Anti-Obesity

CHAPTER 1
INTRODUCTION
One of the greatest impacts of nutrition on health relates to obesity, an excessive accumulation of body fat (Karren et al., 2006). It is a serious problem faced by millions of people worldwide. It is now on the rise among Filipino adults ages 20 and up, and threatens to increase the number of people having degenerative diseases like heart disease, hypertension and diabetes mellitus, as study conducted by the Department of Science and Technology showed (Mabutas, 2010). According to the World Health Oraganization (WHO), more than 1.4 billion adults, 20 and older, were overweight in 2008. Of these, over 200 million men and nearly 300 million women were obese. Studies showed that 65% of the world 's population live in countries where overweight and obesity kills more people than underweight (World Health Organization, 2012). Obesity is one of the greatest banes of our technology centered lives. This problem is evident not only in adults, but also in kids who are spending more time in front of the computer, television or video-game console instead of playing games outside. Since today 's busy families have fewer free moments to prepare nutritious, home-cooked meals, the rapid increase of fast food chains in the country should not come off as surprising. The modern person, though, has started to become aware of this problem. On top of image issues, obesity also poses a number of health-related problems which cannot be taken lightly. The increasing number of weight loss pills, herbal dietary supplements or teas and the fact that Zumba has become extremely popular in Cebu, is proof of that (Sun.Star Cebu Newspaper, 2012). These have not yet been completely effective in eliminating the problem, though.

Obesity has definitely become a growing problem, but a lot of anti-obesity medications, with fen-phen as the best example, have proven to have very damaging side-effects like lung hypertension and heart valve condition (Fen Phen, 2010). Orlistat is also associated with a number of unpleasant intestinal side effects such as loose stools or an urgent need to go to the bathroom and the inability to control bowel movements. It is generally only prescribed as a last resort for weight loss (WebMD, 2013). The researchers have decided to join in developing ways to eliminate obesity by studying the weight loss potential of Snow Pea pods extract. This, if proven effective, could be a natural way of treating obesity. Snow peas are popularly known as “chicharo” or “sitsaro” in the Philippines. They are the most common type of peas found in the country. Peas are low in calories and are good source of fiber, vitamin C, iron, potassium and magnesium (Department of Primary Inductries, November 2009). According to a chart provided by the George Mateljan Foundation, a cup of green peas contains 30.3% fiber and 14.8% protein. Few foods provide us with such substantial amounts of protein and fiber as peas. Dietary fiber, which is found in snow peas, is undoubtedly one of the most talked about nutrients for health promotion and disease prevention (The George Mateljan Foundation, 2009). Dietary fibers promote beneficial physiological effects including laxation, blood cholesterol attenuation and blood glucose attenuation or a combination of the three effects. (American Association of Cereal Chemists, 2001). Diets higher in protein and moderate in carbs are often thought by experts to reduce blood fats. It also helps maintain lean tissue while burning fat for fuel. And this happens without dieters being sidetracked with constant hunger. Researchers have yet to figure out how protein works to reduce appetite, but it is speculated that it may be because protein causes the brain to receive lower levels of appetite-stimulating hormones (WebMD, 2012). With these information, the researchers hope that snow peas would prove to have significant anti-obesity effects.

Objectives of the Study The purpose of the study is to find out the effectiveness of Snow peas Pisum sativum var. saccharatum pod extract as an anti-obesity agent on male albino mice. The study specifically aims to: 1. determine the weight loss or weight gained of each mouse and the difference in the weight of each mouse from day 31 to day 45. 2. determine the percent anti-obesity activity of the test plant extract based on weight loss. 3. determine the number of times each mouse stands or paws during the five minute locomotor test. 4. monitor the quality of fecal activity of each mouse to test the laxative property of Snow peas and check for the presence of fats and oils in the feces through spot test. 5. determine the constituents present in snow peas pod extract using test tube reaction method.

Significance of the Study If proven effective, the research study can serve as one of the basis for future researches, since not much anti-obesity agents were studied by the students of the Department of Pharmacy of the University of San Carlos, Cebu City. The study would be a good step in the isolation and formulation of pharmaceutical products using Philippines’ rich nature, since it considers the potential therapeutic importance of a plant available in the Philippines. The study would promote the planting of Snow peas in the Philippines. Peas are one of the easiest of all vegetables to propagate and grow, giving rather rapid gratification. They will grow satisfactorily in most garden soils. Snow peas are one of the most satisfying vegetables to grow even in small spaces and are ideal as a plant for balconies and courtyards as they could and should be harvested every couple of days. As well as the pods you can also harvest the tender young shoots for salads and stir fried meals. They can be grown just as well in a pot as they can in the ground just as long as they have a support to climb on to. The study would help minimize, if not eliminate, the problems concerning obesity and excessive fat intake from our diets. This will be made possible by encouraging people to include snow peas in their daily diet. Being overweight or obese puts a person at risk for many health problems. The more body fat a person has and the more a person weighs, the more likely for the person to develop diseases such as coronary heart disease, high blood pressure, type 2 diabetes, gallstones, breathing problems, and certain cancers. With the help of modern technology, the study can help improve the quality of life of many people. The study will be beneficial not only to the Filipinos, but also to people outside the country. It will encourage people to eat vegetables, which contain a lot of nutrients. Snow peas have important role inside human nutrients. This plant contains protein, energy, fiber, vitamins and minerals which enhance metabolism. They provide a high dose of pectin, a soluble fiber that makes you feel full which may reduce your appetite. Also, it lowers LDL cholesterol reducing the risk of heart disease, regulates blood sugar which may reduce the onset risk or symptoms of metabolic syndrome and diabetes, and may reduce the risk of colorectal cancer, and help manage diarrhea. They provide a good dose of thiamine, also known as vitamin B1, vitamin that plays a role in energy metabolism, helps maintain normal digestion, appetite, and proper nerve function. Protein of snow peas has good source of amino acid. The study also helps discover the incredible weight loss benefits of Snow peas and how much a person should eat to get maximum benefit from these peas. This research can promote awareness for everyone to pay attention to their lifestyle and food choice.

