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: