Rao and Subramanian (2009) showed that the
ethanolic extract of mengkudu fruit at a dose of 300
mg/kgBW can increase plasma insulin levels in the
group of diabetic rats induced streptozotocin 12.52
μU/ml, while the group given glyclazide (dose 5
mg/kgBW) of 13.27 μU/ml. these research showed
that the ability of mengkudu fruit extract in
increasing the production of insulin comparable with
glyclazide which is one of the oral antidiabetic
sulfonylurea group.
Hartati (2003) had been studied antidiabetic
activity of ethanolic extract of mengkudu fruit by
glucose tolerance method on male rats. These study
showed that glucose tolerance test on rats, 30, 60, 90
and 120 minutes after administration of the extract at
dose of 1200 mg/kgBW, serum glucose
concentration decreased by 13.99%, 31.85%,
44.46% and 56.19 %.
Based on Ramdhini (2005) reseach, fractions of
mengkudu fruit extract can decrease plasma glucose
level of male rats at a dose of 1200 mg/kgBW by
glucose tolerance method. It is known that ethyl
acetate fraction was the best antidiabetic activity
(54.29%), followed by n-hexane fraction (34.18%)
and water fraction (47.42%). Separation and
identification of active compounds that contain in
the ethyl acetate fraction have not been performed.
The aims of this study were : (a). To separate
ethyl acetate fraction from mengkudu (M. citrifolia
Linn.) fruit by Vacuum Liquid Chromatography
method. (b). To study antidiabetic activities from
mengkudu fruit subfractions on male white mice by
glucose tolerance method.
2 METHODS
The fresh ripe fruit of M. citrifolia Linn. were
collected from Lembang, West Java, Indonesia. This
plant was identified at Plants Taxonomy
Laboratorium, Faculty of Mathematics and Natural
Sciences, Universitas Padjajaran.
2.1 Extraction and Phytochemical
Analysis
10 kg mengkudu fruit, mashed and then macerated
in ethanol 70% for 24 hours with 3 times repetitions.
The macerate were filtered, collected, then
concentrated with a rotary evaporator until the
ethanol vaporised. The concentrate extract than
reheated at 55°C by waterbath until obtained a fixed
weight. The extract was subjected to qualitative
chemical tests for the identification of various
phytoconstituents.
2.2 Fractionation of Ethanolic Extract
by Liquid-Liquid Extraction (LLE)
The concentrate extract was dissolved in water, then
filled into a separating funnel and added n-hexane as
the same volume of water added. The two formed
layers are separated, then the water layer was put
back into the separating funnel and added n-hexane
with the same volume. The water portion then
introduced into the separating funnel again, and
fractionated using ethyl acetate in the same way as
fractionation with n-hexane. The three fractions n-
hexane (Ds-I), ethyl acetate (Ds-II) and water (Ds-
III) were evaporated with a rotary evaporator and
continue the evaporation with with water bath until
the weight is obtained constantly.
2.3 Analysis of Ethyl Acetate Fraction
(Ds-II) by Thin Layer
Chromatography (TLC)
Ethyl acetate fraction of mengkudu fruit were
spotted on TLC plate 60 F254 (Merck, Germany)
and developed in mobile phase obtained chloroform
: ethyl acetate : methanol (7:2:1). TLC plate were
visualized under UV light at wavelength 254 nm and
366 nm and spray by using 10% H
2
SO
4
reagent.
2.4 Separation of Ethyl Acetate (Ds-II)
Fraction by Vacuum Liquid
Chromatography (VLC)
The chromatographic column was packed by dry
state in a vacuum conditions to obtain the maximum
packing density. The ethyl acetate fraction (Ds-II)
was dissolved in a suitable solvent, inserted directly
at the top of the column or on the absorbent layer,
sucked slowly into the package by placing it. The
columns were eluted by n-hexane, ethyl acetate and
ethanol with a gradient system. The columns sucked
to dry and every fraction was collected in the bottle.
Result of these separation with VLC obtained 19
fractions.
The fractions of VLC separation were analyzed
by TLC. Eluate which have the same pattern spots
appearance on TLC are combined as one fraction
then concentrated using rotary evaporator. Based on
their similarity spot pattern, five subfractions were
obtained (Ds-II-A, Ds-II-B, Ds-II-C, Ds-II-D and
Ds-II-E).
Separation of Ethyl Acetate Fraction of Mengkudu Fruit (Morinda citrifolia Linn.) and Its Antidiabetic Activity by Glucose Tolerance
Method on Mice
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