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

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 intermediate forms. Some bear miniature fruit of only 6 10 cm in length, which may be served individually as stuffed vegetables. These miniature fruit are popular in Southeast Asia as well as India. The following table shows chemical compounds found in each ampalaya, as well as biological their biological activities based on phytochemical and ethno-botanical databases. Table 1. Chemical Compounds Found in Momordica charantia based on Phytochemical and Ethnobotanical Databases

Chemicals
5--stigmasta-7,22,25-trien-3-ol 5--stigmata-7,25-dien-3-ol 5-hydroxytryptamine

Plant Part
Whole Plant Whole Plant Fruit

Reported Biological Activities

No activity reported No activity reported Allergenic, Antidepressant, Antidote (Mg), Antimutilation, Anti parkinsonian, Cerebrocephilic, Hypertensive, Insectiside, pesticide, spasmogenic, ulcerogenic, vasoconstrictive

Alkaloids -elaostearic acid ascorbigen -sitosterol-D-glucoside

Fruit Seed Fruit Fruit

No activity reported No activity reported No activity reported Anti-spasmodic, antitumor, CNS depressant, convulsant, hypoglycemic

charantin citrulline

Fruit Fruit

Abortifacient, anti-diabetic, hypoglycemic Antiasthemic, diuretic

9
cryptoxantin diospeum Fruit Tissue culture Anti-mutagenic, Vitamin A activity Anti-fatigue, anti-inflammatory, antistress, estrogenic, hepatoprotective, hypocholesterolemic

elasterol flavochrome fluoride

Whole Plant Fruit Fruit

No activity reported No activity reported Anti-carcinogenic, anti-osteoporonic, antiosclerotic, anti-convulsant, antihypertensive, anti-insomniac, antilethargic, anti-stress, diuretic, neuroinhibitor

glacturonic acid lanosterol lutein

Fruit Fruit Fruit

No activity reported Cancer preventive Anti-atherosclerotic, anti-cancer, antioxidant, colorant,, Quinone reductase, inhibitor Anti-cancer, anti-tumor, hypocholesterolemic, pro-oxidant No activity reported No activity reported No activity reported No activity reported No activity reported CNS paralytic, hemostatic, irritant, pesticide No activity reported Herbistat, pesticide Hypoglycemic Colorant No activity reported No activity reported No activity reported Hypoglycemic Anti-cancer, anti-tumor, colorant, hepatoprotective, Quinone Reductase inhibitor

lycopene

Fruit

momordicine momordicoside A, B, C, D, E, K, L momordicoside F1, F2, G, I mutachrome oxalate oxalic acid phylofluene pipecolic acid polypeptide P rubixanthin stigma-5, 25-dien-3--ol sugars urease vicine zeaxanthin

Fruit Seed Fruit Fruit Fruit Fruit Seed Fruit Fruit Fruit Whole plant Fruit Seed Seed Fruit

zeinoxanthin

Fruit

No activity reported

10 As shown by the table above, Momordica charantia has many reported therapeutic activities. Each component corresponded to certain properties, like anti-cancer, antihypertension, diuretic, anti-fatigue, anti-inflammatory, anti-stress, estrogenic,

hepatoprotective, anti-spasmodic, CNS depressant, anti-osteoporonic, antiosclerotic, anticonvulsant, anti-insomniac, anti-lethargic, and many others. However, much of the interest and studies about ampalaya are focused on its hypoglycemic properties. Ampalaya is known to elicit hypoglycemic properties owing to the discovered hypoglycemic compounds shown in the table above. These hypoglycemic compounds include charantin, which is a steroidal glycoside from the alcoholic extract of the fruit; vicine, a pyrimidine nucleoside from the seeds; p-insulin, which is a polypeptide found in fruits, seeds, and tissues; and a bitter steroidal glycoside known as momordicine. Charantin is the earliest identified active constituent of ampalaya fruit which is a steroidal mixture of glucosides of -sitosterol--D-glucoside and 5,25-sigmastadien-3-Dol. This compound was reported to have insulin-like properties. Moreover, polypeptide p, also was also observed to have insulinomimetic characteristics. Lastly, vicine, isolated from the seeds of ampalaya was discovered to induce hypoglycemia in rats at an intraperitoneal dose (Macazo, 2007). As mentioned by Murray (2000), Momordica charantia enhance glucose utilization, following the ingestion of sugar, which, in line may somehow explain the possible mechanism of this plants hypoglycemic activity. Aside from the compounds fore mentioned, many flavonoids are also present in Momordica sp. Flavonoids are widely distributed among plants such as vegetables and

11 fruits. Some flavonoids present are anthocyanins, xanthophylls, and carotene. Flavonoids are reported to have anti-amylase and anti-glucosidase activities. -Amylases are extracellular enzymes which hydrolyze -1,4-glycosidic bonds. These enzymes are endoenzymes, splitting the substrate in the interior of the molecule. Their action is not inhibited by -1,6-glycosidic bonds although such bonds are not split. -Amylases are formed by many bacteria and fungi. They are classified according to their starch-liquefying and/or saccharogenic effect. Saccharogenic amylases produce free sugars, while starch-liquefying amylases break down the starch polymer but do not produce free sugars. Many organisms produce -amylases. Its optimum pH is 6.2 and its optimum temperature is 85C.

