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Determination of Anti--Amylase and Anti--Glucosidase activities of Momordica charantia Galaxy-type and Momordica charantia Bonito-type

A Thesis Submitted to the Department of Biochemistry and Nutrition Institute of Medicine FEU-NRMF

By: SECTION I C2

In Partial Fulfillment of the Requirements For First Year Biochemistry

2

March 2008

Determination of Anti--Amylase and Anti--Glucosidase activities of Momordica charantia Galaxy-type and Momordica charantia Bonito-type

ABSTRACT Momordica charantia, commonly known in the Philippines as Ampalaya, is a vegetable of medicinal value. It is known to treat Diabetes Mellitus by increasing glucose uptake by its Insulin-like properties. However, more than its Insulin-like compounds, Momordica charantia also has phenolic substances which according to studies can inhibit enzymatic activities of alpha-amylase and alpha-glucosidase. These digestive enzymes are involved in the digestion of Carbohydrates thus inhibition of these enzymes could also serve as control point against hyperglycemia. In this study, the inhibitory activity of local types of Momordica charantia (Galaxy-type, Bonito-type) against these enzymes was determined and quantified through Starch-iodine method of enzyme assay. Results have detected significant inhibitory activities. Momordica charantia Galaxy-type obtained a mean percentage inhibitory against alpha-amylase and alpha-glucosidase reaching 72.89% and 72.02% mean inhibition, respectively. The inhibition was higher compared with Momordica charantia Bonito-type which obtained percentage inhibition of 65.77% and 48.43% for alpha-amylase and alpha-glucosidase, respectively.

3 Acknowldgements

The authors would like to acknowledge the enormous guidance and assistance that they had received from the following individuals, for without them, this research undertaking would not have attained its completion and success: To Anna Belen Ignacio Alensuela, MD, their research adviser, for her assistance and coordination; To Professor Noel G. Sabino, University of the Philippines- Los Baos, for his unwavering support throughout the research undertaking, for his intellectual contributions to the project, for sharing his expertise on research methodologies, and for keeping the research project on the right tract; To Dr. Melo Reyes, Institute of Plant Breeding, University of the Philippines- Los Baos for sharing his expertise in M. charantia; To Mark Mendros, RMT and Miko Abella, RMT, staff at the Biochemistry Laboratory FEU-NRMF, for their laboratory assistance; To Dr. Teresita R. Perez, Chairperson, Environmental Science Department, Ateneo de Manila University, Ms. Rowena Argones, Secretary,Environmental Science Department, Ateneo de Manila University; To Mr. Royce Ivan Ilao, Instructor University of the Philippines-Los Banos for helping the authors in searching for the right people who will guide the authors towards uncovering more of ampalaya; Their families, for their love and support Most importantly, to GOD ALMIGHTY.

4 I. INTRODUCTION Momordica charantia (common name: ampalaya, bitter gourd or bitter melon) is a tropical and subtropical vine of the family Cucurbitacea that grows in tropical parts of Asia, Africa, the Caribbean and South America. The plant has a long history of use as a hypoglycemic agent. It has been used in galaxy-type Asian traditional medicine systems for a long time eg. for sluggish digestion, dyspepsia, and constipation. More importantly, it has popular claims for treatment against Diabetes mellitus. Although several constituents of bitter gourd have been found to have hypoglycemic properties, most interest has been centered on a polypeptide fraction, which are claimed to have insulin-like properties. These have been variously described as polypeptide-p and polypeptide-K (http://www.arjunanatural.com). In numerous studies, at least three different groups of constituents found in all parts of bitter melon have clinically demonstrated hypoglycemic (blood sugar lowering) properties or other actions of potential benefit against diabetes mellitus. These chemicals that lower blood sugar include a mixture of steroidal saponins known as charantins, insulin-like peptides, and alkaloids. The hypoglycemic effect is more pronounced in the fruit of bitter melon where these chemicals are found in greater abundance (http://www.rain-tree.com/bitmelon.htm). In a study by Biyani, et. al. (2003), the aqueous extract powder of fresh unripe whole fruits at a dose of 20 mg/kg body weight was found to reduce fasting blood glucose by 48%, an effect comparable to that of glibenclamide, a known synthetic drug. As presented, most studies have focused on the insulin-like compounds found on ampalaya. According to literature, there are other control points for the control of hyperglycemia. For example, there are drugs which reduce glucose absorption in the small