Scope and Delimitation The research study will only focus on the potential anti-obesity property of Snow pea pod extract on male albino mice. The Snow peas pod will undergo ethanol extraction then dried in an oven to obtain its extract. The mice will be grouped into eight, with one mouse per group. Groups one to four will be receiving normal diet, while groups five to eight will be receiving high fat diet for a length of one month before testing. Two doses of extracts (500 mg/ 20 g mouse and 1000 mg/ 20 g mouse) will be used for the experiment and Orlistat will be used for the positive control. Three trials will be conducted for this study. Anti-obesity activity will be determined with the use of body weight, locomotor activity test, and examination of fecal quality. The extraction process, confirmatory test, and phytochemical testing will take place during the first semester of S.Y. 2013-2014 at the Department of Pharmacy, Father Robert Hoeppener Building, University of San Carlos, Talamban Campus, Cebu City, Philippines.
Definition of Terms Anti-Obesity Agent. Substance with possible weight reducing effect administered to the albino mice. Extract. Substance obtained after ethanol extraction, filtration and evaporation of Snow peas pod. Locomotor Activity Test. The mobility test used to determine the capacity of mouse to stand on its rear paws within five minutes. Obesity. A condition characterized by the abnormal or excessive accumulation and storage of fat in the body that presents a risk to health. The test animal is said to be obese if its weight increases by twenty percent of its original weight.

Spot Test. A test used to detect the presence of lipids. Lipids leave translucent spots (grease spots) on filter paper.

Weight Gain. Increase in body weight of test animals which results from the intake of more calories than needed by the body. Weight Loss. Decrease in body weight resulting from intake of weight-reducing agent, as determined by consistent weighing.

CHAPTER 2
REVIEW OF RELATED LITERATURE AND STUDIES

Peas, Pisum sativum Peas are a member of the Fabaceae or legume family, which includes plants that bear pods enclosing fleshy seeds. There are two main kinds of peas: green and edible-pod. Snow peas (See Plate 1) belong to edible pods. Most edible-pod peas have seeds that are not yet fully developed (Calfresh, 2011). The legume family (Leguminosae) takes its name from the Latin legumen, or pod. Thisis a two-valve fruit with seeds attached to the edge of the upper margin. The podmay be straight, coiled, rounded, flattened, winged, woody, flesh, smmoth or hairy. When ripe, they may split open along with the margin or sometimes break into seeded segments. Many seeds are pea- or bean-shaped; all have two cotyledons and an embryo, and can often retain the capacit to germinate for many years. Pods of different species can display a number of interesting features (Millet, 2004).

Snow Peas Pisum sativum var. saccharatum or snow pea is a variety of pea eaten whole in its pod while still unripe. In French, snow peas are called “mange tout,” which means “eat all,” since both the peas and the pods are edible. It is one of the earliest-known cultivated plants, with evidence of having been cultivated in a region that is now along the Thailand-Burma border, 12,000 years ago. Like all legumes, snow peas host beneficial bacteria, rhizobia, that fix nitrogen in the soil, which is considered a mutualistic relationship, and are therefore a useful companion plant, especially useful to grow intercropped with green, leafy vegetables that benefit from high nitrogen content in their soil. The snow peas grow up to three inches long (7.5 cm) with flat, bright green pods that are pointed at both ends. The peas sprout from a blossom that becomes a green leaflet at the stem-end of pods as the peas mature. The pods also have strings along their edges that are usually removed before cooking. The tendrils the vines produce for climbing are also edible and are often used in fine cuisine (Rajeshbac, 2010).
Plate 1. Habit of Growth of Snow Pea Plant Pisum sativum var. saccharatum (Rasbak, 2009)

Inset A: Leaf and Flower (HD Directory, 2012); B: Pods (Burpee, 2012)

Snow peas are rich in nutritional value. They are high in dietary fibre, protein and vitamin C. They are very low in Saturated Fat, Cholesterol and Sodium. They are also a good source of Riboflavin, Vitamin B6, Pantothenic Acid, Magnesium, Phosphorus and Potassium, and a very good source of Vitamin A, Vitamin K, Thiamin, Folate, Iron and Manganese. (The Tucker Patch, 2012) The texture and sweetness of snow peas are at their best when eaten immediately after harvesting. They will also keep for several days in the refrigerator in a plastic bag or container. Snow peas may also be frozen and do not need to be thawed before being added to a dish. Fresh snow peas may be eaten raw as a snack or used as a salad ingredient. They also lend themselves nicely to quick blanching, which enhances their crisp texture and vibrant green color. Snow peas may be added to Asian stir-fry dishes, soups, and pasta. They require very little cooking time and should be added toward the end of the cooking process for optimal texture and color (Heiter, 2003). The extracts from the seeds of snow peas have been shown to have antibacterial properties that may help protect infants and children against certain infections from bacteria such as H. pylori. Studies also shows that a substance contained in snow peas called glycodelin-A has been effective to help overcome one cause of male infertility. Snow peas were proven to increase fertility in some men that are currently considered infertile (Olberding, 2011).

