Determination of Anti-α-Amylase and Anti-α- 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
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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
5
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
6
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
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
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
9
As shown by the table above, Momordica charantia has many reported
therapeutic activities. Each component corresponded to certain properties, like anti-
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)
23
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%).
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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.
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.
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