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European Journal of Scientific Research
ISSN 1450-216X Vol.43 No.3 (2010), pp.384-391
© EuroJournals Publishing, Inc. 2010
http://www.eurojournals.com/ejsr.htm
Antidiabetic Activity of Active Fractions of Leucaena
Leucocephala (lmk) Dewit Seeds in Experiment Model
Syamsudin
Corresponding author Department of Pharmacology, Faculty of Pharmacy
Pancasila University,South Jakarta, Indonesia
E-mail: syamsudin27@yahoo.com
Tel: 6221-7864728; Fax: 6221-7864727
Ros Sumarny
Department of Pharmacology, Faculty of Pharmacy
Pancasila University,South Jakarta, Indonesia
Partomuan Simanjuntak
Research Centre for Biotechnology, Indonesian Institute of Science
Jln. Raya Bogor Km 46, Cibinong 16911, Indonesia
Abstract
A study was conducted on antidiabetic test of active fractions of methanol extract
from Leucaena leucocephala (lmk)DeWit seeds using alloxan-induced rats. Fractionation
was conducted on active fractions using column chromatograph. The most active fractions
in the previous study were analyzed with thin layer chromatography (TLC) using mobile
phase of chloroform-methanol (5:1), chloroform–methanol (2: 1), chloroform-methanol-
water (5: 5: 1) on the isolates yielded with oral glucose tolerance test and identification.
The result findings show that methanol extracts have a greater antidiabetic activity; and 5
isolates resulted from the isolation of methanol extracts. The result of bioactive compound
identification was glycoside compounds with monosaccharide galactose clusters and many
other saccharides. It was concluded that active fractions of Leucaena leucocephala
(lmk)DeWit seeds had antidiabetic activities and their bioactive compounds constitute
glycoside compounds with monosaccharide galactose clusters and many other saccharides.
Keywords: L. leucocephala (lmk)DeWit seeds, antidiabetes, alloxan, oral glucose
tolerance
1. Introduction
Diabetes mellitus is a common degenerative disease among the communities with different age groups
and different socioeconomic levels. Syndromes of diabetes mellitus include chronic hyperglycemia that
is a manifestation of abnormalities in carbohydrate, protein, and fat metabolisms or is correlated with
the deficiency of insulin secretion within the target cell membranes (Alberti and Zimmeti, 1998).
The incidence of diabetes keeps increasing annually. In 2000, the number of diabetic patients in
Indonesia was up to 8.4 millions patients and ranked the fourth worldwide after India, China, and the
United States of America (Wild et al., 2004) Similarly, data obtained from 7 hospitals in Jakarta
Antidiabetic Activity of Active Fractions of Leucaena
Leucocephala (lmk) De Wit Seeds in Experiment Model 385
(RSCM, Fatmawati, Cikini, Pelni, Persahabatan, Husada and St. Carolus) showed that the number of
visits at outpatient services was the greatest for diabetes (Anonym, 2000)
Recently, more than 400 types of plants have been reported to be useful as alternative and
complementary treatments for diabetes; however, relatively few had been studied in terms of their
natural efficacy (Lee et al., 2006). One of the plants recently used as an alternative and complementary
treatment for diabetes is L. leucocephala (lmk)DeWit. The plant has been used by human beings since
many centuries ago for herbal medicine. Indonesia is one of the tropical countries, which are rich of
natural resources. Forest biodiversity as a natural resources remains in need of more exploration to
recognize its potentials as a source of herbal medicine, including for antidiabetes (Syamsudin et al.,
2006). According to the priority of the study, development and implementation of health science and
technology for medicine and health devices for the period of 2005-2025 have included standardized
herbal products and phytopharmaca. The recent study aims at isolating bioactive compounds from L.
leucocephala (lmk)DeWit seeds as an anti-diabetes.
2. Materials and Methods
2.1. Plant Material
L. leucocephala (lmk)DeWit seeds were obtained from BALITRO Bogor and then determined in
Herbarium Bogoriense, Bogor. The experimental models used in the study were rats (Mus musculus)
aged 3-4 months.
2.2. Extraction and Fractionation
L. leucocephala (lmk)DeWit seeds were grounded to powder; then refluxed three times in a gradient
way using solvents like n-hexane, ethylacetate, methanol, and water as well as direct extraction with
methanol. Each extract is let to evaporate and held until fractions are yielded.
2.3. Hypoglycemic Activity Tests
Before treated, the rats were injected with alloxan with a dosage of 70 mg/kg body weight in vitro.
Subsequently, blood samples were taken to figure out the hyperglycemic levels of the experimental
models. Once the rats were hyperglycemic, they were divided into 6 groups of hyperglycemic rats and
1 group of normal rats as a control group; each group contained 6 rats.