Obesity A recent article by Galetta defined obesity as a result of the accumulation of excess fat on the body. It is considered a chronic disease, with many serious long-term consequences on an individual’s health. Along with tobacco use and high blood pressure, it is leading cause of preventable deaths (Galletta, 2012). Department of Science and Technology’s Food and Nutrition Research Institute conducted a study last 2008 which showed that there were about 20 out of 100 Filipino adults of both sexes who were overweight in 1998. The figures have then increased to 24 and 27 out of 100 in 2003 and 2008, respectively (Mabutas, 2010). Obesity is normally perceived as something adults would suffer. But according to Philip Chua, obesity in children has doubled the past couple of decades and tripled the past 30 years. Today, among the affluent people around the world, including those in developing nations, obesity afflicts about 20 percent of children aged 6 to 11, and more alarmingly, about 10 percent of those aged 2 to 5 are obese. Among those between ages 12 and 19, 18 percent are also obese. The more obese the child, the greater the risk of developing a cluster of health problems and their complications early in life, which includes type 2 diabetes, high blood pressure and other cardiovascular diseases. Obesity, in children or in adult, is a significant health danger (Chua, 2012). Obesity and overweight have many causes, including genetic, metabolic, behavioural and environmental. The rapid increase in prevalence suggests that behavioural and environmental influences predominate, rather than biological changes. Direct associations between obesity and several diseases, including diabetes mellitus, hypertension, dyslipidaemia and ischaemic heart disease, are well recognized. Despite this, the relationship between body weight and all-cause mortality is more controversial. A very high degree of obesity (BMI > 35 kg/m2) seems to be linked to higher mortality rates (Malnick and Knobler, 2006). Despite the rapidly increasing cases of obesity worldwide, weight-loss medications should only be considered as an adjunct for patients who are at substantial medical risk because of their obesity and in whom non-pharmacologic treatments have not resulted in sufficient weight loss to improve health or to prevent regain. The safety and efficacy of weight-loss medications beyond two years of use have not been established (Yanovski and Yanovski, 2002).

Health Risks of Obesity (Abrams, et al., 2009) Cancer. Obesity is associated with a higher prevalence of breast, colon, and endometrial cancers. With breast cancer, risks increase in postmenopausal women with increasing body weight. Women who gain more than 20lb from age 18 to midlife have double the risk of breast cancer compared with women who maintain a stable weight during this period of their life. In addition, obesity apprently increases the risk of breast cancer independent of overall obesity. In women with central obesity, this additional risk factor may be related to an excess of estrogen and a deficiency of sex-hormone-binding globulin to combine with the estrogen. Colon cancer seems to be more commone in obese man and women. In addition, a high BMI may be a risk factor for a higher mortality rate with colon cancer. Endometrial cancer is clearly more common in obese women, with adult weight gain again increasing risk. Cardiovascular Diseases. Obesity is a major risk factor for cardiovascular disorders and increased mortality from cardiovascular disease. Studies have confirmed the relationship between obesity and increased risk of coronary heart disease and stroke in both men and women. In addition, obesity during adolescence is associated with higher rates and greater severity of cardiovascular disease as adults. Obesity increases risks by aggravating other risks factors such as hypertension, insulin resistance, low HDL cholesterol, and hypertriglyceridemia. In addition, obesity seems to be an independent risk factor for cardiovascular disorders, and obesity may be more important than BMI as a risk factor for death from cardiovascular disease. The increased mortality rate is seen even with modest excess body weight. Hypertension, dyslipidemia, insulin resistance, and glucose intolerance are known cardiac risk factors that tend to cluster in obese individuals. Hypertension often occurs in obese persons and is thought to play a major role in the increased incidence of cardiovascular disease and stroke observed in patients with obesity. Metabolic abnormalities that occur with obesity and type 2 diabetes mellitus (e.g., insulin resistance and the resultant hyperinsulinemia) aggravate hypertension and increase cardiovascular risks. The combination of obesity and hypertension is associated with cardiac changes (e.g., thickening of the ventricular wall, ischemia, and increased heart volume) that lead to heart failure more rapidly. Weight loss of as little as 4.5 kg (10 lb) can decrease blood pressure and cardiovascular risk in many people with obesity and hypertension. Diabetes Mellitus. Obesity is strongly associated with impaired glucose tolerance, insulin resistance, and diabetes mellitus. In addition, obesity during adolescence is associated with high rates of diabetes as adults as well as more severe complications of diabetes at younger ages. Dyslipidemias. Obesity strongly contributes to abnormal and undesirable changes in lipid metabolism (e.g., increased triglycerides and LDL cholesterol; decreased HDL cholesterol) that increase risks of cardiovascular disease and other health problems. Gallstones. Obesity apparently increases the risk for developing gallstones by altering production and metabolism of cholesterol and bile. The risk is higher in women, especially those who have had multiple pregnancies or who are taking oral contraceptives. However, rapid weight loss with very low-calorie diets is also associated with gallstones. The reason for this may be that rapid weight loss appears to decrease the gallbladder 's ability to contract bile. But, it is unclear whether very low-calorie diets directly cause gallstones or whether the amount of weight loss is responsible for the formation of gallstones. Metabolic Syndrome. Metabolic syndrome is a group of risk factors and chronic conditions that occu together and greatly increase the risks of diabetes mellitus, serious cardiovascular disease, and death. The syndrome is thought to be highly prevalent in the United States. Major characterstics include many of the health problems associated with obesity (e.g., dyslipidemias, hypertension, impaired glucose tolerance, insulin resistance, central obesity). Osteoarthritis. Oesity is associated with osteosrthritis of both weight-bearing joints, such as the hip and knee, and non-weight bearing joints. Extra weight can stress affected bones and joints, contract muscles that abnormally stabilize joints, and may alter the metabolism of cartilage, collagen, and bone. In general, obese people develop osteoarthritis of the knees at earlier age and are more likely than non-obese people to require knee replacement surgery. Sleep apnea. Sleep apnea commonly occurs in obese persons. A possible explanation is enlargement of soft tissue in the upper airways that leads to collapse of the upper airways with inspiration during sleep. The obstructed breathing leads to apnea with hypoxemia, hypercarbia, and a stress response. Sleep apnea is associated with increased risks of hypertension, possible right heart failure, and sudden death. Weight loss leads to improvement in sleep apnea. Miscellaneous Effects. Obesity is associated with numerous difficulties in addition to those described above. These may include • Non-alcoholic fatty liver disease, which is being increasingly recognized and which may lead to liver failure • Poor wound healing • Poor antibody response to hepatitis B vaccine • A negative perception of people who are obese that affects their education, socioeconomic, and employment status • High costs associated with treatment of the medical conditions caused or aggravated by obesity as well as the costs associated with weight-loss efforts • In women, obesity is associated with menstrual irregularities, difficulty in becoming pregnant, and increased complications of pregnancy • In men, obesity is associated with infertility • In children and adolescents, obesity increases risk of bone fractures and muscle and joint pain. Knee pain is commonly reported, and changes in the knee joint make movement and exercise more difficult.

Anti-obesity Drugs Anti-obesity drugs or weight loss drugs include all pharmacological treatments intended to reduce or control weight. The two main categories of anti-obesity drugs are: (1) Anti-obesity drugs that act on the gastrointestinal system (pancreatic lipase inhibitors) and (2) Anti-obesity drugs that act on the central nervous system mainly to suppress appetite. Orlistat is an example of the first kind of anti-obesity drug that inhibits pancreatic and gastric lipase, which reduces dietary fat absorption and in turn leads to weight loss. Sibutramine is the example of second kind of anti-obesity drug that promotes a sense of satiety. It is an anorectic or appetite suppressant that reduces the desire to eat. Rimonabant is a recently developed anti-obesity drug. It also acts centrally on the brain and decreases appetite. There are many anti-obesity drugs but some of them have severe or life-threatening side effects. Therefore, they are recommended to be taken only under medical supervision. Prescription weight loss drugs are approved only for those with a BMI of 30 and above, or 27 and above if they have other risk factors, such as high blood pressure or diabetes (Pharmaceutical Drug Manufacturers, 2012).

Orlistat Orlistat (See Figure 2) is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S, 3S)-3-hexyl-4-oxo-2-oxetanyl] methyl]-dodecyl ester. Its empirical formula is C29H53NO5, and its molecular weight is 495.7. It is a single diastereomeric molecule that contains four chiral centers, with a negative optical rotation in ethanol at 529 nm (Genentech, 2010).

Figure 1. Chemical Structure of Orlistat

Orlistat is a white to off-white crystalline powder. Orlistat is practically insoluble in water, freely soluble in chloroform and very soluble in methanol and ethanol. Orlistat is not capable of ionizing within the physiological pH range (Roche, 2012). Orlistat is used together with a reduced-calorie diet to help a person lose weight and to help keep the lost weight from returning. It is also used in overweight people who may also have diabetes, high blood pressure, high cholesterol, or heart disease. This medicine is available both over-the-counter (OTC) and with doctor 's prescription. This product is available in capsule (Kenny, 2012). Orlistat works by blocking chemicals (enzymes) such as pancreatic and gastric lipases in the gut which digest fat. Nearly a third of the fat that one ingested is blocked by orlistat. The undigested fat is not absorbed into the body, and is passed out with the stools. The normal dose is one capsule - 120 mg, three times a day with each meal. However, it is not needed to take one if there is no fat in the meal or if one misses a meal (MIMS, 2012). Orlistat can also interfere with the absorption of various medicines and vitamins bind to fat (vitamins A, D, E and beta- carotene) and affect the way that they work. Its most notable drug-to-drug interactions happen when it is taken together with a cyclosporin, acarbose, amiodarone, and warfarin. It is also possible with a contraceptive pill. Therefore, if a person takes orlistat he must take a multivitamin supplement at bedtime - a time when he will not be taking orlistat - to help ensure adequate vitamin intake. If a person takes the contraceptive pill and have severe diarrhea he should use other methods of contraception in addition to the pill - for example, condoms. One must tell his doctor or pharmacist about other medicines that they take, before starting on orlistat (Kenny, 2012). The common side effects of orlistat includes: bladder pain, diarrhea, fever, general feeling of discomfort or illness, loss of appetite, muscle aches and pains, nasal congestion, nausea, sweating, unusual tiredness or weakness, abdominal or stomach pain or discomfort, back pain, difficulty with moving, gas with leaky bowel movements, inability to hold bowel movement, increases in bowel movements, loss of bowel control, oily bowel movements, and oily spotting of underclothes (Micromedex, 2012).

Male Albino Mice Most of the mice used in laboratories are white albino house mice (See Figure 3). The mouse has been used in biomedical research since the early 20th century. Several characteristics have made the mouse an appealing research subject. These include the mouse 's genetic similarity to humans (at least 80% of DNA in mice is identical to that of humans), small size, short lifespan and reproductive cycle, low maintenance in captivity, and mild manner. For these reasons, house mice constitute the majority of mammals used in research, testing, and education. Over ten million mice are used each year in U.S. laboratories alone, in tests of new procedures and drugs as well as in research involved in the production of biological products such as vaccines (Animal Liberation Front). The average lifespan of a wild mouse is typically one to two years, while mice kept as pets usually live about three years and have survived up to six years. Mice reach sexual maturity at between five and six weeks of age and have tremendous reproductive potential. They breed throughout the year and may produce as many as eight litters in a single year, with the average litter consisting of four to seven pups. (Animal Liberation Front).

[pic]
Figure 2. Male Albino Mouse
(CharlesRiver, 2013)
Mice should be kept in rooms with the temperature set at about 21.1°C and humidity at 50%. Lights should not be too bright since most white mice are albinos and too much light hurts their eyes. They are diurnal. Their bedding should not be wood shaving since some wood emits toxic fumes to mice. They should have fresh mouse or rat food and water available at all times. Their bedding should be changed 2 or 3 times a week to prevent the buildup of urea (Florida Atlantic University, 2012).
Mice in research facilities are generally fed a pelleted rodent diet ad libitum. Maintenance diets generally contain 4-5% fat and 14% protein. An adult mouse will consume about 15 grams of feed per 100 grams of body weight per day (Schwiebert, 2007). The majority of laboratory rodents are albinos, due to a common mutation in Tyrosinase gene in all albino laboratory rat strains and in at least some of the albino mouse strains. Tyrosinase is the rate-limiting enzyme in the production of melanin pigment. The prevalence of albinism among laboratory rodents is because many of the earliest established strains were albino, and also albinism is an easy selection marker in the early days (Labome, 2012).

