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

, and Ahmad Muhtadi

Department of Biological Pharmacy, Faculty of

Pharmacy, Universitas Padjadjaran

Department of Pharmacology and Clinical Pharmacy,

Faculty of Pharmacy, Universitas Padjadjaran, Indonesia

Faculty of Pharmacy, Universitas Muhammadiyah

Purwokerto, Jl. Raya Dukuh Waluh, Purwokerto,

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

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).

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

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: 10.5220/0009844900002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

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

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).

2.5 Antidiabetic Activity of

Subfractions

2.5.1 Experimental Animals Preparation

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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

spray reagent (Figure 1). Based on TLC analysis,

there were 6 spots have detected with Rf values Rf

= 0.125; Rf

= 0.225; Rf

= 0.425; Rf

= 0.6; Rf

0.7 and Rf

= 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

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

(a)

(b)

Separation of Ethyl Acetate Fraction of Mengkudu Fruit (Morinda citrifolia Linn.) and Its Antidiabetic Activity by Glucose Tolerance

Method on Mice

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.

Table 2. Percent Reduction Relative Blood Glucose Levels

of Mengkudu Fruit Subfractions

Groups

Reduction Relative Blood Glucose Levels (%)

30'

60'

90'

120'

150'

Positive

Control

39.43

58.77

50.49

53.23

58.91

Ds II-A

29.58

48.47

9.07

13.84

39.11

Ds II-B

40.92

56.87

35.72

45.99

52.85

Ds II-C

36.09

52.72

23.01

23.58

35.31

Ds II-D

18.62

21.40

30.10

47.67

43.55

Ds II-E

14.19

53.26

36.24

37.88

33.73

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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- (β-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.

5. 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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BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity