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

Momordica charantia Bonito-type

A Thesis Submitted to theDepartment of Biochemistry and Nutrition

Institute of MedicineFEU-NRMF

By:SECTION I – C2

In Partial Fulfillment of the RequirementsFor First Year Biochemistry

March 2008

Determination of Anti-α-Amylase and Anti-α-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.

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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 Baños, 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 Baños 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.

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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

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small 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

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Bonito-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 α-amylase and

α-glucosidase activities. Local Philippine ampalaya types are used in the study. Both

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types 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.

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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 Plant Part Reported Biological Activities

5-α-stigmasta-7,22,25-trien-3-β-ol

Whole Plant No activity reported

5-α-stigmata-7,25-dien-3β-ol Whole Plant No activity reported

5-hydroxytryptamine Fruit Allergenic, Antidepressant, Antidote (Mg), Antimutilation, Anti parkinsonian, Cerebrocephilic, Hypertensive, Insectiside, pesticide, spasmogenic, ulcerogenic, vasoconstrictive

Alkaloids Fruit No activity reportedα-elaostearic acid Seed No activity reportedascorbigen Fruit No activity reportedβ-sitosterol-D-glucoside Fruit Anti-spasmodic, antitumor, CNS

depressant, convulsant, hypoglycemic

charantin Fruit Abortifacient, anti-diabetic, hypoglycemic

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citrulline Fruit Antiasthemic, diureticcryptoxantin Fruit Anti-mutagenic, Vitamin A activity

diospeum Tissue culture Anti-fatigue, anti-inflammatory, anti-stress, estrogenic, hepatoprotective, hypocholesterolemic

elasterol Whole Plant No activity reportedflavochrome Fruit No activity reportedfluoride Fruit Anti-carcinogenic, anti-osteoporonic,

antiosclerotic, anti-convulsant, anti-hypertensive, anti-insomniac, anti-lethargic, anti-stress, diuretic, neuro-inhibitor

glacturonic acid Fruit No activity reportedlanosterol Fruit Cancer preventivelutein Fruit Anti-atherosclerotic, anti-cancer, anti-

oxidant, colorant,, Quinone reductase, inhibitor

lycopene Fruit Anti-cancer, anti-tumor, hypocholesterolemic, pro-oxidant

momordicine Fruit No activity reportedmomordicoside A, B, C, D, E, K, L

Seed No activity reported

momordicoside F1, F2, G, I Fruit No activity reportedmutachrome Fruit No activity reportedoxalate Fruit No activity reportedoxalic acid Fruit CNS paralytic, hemostatic, irritant,

pesticidephylofluene Seed No activity reportedpipecolic acid Fruit Herbistat, pesticidepolypeptide P Fruit Hypoglycemicrubixanthin Fruit Colorantstigma-5, 25-dien-3-β-ol Whole plant No activity reportedsugars Fruit No activity reportedurease Seed No activity reportedvicine Seed Hypoglycemiczeaxanthin Fruit Anti-cancer, anti-tumor, colorant,

hepatoprotective, Quinone Reductase inhibitor

zeinoxanthin Fruit No activity reported

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As shown by the table above, Momordica charantia has many reported

therapeutic activities. Each component corresponded to certain properties, like anti-

cancer, anti-hypertension, diuretic, anti-fatigue, anti-inflammatory, anti-stress, estrogenic,

hepatoprotective, anti-spasmodic, CNS depressant, anti-osteoporonic, antiosclerotic, anti-

convulsant, 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 β-sitosterol-β-D-glucoside and 5,25-sigmastadien-3β-

D-ol. 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 plant’s 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

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fruits. Some flavonoids present are anthocyanins, xanthophylls, and carotene. Flavonoids

are reported to have anti-amylase and anti-glucosidase activities.

α-Amylases are extracellular enzymes which hydrolyze α-1,4-glycosidic bonds.

These enzymes are endoenzymes, splitting the substrate in the interior of the molecule.

Their action is not inhibited by α-1,6-glycosidic bonds although such bonds are not split.

α-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 α-amylases. Its optimum pH is 6.2 and its

optimum temperature is 85˚C.

