Separation of Ethyl Acetate Fraction of Mengkudu Fruit (Morinda
citrifolia Linn.) and Its Antidiabetic Activity by Glucose Tolerance
Method on Mice
Shintia Lintang Charisma
1,3*
, Yasmiwar Susilawati
1
, and Ahmad Muhtadi
2
1
Department of Biological Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran
2
Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran
Jl. Raya Bandung Sumedang Km.21 Jatinangor, West Java, Indonesia
3
Faculty of Pharmacy, Universitas Muhammadiyah Purwokerto, Jl. Raya Dukuh Waluh, Purwokerto, Central
Java, Indonesia
Keywords: Separation, Morinda citrifolia Linn., Antidiabetic, Glucose tolerance method
Abstract: Mengkudu (Morinda citrifolia Linn.) is one of the medicinal plant that contains alkaloids, carbohydrates,
flavonoids, glycosides, phenols, proteins, amino acids, saponins, steroids, tannins, antraquinone and
terpenoids. Traditionally, mengkudu (M. citrifolia Linn.) fruit has been used for antidiabetic effect.
Previous research showed that ethyl acetate fraction gave significant activity (54.29 %) at dose 1200 mg/kg
body weight on male wistar rats.
The aims of this study were to separate ethyl acetate fraction from mengkudu fruit by Vacuum Liquid
Chromatography method and to study antidiabetic activities on male mice by glucose tolerance method.
This research used separation guided by activity method which ethyl acetate fraction separated by Vacuum
Liquid Chromatography and gave five subfractions (Ds II-A, Ds II-B, Ds II-C, Ds II-D and Ds II-E). The
five subfractions then tested the antidiabetic activity at a dose of 150 mg/kg body weight on male mice by
glucose tolerance method. Blood glucose level measured at 30, 50, 90, 120 and 150 minutes after
administration of subfractions.
Ethyl acetate subfractions from mengkudu fruit (Ds II-A, Ds II-B, Ds II-C, Ds II-D and Ds II-E) showed
antidiabetic activity with percentage of reduction relative blood glucose levels 150 minutes after sample
administration were 39.11%, 52.85%, 35.31% 43.55%, and 33.78% respectively. These research showed
that Ds-II-B subfraction indicate the highest antidiabetic activity by glucose tolerance method. Rutin,
quercetin and scopoletin compounds indicate antidiabetic activity from ethyl acetate subfractions of
mengkudu fruit.
1 INTRODUCTION
Diabetes mellitus is a type of chronic disease that
characterized by elevating blood sugar levels
(Dipiro, 2015). World Health Organization (WHO)
has established several criteria that indicate diabetes
mellitus, including fasting plasma glucose (no
caloric intake of at least 8 hours) ≥126 mg/dL or 2
hours plasma glucose ≥200 mg/dL or random
plasma glucose levels at ≥200 mg/dL (IDF, 2012).
Diabetes mellitus become an important for concern
on health issue because the prevalence of diabetes
has been steadily increasing over the past few
decades (WHO,2016). The incidence rate of diabetes
in Indonesia nearly 6.9 % during 2013 (Depkes RI,
2013).
Mengkudu (Morinda citrifolia Linn.) has a long
been used as medicinal plants in many countries,
especially people in the continent of Polynesia,
South Asia, Southeast Asia, parts of Australia and
the Caribbean continents (Pawlus and Douglas,
2007). Based on the literature, mengkudu has been
used as medicinal plants for diabetes treatment
(Nerurkar, 2015).
Chemical compounds reported on mengkudu
plants were polysaccharides, fatty acids, glycosides,
iridoid, triterpenes, anthraquinones, coumarins,
flavonoids, phytosterols, carotenoids and volatile
compounds (Ahmad, et.al., 2016). Mengkudu fruit
contains caprylic acid, hexanoic acid, caproic acid,
vitamin C, vitamin E, niacin, asperulosidic acid,
quercetin, 2,6-di-O- (b-D-glucopyranosyl 1-O-
octanoyl-b-D-glucopyranose, damnacanthal and
americanin A (Assi, et.al., 2015).
218
Charisma, S., Susilawati, Y. and Muhtadi, A.
Separation of Ethyl Acetate Fraction of Mengkudu Fruit (Morinda citrifolia Linn.) and Its Antidiabetic Activity by Glucose Tolerance Method on Mice.
