphology was assessed by using a microscope (20x objective magnification) with a digital camera as described above in Research Design and Methods. Pictures represent one of three independent experiments. All three experiments showed similar pattern. 48    Figure 12. The effect of fruit CGF on triglyceride accumulation (A) and PPARγ expression (B) in adipocytes. Preadipocyte were induced to differentiate by a cocktail treatment with CGF extract at concentration ranging from 100 – 300 µg/mL added with each medium change during the entire adipogenesis process. The untreated cells acted as control groups. In triglyceride content test (A), three replicates were performed for each treatment and repeated three separated times. Protein expression level of PPARγ (B) was analyzed by western blot. The relative expression of protein was quantified using the software GelPro 32 and calculated according to the reference bands of Beta Actin. The data are expressed as a percentage, relative to PPARγ expression in control group. All values are expressed as means ± standard deviation of three experiments. Means with different letters (a, b, c) are significantly different (p < 0.05). 49    4.5 Inhibited Adiponectin Secretion from Differentiated Adipocytes The secretory amount of adiponectin in medium of mature adipocytes was suppressed after both 12 h (Figure 13 (A)) and 24h treatment (Figure 13 (B)) of fruit CGF when compared to untreated adipocytes. Especially at concentration of 200 µg/mL and 300 µg/mL, the band intensity of adiponectin significantly decreased 29.5% and 61.7% after 12 h incubation; 12.6% and 32.8% for 24 h treatment. In contrast, when differentiated adipocytes were treated with Troglitazone at the concentration of 10 µM for 12 h and 24 h (Figure 13 (C)), adiponectin amount in medium was increased in a time-dependent manner which was in accordance with previous findings by Huypens et al (53). After 12 h, Troglitazone treatment also increased 137.2% band intensity compared to 12 h control (0.02% DMSO in medium) while after 24 h treatment, the band intensity significantly increased 849.5% when compared to 24 h control (0.02% DMSO in medium). 50    Figure 13. Adiponectin secretion is inhibited by fruit CGF in a dose-dependent manner. Differentiated adipocytes on Day 8 were exposed to fruit CGF in indicated concentrations for 12 hours (A) and 24 hours (B). The untreated cells acted as negative control groups while 10 µM TZD treated cells (C) acted as positive controls. Ten µL medium was taken for detection of released adiponectin by Western blotting. The number of viable adipocytes was measured by Trypan Blue exclusion. Band intensity of Adiponectin was quantified using the software GelPro 32 and normalized to viable cell number. Data are expressed relative to untreated control group cells (100%) and represented means ± standard deviation from three independent experiments. * denotes p[...]... investigate whether a concentrated saponin fraction (CGF) extracted from both M charantia fruit and seed show a potential as PPARγ agonist by evaluating their effects and relevant mechanisms of in the 3T3- L1 murine cell line (1) To optimize methods of extracting and concentrating saponin fractions from both M Charantia fruit and seed and to obtain lyophilized CGF in powder for further in vitro 23    experiments... illnesses as well as manage chronic diseases, most notably diabetes, in the traditional medicinal systems of many cultures worldwide, including those of the Asian Indians, Chinese and South Americans [79] Besides anti-diabetic properties, M charantia has also been credited with antiviral, antitumor, 13    antileukemic, antibacterial, anthelmintic, antimutagenic, antimycobacterial, antioxidant, antiulcer, anti-inflammatory,... in deionized water Concentrated aqueous extract Lyophilization Fruit CGF in powder form Figure 6 Schematic representation of methodology used in preparation of concentrated goyasaponin fraction from M charantia fruit 26    2.2 Preparation of Concentrated Goyasaponin Fraction from M charantia Seed Sun-dried M charantia seeds were purchased from a local herbal store (Ban Lee Huat Seed Pte Ltd., Singapore)... of any potential for its development into a nutraceutical product 24    CHAPTER 2 CONCENTRATION OF GOYASAPONIN FRACTIONS 1 Objectives (1) Sample preparation of CGF from both M Charantia fruit and seed for further in vitro experiments (2) Component analysis of CGF by HPLC-MS based on molecular weight confirmation 2 Materials and Methods 2.1 Preparation of Concentrated Goyasaponin Fraction from M charantia... Aqueous phase (Discarded) Removal of n-butanol and dissolved in deionized water Aqueous extract Solid phase extraction using Amberlite® XAD-4; eluted with ethanol Ethanolic extract Removal of ethanol and dissolved in deionized water Concentrated aqueous extract Lyophilization Concentrated Seed Goyasaponin Fraction in powder form Figure 7 Schematic representation of methodology used in preparation of concentrated. .. formulae and structures of selected momordicosides 22    5 Overall Hypotheses, Objectives and Implications of This Study 5.1 Overall Hypotheses A concentrated goyasaponin fraction (CGF) from M Charantia fruit was obtained using an extraction and concentration method It is hypothesized that this fruit CGF shows a potential as a PPARγ agonist and plays a similar role as TZDs which can influence preadipocyte... resistance [67] In addition, TZDs were proved to activate AMPK in rat liver and adipose tissue but it remains unclear whether this is a direct effect and/ or mediated by PPARγ via increasing plasma level of adiponectin [68] However, the safety of earlier generation TZD has been questioned as well as concerns over common side effects of newer generation of TZD such as weight gain, edema and heart failure... Metformin, on the other hand, achieve hypoglycemic effects principally via suppressing hepatic glucose output [54] It was found that metformin might mediate its insulin-sensitizing effect by directly activating AMPK pathway in rat liver and muscle [66] In addition, a clinical study on type 2 diabetes patients demonstrated that metformin caused a significant increase in AMPK α2 activity in muscle after...Table 1 Current therapeutic agents for type 2 diabetes Class Molecular Target(s) Sites of Action Main mode of action Adverse Effects Liver, muscle Decrease hepatic glucose output; Increase peripheral glucose uptake Gastrointestinal Disturbances; lactic acidosis PPARγ Fat, liver, muscle Increase insulin sensitivity Weight gain; Oedema; Anaemia SU receptor/ K+ ATP channel β -cell Increase insulin secretion... activity and other beneficial effects against associated complications [96] However, the active antidiabetic component of M charantia has not been adequately identified, although a wide range of compounds have been isolated, notably steroidal-like saponins such as 14    cucurbitane-triterpene glycosides, oleanane-triterpene saponins [97] and proteins including insulin-like polypeptide-p and napin-like ... their effects and relevant mechanisms of in the 3T3-L1 murine cell line (1) To optimize methods of extracting and concentrating saponin fractions from both M Charantia fruit and seed and to obtain... Preparation of Concentrated Goyasaponin Fraction from M charantia Fruit M charantia fruit was purchased from a local supermarket, washed, separated from the seeds and aerial fibers, cut into small... representation of methodology used in preparation of concentrated goyasaponin fraction from M charantia fruit 26    2.2 Preparation of Concentrated Goyasaponin Fraction from M charantia Seed Sun-dried