Ethanol Extraction Maceration is a widely used procedure which involves leaving the pulverized plant to soak in a suitable solvent in a closed container. Simple maceration is performed at room temperature by mixing the ground drug with the solvent (drug solvent ratio: 1:5 or 1:10) and leaving the mixture for several days with occasional shaking or stirring. The extract is then repeated from the plant particles by straining. The process is repeated for once or twice with fresh solvent. Finally the last residue of extract is pressed out of the plant particles using a mechanical press or a centrifuge (Mahdi and Altikriti, 2010). Ethanol will be the solvent used for the extraction of the plant constituents. Ethanol is a molecule with both a polar and a non-polar end, so its properties are somewhat in between those of water and oil (Lersch, 2008). Ethanolic extracts contain higher amounts of polyphenols as compared to aqueous extracts. It means that they are more efficient in cell walls and seeds degradation which have unpolar character and cause polyphenols to be released from cells. More useful explanation for the decrease in activity of aqueous extract can be ascribed to the enzyme polyphenol oxidase, which degrade polyphenols in water extracts, whereas in methanol and ethanol they are inactive. Ethanol is a good anti-microbial preservative. Additionally, ethanol was found easier to penetrate the cellular membrane to extract the intracellular ingredients from the plant material. Since nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction. Methanol is more polar than ethanol but due to its cytotoxic nature, it is unsuitable for extraction in certain kind of studies as it may lead to incorrect results (Tiwari, et. al., 2011).