Fig. 1. Amylase activity on starch Amylases can also be found in the human saliva produced by the parotid gland and by the pancreas and then distributed to the intestines for digestion. Amyloglucosidase, an Exo-1,4 -D-glucosidase, further degrades following the solubilization of starch by -amylase. This enzyme hydrolyzes -1,4 in addition to 1,6 and 1,3 glycosidic linkages from the non-reducing ends of amylase and amylopectin to produce -D-glucose. The type of linkage and the chain length of the substrate are the basis

12 of hydrolysis. Like -amylase, amyloglucosidase can also be formed by bacteria and fungi. The optimum pH of this microbial enzyme is 3.6 to 4.2; optimum temperature range is 60C. Starch and maltose are the known substrates for this enzyme. Alpha-amylase inhibitors Microorganisms, higher plants, and animals produce a large number of different protein inhibitors of -amylases in order to regulate the activity of these enzymes. Some of these inhibitors act by directly blocking the active centre of the enzyme at various local sites. Plants also use amylase inhibitors as a defence strategy. These inhibitors impede the digestive action of -amylases and proteinases in the insect gut, thereby acting as insect anti-feedants. Tannins like flavonoids, for example are plant polyphenols which are used as a defense mechanism of plants making them unpalatable to predators. This role is attributed to their ability to precipitate plant protein and to inhibit gastrointestinal enzymes such as trypsin and amylase. Most of its biological activities are believed to be related to their protein binding. (Bennick, 2002). As a result, -amylase inhibitors have potential in various fields, including crop protection and the treatment of diabetes. Alpha-amylase and its inhibitors is drug-design targets for the development of compounds for treatment of diabetes Obesity and Hyperlipaemia (Octivio and Rigden, 2000). The reaction mechanisms involved in the inhibition of alpha-amylase by plant protein inhibitors are not clearly understood. But there are suggestion that reducing sugars which are covalently bound to the inhibitor polypeptide chain may play a major role in the

13 mechanism or that the inhibitor may induce conformational changes in the enzyme molecule. Studies have also been done that shows inhibitory action of polyphenols against alpha-amylase. These studies show that plant phenolic substances such as flavonoids, tannins react with proteins or enzymes influencing their in vitro enzymatic activity (Rohn et. al., 2002).

Alpha-Glucosidase Inhibitors Alpha-Glucosidase inhibitors are the most common oral anti-diabetic drugs used for ameliorating post-prandial hypoglycemia that work by preventing the digestion of carbohydrates. Currently, there are three glucosidase inhibitor synthetic drugs namely acarbose, miglitol, and volibone. These alpha-glucosidase inhibitors are saccharides that are said to act as competitive inhibitors of enzymes needed to digest carbohydrates. The membranebound intestinal alpha-glucosidases hydrolyze oligosaccharides, trisaccharides, and disaccharides to glucose and other monosaccharides in the small intestine. Acarbose also blocks pancreatic alpha-amylase in addition to inhibiting membranebound alpha-glucosidases. Pancreatic alpha-amylase hydrolyzes complex starches to oligosaccharides in the lumen of the small intestine. Inhibition of these enzyme systems reduces the rate of digestion of carbohydrates. Less glucose is absorbed because the carbohydrates are not broken down into glucose molecules. In diabetic patients, the short-term effect of these drugs therapies is to decrease

14 current blood glucose levels: the long term effect is a small reduction in hemoglobin level. Currently, studies have been made on the inhibitory properties of different plants against alpha-glucosidase specifically to plant secondary metabolites polyphenols, and flavonoid- rich plants and plant products such as strawberries, mulberries, green tea, red grape, red wine, etc. Their inhibitory activities are attributed to these chemical compounds (McDougall et. al, 2005).
A1c

Fig 2. Molecular Backbone of Flavone The primary structure of flavonoids consists of two moities: benzopyran (A and C rings) and phenyl (B ring) groups. The typeiation in these rings are said to have a relationship in its inhibitory activity (Tadera et. al., 2005).

Description of M. charantia types used Galaxy-type is the common long type of ampalaya available in the market. It measures at average of 30.5 cm. The bonito-type is the common short type of ampalaya available in the market. It measures 7.6 cm (average).

15

Galaxy-type M. charantia Bonito-type

VI. MATERIALS AND METHODS The inhibitory activity of two types of Momordica charantia against the enzymes alpha-amylase and alpha-glucosidase was determined in the study using the starch-iodine method. Alpha-Amylase A. Procurement of Enzyme Ten ml of pure enzyme alpha-amylase was purchased from Sigma-Aldrich. B. Procurement of samples Twenty-five samples of two types of Momordica charantia were purchased from the market. The samples were rinsed and prepared for extraction. C. Extraction Fifteen grams of the fruit of the sample was weighed. Add 30 ml of distilled water. Osterized and filtered using three layers of cheesecloth. The extracts were placed in their respective containers and were labeled accordingly. Momordica charantia type galaxy-type was labeled as V1 and Momordica charantia type. Bonito was labeled as V2.

16 D. Preparation of Enzyme One percent (1%) of enzyme was prepared by diluting 1.0 ml of pure enzyme alpha-amylase in 99 ml of distilled water. E. Incubation a. Test tube B One ml of each sample was placed in test tube (labeled as test tube B). One ml of 1% alpha-amylase was then added. The tubes were incubated at 40C for 24 hours. b. Test tube C One ml of each sample was placed in test tube (labeled as test tube C). The tubes were incubated at 40C for 24 hours. F. Preparation of Substrate One percent (1%) of substrate was prepared by weighing 1.0 g of soluble starch and dissolved it in 100 ml of phosphate buffer. G. Starch-Iodine Method a. Test tube B In test tube B, 1.0 ml of 1% starch solution was added. The tubes are then allowed to incubate in 370C for 10 minutes. After incubation, 1.0 ml of I2KI was added. The absorbance of the resulting solution was read in a spectrophotometer at 600 nm. b. Test tube C 1.0 ml of 1% starch solution and 1.0 ml of I2KI was placed in tube C. The absorbance of the resulting solution was read in a spectrophotometer at 600 nm. H. Preparation of the Control