5 intestines, known as alpha-glucosidase inhibitors. Drugs such as miglitol and acarbose inhibit alpha-glucosidase enzyme, so that maltose products from polysaccharide chain cannot be further broken down to glucose by glucosidase, hence, few glucose products are formed, and there are few glucose to absorb in t he small intestine. This could now prevent post prandial rise in glucose levels, in diabetic patients. In so doing, there were studies conducted whether ampalaya also have inhibitory properties against alpha-glucosidase as well as alpha-amylase, which then cause reduced glucose absorption. In vivo studies by Fonseka, et. al. (2006) showed type dependent significant typeiation in hypoglycemic activity. Some types can reduce blood glucose levels up to 31%. In vitro studies of some types of Momordica charantia showed the highest glucose reduction percentage of (38%) through the inhibition of amylase enzyme, recorded in hybrid H1 followed by Matale Green with glucose reduction percentage of 35.2%. As concluded in this study, there is a type dependent typeiation in hypoglycemic activity of Momordica charantia. In this regard, the researchers would like to determine the presence of such inhibitory activity in local types of Momordica charantia Galaxy-type and Momordica charantia Bonito-type against alpha-amylase and alpha-glucosidase enzymes, and quantify its inhibitory activity.

II. OBJECTIVES The researchers aimed to determine the presence of inhibitory activities of the fruits of local types of Momordica charantia Galaxy-type and Momordica charantia Bonito-

6 type against alpha-amylase and alpha-glucosidase enzymes, and quantify its inhibitory activity. It specifically aimed to: 1. Detect the presence of inhibitory activity of the fruits of Momordica

charantia Galaxy-type and Momordica charantia Bonito-type against Alpha-amylase using Iodine-starch method; 2. Detect the presence of inhibitory activity of the fruits of Momordica

charantia Galaxy-type and Momordica charantia Bonito-type against Alpha-glucosidase using Iodine-starch method; 3. Compare the inhibitory activities of Momordica charantia. Galaxy-type and

Momordica charantia Bonito-type against Alpha-amylase and Alpha-glucosidase;

III. SIGNIFICANCE The results of this study could amplify the use and confirm the hypoglycemic claims of this herb for the therapy of Diabetes Mellitus. Moreover, this could serve as a dietary substitute for drugs which have their own agents specific disadvantages and contraindications, such as renal and liver diseases. It is apparent that due to the side effect of the currently used drugs, there is need for a safe agent with minimal adverse effects, which can be taken for long durations (Biyani et. al, 2003).

IV. SCOPE AND LIMITATIONS As stated earlier, the research intends to investigate the mechanism of hypoglycemic effects of Momordica charantia, specifically its effects on -amylase and glucosidase activities. Local Philippine ampalaya types are used in the study. Both types

7 are commonly sold at produce markets all over the country and are incorporated in various Filipino dishes. These types were chosen over all other types because of their immense availability. The enzymes used are microbial enzymes in order to ensure purity, since these enzymes are readily available in chemical industries. Human or animal enzymes require purification techniques which the authors of the study, as amateurs, might not be able to perform effectively. The experimentations for this research were conducted in vitro. Hence, the results of the experiment does not claim of inhibitory activities of enzymes present in humans; only a possible inhibition which is based on molecular studies and in vitro reaction.

V. REVIEW OF RELATED LITERATURE Momordica charantia is a tropical and subtropical vine of the family Cucurbitaceae, widely grown for edible fruit, which is among the most bitter of all vegetables. English names for the plant and its fruit include bitter melon or bitter gourd. In the Philippines, it is commonly known as ampalaya. The origin of the species is not known, other than that it is a native of the tropics. It is widely grown in South and Southeast Asia, China, Africa, and the Caribbean. The fruit has a distinct warty looking exterior and an oblong shape. It is hollow in cross-section, with a relatively thin layer of flesh surrounding a central seed cavity filled with large flat seeds and pith. It grows in areas where annual precipitation ranges from 480 mm to 4100 mm.

8 Bitter melon comes in a type of shapes and sizes. The typical Chinese phenotype is 20 to 30 cm long, oblong with bluntly tapering ends and pale green in color, with a gently undulating, warty surface. The bitter melon more typical of India has a narrower shape with pointed ends, and a surface covered with jagged, triangular "teeth" and ridges. Coloration is green or white. There are various intermedia