Related Studies Yanovski (2002) conducted a study on obesity. It was proven that obesity is a serious and prevalent disorder whose effective management requires ongoing care. Currently approved prescription medications for weight loss, although moderate in their efficacy, can help carefully selected obese patients lose weight and can reduce the rate of regain. Behavioral interventions to improve diet and increase physical activity are considered the primary means to promote and maintain weight loss. Weight-loss medications should be considered as an adjunct only for patients who are at substantial medical risk because of their obesity and in whom nonpharmacologic treatments have not resulted in sufficient weight loss to improve health or to prevent regain. The safety and efficacy of weight-loss medications beyond two years of use have not been established. In addition, although some risk factors for obesity-related disease are improved with the use of weight-loss medications, the long-term effect of such medications on morbidity and mortality has not been determined. In many ways, the current state of treatment for obesity is similar to the state of the treatment of hypertension several decades ago. Few medications were available, their efficacy was limited, and predictors of response were lacking. Hindawi et al., (2012) revealed that obesity is a serious health problem that increased risk for many complications, including diabetes and cardiovascular disease. The results showed EZA, which found rich in flavonoids and phenolic compounds, exhibited an inhibitory activity on pancreatic lipase in vitro with IC50 of 91.07 μg/mL. In vivo administration of this extract to HFD-rats lowered body weight and serum leptin level; and inhibited lipase activity of obese rats by 37% leading to notable decrease of T-Ch, TGs and LDL-c levels accompanied with an increase in HDL-c concentration in serum and liver of EZA treated HFD-rats. Moreover, the findings revealed that EZA helped to protect liver tissue from the appearance of fatty cysts. Interestingly, supplementation of EZA modulated key enzyme related to hypertension such as ACE by 36% in serum of HFD animals and improve some of serum electrolytes such as Na+, K+, Cl−, Ca2+ and Mg2+. Moreover, EZA significantly protected the liver-kidney function by reverted back near to normal the values of the liver-kidney dysfunction indices AST&ALT, ALP, CPK and GGT activities, decreased T-Bili, creat, urea and uric acid rates. In conclusion, these results showed a strong antihypelipidemic effect of EZA which can delay the occurrence of dislipidemia and hypertension. The whole plant of Stellaria media (family: Caryophyllaceae) has been tested for its anti-obesity activity by using progesterone-induced obesity model in female albino mice. The effect of S. media on food consumption pattern, change in body weight, thermogenesis, lipid metabolism, and histology of fat pad. were examined. Methanolic and alcoholic extracts of the S. media were used in the study. Methanolic extract of S. media (MESM) have prevented the increase in body weight, adipose tissue weight and size, and upturned obesity and associated complications. MESM has also shown promising effects compared with alcoholic extract of S. media may be because of its multiple mechanisms. These findings suggest that antiobesity activity produced by MESM is because of its anorexic property mediated by saponin and flavonoid and partly of by its β-sitosterol content. β-Sitosterol in the plant extract was confirmed by thin-layer chromatography study. β-sitosterol is plant sterol having structural similarity with dietary fat which do the physical competition in the gastrointestinal tract and reduces fat absorption. Before carrying in vivo activity detail pharmacognostic and phytochemical analysis of the extracts was carried out. The plant has shown the presence of saponin, flavonoids, steroids and triterpenoids, glycosides, and anthocynidine. By this study, it can be concluded that, MESM is beneficial in suppression of obesity induced by progesterone (Chidrawar et al., 2011). Another group of researchers from Kyung Hee University, Seoul conducted a study on the "Antiobesity Effects of the Ethanol Extract of Laminaria japonica Areshoung in High-Fat-Diet-Induced Obese Rat". LE administration significantly decreased the body weight gain, fat-pad weights, and serum and hepatic lipid levels in HD-induced obese rats. The histological analysis revealed that LE-treated group showed a significantly decreased number of lipid droplets and size of adipocytes compared to the HD group. To elucidate the mechanism of action of LE, the levels of genes and proteins involved in obesity were measured in the liver and skeletal muscle. LE treatment resulted in an increased expression of fatty acid oxidation and thermogenesis-related genes in obese rats. Conversely, the expression of the fat intake-related gene (ACC2) and lipogenesis-related genes was reduced by LE treatment. Additionally, LE treatment increased the phosphorylation of AMP-activated protein kinase and its direct downstream protein, acetyl coenzyme A carboxylase, which is one of the rate-limiting enzymes in fatty acid synthesis pathway. These findings demonstrate that LE treatment has a protective effect against a high-fat-diet-induced obesity in rats through regulation of expression of genes and proteins involved in lipolysis and lipogenesis (Jang and Choung, 2012). Amin, et al. (2012) conducted a study on the anti-obesity effect of Mushroom (Ganoderma Lucidum) on experimentally induced obese rats. Mushroom was studied because it was abundant in essential macro and micro nutrients, highest in dietary fiber and water, less calorie producing, low in carbohydrate content and a good source of protein and fat. After 28 days of treatment, 5% powder of GL administration significantly reduced body weight in obese rats. The study suggested that GL may serve as a new potential natural product for the prevention of obesity. Rabi et al., (2012) designed a study to evaluate the quality control parameters, quantitative phytochemical analysis (total phenols, total flavonoids, and total saponin content), and the antiobesity effect of ethanol extract of Achyranthes aspera Linn. seed (EAA) by employing in vitro and in vivo models. In in vitro study, the inhibitory activity of EAA on pancreatic amylase and lipase was measured. The in vivo pancreatic lipase activity was evaluated by measurement of plasma triacylglycerol levels after oral administration of EAA along with lipid emulsion to Swiss albino mice. The EAA inhibited pancreatic amylase and lipase activity in vitro and elevations of plasma triacylglycerol level in mice. Furthermore, the antiobesity effect of EAA (900 mg/kg) was assessed in mice fed a high-fat diet with or without EAA for 6 weeks. EAA significantly suppressed the increase in body, retroperitoneal adipose tissue, liver weights, and serum parameters, namely; total cholesterol, total triglyceride, and LDL-cholesterol level. The anti obesity effects of EAA in high-fat-diet-treated mice may be partly mediated through delaying the intestinal absorption of dietary fat by inhibiting pancreatic amylase and lipase activity. Histopathological effects of EAA on the liver of mice were also assessed. Papathanasopoulos and Camilleri (2010) studied the effects in obesity and metabolic syndrome and relationship to gastrointestinal functions of dietary fiber supplements. It is stated from their journal that in many studies, dietary fiber induced greater satiety compared with digestible polysaccharides and simple sugars. Lattimer and Haub conducted a study on the effects of dietary fiber and its components on metabolic health. They defined dietary fiber as a carbohydrate that resists digestion and absorption and may or may not undergo microbial fermentation in the large intestine. According to their research, arabinoxylan, insulin, B-glucan, pectin, bran and resistant starches, which are components of dietary fiber, have been shown to significantly play an important role in improving human health. Further research is needed to better understand the mechanisms involved, though. It was also stated that although not as yet adopted by the FDA, dietary fiber is suggested to play a role in other conditions such as obesity and diabetes. The digestive and viscosity characteristics of dietary fiber are the likely modes of action which affect diabetes and obesity risk. These mechanisms appear to decrease nutrient absorption, therefore, decreasing metabolisable energy (Lattimer and Haub, 2010). A study was done by Sacks, et al., (2009) to compare the weight-loss diets with different compositions of fat, protein, and carbohydrates. Eight hundred eleven overweight adults were assigned to one of four diets. The targeted percentages of energy derived from fat, protein, and carbohydrates in the four diets were 20, 15, and 65%; 20, 25, and 55%; 40, 15, and 45%; and 40, 25, and 35%. The diets consisted of similar foods and met guidelines for cardiovascular health. The participants were offered group and individual instructional sessions for 2 years. The primary outcome was the change in body weight after 2 years in two-by-two factorial comparisons of low fat versus high fat and average protein versus high protein and in the comparison of highest and lowest carbohydrate content. At 6 months, participants assigned to each diet had lost an average of 6 kg, which represented 7% of their initial weight; they began to regain weight after 12 months. By 2 years, weight loss remained similar in those who were assigned to a diet with 15% protein and those assigned to a diet with 25% protein (3.0 and 3.6 kg, respectively); in those assigned to a diet with 20% fat and those assigned to a diet with 40% fat (3.3 kg for both groups); and in those assigned to a diet with 65% carbohydrates and those assigned to a diet with 35% carbohydrates (2.9 and 3.4 kg, respectively) (P>0.20 for all comparisons). Among the 80% of participants who completed the trial, the average weight loss was 4 kg; 14 to 15% of the participants had a reduction of at least 10% of their initial body weight. Satiety, hunger, satisfaction with the diet, and attendance at group sessions were similar for all diets; attendance was strongly associated with weight loss (0.2 kg per session attended). The diets improved lipid-related risk factors and fasting insulin levels. Reduced-calorie diets result in clinically meaningful weight loss regardless of which macronutrients they emphasize. A study was done by Renna et al., (2010) to evaluate the use of home-grown pea seeds as protein source in diets for lactating sheep. Two isonitrogenous and isoenergetic diets were fed to 12 mid-lactating Delle Langhe ewes for 73 days. The animals were fed with 1.5[pic]kg alfalfa hay and either 0.7[pic]kg commercial concentrate (control group, C) or 0.6[pic]kg home-grown pea–barley mix (experimental group, PB). The main protein sources in the supplements were sunflower meal and soybean seeds for C group, and pea seeds for PB group. Milk yield was recorded and milk samples were analysed for fat, protein, lactose, casein, solids non-fat, somatic cell count, total bacterial count and fatty acids. Results showed that milk yield and gross composition were not significantly affected by the supplementation types. Differences were instead observed in milk fatty acid profile essentially as a consequence of variations in dietary fatty acids supplies. Milk from the PB group had higher concentrations of short-chain (P[pic]≤[pic]0.05) and saturated fatty acids (P[pic]≤[pic]0.01) and lower concentrations of long-chain (P[pic]≤[pic]0.05), monounsaturated (P[pic]≤[pic]0.01), trans fatty acids (P[pic]≤[pic]0.001) and total conjugated linoleic acids (P[pic]≤[pic]0.001). The use of home-grown Pisum sativum in diets for dairy ewes could enhance farm sustainability without affecting milk production, but possible modifications in milk fatty acid composition have to be taken into consideration. Lanza, et al., (2002) did a study on peas. Thirty weaned Barbaresca lambs were divided into three groups and fed diets that differed in their protein supplement source; the main protein source was soybean meal in SBM diet treatment, totally or partially replaced by peas. Soybean meal and peas were present in the diets in the following proportions (on as fed basis): 16:0%, 0:39%, 9:18%. The diets were of different crude protein content. Carcass fatness was comparable among treatments. Meat pH, colour parameters and cooking losses were comparable among groups. In the present trial, the replacement of soybean meal with peas did not significantly affect growth and slaughter performances and preserved meat quality. Researchers from the University of San Carlos studied the anti-obesity properties of Breadfruit (Artocarpus altilis) leaf extract, which was believed to have laxative properties. They divided their test subjects into 10 groups, Groups 1-5 receiving the normal diet and Groups 6-10 having the High-Fat diet. The parameters for their study were - locomotor activity, body weight and fecal quality. Doses of 200 mg, 350 mg and 500 mg have shown to reduce weight of those mice receiving a normal diet. However, only the 500 mg have shown a positive effect towards mice receiving high fat diet (Satorre and Uy, 2011).