17 For the control, two tubes were prepared. The first tube, labeled as tube A2, consisted of 1.0ml of 1% starch and 1.0 ml of 1% alpha-amylase. The tube was then incubated for 10 minutes at 370C. The second tube consisted of 1% starch solution only (A1). 1.0ml of I2KI was added to both tubes. The absorbance of the resulting solution was read in a spectrophotometer at 600 nm. Alpha-Glucosidase For -glucosidase, amyloglucosidase (exo-1,4--glucosidase) enzyme was used as the representative enzyme. The enzyme was purchased at Sigma-Aldrich. The same procedure was used for alpha-glucosidase (as in alpha-amylase) only that the enzyme was diluted to 0.1% and the incubation time with the substrate was 1 hour at 650C. III. Preparation of Standards A. For Control Five test tubes were prepared and were labeled respectively. Test tube 1 contained 3.0ml of Phosphate buffer. In test tube 2, 2.75 ml of phosphate buffer was mixed with 0.25 ml of 1% starch solution. In tube 3, 2.50 ml of phosphate buffer was added with 0.50 ml of 1% starch solution. Tube 4 contained 2.25 ml of phosphate buffer and 0.75 ml of 1% starch solution. Tube 5 was a mixture of 2.0 ml phosphate buffer and 1.0 ml of 1% starch solution. One ml of I2KI was then added to all the five tubes. The absorbance of the resulting mixture was read in a spectrophotometer at 600 nm.

Table 2. Standards for the Control

18

TUBE 1 2 3 4 5

1% Phosphate Starch Buffer Solution (mL) (mL) 3.00 0.00 2.75 0.25 2.50 0.50 2.25 0.75 2.00 1.00

I2KI (mL) 1.00 1.00 1.00 1.00 1.00

B. For Inhibitor Standards for the inhibitor for each type were prepared. Five test tubes were labeled accordingly. Test tube 1b contained 2.0 ml of phosphate buffer, 1.0 ml ampalaya extract. In test tube 2b, 1.75ml of Phosphate buffer and 1.0 ml of ampalaya extract was added with 0.25 ml of 1% starch solution. Test tube 3b was a mixture of 1.50 ml of phosphate buffer, 1 ml ampalaya extract, and 0.50ml 1% starch solution. Test tube 4b contained 1.25 ml of phosphate buffer, 1.0 ml of ampalaya extract and 0.50 ml of 1% starch solution. Test tube 5b had 1.0 ml of phosphate buffer, 1.0 ml ampalaya extract, and 1.0 ml of 1% starch solution. One ml of I2KI was then added to all the five tubes. The absorbance of the resulting mixture was read in a spectrophotometer at 600 nm.

Table 3. Standards for tubes with inhibitor Phosphate Buffer (ml) 2.00 1.75 1% Starch (ml) 0.00 0.25 Ampalaya I2KI Extract (ml) (ml) 1.00 1.00 1.00 1.00

TUBE 1b 2b

19 3b 4b 5b 1.50 1.25 1.00 0.50 0.75 1.00 1.00 1.00 1.00 1.00 1.00 1.00

The resulting concentration of starch was computed using the formula: [starch] = [stock] x ml added 4ml The computed starch concentration versus its absorbance was plotted. H. Interpretation of Data The two control tubes (A1:starch only, A2:starch +enzyme) were for the measurement of normal reduction of starch concentration without the presence of ampalaya extract. Tube A1 contained the total initial amount of starch, whereas tube A2 contained only the remaining amount of starch that were not broken down by enzymatic action. In this regard enzymatic activity was measured by determining the concentration of starch in Tube A1 minus the concentration of starch in Tube A2, divided by concentration of starch in Tube A1. This now corresponded to percent reduction of starch concentration in normal condition. % Reduction of [Starch] = (without ampalaya extract) (% starch broken down by enzymatic action) [Starch] A1 [Starch] A2 x 100 [Starch] A1

In the presence of inhibitor, percent of starch concentration was also used as a measure of enzyme activity. In so doing each sample had two test tubes each one tube contain starch + ampalaya extract ( tube C = starch + ampalaya extract); the other tube contain starch + enzyme + inhibitor ( tube B = starch + inhibitor). Tube C contained the total initial amount of starch, whereas tube B contained only the remaining amount of

20 starch that was not broken down by enzyme action. Percent reduction of starch was obtained by the following equation: % Reduction of [Starch] = (in the presence of ampalaya extract) (% starch broken down by enzymatic action) where: [Starch] C - [Starch] B x 100 [Starch] C

C = Starch + ampalaya extract B = Starch + ampalaya extract + alpha-amylase The obtained percent reduction which corresponded to the enzymatic activity or to

the percent starch broken down by enzymatic reactions were compared in the presence and absence of ampalaya extract. I. Statistical analysis The data was analyzed using 2 sample t-test for unequal typeiances.