(1) Normal control group: a group of rats which were not treated and were normally fed;
(2) Negative control group: A group of hyperglycemic rats, which were treated with distilled
water of 2 mL orally for 14 days;
(3) Group A: A group of hyperglycemic rats which were treated with n-hexane extract with an
oral dosage of 0.5 g/kg body weight on daily basis for 14 days.
(4) Group B: A group of hyperglycemic rats which were treated with ethylacetate extract with an
oral dosage of 0.5 g/kg body weight on daily basis for 14 days.
(5) Group C: A group of hyperglycemic rats which were treated with indirect methanol extract
with an oral dosage of 0.5 g/kg body weight on daily basis for 14 days.
(6) Group D: A group of hyperglycemic rats which were treated with water extract with an oral
dosage of 0.5 g/kg body weight on daily basis for 14 days.
(7) Group E: A group of hyperglycemic rats which were treated with direct methanol extract with
an oral dosage of 0.5 g/kg body weight on daily basis for 14 days.
(8) On Day 0, 3, 7, and Day 14 blood samples were taken through tail vein; the level of blood
glucose was then measured using a glucometer.
386 Syamsudin, Ros Sumarny and Partomuan Simanjuntak
2.4. Purification with Column Chromatography
Methanol extracts were fractionated with column chromatography using chloroform-methanol eluents
in a gradient way, ranging from (5:1), (4:1), (3:1), (2:1), and (1:1) to categorize the compounds
contained in the methanol extracts based on their polarity.
2.5. Oral Glucose Tolerance (OGT) Method
With this method, 35 rats were initially acclimatized before the experimental phase. The rats were
grouped into seven, each with 5 rats.
(1) Negative control group: a group that was only treated with oral suspension of CMCNa 0,1%
with a volume of 0.1 mL.
(2) Positive control group: a group that was only treated with oral quercetin with a dosage 10
mg/kg body weight. An hour after the treatment, the rats were given glucose solution with a
dosage of 1,5 mg/kg body weight.
(3) Isolate Group A-1: a group which was treated with oral A-1 isolation solution with a dosage
of 10 mg/kg body weight. An hour after the treatment, the rats were given glucose solution
with a dosage of 1,5 mg/kg body weight.
(4) Isolate Group A-2: a group which was treated with oral A-2 isolation solution with a dosage
of 10 mg/kg body weight. An hour after the treatment, the rats were given glucose solution
with a dosage of 1,5 mg/kg body weight.
(5) Isolate Group A-3: a group which was treated with oral A-3 isolation solution with a dosage
of 10 mg/kg body weight. An hour after the treatment, the rats were given glucose solution
with a dosage of 1,5 mg/kg body weight.
(6) Isolate Group A-4: a group which was treated with oral A-4 isolation solution with a dosage
of 10 mg/kg body weight. An hour after the treatment, the rats were given glucose solution
with a dosage of 1,5 mg/kg body weight.
(7) Isolate Group A-5: a group which was treated with oral A-5 isolation solution with a dosage
of 10 mg/kg body weight. An hour after the treatment, the rats were given glucose solution
with a dosage of 1,5 mg/kg body weight.
An hour following the administration of preparations (hour 0), blood samples of the rats were
immediately taken through their tail vein. Blood taking were repeated with an interval of 0.5 hour
starting from hour 0 through hour 2,5 using a glucometer.
2.6. Identification of Active Compounds
Purified isolates were identified using a spectrophotometer UV-Vis, IR and RMI.
2.7. Data Analysis
The data were initially tested for their normality and homogeneity. When the data were normally
distributed and homogenous, they were further analyzed with one-way ANNOVA using a level of
significance p = 0.05 and 0.01. When a significant difference was noted, the further step was Turkey
test to find out the existence of real difference among the treatment groups.
3. Results and Discussion
Obtaining animal models with diabetes mellitus could be done with pancreatomy and administration of
certain chemical substances. The chemical substances used were allocsan due to its greater selectivity
on rats compared to mice within the merusal of pancreatic cell β. In addition, allocsan increases the
hyperglycemic effect within 2 to 3 days. Before used for experimental purposes, all rats from each
Antidiabetic Activity of Active Fractions of Leucaena
Leucocephala (lmk) De Wit Seeds in Experiment Model 387
group were measured for their body weight and subsequently taken care for a week. The result of body
weight measurement is presented in the following Table 1.