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 α-D-glucosidase, further degrades following the

solubilization of starch by α-amylase. This enzyme hydrolyzes α-1,4 in addition to α1,6

and α1,3 glycosidic linkages from the non-reducing ends of amylase and amylopectin to

produce β-D-glucose. The type of linkage and the chain length of the substrate are the

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basis of hydrolysis. Like α-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 60˚C. 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 α-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 α-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, α-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

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role in the 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 membrane-

bound 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

membrane-bound 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

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decrease current blood glucose levels: the long term effect is a small reduction in

hemoglobin A1c 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).

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).

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Galaxy-typeM. 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.

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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

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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 α-glucosidase, amyloglucosidase (exo-1,4-α-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

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TUBE

Phosphate Buffer (mL)

1% Starch

Solution (mL)

I2KI (mL)

1 3.00 0.00 1.002 2.75 0.25 1.003 2.50 0.50 1.004 2.25 0.75 1.005 2.00 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

TUBE

Phosphate Buffer (ml)

1% Starch

(ml)I2KI (ml)

Ampalaya Extract

(ml)1b 2.00 0.00 1.00 1.002b 1.75 0.25 1.00 1.00

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3b 1.50 0.50 1.00 1.004b 1.25 0.75 1.00 1.005b 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] = [Starch] A1 – [Starch] A2 x 100(without ampalaya extract) [Starch] A1

(% starch broken down by enzymatic action)

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

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starch that was not broken down by enzyme action. Percent reduction of starch was

obtained by the following equation:

% Reduction of [Starch] = [Starch] C - [Starch] B x 100 (in the presence of ampalaya extract) [Starch] C(% starch broken down by enzymatic action)

where: C = Starch + ampalaya extractB = 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

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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

Sigma-Aldrich.

Inhibition of α-Amylase

In this study, 25 samples of two types of Momordica charantia (Galaxy-type and

type. Bonito) were collected. Anti-α-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 alpha-amylase 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%,

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N= 21) was significantly smaller than 88.72% (control value), with t(44)=33.72, two-tail

p = <0.0001, at α = 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 α = 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 α =

0.05.

Table 4. Anti alpha-Amylase activity of two types of Momordica charantia at 1% [starch] and 1% [α-amylase]

Type% Starch Reduction

% Inhibition

of Enzyme Activity

Type 1 15.83 72.89Type 2 22.95 65.77Control 88.72 n.a.

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

100%

Treatment

% S

tarc

h Re

duct

ion

Control:w /o

inhibitor

Treatment A: w ith M. charantia

Var. 1 extract

Treatment B: w ith M. charantia

Var. 2 extract

Fig. 3. Mean percent reduction of starch by α-amylase. 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)

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

100%

Treatment

% α

-am

ylas

e ac

tivity

inhi

bite

d

Treatment A: w ith M. charantia

Var. 1 extract

Treatment B: w ith M. charantia

Var. 2 extract

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

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Inhibition of α-Glucosidase

In this study, 25 samples of two types of Momordica charantia (Galaxy-type and

Bonito-type) were collected. Anti-α-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 alpha-glucosidase 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 α = 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 α = 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%).

24

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 α = 0.05.

Table 5. Anti alpha-Glucosidase activity of two types of Momordica charantia at 1% [Starch] and 0.1% [α-glucosidase]

Type% StarchReduction

% Inhibition

of Enzyme Activity

Type 1 16.7 72.02Type 2 40.29 48.43Control 82.13 n.a.

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

100%

Treatment

% S

tarc

h Re

duct

ion

Control:w /o

inhibitor

Treatment A: w ith M. charantia

Var. 1 extract

Treatment B: w ith M. charantia

Var. 2 extract

Fig. 5. Mean percent reduction of starch by α-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)

25

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

100%

Treatment

% α

-glu

cosi

dase

act

ivity

inhi

bite

d

Treatment A: w ith M. charantia

Var. 1 extract

Treatment B: w ith M. charantia

Var. 2 extract

Fig. 6. Mean percent α-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.

Fig 7. Primary structure of Flavonoids

26

C - Ring

B - RingA - Ring

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

27

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,

p-nitrophenol 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

28

inhibition. A 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 non-competitive 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.

29

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 α-Glucosidase and β-Fructosidase from yeast. Department of Food and Nutrition Science, Faculty of Life Sciences, Kumamoto Women’s 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 Women’s University, Tokyo.

Murray, F. (2000). Ampalaya: Nature’s 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 α-Glucosidase and α-Amylase by Flavonoids. Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Japan.

30

APPENDIX

I. Standards      

 Starch Only

A. Control tube

StandardsB. Typeiant 1 Standards

C. Typeiant 2 Standards

  {Starch} (mg/mL) Absorbance Absorbance Absorbance1 0.000 N/A N/A N/A2 0.625 1.09 0.375 lab accident3 1.250 1.757 0.775 0.4374 1.860 2.196 1.041 0.7585 2.500 2.465 1.596 1.386

31

Standard Curve ofStarch Concentration vs. Absorbance

using Starch-Iodine Method for tubes withMomordica charantia Galaxy type (type 1)

y = 0.6309x - 0.0366

0

0.5

1

1.5

2

0.000 0.500 1.000 1.500 2.000 2.500 3.000

Starch concentration (mg/mL)

Abso

rban

ce (O

D)

32

Standard Curve of Starch Concentration vs. Absorbance

using Starch-Iodine Method for Momordica charantia Bonito-type(type 2)

for investigation of amylase activity

y = 0.761x - 0.5628

0

0.5

1

1.5

0.000 0.500 1.000 1.500 2.000 2.500 3.000

Starch Concentration (mg/mL)

Abso

rban

ce (O

D)

Standard Curve of Starch Concentration vs. Absorbance

using Starch-Iodine Methodfor control tubes

y = 0.7312x + 0.7372

0

0.5

1

1.5

2

2.5

3

0.000 0.500 1.000 1.500 2.000 2.500 3.000

Starch concentration (mg/mL)

Abso

rban

ce (O

D)

z

II. Control Tubes          

   {Starch} (mg/mL) Abs

Reduction in Abs

Reduction in Starch

% Reduction

Starch1 S 2.363 2.465        S + E 0.217 0.896 1.569 2.145787746 90.81%2 S 2.254 2.385        S + E 0.280 0.942 1.443 1.973468271 87.57%3 S 2.356 2.46        S + E 0.194 0.879 1.581 2.162199125 91.77%4 S 2.371 2.471        S + E 0.224 0.901 1.57 2.147155361 90.55%5 S 2.327 2.439        S + E 0.220 0.898 1.541 2.10749453 90.55%6 S 2.371 2.471        S + E 0.273 0.937 1.534 2.097921225 88.48%7 S 2.323 2.436        S + E 0.283 0.944 1.492 2.0404814 87.83%8 S 2.397 2.49        S + E 0.247 0.918 1.572 2.149890591 89.69%9 S 2.322 2.435        S + E 0.406 1.034 1.401 1.916028446 82.52%