VII. RESULTS AND DISCUSSION Starch-iodine method of enzyme assay was used in the experiment. I2KI was the primary reagent. Free iodine complexes with the amylase component of the starch polymer and the starch-iodine complex structure formed exhibits a strong absorption of light. The greater is the reduction in the blue-black color in the tubes, the lesser is the starch present, hence, the greater was the enzyme activity. The experiment determined the inhibitory activity of two types of Momordica charantia (type. Galaxy-type, type. Bonito) against alpha-amylase and alpha-glucosidase using Starch-Iodine method. The standards prepared was for the quantitative measurement of the concentration of starch. Tubes with known concentration of starch was prepared and its corresponding absorbance was read. The results were plotted in a graph. Through this graph, absorbance

21 readings from samples can be quantitatively converted to its corresponding starch concentrations. The ratio of starch concentration (1%) to alpha-glucosidase concentration used (0.1%) was based on the theoretical optimum breakdown of starch by this enzyme: 0.1 ml of pure enzyme breaks down 1 gram wheat starch to glucose as cited by Sigma Aldrich product information. The ratio 1% starch concentration to 1% alpha-amylase concentration was determined as the optimum ratio, based on the experimentations done by the researchers, since there was none cited in the product information provided by SigmaAldrich. Inhibition of -Amylase In this study, 25 samples of two types of Momordica charantia (Galaxy-type and type. Bonito) were collected. Anti--amylase activities of these samples are shown in Table 4. All the aqueous extracts of both types showed a percentage inhibition of enzyme activity. The first type obtained a mean starch reduction of 15.83%. This caused a 72.89% mean inhibition of normal enzymatic activity of alpha-amylase. The second type on the other hand obtained a mean starch reduction of 22.95% and caused a smaller 65.77% mean inhibition of normal alpha-amylase activity. The percentage inhibition of enzyme activity was determined by comparing it with the mean percent starch reduction of normal alphaamylase activity of 88.72%. The comparison of mean percent starch reduction and mean percent inhibition of enzyme activity of two types are shown in figure 3. An unpaired t-test was performed to determine if the inhibition was effective. Results show that the mean % reduction in starch for Type 1 (M=15.83%, SD =9.57%, N= 21) was significantly smaller than 88.72% (control value), with t(44)=33.72, two-tail p =

22 <0.0001, at = 0.05, providing evidence that type 1 is effective in inhibiting the activity of the amylase. A 95% C.I. about mean starch reduction is (11.48%, 20.19%). The mean % reduction in starch for Type 2 (M=22.95%, SD =10.67%, N= 24) was significantly smaller than 88.72% (control value), with t(47)=29.27, two-tail p = <0.0001, at = 0.05, providing evidence that type 2 is effective in inhibiting the activity of the amylase. A 95% C.I. about mean starch reduction is (18.45%, 27.46%,) The mean percentage for Type 1 (M=72.89%, SD= 9.57%, N= 21) was significantly greater than the percentages for Type 2 (M=65.77%, SD=10.67%, N= 24) using the two-sample t-test for unequal typeiances, t(43) = 2.36, two-tail p = 0.02, at = 0.05. Table 4. Anti alpha-Amylase activity of two types of Momordica charantia at 1% [starch] and 1% [-amylase] % Inhibition of % Starch Enzyme Type Reduction Activity Type 1 15.83 72.89 Type 2 22.95 65.77 Control 88.72 n.a.

23

% Starch Reduction

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Control: w /o inhibitor

Treatment A: Treatment B: w ith M. charantia w ith M. charantia Var. 1 extract Var. 2 extract

Treatment
Fig. 3. Mean percent reduction of starch by -am ylase. Bars represent standard deviation. Control (S + E w ithout inhibitor), Treatment A (S + E + M. charantia Galaxy-type), Treatment B (S + E + M. charantia Bonito-type)

% -amylase activity inhibited

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Treatment A: w ith M. charantia Var. 1 extract

Treatment B: w ith M. charantia Var. 2 extract

Treatment
Fig. 4. Mean percent -am ylase activity inhibited. Bars represent standard deviation. Treatment A (S + E + M. charantia Galaxy-Type), Treatment B (S + E + M. charantia Bonito-Type)

24 Inhibition of -Glucosidase In this study, 25 samples of two types of Momordica charantia (Galaxy-type and Bonito-type) were collected. Anti--glucosidase activities of these samples are shown in Table 5. All the aqueous extracts of both types showed a percentage inhibition of enzyme activity. The first type obtained a mean starch reduction of 16.70%. This caused a 72.02% mean inhibition of normal enzymatic activity of alpha-glucosidase. The second type on the other hand obtained a mean starch reduction of 40.29% and caused a smaller 48.43% mean inhibition of normal alpha-amylase activity. The percentage inhibition of enzyme activity was determined by comparing it with the mean percent starch reduction of normal alphaglucosidase activity of 82.13%. The comparison of mean percent starch reduction and mean percent inhibition of enzyme activity of two types are shown in figures 5. An unpaired t-test was performed to determine if the inhibition was effective. Results show that the mean % reduction in starch for Type 1 (M=16.71%, SD =29.89%, N= 21) was significantly smaller than 82.13% (control value), with t(44)=9.96, two-tail p = <0.0001, at = 0.05, providing evidence that type 1 is effective in inhibiting the activity of the glucosidase. A 95% C.I. about mean starch reduction is (3.10%, 30.31%) The mean % reduction in starch for Type 2 (M=40.29%, SD =19.93%, N= 24) was significantly smaller than 82.13% (control value), with t(47)=10.11, two-tail p = <0.0001, at = 0.05, providing evidence that type 2 is effective in inhibiting the activity of the glucosidase. A 95% C.I. about mean starch reduction is (31.87%, 48.71%). The mean percentage for type 1 (M=72.02%, SD= 29.89%, N= 21) was significantly greater than the percentages for type 2 (M=48.43, SD=19.93, N= 24) using the two-sample t-test for unequal typeiances, t(43) = 3.07, p = 0.004, at = 0.05.

25

Table 5. Anti alpha-Glucosidase activity of two types of Momordica charantia at 1% [Starch] and 0.1% [-glucosidase] % Inhibition of % Starch Enzyme Type Reduction Activity Type 1 16.7 72.02 Type 2 40.29 48.43 Control 82.13 n.a.

% Starch Reduction

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Control: w /o inhibitor

Treatment A: Treatment B: w ith M. charantia w ith M. charantia Var. 1 extract Var. 2 extract

Treatment
Fig. 5. Mean percent reduction of starch by -glucosidase. Bars represent standard deviation. Control (S + E w ithout inhibitor), Treatment A (S + E + M. charantia Galaxy-type), Treatment B (S + E + M. charantia Bonito-type)

26

% -glucosidase activity inhibited

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

Treatment A: w ith M. charantia Var. 1 extract

Treatment B: w ith M. charantia Var. 2 extract

Treatment
Fig. 6. Mean percent -glucosidase activity inhibited. Bars represent standard deviation. Treatment A (S + E + M. charantia Galaxy-type), Treatment B (S + E + M. charantia Bonito-type)

Polyphenols specifically flavonoids are secondary plant metabolites that are abundant in Momordica charantia. The primary structure of flavonoids consists of three rings: 2 benzopyran (A nd C ring) and a phenyl group (B ring). Flavonoids are classified into six groups based on the typeiation in the C ring and the linkage between the benzopyran and phenyl groups: flavone, flavonol, flavanone, isoflavone, flavan-3-ol, and anthocyanidine.

A - Ring C - Ring

B - Ring

Fig 7. Primary structure of Flavonoids

27 A study done by Tadera, et. al (2006) correlated the hydroxyl substitution on the A, B, C rings on the inhibitory effects of Flavonoids against the enzymes alpha-amylase and alpha-glucosidase. It was showed that 4-OH groups on B ring was important to the inhibitory activity. The inhibitory activity increased considerably with the increase in the number of hydroxyl groups on the B ring. As to the A and C rings, hydroxylations at the 3 and 5 positions of flavone enhanced the inhibitory activity. Hanamura et. Al (2005) also conducted a research on the hypoglycemic effect of polyphenols. Their results support the possible influence of the number of hydroxyl groups on B ring to the inhibitory activities of the polyphenols although the exact mechanism is still unclear. According to Rohn et. al. (2000), phenolic substances generally may be readily oxidized in alkaline solutions or in the presence of polyphenoloxidase to the respective quinones, which in turn represent a reactive species capable of undergoing interactions in the free amino groups of proteins. The stepwise addition of protein-bound amino groups to an oxidized o-diphenol has already been cited. The rate of oxidation depends on pH. Likewise, further reaction with nucleophilic groups is also dependent on pH value. As a consequence, these reactions of phenolic compounds with the reactive side chains of the enzymes result in changes of physicochemical properties such as solubility, electrophoretical behavior, hydrophobicity, molecular weight, secondary and tertiary structure as well as thermodynamic parameters as already shown for selected food proteins in model studies. In this study, the determination kinetics of enzyme activity was not conducted. However, previous studies have established the effects of polyphenolic compounds on the

28 kinetics of alpha-amylase and alpha-glucosidase activity. Iio et. al. (1983), conducted a research on the effects of flavonoids on alpha-glucosidase and beta-fructosidase from yeast. Their results showed that most of the flavonoids act as mixed type of enzyme inhibitor. The inhibitor therefore can bind with both the free anzyme and enzyme-substrate complex. Some flavonoids have glycosyl constituents which may cause competitive inhibition of alpha-glucosidse through the glycosyl groups. The other is that the product, pnitrophenol has, at least some structural resemblance to aglycons, which may cause binding of the aglycons to the substrate-binding site of the enzyme. But the results suggests that flavonoids can bind both free enzyme and enzyme-sustrate complex. This means that flavonoids are able to combine with the enzyme at sites other than the substrate binding site. A separate study was conducted by McDougall et. al., (2005) which related specifically anthocyanin, a flavonoid, to a greater inhibitory activity against alpha-amylase and alpha-glucosidase causing a lower Ki value. Ki value is the dissociation constant for inhibitor binding. The lower the Ki value, the stronger is the binding of the inhibitor with the enzyme, thus the stronger inhibitory action.

VIII. Conclusion In vitro assay confirmed the effect of Momordica charantia type. Galaxy-type and type. Bonito on the reduction of enzyme activity of alpha-amylase and alpha-glucosidase. Galaxy-type typeiant obtained higher inhibitory activity against alpha-amylase with a mean percentage inhibition of 72.89%. type. Bonito had a 65.77% mean percentage inhibition. A

29 higher inhibitory activity for the enzyme alpha-glucosidse was also obtained in Sta, Rita type than the Bonito type having percentage reductions of 72.02% and 48.43%, respectively. The inhibitory property of Momordica charantia was associated to its polyphenolic contents especially to its flavonoid components. Previous studies indicated that hydroxyl groups of flavonoids is directly proportional to its inhibitory activity. It is also elucidated in other studies that polyphenolic compounds can act as both competitive and noncompetitive inhibitors of alpha-amylase and alpha-glucosidase. A decrease in Ki was also noted in some studies,related to the presence of anthocyanin, which is an indicator of higher enzyme inhibition.

IX. RECOMMENDATIONS Based on the results the study, the researchers would like to recommend the following for further investigation: 1. Identify and utilize specific varieties of M. charantia that would incur optimum inhibitory activities to the enzymes. 2. Determination of optimum concentration of ampalaya extract that would effect maximum inhibitory activity to alpha-amylase and alpha-glucosidase enzymes; 3. Isolation, identification, and determination of concentration of flavonoids present in ampalaya that impart inhibitory activity the most. 4. Experimentation using animal enzymes, and then eventually, human enzymes.

