METHOD Collection of Plant Material

CHARACTERIZATION COMPOUND FROM EXTRACT CHLOROFORM

LEAVES

M. umbellata

_{(Houtt.) Stapf var. Degrabrata K. AND}

TEST ANTIHYPERGLYCEMIC ACTIVITY

Imran

1

, Nunuk Hariani S.

1

, and Hanapi Usman

1

1

Chemistry Department, Science Faculty, Hasanuddin University, Makassar, South Sulawesi,

90245

Abstrak.

Penelitian karakterisasi senyawa dari ekstrak kloroform daun

M. umbellata

(Houtt.) stapf

var. degrabrata K. dan uji aktivitas antihiperglikemik telah dilakukan. Penelitian ini bertujuan

untuk mengisolasi dan mengkarakterisasi senyawa dari ekstrak kloroform serta menentukan

konsentrasi optimum dari ekstrak kloroform dalam menurunkan kadar glukosa darah. Isolasi

senyawa dari ekstrak kloroform dilakukan dengan cara maserasi, fraksinasi, dan pemurnian.

Berdasarkan hasil analisis spektroskopi IR dan NMR, senyawa yang berhasil diisolasi dari

penelitian ini adalah

β

-

sitosterol dan senyawa asam lemak yang terdiri dari 25 atom karbon.

Pengujian toleransi kadar glukosa, konsentrasi 6% memperlihatkan penurunan kadar glukosa yang

hampir setara dengan obat glibenklamid yang dijadikan pembanding.

Kata kunci: β

-sitosterol, ekstrak kloroform, Melochia umbellata, Uji toleransi kadar glukosa.

Abstract

. A research about characterization of the chloroform extract of leaves of

M. umbellata

(Houtt.) stapf var. degrabrata K. and activity test as anti hyperglycemic has been done. This study

aimed to isolate and characterize compounds from chloroform extract and determine the optimum

concentration of chloroform extract in reducing blood glucose levels. Isolation compound from

chloroform extract has been done by maceration, fractionation, and purification. Based on the data

of IR and NMR spectroscopic, compounds isolated from this research were

β

-

sitosterol and fatty

acid compound consisting of 25 carbon atoms. The glucose tolerance test, concentration of 6%

showed a decrease in glucose levels almost equivalent with drug glibenclamide as comparator.

Keywords:

β

-sitosterol, chloroform extract, Glucose tolerance test, M. umbellata

1

INTRODUCTION

Exploration of medicinal plants as a

source of material have been carried out. Many

plants that have efficacy as traditional

medicine, one of which is paliasa. Paliasa is one

of the plants of the family Malvaceae used by

the society, especially in South Sulawesi as a

drug capable of treating liver disease,

hypertension, diabetes, high cholesterol, and

hepatitis by drinking boiled water leaves

(Raflizar et al., 2006).

Paliasa known in two different plant

species that

Kleinhovia hospita

L. and

M.

umbellata

(Houtt.) Stapf.

M. umbellata

(Houtt.)

Stapf consists of two varieties, namely

Stapf var. degrabarata K. showed significant

improvement of liver function. Similarly of the

toxicity against

Artemia salina

larvae, the

toxicity

of

methanol extract

of leaves

M. umbellata

(Houtt.) Stapf var. degrabata K.

showed

highest.

Bioactivity

screening

conducted by Nuvita (2006) from the methanol

extract of free radicals and BHT as controls also

showed that the leaves of

M. umbellata

(Houtt.)

Stapf var. degrabrata K. has a higher

antioxidant effect compared with the other plant

species of paliasa.

Based on the literature, chemical

compounds in the plants of

M. umbellata

(Houtt.) Stapf var. degrabrata K. are essential

oils, triterpenoids, alkaloids, and flavonoids

(Heyne, 1987); and saponin compounds and

antarkuinon. The lattest two compounds can be

prevent liver inflammation in mice (Lalo and

Tayeb, 2003).

Research on paliasa have been done.

There

are

many

secondary

metabolites

compounds that have been isolated from this

plant, either from the extract with a polar

solvent, non-polar or semi-polar solvents such

as

chloroform.

Two

quinolone

alkaloid

compounds have been isolated from the

chloroform extract

M. umbellata

(Houtt.) Stapf

var.

degrabrata

K.

namely

7,8-epoxy

melochinon (3) (Erwin, 2010) and 9,10-epoxy

melochinon (4) (Ridhay, 2012). Melochinon

was active against leukemia cancer cells P-388

with LC

50

= 0.83 µg/mL. Secondary metabolites

steroids also isolated from the chloroform

extract

Kleinhovia hospita

Linn. (Soekamto,

2008) whereas for the group of terpenoid

compounds 3-hydroxy-12-oleanan-28-oats (5)

was also isolated from the chloroform extract of

K. hospita Linn. (Purwaningsih, 2010).

Further studies of bioactive compounds

contained in extracts chloroform

M. umbellata

(Houtt.) Stapf var. degrabata K. has done.

that can lower blood glucose levels.

METHOD

Collection of Plant Material

Leaves of plants

M. umbellata

(Houtt.)

Stapf var. degrabata K. collected on April 2012

were

taken

in

Hasanuddin

University,

Tamalanrea Campus, Jl. Perintis Kemerdekaan

Km. 10, Makassar, South Sulawesi. This plant

is identified by Herbarium Bogoriense, Center

of Research and Development Biology, LIPI

Bogor with specimen number BO-1912171.

Materials

To

extraction,

fractionation,

and

recrystallization used solvent with the quality of

pure analys (p.a) and technis such as

chloroform, methanol, n-hexane, ethyl acetate

and acetone. In the process of chromatography

used silica gel Merk. 7730 catalog for vacuum

column chromatography (KKV), silica gel

chromatography (KKT), silica gel Merk. 7733

catalog for gravity column chromatography

(KKG), and for thin-layer chromatography

(TLC) performed

with silica

gel-coated

aluminum plate Brand Keselgel 60 F254

scales

(O'hauss),

scissors,

oral

syringe,

distillation, rotary evaporator, UV lamps, tools

for thin layer chromatography, vacuum column

chromatography,

press

column

chromatography,

gravity

column

chromatography,

and

tools

glasses

are

commonly used in laboratories. For the

measurement of the melting point used a Fisher

John. While IR spectrometer for the analysis of

variants with Shimadzu FTIR 8501, JEOL JMN

A 5000 working on 500.0 MHz for

1

H-NMR

Subsequently fractionated by liquid-liquid

extraction with n-hexane and chloroform.

Chloroform extracts were evaporated to dryness

obtained is then weighed and then fractionated

by column chromatography vacuum, press, and

gravity by using the appropriate eluent. Further

isolates obtained purified by recromatography,

crystallization, and recrystallization. Structure

determination is based on the data of IR spectra

and

1

H and

13

C-NMR.

Glucose Tolerance Test

Chloroform fractions obtained tested

their toxicity to mice adult male (

Mus

musculus

). The mice had been treated

previously and had been given the originator of

diabetes. Concentration varied dosage. The data

obtained and recorded for further analysis.

RESULT AND DISCUSSION

In

isolation

study

of

secondary

metabolites from chloroform extract of the

leaves

M. umbellata

(Houtt.) Stapf var.

degrabata K. obtained two compounds. The

chemical structure of the two compounds

determined by IR and NMR spectroscopic data

of the first compound is suspected

β

-sitosterol

and the second compound suspected as fatty

acid compound consisting of 25 carbon atoms.

Extraction

Dried leaves powder

M. umbellata

(Houtt.) Stapf var. degrabata K. macerated with

methanol 1 x 24 hours 4 times resulted 39.1 g

of methanol extract of dark green. Then, the

methanol extract was partitioned by

liquid-liquid extraction with n-hexane, chloroform,

and ethyl acetate that produce n-hexane

extracts, chloroform extract, and ethyl acetate

extract. Further isolation and purification of the

chloroform extract.

Isolation and Purification

15.99 g of chloroform extract was

fractionated

by

vacuum

column

chromatography

with

eluent

n-hexane,

n-hexane: ethyl acetate, ethyl acetate, acetone,

and methanol with increased the polarity. The

process

begins

with

a

search

eluent

Figure 1

. Chromatogram of TLC analysis of

chloroform extract, (a) the results of

fractionation

KKV;

(b)

the

combined fractionation.

Furthermore, the purity test through

analysis with 2-dimensional TLC with eluent

[n-hexane (7): ethyl acetate (3)] also showed a

stain that appears after heated but not fluoresce

under UV light of short wave and long wave as

shown in Figure 3 which indicates that the

crystal is a pure isolate expressed as compound

1 Test groups to these compounds showed that

steroids purplish blue color after the addition of

sulfuric acid and acetic anhydride.

Figure 2.

Chromatogram of TLC analysis of

compound 1, (a) [n-heksan(9) : etil

asetat(1)];

(b)

[n-heksan(8,5)

:

CHCl

3

(1)

:

metanol

(0,5)];

(c) [Aseton(2) : CHCl

3

(8)].

596 mg Fraction A further fractionated

by press column chromatography with eluent

n-hexane: ethyl acetate, ethyl acetate, acetone,

and methanol yield 28 major fractions as in

Figure 4.

Figure 3.

Chromatogram of TLC analysis 2

dimention compound 1 after heated

Figure 4.

Chromatogram of TLC analysis

fraction A, [

n-

heksan(9) : etil

asetat(1)].

