Prosiding Seminar Kimia Bersama UKM-ITB VIII

9-11 Jun 2009

ANTIHYPERGLICEMIC EFFECT OF FLESHED AND SEEDED EXTRACT OF

MOMORDICA CHARANTIA
SITI AISYAH1*, HAYAT SHOLIHIN1, IQBAL MUSTHAPA1, ATUN QOWIYAH2
1
Department of Chemistry Education, Faculty of Mathematics and Natural Sciences, Indonesia
University of Education, Jl. Setiabudi 229 Bandung
2
Faculty of Pharmacy, University of Garut, Jl. Jati 42 Tarogong Garut
*email: [email protected]

ABSTRACT
The research is conducted in order to discover active fraction toward the reduction of blood glucose
(antihyperglicemic) from fleshed and seed of Momordica charantia or pare. Extraction is carried out
using methanol solvent proceeded with fractionation using organic solvent such as hexane, ethyl
acetate, and n-butanol. Entire extracts and fractions are examined to obtain the information of blood
glucose intensity reduction activities and the major compound group characteristics with Liebermann-
Burchad methods and FT-IR spectroscopy. Based on the glucose tolerance test with in vivo way on
Wistar rats it is discovered that hexane fraction of the fleshed is the most active and the fraction
insertion causes the mouse with lower blood glucose intensity differs significantly from the positive
control. Based on the characteristic test with color method Lieberman-Burchad and spectroscopy IR,
active fraction hexane is presumed to contain the major compound such as unglycosides terpenes.

Keywords : antihyperglicemic, Momorica charantia, glucose tolerance test.

INTRODUCTION
Diabetes mellitus is the commonest endocrine disorder attacking more than 4% of people
around the world. World Health Organization (WHO) assume that the disease will affect five times
more people in th world (Grover, J.K., et al. 2002). The disease is indicated by the increase of blood
glucose intensity. The intensity of blood glucose alone to normal people varies in a day. It is
increasing after having meal and back to normal in 2 hours. The increasing of blood glucose after
having meal or drink stimulate the pancreas to produce insulin that preventing the continued increase
of blood glucose and even more decreasing it gradually. In the diabetes mellitus sufferer the blood
glucose intensity is high and difficult to get back to normal the body is unable to release or use
sufficient insulin. It causes the sufferer lose his/her weight. The serious matter of the disease is the
proceeding complication it causes including arterosclerosis, retinopati, nefropati and heart coroner
(Vats, V., 2004; Malik S., 2007).

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The disease is divided into two types, namely insulin-dependent diabetes mellitus (T1DM,
Type 1 Diabetes Mellitus) and insulin-independent diabetes mellitus (T2DM, Type 2 Diabetes
Mellitus). Just about 90% of the diabetes mellitus sufferers are type T2DM (Giorgio et al., 2005). In
dealing with the second type (T2DM), there are various oral medicines, such as metformin. However,
in some cases, the existing medicines fail to turn back the blood glucose to normal level, or need
higher dozes. The increase of the doze surely causes the origination of some side effects.
Consequently, there is a need to find the alternative or additional remedy in order to turn the blood
glucose to the normal level such as by the use of traditional medicinal plants.
There are more than 400 plants are recognized to produce the effect of blood glucose intensity
reduction (Ernst, 1997). One of them largely traditionally used to diabetes treatment is ―paria‖ or
―pare‖ (Momordica charantia). Antidiabetes studies of M. charantia have been carried out many times
and they have proven that it is able to reduce blood glucose intensity to normal animal, aloksan
inducted diabetes animal, streptozotosin inducted diabetes animal and genetical diabetes animal (Rao,
BK., 2001; Grover and Yadav, 2004; Virdi, J., 2003). Furthermore, the ethanol extract of the fruit is
also proven to decrease the blood glucose as good as glikenklamid effect in experiment using little
mouse.
Instead of the antidiabetes study, the phitochemistry aspect of the plant has intensively been
observed. Based on completed literature study, it contains compounds of glycoside, saponin, alkaloid,
triterpen, protein, and steroi. (Raman and Lau, 1996). From those compounds, at least, there are 50
secondary metabolite compounds of triterpen aglikon and triterpen glycoside. (Buckinghham, 2006).
Some chemistry compounds such as momorcharins, momordenol, momordicilin, momordicins,
momordicinin, momordin, momordolol, charantin, charine, cryptoxanthin, cucurbitins, cucurbitacins,
cucurbitanes, cycloartenols, diosgenin, erythrodiol, galacturonat acid have been isolated successfully
from the plant (Grover and Yadav, 2004).
Although the two studies, namely antidiabetes effect and phytochemistry, have been carried
out frequently, the study of the correlation between them are rarely conducted. Based on the result of
literature investigation, there is only one given study, resulting in the identification of some
hypoglycemidal peptide. (Lotlikar and Rao, 1996; Khanna et al., 1981). In fact, M. charantia contains,
instead of peptide, a number of secondary metabolite compounds of triterpana inheritor, as well as
aglikon or glycoside (Buckingham, 2006). As the result, the research proposed attempts to answer a
hypotheses that one of the secondary metabolite compounds or more could contribute to hypoglycemia
effect of M. charantia fruit.

MATERIALS AND METHODS

Collection of plant material

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Green, unriped fresh fruits of Momordica charantia were purchased from Kampung Pamahan, Jati
Asih, Bekasi. Part of fleshed and seeded were sun dried to get Momordica charantia fleshed and
seeded dried powder.

Preparation of extracts
The active principle/s of M. charantia fleshed and seeded were extracted into three different
solvents, hexane, ethyl acetate, and n-butanol. M.charantia fleshed and seeded powder was soaked in
methanol solvents in plastic jars for 1 day at room temperature and the solvent was filtered. This was
repeated three times until the extract gave no coloration. The extracts were distilled and concentrated
under reduced pressure in the Buchi, rotavapour R-114 and finally freeze dried. These extracts were
extracted into three different solvents, hexane, ethyl acetate, and n-butanol. These extract was used for
further studies.
Experimental animals
Females Wistar rats weighing 150–180 g were purchased from Sekolah Ilmu dan Teknologi
Hayati ITB. The rats were housed in air-conditioned animal house. Each rat was kept in a separate
cage.
The methode of glucose tolerance
The methode of glucose tolerance was used in the test of antihyperglikemic effects. Diabetes
was induced in Wistar rats which is orally fed glucose by gastric intubation. The extract powders were
orally fed at a dosage of 0.25 g/Kg body weight and glibenclamide at a dosage of 0.45 mg/kg body
weight. In every batch the rats were divided into 12 groups and each group consisted of 3 rats.
Group 1—Diabetic untreated rats
Group 2— Diabetic rats treated with 0.25 g/kg b.w. of fleshed methanol extract.
Group 3— Diabetic rats treated with 0.25 g/kg b.w. of fleshed hexane extract.
Group 4— Diabetic rats treated with 0.25 g/kg b.w. of fleshed ethyl acetate extract.
Group 5— Diabetic rats treated with 0.25 g/kg b.w. of fleshed n-butanol extract.
Group 6— Diabetic rats treated with 0.75 g/kg b.w. of fleshed methanol-water extract.
Group 7— Diabetic rats treated with 0.25 g/kg b.w. of seeded methanol extract.
Group 8— Diabetic rats treated with 0.25 g/kg b.w. of seeded hexane extract.
Group 9— Diabetic rats treated with 0.25 g/kg b.w. of seeded ethyl acetate extract.
Group 10— Diabetic rats treated with 0.25 g/kg b.w. of seeded n-butanol extract.
Group 11— Diabetic rats treated with 0.75 g/kg b.w. of seeded methanol-water extract.
Group 12—Diabetic rats treated with 0.45 mg/kg b.w. of Glibenclamide.

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After an overnight fast, the plant extract suspended in tragakan was fed by gastric intubation, using a
force feeding needle. Group 1 rats were fed tragakan alone. Blood samples were collected for the
measurement of blood glucose from the tail vein at 0, 30, 60, 90 and 120 minutes after feeding
glucose. The results were compared with those of the 12th group of rats which was treated
glibenclamide (OHA). Blood glucose levels were determined by using Optimum Omega®
Glucometer.

Statistical analysis
Data are expressed as mean S.D.
Characterisation of mayor compounds in each extract
Each extract was measured by Liebermann-Burchad methods and FT-IR spectroscopy

RESULTS AND DISCUSSIONS

The fruit of M. charantia (11 Kg) was separated from flesh (8 Kg) and seeds (3 Kg). Then
they were dried to get powder of flesh (770 g, 9.6%) and seed powder (605 g, 20.16%). The powder
were then extracted by methanol (3x5L). The solvent from both of extracts were evaporated by rotary
evaporator and yielded fleshed extract (28 g, 3.6%) and seeded extract (26 g, 4.3%). The concentrated
of fleshed extract (11,4 g) was diluted by methanol then extracted in succession with hexane to yield
(0.8 g, 7%), ethyl acetate to yield (4.9 g, 43%), n-butanol to yield (5.2 g, 45.6%), and the rest fraction
of methanol-water (0.5 g, 4.4%). As well as the flesh part, the seeded extract (29.2 g) was diluted by
methanol, then extracted in succession with hexane, to yield (0.2 g, 0.7%), ethyl acetate to yield (3.2 g,
11%), n-butanol to yield (3.4 g, 11.6%), and the rest fraction of methanol-water (22.4 g, 4.4%).
Test of glucose tolerance was carried out in vitro to white Wistar rats. The methode of glucose
tolerance was used in the test of antihyperglikemic effects which was meassured every 30 minutes.
First meassurement was carried out after 30 minutes induction of test preparations and comparison.
This activity was indivated as zero time. On the tests of induction of the extract fractions showed that
the rate of glucose concentration in the blood of animals were 80-90 mg/dl on the first minute. It was
higher than comparison group which stated at 74 mg/dl. It means that hypoglicemia was caused by
introduction of gibenklamide. 30 minutes induction, all of animal blood glucose concentrations had
increased. Then after the following 60 minutes some groups were still high, therefore most of them
had decreased. Accordingly, the glucose concentration all of groups had decreased during 90 minutes.
Meanwhile the data of the rate glucose concentrations before and after treatment with fleshed extract
then compared to both of positif control and comparison group can be shown on the figure 1. Base on
statistical count resulted that induction of fleshed extract of M charantia on minute of 90 and 120 lead
to inhibit increasing of glucose concentration. Introduction this fraction caused glucose concentration
of rat blood was lower than positif control group (p < 0,05).