10 S 2.344 2.451        S + E 0.212 0.892 1.559 2.132111597 90.97%

11 S 2.396 2.489        S + E 0.243 0.915 1.574 2.152625821 89.85%

12 S 2.405 2.496        S + E 0.216 0.895 1.601 2.189551422 91.03%

13 S 2.370 2.47        S + E 0.221 0.899 1.571 2.148522976 90.66%

33

14 S 2.336 2.445        S + E 0.253 0.922 1.523 2.082877462 89.18%

15 S 2.363 2.465        S + E 0.251 0.921 1.544 2.111597374 89.36%

16 S 2.393 2.487        S + E 0.272 0.936 1.551 2.121170678 88.64%

17 S 2.080 2.258        S + E 0.283 0.944 1.314 1.797045952 86.40%

18 S 2.259 2.389        S + E 0.281 0.943 1.446 1.977571116 87.54%

19 S 2.109 2.279        S + E 0.240 0.913 1.366 1.868161926 88.60%

20 S 2.197 2.344        S + E 0.182 0.87 1.474 2.015864333 91.74%

21 S 2.271 2.398        S + E 0.312 0.965 1.433 1.959792123 86.28%

22 S 2.263 2.392        S + E 0.245 0.916 1.476 2.018599562 89.20%

23 S 2.342 2.45        S + E 0.284 0.945 1.505 2.058260394 87.87%

24 S 2.344 2.451        S + E 0.212 0.892 1.559 2.132111597 90.97%

25 S 2.386 2.482        S + E 0.481 1.089 1.393 1.905087527 79.84%

AVERAGE           88.72%

III. Samples with Ampalaya (Possible Inhibitor)  

   A. Variant 1 [Momordica charantia Galaxy-type]  

Tubes  (Starch)(mg/mL) Abs

% Reduction Starch

% Starch Breakdown

Inhibited

1S + Ampalaya + E 1.933 1.183 3.63% 85.09%

  S + Ampalaya 2.006 1.229    

2S + Ampalaya + E 1.852 1.132 14.05% 74.67%

  S + Ampalaya 2.155 1.323    

3S + Ampalaya + E 1.781 1.087 5.71% 83.01%

  S + Ampalaya 1.889 1.155    

4S + Ampalaya + E 1.892 1.157 11.10% 77.62%

  S + Ampalaya 2.128 1.306    

5S + Ampalaya + E 1.838 1.123 14.27% 74.45%

  S + Ampalaya 2.144 1.316    

6S + Ampalaya + E 1.367 0.826 26.44% 62.28%

34

  S + Ampalaya 1.859 1.136    

7S + Ampalaya + E 1.275 0.768 33.70% 55.02%

  S + Ampalaya 1.924 1.177    

8S + Ampalaya + E 1.177 0.706 39.70% 49.02%

  S + Ampalaya 1.952 1.195    

9S + Ampalaya + E 1.911 1.169 lab accident  

  S + Ampalaya 1.800 1.099    

10S + Ampalaya + E 2.031 1.245 8.04% 80.68%

  S + Ampalaya 2.209 1.357    

11S + Ampalaya + E 2.321 1.428 lab accident  

  S + Ampalaya 2.204 1.354    

12S + Ampalaya + E 1.817 1.110 3.45% 85.27%

  S + Ampalaya 1.882 1.151    

13S + Ampalaya + E 1.901 1.163 16.79% 71.93%

  S + Ampalaya 2.285 1.405    

14S + Ampalaya + E 1.955 1.197 15.94% 72.78%

  S + Ampalaya 2.326 1.431    

15S + Ampalaya + E 1.857 1.135 20.71% 68.01%

  S + Ampalaya 2.342 1.441    

16S + Ampalaya + E 1.968 1.205 6.34% 82.38%

  S + Ampalaya 2.101 1.289    

17S + Ampalaya + E 1.545 0.938 13.42% 75.30%

  S + Ampalaya 1.784 1.089    

18S + Ampalaya + E 1.687 1.028 23.11% 65.61%

  S + Ampalaya 2.195 1.348    

19S + Ampalaya + E 1.857 1.135 8.51% 80.21%

  S + Ampalaya 2.030 1.244    

20S + Ampalaya + E 1.618 0.984 8.27% 80.45%

  S + Ampalaya 1.764 1.076    

21S + Ampalaya + E 1.454 0.881 19.05% 69.67%

  S + Ampalaya 1.797 1.097    

22S + Ampalaya + E 1.515 0.919 21.84% 66.88%

  S + Ampalaya 1.938 1.186    

23S + Ampalaya + E 1.616 0.983 lab accident  

  S + Ampalaya 1.126 0.674    

24S + Ampalaya + E 1.949 1.193 18.44% 70.28%

35

  S + Ampalaya 2.390 1.471    

25S + Ampalaya + E 1.737 1.059 lab accident  

  S + Ampalaya 1.678 1.022    AVERAGE       15.83% 72.89%

III. Samples with Ampalaya (Possible Inhibitor)      B. Variant 2 [Momordica charantia Bonito-type

Tubes  (Starch)(mg/mL) Abs

% Reduction Starch

% Starch Breakdown

Inhibited

1S + Ampalaya + E 1.483 0.566 33.24% 55.48%

  S + Ampalaya 2.222 1.128    

2S + Ampalaya + E 1.380 0.487 28.14% 60.58%

  S + Ampalaya 1.920 0.898    

3S + Ampalaya + E 1.843 0.840 28.24% 60.48%

  S + Ampalaya 2.569 1.392    

4S + Ampalaya + E 1.390 0.495 35.18% 53.54%

  S + Ampalaya 2.144 1.069    

5S + Ampalaya + E 1.750 0.769 15.86% 72.86%

  S + Ampalaya 2.080 1.020    

6S + Ampalaya + E 2.272 1.166 15.54% 73.18%

  S + Ampalaya 2.690 1.484    

7S + Ampalaya + E 1.866 0.857 17.40% 71.32%

  S + Ampalaya 2.259 1.156    

8S + Ampalaya + E 1.828 0.828 22.55% 66.17%

  S + Ampalaya 2.360 1.233    

9S + Ampalaya + E 2.083 1.022 15.02% 73.70%

  S + Ampalaya 2.450 1.302    

10S + Ampalaya + E 1.482 0.565 27.83% 60.89%

  S + Ampalaya 2.054 1.000    

11S + Ampalaya + E 1.761 0.777 22.68% 66.04%

  S + Ampalaya 2.277 1.170    

12S + Ampalaya + E 1.866 0.857 16.96% 71.76%

  S + Ampalaya 2.247 1.147    

13S + Ampalaya + E 1.787 0.797 20.33% 68.39%

  S + Ampalaya 2.243 1.144    

14S + Ampalaya + E 2.235 1.138 lab accident  

36

  S + Ampalaya 2.220 1.127    

15S + Ampalaya + E 2.060 1.005 15.29% 73.43%

  S + Ampalaya 2.432 1.288    

16S + Ampalaya + E 1.792 0.801 20.33% 68.39%

  S + Ampalaya 2.249 1.149    

17S + Ampalaya + E 2.094 1.031 9.38% 79.34%

  S + Ampalaya 2.311 1.196    

18S + Ampalaya + E 2.123 1.053 16.96% 71.76%

  S + Ampalaya 2.557 1.383    

19S + Ampalaya + E 1.479 0.563 28.56% 60.16%

  S + Ampalaya 2.071 1.013    

20S + Ampalaya + E 2.131 1.059 7.95% 80.77%

  S + Ampalaya 2.315 1.199    

21S + Ampalaya + E 1.914 0.894 25.51% 63.21%

  S + Ampalaya 2.570 1.393    

22S + Ampalaya + E 1.881 0.869 17.23% 71.49%

  S + Ampalaya 2.273 1.167    

23S + Ampalaya + E 2.165 1.085 24.75% 63.97%

  S + Ampalaya 2.878 1.627    

24S + Ampalaya + E 1.678 0.714 24.89% 63.83%

  S + Ampalaya 2.234 1.137    

25S + Ampalaya + E 0.847 0.0817 61.05% 27.67%

  S + Ampalaya 2.175 1.092    AVERAGE       22.95% 65.77%

II. ALPHA-GLUCOSIDASE

I. Standards      

  A. Starch Only  B. Variant 1 Standards

C. Variant 2 Standards

  {Starch} (mg/mL) Absorbance Absorbance Absorbance1 0 N/A N/A N/A2 0.625 1.09 0.441 lab accident3 1.250 1.757 1.113 0.9154 1.860 2.196 1.525 1.3535 2.500 2.465 2.018 1.677

II. Control Tubes          

37

   {Starch} (mg/mL) Abs

Reduction in Abs

Reduction in Starch

% Reduction

Starch1 S 2.292 2.413        S + E 0.403 1.032 1.381 1.888676149 82.41%

2 S 2.152 2.311        S + E 0.239 0.912 1.399 1.913293217 88.89%

3 S 2.392 2.486        S + E 0.550 1.139 1.347 1.842177243 77.02%

4 S 2.301 2.42        S + E 0.491 1.096 1.324 1.810722101 78.68%

5 S 2.338 2.447        S + E 0.383 1.017 1.43 1.955689278 83.64%

6 S 2.271 2.398        S + E 0.410 1.037 1.361 1.861323851 81.95%

7 S 2.260 2.39        S + E 0.245 0.916 1.474 2.015864333 89.18%

8 S 2.191 2.339        S + E 0.383 1.017 1.322 1.807986871 82.53%

9 S 2.363 2.465        S + E 0.493 1.098 1.367 1.86952954 79.12%

10 S 2.290 2.412        S + E 0.362 1.002 1.41 1.92833698 84.19%

11 S 2.260 2.39        S + E 0.484 1.091 1.299 1.776531729 78.59%

12 S 2.286 2.409        S + E 0.385 1.019 1.39 1.900984683 83.14%

13 S 2.002 2.201        S + E 0.280 0.942 1.259 1.721827133 86.01%

14 S 2.143 2.304        S + E 0.406 1.034 1.27 1.736870897 81.06%

15 S 2.074 2.254        S + E 0.500 1.103 1.151 1.574124726 75.88%

16 S 2.312 2.428        S + E 0.398 1.028 1.4 1.914660832 82.80%

17 S 2.154 2.312        S + E 0.487 1.093 1.219 1.667122538 77.41%

18 S 2.262 2.391        S + E 0.366 1.005 1.386 1.895514223 83.81%

19 S 2.318 2.432        S + E 0.500 1.103 1.329 1.817560175 78.42%

20 S 2.260 2.39        S + E 0.376 1.012 1.378 1.884573304 83.37%

21 S 2.288 2.41        S + E 0.428 1.05 1.36 1.859956236 81.30%

22 S 2.330 2.441        S + E 0.265 0.931 1.51 2.065098468 88.63%

23 S 2.396 2.489        S + E 0.504 1.106 1.383 1.891411379 78.95%

38

24 S 2.288 2.41        S + E 0.286 0.946 1.464 2.002188184 87.52%

25 S 2.383 2.48        S + E 0.506 1.107 1.373 1.87773523 78.78%

AVERAGE           82.13%

III. Samples with Ampalaya (Possible Inhibitor)  

   A. Variant 1 [Momordica charantia galaxy-type]  

Tubes  Starch

(mg/mL) Abs% Reduction

Starch

% Starch Breakdown

Inhibited

1S + Ampalaya + E 2.954 2.425 -28.20% 116.92%

  S + Ampalaya 2.304 1.889  

2S + Ampalaya + E 2.953 2.424 -20.18% 108.90%

  S + Ampalaya 2.457 2.015  

3S + Ampalaya + E 1.418 1.158 42.69% 46.03%

  S + Ampalaya 2.474 2.029  

4S + Ampalaya + E 1.086 0.884 44.60% 44.12%

  S + Ampalaya 1.960 1.605  

5S + Ampalaya + E 1.265 1.032 13.08% 75.64%

  S + Ampalaya 1.456 1.189  

6S + Ampalaya + E 1.115 0.908 43.92% 44.80%

  S + Ampalaya 1.988 1.628  

7S + Ampalaya + E 1.651 1.35 30.52% 58.20%

  S + Ampalaya 2.376 1.948  

8S + Ampalaya + E 1.692 1.384 35.68% 53.04%

  S + Ampalaya 2.630 2.158  

9S + Ampalaya + E 1.681 1.375 lab accident

  S + Ampalaya 2.593 2.127  

10S + Ampalaya + E 2.943 2.416 -68.75% 157.47%

  S + Ampalaya 1.744 1.427  

11S + Ampalaya + E 1.327 1.083 lab accident

  S + Ampalaya 2.008 1.645  

12S + Ampalaya + E 1.247 1.017 50.04% 38.68%

  S + Ampalaya 2.496 2  13 S + Ampalaya 1.881 1.54 5.31% 83.41%

39

+ E  S + Ampalaya 1.987 1.627  

14S + Ampalaya + E 1.583 1.294 32.20% 56.52%

  S + Ampalaya 2.335 1.914  

15S + Ampalaya + E 1.242 1.013 38.63% 50.09%

  S + Ampalaya 2.024 1.658  

16S + Ampalaya + E 1.784 1.46 15.79% 72.93%

  S + Ampalaya 2.119 1.736  

17S + Ampalaya + E 1.453 1.187 28.13% 60.59%

  S + Ampalaya 2.022 1.656  

18S + Ampalaya + E 1.662 1.359 28.86% 59.86%

  S + Ampalaya 2.336 1.915  

19S + Ampalaya + E 2.879 2.363 -17.29% 106.01%

  S + Ampalaya 2.455 2.013  

20S + Ampalaya + E 1.893 1.55 21.08% 67.64%

  S + Ampalaya 2.399 1.967  

21S + Ampalaya + E 1.651 1.35 -4.13% 92.85%

  S + Ampalaya 1.585 1.296  

22S + Ampalaya + E 1.640 1.341 18.20% 70.52%

  S + Ampalaya 2.005 1.642  

23S + Ampalaya + E 1.282 1.046 lab accident

  S + Ampalaya 1.830 1.498  

24S + Ampalaya + E 1.409 1.151 40.64% 48.08%

  S + Ampalaya 2.375 1.947    

25S + Ampalaya + E 1.077 0.877 lab accident  

  S + Ampalaya 2.668 2.189    AVERAGE       16.70% 72.02%

III. Samples with Ampalaya (Possible Inhibitor)  

   B. Variant 2 [Momordica charantia Bonito-type]  

Tubes  Starch

(mg/mL) Abs% Reduction

Starch

% Starch Breakdown

Inhibited

1S + Ampalaya + E 0.640 0.566 59.07% 29.65%

  S + Ampalaya 1.563 1.128    

2S + Ampalaya + E 0.510 0.487 56.97% 31.75%

  S + Ampalaya 1.185 0.898    

40

3S + Ampalaya + E 1.090 0.840 45.42% 43.30%

  S + Ampalaya 1.996 1.392    

4S + Ampalaya + E 0.523 0.495 64.32% 24.40%

  S + Ampalaya 1.466 1.069    

5S + Ampalaya + E 0.973 0.769 29.76% 58.96%

  S + Ampalaya 1.385 1.020    

6S + Ampalaya + E 1.625 1.166 24.32% 64.40%

  S + Ampalaya 2.148 1.484    

7S + Ampalaya + E 1.118 0.857 30.53% 58.19%

  S + Ampalaya 1.609 1.156    

8S + Ampalaya + E 1.070 0.828 38.34% 50.38%

  S + Ampalaya 1.735 1.233    

9S + Ampalaya + E 1.389 1.022 24.88% 63.84%

  S + Ampalaya 1.849 1.302    

10S + Ampalaya + E 0.638 0.565 52.83% 35.89%

  S + Ampalaya 1.352 1.000    

11S + Ampalaya + E 0.986 0.777 39.56% 49.16%

  S + Ampalaya 1.632 1.170    

12S + Ampalaya + E 1.118 0.857 29.88% 58.84%

  S + Ampalaya 1.594 1.147    

13S + Ampalaya + E 1.019 0.797 35.87% 52.85%

  S + Ampalaya 1.589 1.144    

14S + Ampalaya + E 1.579 1.138 lab accident  

  S + Ampalaya 1.561 1.127    

15S + Ampalaya + E 1.361 1.005 25.46% 63.26%

  S + Ampalaya 1.826 1.288    

16S + Ampalaya + E 1.026 0.801 35.79% 52.93%

  S + Ampalaya 1.597 1.149    

17S + Ampalaya + E 1.403 1.031 16.19% 72.53%

  S + Ampalaya 1.674 1.196    

18S + Ampalaya + E 1.440 1.053 27.35% 61.37%

  S + Ampalaya 1.982 1.383    

19S + Ampalaya + E 0.635 0.563 53.80% 34.92%

  S + Ampalaya 1.374 1.013    

20S + Ampalaya + E 1.449 1.059 13.69% 75.03%

  S + Ampalaya 1.679 1.199    

41

21S + Ampalaya + E 1.178 0.894 41.02% 47.70%

  S + Ampalaya 1.998 1.393    

22S + Ampalaya + E 1.137 0.869 30.09% 58.63%

  S + Ampalaya 1.627 1.167    

23S + Ampalaya + E 1.492 1.085 37.37% 51.35%

  S + Ampalaya 2.382 1.627    

24S + Ampalaya + E 0.883 0.714 44.04% 44.68%

  S + Ampalaya 1.578 1.137    

25S + Ampalaya + E -0.156 0.0817 110.37% -21.65%

  S + Ampalaya 1.504 1.092    AVERAGE       40.29% 48.43%

42