Study on Biological Active Components of Eurycoma Longifolia
Nik Nur Shamiha N. D.
1,3*
, Siti Shukriyah S.
1,2
, Ryuichiro S.
2
, Yoshiaki S.
2
, Jiyauddin Khan
1,3
,
Ibrahim A.
1,3
*, Mohd Nizam A. G.
1,3
, Mohd Fadli Asmani
1,3
and Eddy Yusuf
3
1
School of Pharmacy, Management and Science University,
40100 Shah Alam, Selangor Darul Ehsan, Malaysia
2
Faculty of Pharmaceutical Sciences, Josai University,
1-1 Keyakidai, Sakado, Saitama 350-0295, Japan
3
ICHLAS, Management & Science University,
40100 Shah Alam, Selangor, Malaysia
Keywords: Eurycoma longifolia, Anti-glycation activity, % of inhibition, Tongkat Ali, AGEs formation
inhibition in vitro
.
Abstract: Background and aims: Constant hyperglycemia in diabetic patient may lead to excess glycation and
thus is believed to cause diabetic complications. Eurycoma longifolia (Simaroubaceae) is tested for
inhibitory activity of advanced glycation end-products (AGEs) formation in vitro. Materials and
methods: Three concentration of methanolic extract were tested together with bovine serum albumin
in anti-CML antibody. HRP- conjugated anti-mouse IgG antibodies were introduced and sample
were reacted with phenyldiamine dihydrochloride. Absorbance were read by using micro-ELISA and
percentage of inhibition were calculated. Results: The calculated percentage of AGEs formation
inhibition by E. longifolia root are -3.62 % (0.1 mg/mL), 58.38 % (1 mg/mL) and 92.28 % (10
mg/mL) as compared to aminoguanidine 5.55 % (0.1 mg/mL), 39.32 % (1 mg/mL), 72.92 % (10
mg/mL) as referring to the concentration. Since the biological activity was tested on the whole
methanolic extract, the activity is suggested to be due to synergistic activity of the extract.
Conclusion: New biological activity of E. longifolia methanolic extract which is inhibition of AGEs
formation in vitro is seen. However, isolation of Fr.8-2, m/z:381 does not lead to any compound
isolated related to the plant.
1 INTRODUCTION
This study focus on the antiglycation activity of
Eurycoma longifolia (Simaroubaceae). To date,
there is no known activity on inhibition of advanced
glycation end products (AGEs) formation by E.
longifolia. The plant is widely known for anti-tumor
promoting activities, antischistosomal,
plasmodicidal activities (Jiwajinda, S. et al., 2002),
potent antiulcer activity (Tada, H. et al., 1991),
helps to improves stress hormone profile and certain
mood state parameters (Talbott, S. M., Talbott, J. A.,
George, A., & Pugh, M., 2013), cytotoxic activity
(Kuo, P.C., Damu, A.G., Lee, K.H., & Wu, T.S.,
2003), antibacterial action (Farouk, A., & Benafri, A.,
2007) as well as antimalarial activity (Ang, H., Chan, K.,
& Mak, J., 1995). Chronic use of E. longifolia is said to
increase the testosterone level in men.
AGEs formation lead to kaput protein. AGEs
formation in normal healthy people may not be
detrimental to their health compared to diabetic patient.
It is believed that major complication of diabetes;
nephropathy, neuropathy and retinopathy are augmented
by AGEs formation as well as constant hyperglycemia.
This study utilizes the method of evaluation based on a
previous study conducted by Okada, Y., Ishimaru, A.,
Suzuki, R. and Okuyama, T. in 2004. Inhibition of AGEs
formation is based on the carboxymethyl lysine level
inhibition.
Shamiha, N., Shukriyah, S., S., R., S., Y., Khan, J., A., I., Nizam, M., Asmani, M. and Yusuf, E.
Study on Biological Active Components of Eurycoma Longifolia.
DOI: 10.5220/0008361803050309
In Proceedings of BROMO Conference (BROMO 2018), pages 305-309
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
305
By determining the biological activity, it
may leads to isolation of compounds responsible for
the said activity. This study suggest for further
progress in formulating therapeutic agents to counter
diabetic complications and thus improve the
patients’ quality of life and reduce the mortality rate
among diabetic due to complications.