30 REFERENCES Champe, P. C. & Harvey, R. A. (1994). Biochemistry, 2nd Ed. Philadelphia: J.B. Lippincott Company. Cruger & Wulf (1982). Biochemistry: A Textbook of Industrial Microbiology, 161-169. Foneska, H.H. (2007). Determination of Anti-Amylase and Anti-Glucosidase activity of different genotype of Bitter Gourd (Momordica Charantia L.) and Thumba karavila (Momordica) Dioca L. Faculty of Agriculture, University of Peradeniya, Perandeniya. Sri Lanka. Hanamura, T., Mayama, C., Aoki, H. (2005). Antihyperglycemic effect of Polyphenols from Acerola (Malphigia emarginata DC.) Fruit. Research and Development Division, Nichrei Foods, Inc. Japan. Horton, E. S. (1995). NIDDM. The Devastating Disease. Diabetes Research and Clinical Practice, 28, s3-s11. Iio, M., et. Al (1983). Effects of Flavonoids on -Glucosidase and -Fructosidase from yeast. Department of Food and Nutrition Science, Faculty of Life Sciences, Kumamoto Womens University, Kumamoto, Japan. May, T. T. and Chungyen, N. V. (2006). Anti-Hyperglycemic Activity of an aqueous extract from flower buds of Cleistocalyx Operculatus (Rbx) Merry and Perry. Department of Food and Nutrition, Japan Womens University, Tokyo. Murray, F. (2000). Ampalaya: Natures Remedy for Type 1 and Type 2 Diabetes. CA: Basic Health Publications, Inc. Nobre, C.P. (2005). Standardization of extracts from Momordica Charantia L. (Cucurbitaceac) by total flavonoids content determination. Departamento de Farmacia, Universidade Federal do Rio Grande do Norte. Rio Grande Ringpfeil, M. & Gerhardt, I. M. (n.d). Determination of Amyloglucosidase Activity. Retrieved February 29, 2008, from http://www.biopract.de/Downloads/news013.pdf Rohn, S., Rawel, H. M., & Kroll, J. (2000). Food Chemistry, 72 (1), 59-71. Tadera, K., Minami, Y., Takamatsu, K., & Matsuoka, T. (2005). Inhibition of Glucosidase and -Amylase by Flavonoids. Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Japan.

31 APPENDIX

I. Standards
Starch Only {Starch} (mg/mL) 0.000 0.625 1.250 1.860 2.500 A. Control tube Standards Absorbance N/A 1.09 1.757 2.196 2.465 B. Typeiant 1 Standards Absorbance N/A 0.375 0.775 1.041 1.596 C. Typeiant 2 Standards Absorbance N/A lab accident 0.437 0.758 1.386

1 2 3 4 5

32

1.5 Absorbance (OD) 1 0.5 0 0.000

y = 0.761x - 0.5628

0.500

1.000

1.500

2.000

2.500

3.000

Starch Concentration (mg/mL) Standard Curve of Starch Concentration vs. Absorbance using Starch-Iodine Method for Momordica charantia Bonito-type(type 2) for investigation of amylase activity

33

2 Absorbance (OD) 1.5 1 0.5 0 0.000 y = 0.6309x - 0.0366

0.500

1.000

1.500

2.000

2.500

3.000

Starch concentration (mg/mL) Standard Curve of Starch Concentration vs. Absorbance using Starch-Iodine Method for tubes with M omordica charantia Galaxy type (type 1)

3 Absorbance (OD) 2.5 2 1.5 1 0.5 0 0.000 z 0.500 1.000 1.500 2.000 2.500 3.000 Starch concentration (mg/mL) Standard Curve of Starch Concentration vs. Absorbance using Starch-Iodine Method for control tubes y = 0.7312x + 0.7372

II. Control Tubes {Starch} (mg/mL) 2.363 0.217 2.254 0.280 2.356 0.194 2.371 0.224 Reduction in Abs 1.569 1.443 1.581 1.57 Reduction in Starch 2.145787746 1.973468271 2.162199125 2.147155361 % Reduction Starch 90.81% 87.57% 91.77% 90.55%

1 2 3 4

S S+E S S+E S S+E S S+E

Abs 2.465 0.896 2.385 0.942 2.46 0.879 2.471 0.901

34
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 AVERAGE S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E 2.327 0.220 2.371 0.273 2.323 0.283 2.397 0.247 2.322 0.406 2.344 0.212 2.396 0.243 2.405 0.216 2.370 0.221 2.336 0.253 2.363 0.251 2.393 0.272 2.080 0.283 2.259 0.281 2.109 0.240 2.197 0.182 2.271 0.312 2.263 0.245 2.342 0.284 2.344 0.212 2.386 0.481 2.439 0.898 2.471 0.937 2.436 0.944 2.49 0.918 2.435 1.034 2.451 0.892 2.489 0.915 2.496 0.895 2.47 0.899 2.445 0.922 2.465 0.921 2.487 0.936 2.258 0.944 2.389 0.943 2.279 0.913 2.344 0.87 2.398 0.965 2.392 0.916 2.45 0.945 2.451 0.892 2.482 1.089

1.541 1.534 1.492 1.572 1.401 1.559 1.574 1.601 1.571 1.523 1.544 1.551 1.314 1.446 1.366 1.474 1.433 1.476 1.505 1.559 1.393

2.10749453 2.097921225 2.0404814 2.149890591 1.916028446 2.132111597 2.152625821 2.189551422 2.148522976 2.082877462 2.111597374 2.121170678 1.797045952 1.977571116 1.868161926 2.015864333 1.959792123 2.018599562 2.058260394 2.132111597 1.905087527

