Asian Journal of Pharmaceutical and Clinical Research

INHIBITORY ACTIVITY ON ADVANCED GLYCATION END PRODUCTS (AGES)

FORMATION OF COMPOUND OF THE SEEDS OF LEUCAENA
LEUCOCEPHALA (LMK) DE WIT AND POSTPRANDIAL HYPERGLYCAEMIA
1SYAMSUDIN, 2DIAH WIDOWATI, 3PARTOMUAN SIMANJUNTAK,

The compound of the seeds of Leucaena leucocephala (Lmk) De Wit that could inhibit on
Advanced Glycation End Products (AGEs) Formation are potentially used for antidiabetic
by suppressing postprandial hyperglycaemia. This research aimed to investigate the
hypoglycaemic activity in compounds of the seeds of Leucaena leucocephala (lmk) De Wit
using an in vitro bioassay based on the inhibition of the formation of AGEs. The active
constituent showed potential as inhibit on AGEs with IC50 28,32 mg/mL. In animal
experiment of active constituent suppressed the increase of postprandial blood glucose
level compare to the control (p<0,05). The structure of active constituent galactomannan
was determined by spectroscopic data interpretation and by comparison of these data with
vqlues in the literature.

Key words : Leucaena leucocephala (Lmk), Advanced Glycation End Product, active
constituent, postprandial hyperglycaemia

INTRODUCTION : activity. A preliminary research showed that the

Diabetes mellitus is a common endocrine
disorder characterized by hyperglycemia and
long term complications affecting the eyes,
nerves, blood vessels, skin and kidneys. Increased
glycation of protein and accumulation of
Advanced Glycation End Products (AGEs) have
been implicated in the pathogenesis of diabetic
complication. 1
Increased glycation and accumulation of tissue on
AGEs formation can alter protein conformation and
impair function by altering enzyme activity,
modifying protein half life, altering immunogenicity
and cross-linking of structural protein. 2,3 More than
400 plants species having hypoglycaemic activity
have been available in literature, howerer searching
for new antidiabetic drugs from natural planst is still
attractive because they contain substances which
take alternative and safe effect on diabetes mellitus.
Most of plants contain glycosides, alkaloids,
terpenoids, flavonoids, carotenoids etc., that are
frequently implicated as having antidiabetic effect.4
Leucaena leucocephala (lmk) De Wit is medicine
plants reputed for antidiabetic properties in the
traditional medicine in Indonesia. The seeds of this
plants was subjected to studies for hypogglycaemic
methanol fraction of Leucaena leucocephala (lmk)
De Wit seed extract was the most active extract to
decrease the blood glucose levelof mice induced by
alloxane.5 The isolation using column
chromatography of this active fraction produced six
isolates which in turn were purified with HPLC. The
purified form of these isolates were, then, in-vitro
bioassay tested to know their ability to inhibit AGEs
formation. Besides, the isolates were also in-vivo
tested on mice using Oral Tolerance Glucose. Thus
the design and discovery of inhibitors of the
glycation cascade can offer a promising therapeutic
approach for the prevention of diabetic or other
pathogenic complications.6 In thispaper we report
the isolation and structure elucidation of active
compound of the seeds of Leucaena leucocephala
(lmk) De Wit that could inhibit on AGEs Formation
are potentially used for antidiabetic by suppressing
postprandial hyperglycaemia.
Material and Methods:
Plant Material. The seed powder of L.
leucocephala was obtained from plant grown in the
Balitro in Bogor, West Java. The plant was
identified by Dr. Eko Baroto and authenticated
material was deposit in the Herbarium Bogoriense,