DOI: 10.5220/0008360302180223
In Proceedings of BROMO Conference (BROMO 2018), pages 218-223
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
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
219
2.5 Antidiabetic Activity of
Subfractions
2.5.1 Experimental Animals Preparation
Animal experiments were reviewed and approved by
Research Ethics Comittee Universitas Padjadjaran
(approval no. 132/ UN6.KEP/EC/ 2018). Swiss
Webster male mice weighing 20-30 g procured from
Center for Life Sciences Institut Teknologi
Bandung. The mice were adapted in the cage for
approximately 7 days. During the adaptation, mice
were given drink and food in the form of special
feed of livestock with adequate nutrient content.
2.5.2 Experimental Design
The glucose tolerance method was performed in
overnight fasted (16 hours) normal mice, ad libitum.
Mice were divided into seven groups, each
consisting of four mice were administered 2% (w/v)
PGA, glibenclamide 0.65 mg/kgBW, and
subfractions (Ds-II-A, Ds-II-B, Ds-II-C, Ds-II-D and
Ds-II-E) 150 mg/kgBW, respectively. Glucose (2
g/kgBW) was fed 30 min after the administration of
subfractions. Blood sample was collected at 0, 30,
60, 90, 120 and 150 minutes of glucose
administration and blood glucose level was
estimated using electronic glucometer (Accu-Chek
Active Glucometer) and glucostrips (Accu-Chek
Active Diabetic Test Strips).
2.6 Data Analysis
Data from blood glucose measurement, further
analyzed by statistics using variant analysis
(ANAVA) design with fixed model at level of 0.05
and 0.01 and further analyzed by Tukey test method
at level 0.05.
3 RESULTS
This plant was taxonomically determined at Plants
Taxonomy Laboratorium, Faculty of Mathematics
and Natural Sciences, Universitas Padjajaran as
Morinda citrifolia Linn. of the family Rubiaceae,
collection number 444/HB/09/2017.
3.1 Extraction and Preliminary
Phytochemical Analysis
The percentage yield of ethanol extract was found to
be 22.46% w/w. The qualitative phytochemical
analysis of the ethanolic extract showed the presence
of saponins, triterpenoids, flavonoids, alkaloids and
phenolic compounds.
3.2 Fractionation of Ethanolic Extract
by Liquid-Liquid Extraction (LLE)
The yield of fractions obtained by LLE method
were n-hexane fraction (Ds-I) 103.18 g (5.15%);
ethyl acetate fraction (Ds-II) 54.18 g (2.85%) and
water fraction (Ds-III) 334.86 g (16.74 %).
3.3 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 (a) 254 nm
and (b) 366 nm then detected by using 10% H
2
SO
4
spray reagent (Figure 1). Based on TLC analysis,
there were 6 spots have detected with Rf values Rf
1
= 0.125; Rf
2
= 0.225; Rf
3
= 0.425; Rf
4
= 0.6; Rf
5
=
0.7 and Rf
6
= 0.825 respectively.
Figure 1. TLC profile of ethyl acetat fraction (Ds-II) after
being developed with chloroform : ethyl acetate :
methanol (7:2:1) as mobile phase. TLC plate were
visualized under UV light at wavelength (a). 254 nm and
(b) 366 nm.
3.4 Fractionation of Hs-II fraction by
Vacuum Liquid Chromatography
(VLC)
A total of 50 g fraction (Ds-II) was separated by
Vacuum Liquid Chromatography (VLC). The
principle of separation in VLC is based on
adsorption chromatography. Higher polar
compounds will be more attached to silica gel and
(a)
(b)
2
1
3
6
5
4
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
220
more retained in the VLC column. Polar silica gel
will bind compounds that are relatively more polar.
Compounds with lower levels of polarity will come
out first carried by eluen so that the separation of the
compound occurs based on differences in polarity.
The mobile phase used in VLC is n-hexane:
EtOAc: EtOH with gradient system. From VLC
obtained 19 fractions, which then monitored the
pattern of spot through TLC. This monitoring was
done to see the similarity of pattern to be combined
into subfractions.
Based on the similarity of spot pattern, five
subfractions were obtained, namely Ds-II-A
(fractions 1-3), Ds-II-B (fractions 4-6), Ds-II-C
(fraction 7-9), Ds-II-D (fractions 10-12) and Ds-II-E
(fractions 13-16). The weight of Ds-II-A, Ds-II-B,
Ds-II-C, Ds-II-D and Ds-II-E were obtained 2.65 g,
10.89 g, 13.65 g, 11.94 g and 2.58 g respectively. So
that the yield obtained of each subfraction were
0.118%, 0.488%, 0.612%, 0.535% and 0.116%
respectively.
3.5 Antidiabetic Activity of Subfraction
Antidiabetic activity of mengkudu fruit subfractions
were performed by glucose tolerance method. Mice
were divided into seven groups, each consisting of
four mice were administered 2% (w/v) PGA,
glibenclamide 0.65 mg/kgBW, and subfractions (Ds-
II-A, Ds-II-B, Ds-II-C, Ds-II-D and Ds-II-E) 150
mg/kgBW, respectively. Glucose (2 g/kgBW) was
fed 30 min after the administration of subfractions.
Blood sample was collected at 0, 30, 60, 90, 120 and
150 minutes of glucose administration and blood
glucose level was estimated using electronic
glucometer (Accu-Chek Active Glucometer) and
glucostrips (Accu-Chek Active Diabetic Test
Strips).Based on Figure 2, the relative blood glucose
levels of mice reached a peak at 30 minutes after
oral administration of glucose (2 g/kgBW) and
gradually decrease in 150 minutes.
Figure 2. Antidiabetic Activity of Mengkudu Fruit Subfractions
Subfractions of mengkudu fruit at dose 150
mg/kgBW showed antidiabetic activity at minute 30
until minute 150 after administration.The best
antidiabetic activity of subfractions (Ds-II-A, Ds-II-
B, Ds-II-C, Ds-II-D and Ds-II-E) at a dose 150
mg/kgBW was demonstrated by Ds-II-B subfraction
with percent reduction relative blood glucose levels
at minute 30, 60, 90, 120 and 150 were 40.92%,
56.87%, 35.72%, 45.99% and 52.85% respectively.
Percentage reduction relative blood glucose levels
each group shown in Table 2 and Figure 3.
Separation of Ethyl Acetate Fraction of Mengkudu Fruit (Morinda citrifolia Linn.) and Its Antidiabetic Activity by Glucose Tolerance
Method on Mice
221
Table 2. Percent Reduction Relative Blood Glucose Levels of Mengkudu Fruit Subfractions
Figure 3. Percent Reduction Relative Blood Glucose Levels of Mengkudu Fruit Subfractions
Based on the analysis of Anava with 95%
confidence level can be concluded that there was a
significant difference between the treatment given to
decreased blood glucose levels of mice. Then a
follow-up test with Tukey was conducted to
determine the significant differences between the
positive control group, Ds-II-A, Ds-II-B, Ds-II-C,
Ds-II-D and Ds-II-E compared with the negative
control group. The results obtained that the decrease
in relative blood glucose levels of the positive
control group and all the test groups significantly
different compared with the negative control group
(sig> 0.05).
Antidiabetic activity of ethyl acetate subfractions
caused by enrichment of phytochemical constituents
in mengkudu fruit such as caprylic acid, hexanoic
acid, caproic acid, vitamin C, vitamin E, niacin,
asperulosidic acid, quercetin, 2,6-di-O- -D-
glucopyranosyl 1-O-octanoyl- β-D glucopyranose,
damnacanthal, americanin A, xeronin and scopoletin
(Yashaswini et al., 2014; Assi, et. al., 2015). These
compounds are possible to have antidiabetic activity.
An HPLC analysis from previous study showed
that the ethyl acetate fraction of mengkudu fruit
contained rutin and quercetin from flavonoid grup,
and scopoletin compund from coumarin group
(Pandy, et.al., 2017). These chemical compounds
indicate antidiabetic activity from ethyl acetate
subfractions of mengkudu fruit.
4 CONCLUSION
Ethyl acetate subfractions from Mengkudu (M.
citrifolia Linn.) fruit (Ds-II-A, Ds-II-B, Ds-II-C,
Ds-II-D and Ds-II-E) showed antidiabetic activity
with percentage of reduction relative glucose levels
were 39.11%; 52.85%; 35.31%; 43.55% and 33.73%
at 150 minutes after sample administration.
These research showed that Ds-II-B subfraction
indicate the highest antidiabetic activity by glucose
tolerance method. Rutin, quercetin and scopoletin
compounds indicate antidiabetic activity from ethyl
acetate subfractions of mengkudu fruit. However,
the research for the active antidiabetic compound of
Ds-II-B subfraction needs to be explored further.
Groups
Reduction Relative Blood Glucose
Levels (%)
30'
60'
90'
150'
Positive
Control
39.43
58.77
50.49
58.91
Ds II-A
29.58
48.47
9.07
39.11
Ds II-B
40.92
56.87
35.72
52.85
Ds II-C
36.09
52.72
23.01
35.31
Ds II-D
18.62
21.40
30.10
43.55
Ds II-E
14.19
53.26
36.24
33.73
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
222
ACKNOWLEDGEMENT
The author would like to thank DRPMI Universitas
Padjadjaran and Prof. Dr. Ahmad Muhtadi, MS.,Apt.
for financial support by Academic Leadership Grant
(ALG) scheme in the year of 2017.
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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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