2 MATERIALS AND METHODS
2.1 Materials
E. longifolia sliced, dried roots were procured from
Malaysia, 99.5 % Methanol (Wako Pure Chemical
Industries, LTD, Lot: DSH3947), 99.9 % Methanol
(Wako Pure Chemical Industries, LTD, Lot:
DSJ0787), 99.7 % Methanol (Wako Pure Chemical
Industries, LTD, Lot: DSF3711), ethyl acetate
(Kanto Chemical Co. Inc. Lot: 51B1137), 99.0 % 1-
butanol (Wako Pure Chemical Industries, LTD, Lot:
DSR6754), 99.0 % Chloroform (Wako Pure
Chemical Industries, LTD, Lot: DSF3237), 99.8 %
Chloroform D + Silver Foil (Cambridge Isotope
Laboratories, Inc., Lot: PR-27572/04086CL1),
dimethyl sulfoxide, bovine serum albumin,
phosphate buffer solution, glucose, PBS containing
0.05% Tween 20, 0.5% gelatin in 100 mL coating
buffer, HRP-conjugated anti-mouse IgG antibodies,
1,2-phenyldiamine dihydrochloride, 100 µL of 1 M
sulfuric acid.
2.2 Apparatus
Evaporating flask, mantel heater, rotary evaporator
(EYELA NVC, No: 038006204), desiccator with
silica gel, TLC ODS plate, TLC silica gel plate. UV
light transmitter, microtube, micropipette, 96-well
plate, micro-ELISA plate reader, HPLC-RI detector
(Waters 600 Pump, Waters 600 Controller, Shodex
RI-201H Refractive Index Detector), MPLC Micro
pump KPW-20 (Kusano, Kagakukikai Co.),
Advantec Fraction Collector (CHF122SC), H-NMR
Varian (Agilent, 400 Hz), EI-MS (JEOL), HPLC
ODS-4151-N column (Senshu Pak, 10 x 150 mm,
No: 1110201H), MPLC column (MERCK,
LiChroprep Si 60 (40-63 µm), No: 540087666).
2.3 Preparation of Methanolic Extract
Methanolic extract of the roots of E. Longifolia (200 g)
was obtained by extraction with MeOH (5.4 L) three
times under reflux for 3 hours. The solvent was
evaporated in vacuo to give MeOH extract (6.74 g).
2.4 Isolation of Components from
Methanolic Extract
The methanolic extract was suspended in water, then
extracted with EtOAc and n-BuOH, sequentially. Each
soluble portion was evaporated in vacuo to give EtOAc
(1.07 g) and n-BuOH (1.79 g) fractions, respectively.
The EtOAc fraction was chromatographed on a
prepacked silica gel column (LiChroprep Si60 (40-63
µm) Merck Co. serial number: 540087666, 140987)
eluting with CHCl
3
to give 15 fractions. Fr.8, Fr.9, and
Fr.10 was further purified with HPLC-RI (Detector: RI-
201H, SHODEX, Column: ODS-4151-N; size: 10 x 150
mm; number: 1110201H, Senshu Scientific Co. Ltd.)
detector. Fr.8 (0.0123 g) was further purified with
HPLC-RI detector using MeOH to provide four fraction
Fr.8-1 (0.0001 g) Fr.8-2 (0.0006 g) Fr.8-3 (0.0001 g)
and Fr.8-4 (0.0001 g). Fr.9 (0.0148 g) was further
purified with MeOH-H
2
O mixture (MeOH : H
2
O = 10 :
1) to gives four fraction Fr.9-1 (0.0014 g) Fr.9-2
(0.0010 g) Fr.9-3 (0.0014 g) and Fr.9-4 (0.0007 g).
Fr.10 (0.0074 g) was further purified with MeOH-H
2
O
mixture (MeOH : H
2
O = 10 : 1) to gives three fraction
Fr.10-1 (0.0002 g) Fr.10-2 (0.0003 g) Fr.10-3 (0.0001
g).
2.5 Inhibition Test on AGE Formation in
vitro
BSA was incubated with 200 mmol/L glucose in both
presence and absence of test compound for 7 days in 0.1
M of phosphate buffer (pH 7.4) at 37 °C. After
incubation, coating buffer, blocking buffer and anti-
CML antibody were introduced to the cell. HRP-
conjugated anti-mouse IgG antibodies was treated to the
cells. 1 M sulfuric acid was used to stop the reaction.