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100

80
persen perubahan kadar gula darah (%)

60

40 kontrol
pembanding
total
20 heksan
etil asetat
n-butanol
0 sisa metanol-air
30 60 90 120

-20

-40

-60
menit

Figure 1. Percentage of glucose concentration change before and after treatment with fleshed extract of
M. charantia
On the tests of induction of the seeded extract fractions showed that the rate of glucose
concentration in the blood of animals were 80-110 mg/dl on the first minute. It was higher than
comparison group which stated at 74 mg/dl. After 30 minutes induction, all of animal blood glucose
concentrations had increased. Then after the following 60 minutes some groups were still high,
therefore most of them had decreased. Accordingly, the glucose concentration all of groups had
decreased during 90 minutes except on the total methanol and hexane fractions. Meanwhile the data of
the percentage change of rate glucose concentrations before and after treatment with seeded extract
compared to both of positif control and comparison group can be shown on the figure 2. Base on
statistical count resulted that induction of methanol seeded extract of M charantia on minute of 60 and
hexane fraction on minute of 90 lead to inhibit increasing of glucose concentration. Introduction this
fraction caused glucose concentration of rat blood was lower than positif control group (p < 0,05).
80

60
persen perubahan kadar gula darah (%)

40

kontrol
20
pembanding
total
heksan
etil asetat
0
n-butanol
30 60 90 120
sisa metanol-air

-20

-40

-60
menit

Figure 2. Percentage of glucose concentration change before and after treatment with seeded extract of
M. charantia

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Based on the experiment data, showed that antihyperglikemic effect of flashed and seeded
extract on dosage of 250mg/Kg b.w. was lower than comparison group (gibenklamid). However, the
hexane fractions had decreased glucose concentration significantly.
Based on identification of phytochemistry showed that all of flashed and seeded extract
fraction (except of fleshed methanol-water residue) contain terpenoide groups. However, this test
cannot distinguish between terpens itself and glycosides terpens. Meanwhile on the flashed extract was
also found flavonoid groups. The phytochemistry test proved that flesh and seed of M charantia is a
source of terpenoid which also been indicated by its IR spectrum.
Its spectrum IR showed two models of spectrum. Fistly was shown in hexane fraction which
has strong absorbance at wave number of 2923.9 cm-1 and 2854.5cm-1correspond to streching of
alifatic C-H bondings. This data was also supported with strong absorbance at 1465.8 dan 1377.1 cm-
1
due to bending of alifatic C-H bondings. As weel maximum absorbance at 1735,8 cm-1 indicated of
presence of karbonil group as an ester. Based on the spectrum can be resulted that both of flashed and
seeded hexane fractions was predicted contain major group of unglycosides terpenes. The second IR
spectrum model was presented by flashed and seeded polar fractions. The flashed ethyl acetate fraction
spectrum absorbed wave number at 3402.2 cm-1 correspond into hydroxyl groups which predicted
come from –OH sugar binding into terpenoid structures. The presence of terpenoid structures was
shown by strong absorbance at 2935.5 cm-1 correspond to alifatic C-H streching and C-H bendings at
1384.8 and 1222.8 cm-1. The presence of carbonyl group was also indicated by absorbance at 1778.2
and 1720.4 cm-1. Based on this spectrum can be predicted that fraction of ethyl acetat and other polar
contain glycosides terpenes as the major compounds. Related on sugar tolerance and phytochemistry
tests from each fractions can be identified that componds active which lead to decrease glucose
concentration on rat blood is unglycosides terpenes.

CONCLUSION
Based on sugar tolerance and phytochemistry tests into ten fractions of extract M charantia
can be identified that hexane fraction is the most active part. It contain active componds which lead to
decrease glucose concentration on rat blood. Moreover correspond to IR spectrum and colored
Lieberman Buchard method, this active fraction contain unglycosides terpenes compound as an active
ones.

ACKNOWLEDGEMENTS
The author thank to the UPI Rector who has given financial support through Dana Hibah
Kompetitif Internal UPI 2008.

REFERENCES

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