Table 1: Mean body weights of rats after the treatment with test preparations
normal diabetes n-hexane ethylacetae methanol A water methanol B
Baseline 31.7 ± 2.4 32.4 ± 2.4 34.1 ± 3.4 30.4 ± 4.5 36.8 ± 3.2 37.6 ± 3.9 35.0 ± 6.4
Day-0 32.8 ± 4.3 24.8 ± 2.8 28.3 ± 4.2 24.7 ± 6.4 32.2 ± 6.3 33.2 ± 8.3 29.9 ± 4.2
Day -3 33.9 ± 5.4 26.2 ± 4.3 29.2 ± 4.3 25.1 ± 3.4 34.2 ± 4.2 34.9 ± 3.8 31.1 ± 4.5
Day-7 34.7 ± 3.2 26.9 ± 4.3 30.1 ± 5.7 27.6 ± 7.3 34.1 ± 5.7 35.7 ± 5.4 32.5 ± 6.9
Day-14 35.8 ± 4.3 28.4 ± 2.5 30.2 ± 6.4 28.1 ± 4.8 35.2 ± 8.6 36.5 ± 7.8 33.3 ± 5.4
Table 1 shows that on Day 0, body weights of hyperglycemic states of rats in control group,
treatment groups n-hexane and ethylacete decreased. It may be due to abnormalities in glucose
metabolism in which energy supply were not sufficient, causing depletion of fatty cells and protein in
order to meet energy requirements that could be sufficiently supplied from glucose metabolism. On
Day 14, body weights of all rats in the treatment groups were restored even though they had not gained
their baseline body weight. This was perhaps since energy had been sufficiently supplied and glucose
metabolism had been adequate. Results of observations on amounts of feed, drinking water volume,
and urine volume are presented in Table 2.
Table 2: Mean drinking water volume, urine volume, and amounts of feed
Groups Drinking Water Volume (mL) Urine Volume (mL) Amount of feed (g)
Normal 7.60 ± 1.23 1.10 ± 0.92 3.67 ± 1.92
Diabetes 13.98 ± 3.41 3.46 ± 1.42 11.37 ± 1.32
n-hexane 7.80 ± 1.42 2.40 ± 1.31 5.37 ± 2.41
ethylacetate 7.51 ± 1.34 2.44 ± 0.98 5.24 ± 1.52
methanol A 6.14 ± 2.41 2.14 ± 1.34 4.73 ± 1.31
water 5.92 ± 2.45 2.06 ± 1.45 4.37 ± 2.34
methanol B 5.98 ± 0.89 1.89 ± 1.03 4.95 ± 7.59
Table 2 shows that in diabetic groups, drinking water volume and urine volume were greater
than those in normal and treatment group. It was probably attributable glucosaria among the diuretic
osmotic hyperglycemic groups; hence resulting in dyuresis followed by a depletion of electrolyte since
the body tried to overcome the dyuresis by drinking more (polydipsia). Amount of feedings among
diabetic groups increased more greatly (polyphagia) than those among normal and treatment groups.
This is perhaps resulted from stimulation in the center of appetite within the hypothalamus because of
insufficiency in the utilization of glucose within the cells due to hyperglycemia. Syndromes like
polyuria, polydipsia, and polyphagia were commonly found among diabetic. After the extract
administration to all treatment group A-E, an increase in the level of blood glucose was observed both
on Day 3, Day 7, and Day 14.
388 Syamsudin, Ros Sumarny and Partomuan Simanjuntak
Figure 2: Potential decrease in the level of blood glucose among all extract groups
Figure 2 shows that group direct methanol extract (meth B) had greater decrease in the level of
blood glucose than any other groups, i.e. a decrease of 44.38%, n-hexane of 21.29%, ethylacetate of
19.67%, indirect methanol extract of (meth A) 32.42%, and water extract of 36.11%. Group meth B
and water had the greatest capability in decreasing the level of blood glucose compared to any other
groups. This was perhaps because group meth B and water constituted polar solvent and contained
compound groups that are found in indirect methanol extracts (meth A) and water extract. It was
estimated that groups with the greatest capability of reducing the level of blood glucose were groups
with polar solvent. Table 3 presents rendement or yields of each extract.
Table 3: Rendements of each extract
Extract Rendement (%)
n-hexane 2.19%
ethylacetate 1.14%
methanol 5.42%
water 5.67%
direct methanol 6.32 %
Table 3 shows that rendement extract with polar solvent like methanol, water, and direct
methanol were more considerable than non-polar solvents (n-hexane) and semi-polar solvents
(ethylacetate). Graded extractions were conducted by using solvents with different polarity. The
objective was for initial pre-fractionation since it could isolate chemicals properties contained in the L.
leuco cephala (lmk)DeWit seeds based on their polarity.
Fractionation with column chromatograms aimed at isolating compounds within the methanol
fractions with the expectation that a pure compound could be obtained. Methanol extract at preliminary
test was subsequently fractionated with column chromatography by using appropriate eluents.
Fractionation was done with chloroform-methanol eluents in a gradient way with respective solvent
proportions of 5:1, 4:1, 3:1, 2:1, 1:1. Eluate from each extract was retained well. Every fraction was
also treated with TLC. Fractions with similar pattern of isolation with chromatograms were combined;
yielding simpler fractions. The results of combining TLC examination could be summerized as shown
in the following Table 4.