90.55% 88.48% 87.83% 89.69% 82.52% 90.97% 89.85% 91.03% 90.66% 89.18% 89.36% 88.64% 86.40% 87.54% 88.60% 91.74% 86.28% 89.20% 87.87% 90.97% 79.84% 88.72%

III. Samples with Ampalaya (Possible Inhibitor) A. Variant 1 [Momordica charantia Galaxy-

35
type] (Starch) (mg/mL) S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya 1.933 2.006 1.852 2.155 1.781 1.889 1.892 2.128 1.838 2.144 1.367 1.859 1.275 1.924 1.177 1.952 1.911 1.800 2.031 2.209 2.321 2.204 1.817 1.882 1.901 2.285 1.955 2.326 1.857 2.342 1.968 2.101 1.545 % Reduction Starch 3.63% 14.05% 5.71% 11.10% 14.27% 26.44% 33.70% 39.70% lab accident 8.04% lab accident 3.45% 85.27% 80.68% % Starch Breakdown Inhibited 85.09% 74.67% 83.01% 77.62% 74.45% 62.28% 55.02% 49.02%

Tubes 1 2 3 4 5 6 7 8 9 10 11 12

Abs 1.183 1.229 1.132 1.323 1.087 1.155 1.157 1.306 1.123 1.316 0.826 1.136 0.768 1.177 0.706 1.195 1.169 1.099 1.245 1.357 1.428 1.354 1.110 1.151 1.163 1.405 1.197 1.431 1.135 1.441 1.205 1.289 0.938

13 14 15 16 17

16.79% 15.94% 20.71% 6.34% 13.42%

71.93% 72.78% 68.01% 82.38% 75.30%

36
+E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya

1.784 1.687 2.195 1.857 2.030 1.618 1.764 1.454 1.797 1.515 1.938 1.616 1.126 1.949 2.390 1.737 1.678

1.089 1.028 1.348 1.135 1.244 0.984 1.076 0.881 1.097 0.919 1.186 0.983 0.674 1.193 1.471 1.059 1.022 23.11% 8.51% 8.27% 19.05% 21.84% lab accident 18.44% lab accident 15.83% 72.89% 70.28% 65.61% 80.21% 80.45% 69.67% 66.88%

18 19 20 21 22 23 24 25 AVERAGE

III. Samples with Ampalaya (Possible Inhibitor) B. Variant 2 [Momordica charantia Bonito-type (Starch) (mg/mL) S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya 1.483 2.222 1.380 1.920 1.843 2.569 1.390 2.144 1.750 2.080 2.272 2.690 % Reduction Starch 33.24% 28.14% 28.24% 35.18% 15.86% 15.54% % Starch Breakdown Inhibited 55.48% 60.58% 60.48% 53.54% 72.86% 73.18%

Tubes 1 2 3 4 5 6

Abs 0.566 1.128 0.487 0.898 0.840 1.392 0.495 1.069 0.769 1.020 1.166 1.484

37
S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya S + Ampalaya + E S + Ampalaya

7 8 9 10 11 12

1.866 2.259 1.828 2.360 2.083 2.450 1.482 2.054 1.761 2.277 1.866 2.247 1.787 2.243 2.235 2.220 2.060 2.432 1.792 2.249 2.094 2.311 2.123 2.557 1.479 2.071 2.131 2.315 1.914 2.570 1.881 2.273 2.165 2.878 1.678 2.234

0.857 1.156 0.828 1.233 1.022 1.302 0.565 1.000 0.777 1.170 0.857 1.147 0.797 1.144 1.138 1.127 1.005 1.288 0.801 1.149 1.031 1.196 1.053 1.383 0.563 1.013 1.059 1.199 0.894 1.393 0.869 1.167 1.085 1.627 0.714 1.137

17.40% 22.55% 15.02% 27.83% 22.68% 16.96%

71.32% 66.17% 73.70% 60.89% 66.04% 71.76%

13 14 15 16 17 18 19 20 21 22 23 24

20.33% lab accident 15.29% 20.33% 9.38% 16.96% 28.56% 7.95% 25.51% 17.23% 24.75% 24.89%

68.39%

73.43% 68.39% 79.34% 71.76% 60.16% 80.77% 63.21% 71.49% 63.97% 63.83%

38
S + Ampalaya + E S + Ampalaya

25 AVERAGE

0.847 2.175

0.0817 1.092

61.05% 22.95%

27.67% 65.77%

II. ALPHA-GLUCOSIDASE I. Standards A. Starch Only {Starch} (mg/mL) 1 2 3 4 5 0 0.625 1.250 1.860 2.500 B. Variant 1 Standards Absorbance N/A 0.441 1.113 1.525 2.018 C. Variant 2 Standards Absorbance N/A lab accident 0.915 1.353 1.677

Absorbance N/A 1.09 1.757 2.196 2.465

II. Control Tubes {Starch} (mg/mL) 2.292 0.403 2.152 0.239 2.392 0.550 2.301 0.491 2.338 0.383 2.271 0.410 2.260 0.245 2.191 0.383 2.363 0.493 2.290 0.362 2.260 0.484 2.286 0.385 2.002 Reduction in Abs 1.381 1.399 1.347 1.324 1.43 1.361 1.474 1.322 1.367 1.41 1.299 1.39 Reduction in Starch 1.888676149 1.913293217 1.842177243 1.810722101 1.955689278 1.861323851 2.015864333 1.807986871 1.86952954 1.92833698 1.776531729 1.900984683 % Reduction Starch 82.41% 88.89% 77.02% 78.68% 83.64% 81.95% 89.18% 82.53% 79.12% 84.19% 78.59% 83.14%

1 2 3 4 5 6 7 8 9 10 11 12 13

S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S

Abs 2.413 1.032 2.311 0.912 2.486 1.139 2.42 1.096 2.447 1.017 2.398 1.037 2.39 0.916 2.339 1.017 2.465 1.098 2.412 1.002 2.39 1.091 2.409 1.019 2.201

39
S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E S S+E 0.280 2.143 0.406 2.074 0.500 2.312 0.398 2.154 0.487 2.262 0.366 2.318 0.500 2.260 0.376 2.288 0.428 2.330 0.265 2.396 0.504 2.288 0.286 2.383 0.506 0.942 2.304 1.034 2.254 1.103 2.428 1.028 2.312 1.093 2.391 1.005 2.432 1.103 2.39 1.012 2.41 1.05 2.441 0.931 2.489 1.106 2.41 0.946 2.48 1.107 1.259 1.27 1.151 1.4 1.219 1.386 1.329 1.378 1.36 1.51 1.383 1.464 1.373 1.721827133 1.736870897 1.574124726 1.914660832 1.667122538 1.895514223 1.817560175 1.884573304 1.859956236 2.065098468 1.891411379 2.002188184 1.87773523 86.01% 81.06% 75.88% 82.80% 77.41% 83.81% 78.42% 83.37% 81.30% 88.63% 78.95% 87.52% 78.78% 82.13%

14 15 16 17 18 19 20 21 22 23 24 25 AVERAGE

III. Samples with Ampalaya (Possible Inhibitor) A. Variant 1 [Momordica charantia galaxytype] Starch (mg/mL) S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E 2.954 2.304 2.953 2.457 1.418 2.474 1.086 1.960 1.265 % Reduction Starch -28.20% -20.18% 42.69% 44.60% 13.08% % Starch Breakdown Inhibited 116.92% 108.90% 46.03% 44.12% 75.64%

Tubes 1 2 3 4 5

Abs 2.425 1.889 2.424 2.015 1.158 2.029 0.884 1.605 1.032

40
S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E 1.456 1.115 1.988 1.651 2.376 1.692 2.630 1.681 2.593 2.943 1.744 1.327 2.008 1.247 2.496 1.881 1.987 1.583 2.335 1.242 2.024 1.784 2.119 1.453 2.022 1.662 2.336 2.879 2.455 1.893 2.399 1.651 1.585 1.640 2.005 1.282 1.189 0.908 1.628 1.35 1.948 1.384 2.158 1.375 2.127 2.416 1.427 1.083 1.645 1.017 2 1.54 1.627 1.294 1.914 1.013 1.658 1.46 1.736 1.187 1.656 1.359 1.915 2.363 2.013 1.55 1.967 1.35 1.296 1.341 1.642 1.046 43.92% 30.52% 35.68% lab accident -68.75% lab accident 50.04% 38.68% 157.47% 44.80% 58.20% 53.04%

6 7 8 9 10 11 12

13 14 15 16 17 18 19 20 21 22 23

5.31% 32.20% 38.63% 15.79% 28.13% 28.86% -17.29% 21.08% -4.13% 18.20% lab accident

83.41% 56.52% 50.09% 72.93% 60.59% 59.86% 106.01% 67.64% 92.85% 70.52%

41
S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya 1.830 1.409 2.375 1.077 2.668 1.498 1.151 1.947 0.877 2.189 40.64% lab accident 16.70% 72.02% 48.08%

24 25 AVERAGE

III. Samples with Ampalaya (Possible Inhibitor) B. Variant 2 [Momordica charantia Bonitotype] Starch (mg/mL) S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya 0.640 1.563 0.510 1.185 1.090 1.996 0.523 1.466 0.973 1.385 1.625 2.148 1.118 1.609 1.070 1.735 1.389 1.849 0.638 1.352 0.986 1.632 1.118 1.594 % Reduction Starch 59.07% 56.97% 45.42% 64.32% 29.76% 24.32% 30.53% 38.34% 24.88% 52.83% 39.56% 29.88% % Starch Breakdown Inhibited 29.65% 31.75% 43.30% 24.40% 58.96% 64.40% 58.19% 50.38% 63.84% 35.89% 49.16% 58.84%

Tubes 1 2 3 4 5 6 7 8 9 10 11 12

Abs 0.566 1.128 0.487 0.898 0.840 1.392 0.495 1.069 0.769 1.020 1.166 1.484 0.857 1.156 0.828 1.233 1.022 1.302 0.565 1.000 0.777 1.170 0.857 1.147

42
S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya S + Ampalaya +E S + Ampalaya

13 14 15 16 17 18 19 20 21 22 23 24 25 AVERAGE

1.019 1.589 1.579 1.561 1.361 1.826 1.026 1.597 1.403 1.674 1.440 1.982 0.635 1.374 1.449 1.679 1.178 1.998 1.137 1.627 1.492 2.382 0.883 1.578 -0.156 1.504

0.797 1.144 1.138 1.127 1.005 1.288 0.801 1.149 1.031 1.196 1.053 1.383 0.563 1.013 1.059 1.199 0.894 1.393 0.869 1.167 1.085 1.627 0.714 1.137 0.0817 1.092

35.87% lab accident 25.46% 35.79% 16.19% 27.35% 53.80% 13.69% 41.02% 30.09% 37.37% 44.04% 110.37% 40.29%

52.85%

63.26% 52.93% 72.53% 61.37% 34.92% 75.03% 47.70% 58.63% 51.35% 44.68% -21.65% 48.43%