CHAPTER 3
MATERIALS AND METHODS

Procurement of Plant Material Fresh Snow pea pods will be purchased from a local supermarket in Banawa, Cebu City. The pods will thoroughly be washed in running water to remove dirt before being cut into small pieces.

Preparation of Snow Pea Pod Extract Five hundred grams of Snow pea pods will be cut, washed and placed in a beaker. These will be macerated using 500 mL ethanol and the mixture will be allowed to stand for 72 hours. After which, it will be strained using cheesecloth. The solid material or marc will be discarded. The liquid extractive collected will be concentrated on a rotary evaporator and evaporated to dryness in an evaporating dish using direct heat. This residue will be weighed and labelled as the extract.

Preparation of the Test Solutions The Snow pea pod extract obtained will be dissolved in 50 mL distilled water. It will be administered in two different doses: 500 mg / 20g mouse and 1000 mg / 20 g mouse (Appendix E). The different doses of the extract will be given twice daily for 15 days.

Preparation of the Test Animal Twenty four healthy male albino mice will be used. The mice will be placed in individual cages to make sure they eat the right amount of feeds per day. They will be divided into eight groups, with one mouse per group. Each will receive a measured amount of feeds. Three trials will be performed. After procurement of test animals, a study gap of 5 days will be allowed for acclimatization.

Macerate for 72 hours and strain

Figure 3. Preparation of Snow Pea Pod Extract
Preparation of the Positive Control Orlistat will serve as the positive contol for this study. It will be purchased from a licensed pharmacy in Cebu City. The capsule contents will be dissolved in 50 mL distilled water, giving a 0.24% (g/mL) solution. The amount of drug each mouse will receive will be calculated based on the weight of the mouse. The 120mg (0.312 mg/ 20 g mouse) standard drug Orlistat will be given twice daily for 15 days. (Appendix F)

Preparation of Negative Control Distilled water will be used as the negative control. The dose of distilled water given will be based on the calculated volume of the positive control.

Composition of Normal and High Fat Diet Each mouse belonging to groups one to four (normal diet) will be given 15 grams of pellet per 100 grams body weight per day. Groups five to eight will be given a high fat diet which will be composed of 1.5 butter: 0.5 coconut oil: 0.5 raw quail egg at a 1:1 ratio with pellets. Honey will be used as binder and sweetening agent. Each mouse will be receiving 15 grams of high fat diet per 100 grams of body weight per day. Distilled water will be provided for all the mice ad libitum. Ten grams of butter provides more than five grams of saturated fat (Collins, 2009). Pure virgin coconut oil contains 92% saturated fat -- the highest amount of saturated fat of any fat (Zelman, 2011). A quail egg contains 1 g of fat and 76 mg of cholesterol, or about 25 percent of the daily value for cholesterol (Robinson, 2011).

Test Groups Eight male Albino mice will randomly be assigned a group, with one mouse per group. Groups one to four will receive a normal diet, and Groups five to eight will receive high fat diet. Group one will only receive the normal diet while Group five will only receive high fat diet. Groups four and eight, the positive controls, will receive Orlistat in addition to their diets. Groups two, three, six and seven will be the experimental groups which will receive different doses of Snow pea pod extract in addition to their diets. A mouse will be considered obese if there is a 20% increase in the mouse’s weight on Day 30.

Table 1. Diet and Treatment of the Test Groups
|Group Number |Diet |Treatment |
| |Normal Diet |High Fat Diet |Untreated Group |
|Keller Killiani’s Test |Deoxysugars |FeCl3 + H2SO4 |reddish brown solution with |
| | | |purple ring |
|Test for the Presence of Tannins |Tannins |5% FeCl3 + H2SO4 |brownish green or blue black |
| | | |solution |
|Mayer’s Test |Alkaloids |1% HCl + Mayer’s Reagent |orange precipitate |
|Liebermann-Burchard Test |Sterols |Acetic anhydride + H2SO4 |green color turns to red or blue|
| | | |tones |
|Test for the Presence of Flavonoids |Flavonoids |Conc. HCl |Strong red violet color |
|Kedde Test |Unsaturated Lactones |Kedde’s Reagent |blue violet color |
|Salkowski Test |Terpenes |Chloroform + H2SO4 |red brown color at the interface|
|Fehling’s Test |Reducing Sugars |Fehling’s A & B |Red to pink precipitate |

APPENDIX E
CALCULATION OF DOSES OF SNOW PEA POD EXTRACT TO BE ADMINISTERED
Dose: 500mg per 20 g mouse

Dose for experimental mouse (x)
Assuming the experimental mouse weighs 21.0g. 500 mg plant extract Amount of plant extract ___________________ = _________________________

20 g mouse Weight of experimental mouse

500 mg x mg _____________ = ________________

20.0 g 21.0 g

x = 525 mg

Volume to be Administered (y)

Assuming the amount of extract obtained will be 20 g (20,000 mg).