The level of inhibition is measured by calculating the
level of CML measured by CML-specific micro-ELISA
plate reader at 492 nm (SpectraMax PLUS 190PC ROM
v1.23). Percentage of inhibition was calculated as in
following equation:
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
306
Inhibition (%) = [1-(A
s
– A
b
)/(A
c
– A
b
)] x 100,
where A
s
is the CML level in the incubated mixture
with sample, A
c
is the CML level in the incubated
mixture without sample, and A
b
is the CML level in
the incubates mixture without sample and glucose
that served as blank control.
3 RESULTS
Methanolic extraction of E.longifolia root (200 g)
yielded 6.74 g of dried extract. Repeated extraction
under reflux ensures complete extraction from the
root. 20 mg of methanolic extract were subjected to
inhibition of AGEs formation in vitro by measuring
CML level using microELISA at 492 nm. Three
reading were recorded for each concentration. The
average reading is tabulated in Table 1.
Table 1: Average reading of absorbance of AGEs
inhibition by microELISA.
Sample
Reading
1
Reading
2
Reading
3
Average
reading
Blank 0.755 0.765 0.663 0.728
Control 2.592 2.280 2.287 2.386
AG 0.1 2.522 2.240 2.119 2.294
AG 1 1.657 1.758 1.787 1.734
AG 10 1.260 1.069 1.201 1.177
EL 0.1 3.146 2.220 1.972 2.446
EL1 1.344 1.683 1.226 1.418
EL 10 0.842 0.993 0.733 0.856
Upon calculating the average reading of
each sample, the percentage of AGEs formation
inhibition were determined by substituting the
absorbance obtained by Micro-ELISA plate reader
in the formula. -3.62 %, 58.38 % and 92.28 % of
inhibition were calculated in E. longifolia with
concentration of 0.1 mg/mL, 1 mg/mL and 10
mg/mL respectively. The percentage of inhibition of
AGEs by E. longifolia compared to Aminoguanidine
is as in Figure 1.
Figure 1: Comparison between percentages of inhibition
of AGEs formation between aminoguanidine (positive
control) and E. longifolia root extract.
4 DISCUSSION
Three concentration of E. longifolia root were tested; 0.1
mg/mL, 1 mg/mL, and 10 mg/mL. The calculated
percentage of AGEs formation inhibition by E.
longifolia root are -3.62 % (0.1 mg/mL), 58.38 % (1
mg/mL) and 92.28 % (10 mg/mL) as compared to
aminoguanidine 5.55 % (0.1 mg/mL), 39.32 % (1
mg/mL), 72.92 % (10 mg/mL) as referring to the
concentration. The activity of E. longifolia is suggested
to match the activity of aminoguanidine. The percentage
of inhibition is concentration dependent. Since the
biological activity was tested on the whole methanolic
extract, the activity is suggested to be due to synergistic
activity of the extract. Due to the small amount of root,
the isolated fractions yield are very small. Thus, we are
unable to test the biological activity on each fraction.
Based on EI-MS analysis of Fr.8-2, the molecular weight
is suggested to be m/z: 381 as attached in Appendix.
However, based on this data alone, we are not able to
relate the finding to any compounds reported to be
having relationship with the plant E. longifolia.
-20
0
20
40
60
80
100
120
0.1 mg/mL 1 mg/mL 10 mg/mL
Percentage (%) of inhibition
Concentration
Aminoguanidine E. longifolia
Study on Biological Active Components of Eurycoma Longifolia
307
5 CONCLUSIONS
Methanolic extract of E. longifolia is suggested to be
having the inhibitory activity of AGEs formation.
The antiglycation activity is believed to be
synergistic action between compounds in the extract.
Further isolation of fractions however does not lead
to identification of isolates. In the future study, it is
strongly suggested that large amount of plant sample
should be used and care attention to work procedure
must be applied to prevent possible impurity to the
components.
ACKNOWLEDGEMENTS
The authors would like to thank all members of
School of Pharmacy MSU and Josai university for
their support.
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APPENDIX
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