Antidiabetic Activity of Active Fractions of Leucaena
Leucocephala (lmk) De Wit Seeds in Experiment Model 389
Table 4: The Result of combining TLC examination of all Fractions
Sub-fraction Combination spot color Number of spots Rf
A-1 1-10 brown 1 0.88
A-2 11-17 tosca green 1 0.48
brown 0.29
tosca green 0.19
A-3 13-27
brown
3
0.097
bright green 0.25
dark green 0.19
A-4 37-60
brown
3
0.097
A-5 61-90 dark green 1 0.19
A-6 91-137 dark green 1 0.16
In relation to 6 sub-fractions that had been obtained, oral glucose tolerance test was conducted
to rats treated with glucose in an oral dosage of 1.5 g/kg body weight; then, the level of blood glucose
was measured through tail vein on minutes 0, 30, 60, 90, 120, and 180 using glucometer. To find out
their potentials in reducing the level of blood glucose, the size of area under the curve was measure to
be subsequently used to figure out its ability to reduce the level of blood glucose in experimental rats
following the administration of test preparation. The results are presented in Figure 3.
Figure 3: Diagram of the potential decrease in the level of blood glucose from test preparations
Figure 3 shows that the potential decrease in the level of blood glucose of isolate A-4 is higher
(15.15%) than isolate A-1 (6.63%), A-2 (5,90%), A-3 (7.48%), A-5 (5,74%) and A-6 (7.54%).
Statistical analysis shows no significant difference between quercetin (positive control) and isolate A-4
in reducing the level of blood glucose (p>0.05). Isolate A-4 or active isolates were purified with High-
Performance Liquid Chromatography (HPLC) using the column of reversed phase C18, with mobile
phase of methanol-water (5:1); hence pure isolate compound could be obtained. In the chromatogram
of HPLC, in the isolate A-4 were observed two main peaks; and the sharpest one is peak 2 which in on
16.876 minutes. Then, isolates within the minute were retained based on the chromatogram detected by
HPLC. Purification was done with repeated injection. Pure isolate was subsequently identified.
Investigation on the spectrum of isolate compounds A-4 with proton RMI showed that chemical shift
between 0,88 bpj ~ 1,27 was estimated to contain single bonded CH3- ; -CH2- dan -CH-clusters. An
anomeric proton of a glycoside was found at δH 3, 20 ~ 4,47 bpj and 5,37 bpj. Investigation on the
spectrum of isolate compounds A-4 on carbon RMI indicated that the signals of isolate compounds A-4
had 40 carbon atoms. Chemical shift at δC 83,84 bpj ~ 105,37 bpj indicated the existence of glycoside;
meanwhile, chemical shift at δC 54,79 ~ 79,67 bpj indicated the existence of oxygenated carbons.
Therefore, based on the data isolate A-4 could only be predicted as a glycoside compound with
galactose monosaccharide clusters and other saccharides.
390 Syamsudin, Ros Sumarny and Partomuan Simanjuntak
Figure 4: The structure of galactomannan
In another study on L. leucocephala (lmk)DeWit seeds, it was indicated that L. leucocephala
(lmk)DeWit seeds contain galactomannan and lectin galactomannan that constitutes a glycoside
(Lesniak and Liu, 1981). A study conducted by Ali et al. (1995) on antidiabetic test on active fractions
of Trigonela foenum graecum seeds of Leguminosae family showed that the plants might reduce the
level of blood glucose in streptozotozin-fed rats. The results of identification on molecular structures
assumed to be responsible for the antidiabetic effects were galactomannan. Galactomannan were
mostly dispersed within the plants of Leguminosae family. Based on the similarity among molecular
formulas, it could be temporarily assumed that the molecular structures of isolate A-4 and
galactomannan resulted from the isolation of Trigonela foenum graecum seeds, it is possible that on
isolate A-4 the one responsible for the antidiabetic activity of Leucaena leucocephala (lmk)DeWit
seeds was galactomanna. It is highly possible due to a similarity in the chemical structures of isolate A-
4 and galactomannan.
4. Conclusion
Based on the findings of the study, it can be concluded that Leucaena leucocephala (lmk)DeWit seeds
have an effect for reducing the level of blood glucose. The results of identification on the bioactive
compounds indicate that glycoside compounds have galactose monosaccharide clusters and other
saccharides.
Acknowledment
Our gratitude goes to the Directorate of Research Development and Public Dedication, Directorate
General of Higher Education, Department of National Education that has provided research fund
through Competitive Grant with a Research Contract for the Fiscal Year of 2009.
Antidiabetic Activity of Active Fractions of Leucaena
Leucocephala (lmk) De Wit Seeds in Experiment Model 391
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