Weight of extract Dose for experimental mouse ___________________ = _________________________

Volume used to dissolve Volume to be administered

20,000 mg 525 mg _____________ = ________________

50.0 ml y mL

y = 1.31 mL

Dose: 1000mg per 20 g mouse

Dose for experimental mouse (x)
Assuming the experimental mouse weighs 21.0g. 1000 mg plant extract Amount of plant extract ___________________ = _________________________

20 mouse Weight of experimental mouse

1000 mg x mg _____________ = ________________

20.0 g 21.0 g

x = 1,050 mg

Volume to be Administered (y)

Assuming the amount of extract obtained will be 20 g (20,000 mg).

Weight of extract Dose for experimental mouse ___________________ = _________________________

Volume used to dissolve Volume to be administered

20,000 mg 1,050 mg _____________ = ________________

50.0 ml y mL

y = 2.625 mL
APPENDIX F

CALCULATION OF DOSE OF POSTIVE CONTROL

Postive Control: Orlistat

Each capsule of Orlistat (Xenical) contains 120 mg (dissolved in 50ml distilled

water).

After dissolving in 50ml water, the strength of the Positive Control Drug will be:

120 mg

Strength (mg/ml) = --------------------- = 2.4 mg/mL

50 ml water

CF will be 0.0026 if a dose intended for a 70.0kg man is administered to a 20.0g mouse

Dose of Orlistat for 20.0g mouse

= (dose of Orlistat for 70.0kg man) X (surface ration of man and mouse)

= (120 mg) X (0.0026)

= 0.312 mg/ 20g mouse

Dose of Orlistat for Experimental Mouse (x)

Assuming the experimental mouse weighs 21.0g

Dose of Orlistat for 20.0g mouse Dose of Orlistat for experimental mouse

---------------------------------------- = -------------------------------------------------

Weight of standard mouse Weight of experimental mouse

0.312mg x mg

---------------- = ------------------

20. 0 g 21.0g

x = 0.3276 mg

Volume to be Administered

Volume to be administered = Dose for experimental mouse / Strength of positive control

= 0.3276mg / 2.4mg/mL

= 0.1365 mL

APPENDIX G

CALCULATION OF DOSE OF NEGATIVE CONTROL

Negative Control: Distilled water

The amount of negative control to be given will be based on the amount of positive control. Assuming the experimental mouse weighs 23.0g. In Appendix E, a 21.0g mouse will receive 0.1365mL of positive control.

Volume to be administered (positive) Volume to be administered (negative)

---------------------------------------- = -------------------------------------------------

Weight of experimental mouse Weight of experimental mouse

0.1365mL x mg

---------------- = ------------------

21. 0 g 23.0g

x = 0.1495mL

APPENDIX H

COMPARISON OF PERCENT WEIGHT LOSS OR GAINED

Test groups

Percent Weight Loss or Gained of Test Group

% = ____________________________________________ x 100

Percent Weight Loss or Gained of Untreated Group

Positive Control

Percent Weight Loss or Gained of Positive Control

% = ____________________________________________ x 100

Percent Weight Loss or Gained of Untreated Group

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Zelman, 2011. The Truth About Coconut Oil. Retrieved March 25, 2013 from the World Wide Web: http://www.webmd.com/diet/features/coconut-oil-and-health?page=3 BIOGRAPHICAL DATA

NAME: Joy Edith Go Chua
AGE: 19 years old
DATE OF BIRTH: April 16, 1993
PLACE OF BIRTH: Manila, Philippines
HOME ADDRESS: #25 L. Bacayo Street, Guadalupe, Cebu City
TELEPHONE NO: (032) 2543936
MOBILE NO: 09237297230
RELIGION: Protestant
FATHER’S NAME: Edison Lim Chua
MOTHER’S NAME: Jennifer Ann Go Chua

EDUCATIONAL BACKGROUND
LEVEL NAME OF SCHOOL YEAR
Elementary Grace Christian College 2000-2006
Secondary Bethany Christian School 2006-2010
College University of San Carlos 2010-present
Degree Pursued Bachelor of Science in Pharmacy

MEMBERSHIP IN ORGANIZATION
Junior Philippine Pharmacists Association –Pi Chapter BIOGRAPHICAL DATA

NAME: Kimberly Mae B. Siao
AGE: 19 years old
DATE OF BIRTH: May 23, 1993
PLACE OF BIRTH: Indiana, USA
HOME ADDRESS: #21 Sunbeam Drive, Sunny Hills Subdivision, Talamban, Cebu City
TELEPHONE NO: (032) 5050100
MOBILE NO: 09228832459
RELIGION: Roman Catholic
FATHER’S NAME: Mario U. Siao
MOTHER’S NAME: Emma B. Siao

EDUCATIONAL BACKGROUND
LEVEL NAME OF SCHOOL YEAR
Elementary Bethany Christian School 2000-2006
Secondary Bethany Christian School 2006-2010
College University of San Carlos 2010-present
Degree Pursued Bachelor of Science in Pharmacy

MEMBERSHIP IN ORGANIZATION
Junior Philippine Pharmacists Association –Pi Chapter
Student Power Party
-----------------------
A

BA

Snow Pea Pods

500g of washed, cut and finely divided pods placed in a beaker with 95%ethanol

Extractive

Marc

Discard

Heat to dryness on an evaporating dish

Concentrate in a Rotary Evaporator

Extract

Weigh

Statistical analysis

Determination of anti-obesity property

Administration of varying doses of extract (500mg and 1000mg) and standard drug (0.312mg)

Determination of constituents present using test tube reaction method

Preparation of snow pea pod extract

Preparation of different diets

Weighing and recording of each mouse’s weight

Divide mice into 8 groups,
1 mouse per group

Preparation of 8 male albino mice

2.Locomotor activity test

1.Determining each mouse’s body weight

3.Fecal examination

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