Volume 1, Issue 2, 2008 18

 Asian Journal of Pharmaceutical and Clinical Research
West Java. The seeds were collected during the administration of compounds and metformnine for t
month of March 2007, 2 kg of seeds dried in h glucose (2gkg-1 b.w. was
sunlight yielded 850 g of L. leucocephala seed administered orally of these mice. Blood glucose
powder. levels were measured from the tail vein at 0, 30, 60,
Extraction and isolation. Two kilograms of dried 90 and 180 min after the glucose load7.
seed powder of L. leucocephala was extracted three Results and Discussion:
times using methanol (6 L) reflux method for two
days. The filtrate was evaporated under pressure at Some glycocydes from the family of
the temperature of 40º C to produce 123 gram Leguminoceae, like Cassia tora,were reported to
methanol extract. This extract was fractionated with have a quite strong inhibitory activity against AGE
column chromatography, using chloroform- compound formation.8 In the Table l, we can see that
methanol-water (10:10:1) to give 7 fraction. The (FS6) has lC50 28.32 μg/mL, less than other
most active fraction (Fr 5) was fractioned using components of the compound. It shows that
column chromatography with the elusion liquid of component-6 is able to inhibit AGE compound
chloroform-methanol-water (10:10:1) to give six formation, stronger than FS1-FS5, but as strong as
subfractions FS1 (0.451 g), FS2 (0.703 g ) , FS3 the aminoguaidine (a positive control) (p<0.05).
(0.791 g), FS4 (0.804 g), FS5 (5.354 g) and FS6 Although, the synthetic glycation inhibitor,
(0.336 g). These subfractions (FS6) were purified aminoguanidine attenuares the development of range
using HPLC (C 18 Bondapak, methanol-water = 5:1) of diabetic vascular complications.3 The mechanism
and bioassay tested to know their ability to inhibit of action of aminoguanidine is still unclear, but it
AGEs formation. The subfractions were also in-vivo may act by blocking carbonylgorups on free sugars,
on mice using Oral Tolerance Glucose. Amadori products or dicarbonyl intermediates such
as 3- DG. Some toxiciry problems have been
Compound (FS6) encountered in clinical trials with this drug. On the
Brown-yellow, UV λ max 277.5 nm; IR ν max cm-1 other hand, natural products have proven to be
933 (C-H), 1138 (C-O), 1407 (C-H), 3322 (OH); 1H- somewhat effective for inhibiting AGEs
NMR (500 MHz, DMSOdet) δ H (ppm) 4.47;5.10; formation.4,9
and 5.37 (anomeric proton) and 3.20 - 4,62 (CH and Table 1 , lnhibitory activity of compounds from the seed of
CH2) ;13C – NMR (75 MHz, DMSOdet 13C (ppm) Leucaena leucocephala (lmk) De Wit on AGEs formation itro.
62.91; 63.45; 64.05; 71.26, 71.36; 71.47; 71.47;
73.27; 75.72; 76.36; 76.56; 78.11; 78.49; 83.84;
85.20; 93.71; 98.19; and 105.37.
Determination of AGEs formation
According to well established method Lee et al.
(2006), the reaction mixture 10 mg/mL of bovine
serum albumin (Singma, St Louis, MO, USA) in 50
m phosphate buffer (pH 7.4) with 0.02% sodium
azide to prevenr bacterial growth was added to 0.2
M fructose and glucose. The
reaction mixture was then mixed with compounds or
aminoguanidine (Sigma, St Louis, MO, USA). After
incubating at 37ºC for 7 days, rhe fluoresecenr
reaction products were assayed on a
spectrofluorometric detector (BIO-TEK, Synergy
HT, USA: Ex 350, Em: 450nm). AGEs assay was
performed in quadruplicate. The concentration of
each test sample giving 50% inhibition of the
activities (lC50) was esrimated from the least-squares
regression line of the logarithmic concenrration
plotted agains the remaining activity.4,8
Oral Glucose Tolerance Test (OGTT)
After fasting for 16 h, normal mice divided into
seven groups, normal conrrol, compounds FS1, FS2,
FS3, FS4, FS5, FS610 mgkg-1 b.w., orally and Inhibitory effect was expressed as mean ± SD of quadruplicate
metformin 175 mgkg-1 b.w., orally. After the experiments IC50 values were calculated from the dose inhibiton curve.

Volume 1, Issue 2, 2008 19

 Asian Journal of Pharmaceutical and Clinical Research
Postplandial blood glucose levels of dose 10 mgkg-1
b.w of mice show significant degradation relative
higher at 30 min, at 60 min after administered rate
downhill and constant relative until 180 min. To
know the effectiveness of the component in
decreasing the blood glucose level in mice, we can
analyze the AUC (area under curve) level in Fig 2,
componenr-6 has the highest AUC value, compared
to other components, and its AUC value is the same
as the positive control’s. The result of this
postprandial tesr, using OTGT method, is the same
as the result of the in-vitro test, where the (FS6) is
the most active compared to others.
REFERENCES
1. Bennet, P.H., In; Joslin Diabetes mellirus Kahn,
C.R- Gordon, C. l3th edition, Lea &-Febiger
Philadelphia, 1994.
2. Forbes, JM., Cooper, ME., Oldfield, MD and
Thomas, MC. J. Am. Soc. Nephrol
2003,14,5254-59
3. Kalousova er al., Kidney Blood Press Res,
2004,27:18-28.
4. Kim, JM er d., Arch Pharm Res, 2006, 29(10),
821-825.
5. Syamsudin., Darmono and Simanjuntak, P, Int J
of Science and Res,2006, 2(l), 49-52.
6. Ahmad, MS and Ahmed, N., J of Nutrition
2006,796S-799S.
7. Shan,JJ.,Yang, MY, and Ren, Biol Pharm
Bull,2006,29(3), 455-459.
8. Lee, GY., Jong, DS., Lee, S and Kim, JS., Arch
Pharm Res, 2006,29(7), 587-590,
9. Matsuda. H. et al., Bioorg Med Chem, 2003, ll,
5317- 5323.
10. Garcia. MJ. et al, Current Therapy Research,
1988, 43, l0l0-62.
11. Ali. L. et al., Planta Med, 1995, 61, 358-360.

Fig 2. Effect of compounds from the seeds of L. leucocephala

on oral glucose tolerance test in mice (AUC)

The identificarion of (FS6) with adsorbence-based

IR-spectrophotometry showed alkana on 1407.94
cm--1 and 2932 cm-1 ; and O-H 3322 cm-1. The
identification using 1H-NMR showed a chemical
shift at aH 4.47; 5.10; and 537 showed anomeric
proton, An identification using 13C-NMR showed
glycoside carbon at 93.71;98.19; and 105.37 ppm.
By comparing the spectra data of the (FS6), we are
sure that the (FS6) is galacromannan.10
The antidiabetic activity of galactomannan, isolated
from Amorphophalus konjack, was studied by
Garcia et al (l988). Ali et al. (1995) the antidiabetic
activiry of Trigonella foenum graenum seed on
ailoxane-induced mice. One of the plant isolated
compounds is galactomannan. In conclusion,
galactomanan showed inhibitory activity of inhibit
on Advanced Glycation End products (AGEs)
Formation in vitro assay and show hypoglycaemic
activiry on animal experiment.

ACKNOWLEDGMENTS
We would like thank to Dr. M. Hanafi, The
Research Centre for Chemistry, Indonesian Institute
of Science, Serpong for running 1H & 13C-NMR
measurements.

Volume 1, Issue 2, 2008 20