Fraction

4

was

followed

by

recrystallization with warm acetone. The results

were then analyzed recrystallization fraction A4

TLC with eluent ethyl acetate (2): n-hexane (8)

and showed a single stain was also supported by

the results of measurements of the melting point

of 67-69 °C (Figure 5).

Figure 5.

Chromatogram of TLC analysis

compound 2 under UV long wave

Compound 1

Compound 1 in the form of a white

powder with a weight of 10.9 mg showed a

positive steroid test with Liebermann-Burchard

reagent. This compound does not fluoresce

under UV light which indicates that these

compounds do not have conjugated double

bonds. This is consistent with the IR spectrum

of the data does not show any absorption band

in the region 3100-3000 cm

-1

(Lambert et al.,

1998) as the wavelength for the aromatic group.

IR spectral data showed only aliphatic

CH absorption band area at 2956, 2926, and

2852 cm

-1

which is supported by the presence

of absorption at 1462 and 1377 cm

-1

for CH

2

and CH

3

. Absorption band at wave number

Table 1.

IR spectrum data compound 1

Groups

Function

Wavelength (cm

-1

)

Compound 1

β

-sitosterol

(Sari, 2011)

Wave numbers in the IR spectrum of compound

1 did not show any significant difference with

the IR spectra of compound

β

-sitosterol

standard, so it can be concluded that compound

1 is a

β

-sitosterol.

Figure 6.

Structure compound 1 (

β

-sitosterol)

Compound 2

Compound 2 in the form of a white

powder with a weight of 29.2 mg and a melting

point of 67-69

o

C. IR spectroscopy showed

absorption band at a wavelength of 3448 cm-1

which indicates the presence of OH groups

which are supported by the bending of CO at a

wavelength of 1172 cm

-1

. Area 2918 cm

-1

and

2848 cm

-1

indicating the presence of aliphatic

CH bending backed by methylene (CH

2

) and

methyl (CH

3

) at a wavelength of 1467 cm

-1

and

1379 cm

-1

_{. Absorption at region 1636 cm}

-1

indicates the presence of an olefin group (C=C).

IR spectral data of compounds 2 can be seen in

the Table 2.

Table 2.

Data spectrum IR compound 2

Groups function

Wavelength (cm

-1

)

O-H

3448

H-NMR spectroscopic data are summarized in

Table 3 shows the 5 peaks. The first peak with a

chemical shift of 0.88 ppm is a triplet form CH

3

group that interacts with the binding carbon 2

protons (CH

2

). Chemical slider with a

multiplicity of 1.29 ppm multiplet with a very

high intensity, demonstrating some of CH

2

groups in the wave number that has the same

environmental conditions that led to the second

peak shows a very high peak. The third peak

with a chemical shift of 1.61 ppm multiplet

shaped and fourth peaks with a chemical shift

of 2.29 ppm triplet shaped carbon interacts with

oxygen binding. Fifth peak is the peak with a

chemical shift of 4.05 ppm which has a triplet

multiplicity is far away from the TMS peak is

caused by a group that binds oxygen.

13

C-NMR spectral data of compound 2

(Table 3) showed nine carbon signals consisting

of a methyl group with a chemical shift of

14.31 ppm and 5 methylene on the chemical

shift of 22.89; 25; 24; 26.14; 28.86, and

29.85 ppm, and 2 metin groups on the chemical

shift of 32.12 and 34.63 ppm and also there is

the carbon bonded to the OH group on the

chemical shift of 64.59 ppm.

Bioactivity Test

Effect of leaves extract of CHCl

3

M. umbellata

(Houtt.) Stapf var. degrabata K. in mice for

3 hours showed a decrease in blood glucose levels (Table 4).

Table 4.

Average blood glucose levels of mice caused by inject of the chloroform extract of leaves

of

M. umbellata

(Houtt.) Stapf var. K. degrabrata with control and comparison.

Treatment

Blood glucose

levels after

induction

(mg/dl)

Blood glucose levels every

hour after inject of the test

preparation treatment

(mg/dl)

The rate of

decline in

glucose levels

(mg/dl. jam)

1

2

3

Na-CMC

1 % b/v

202,00

132,00

141,00

101,67

29,20

Glibenklamid

0,0195 mg/ml

237,00

137,00

90,67

73,67

53,63

Chloroform extract

M. umbellata

2% b/v

233,67

136,33

129,67

108,67

38,17

Chloroform extract

M. umbellata

4% b/v

248,67

172,67

125,67

105,00

47,80

Chloroform extract

M. umbellata

6% b/v

258,33

129,67

119,67

94,67

50,10

The data of Table 4 obtained diagrams mice blood glucose levels decrease as the effect of

chloroform extract

M. umbellata

(Houtt.) Stapf var. degrabrata K. leaves accompanied by a

negative control and comparison as shown in Figure 7 below:

Hyperglycemia was defined as fasting

glucose levels higher than 110 mg/dL.

Hyperglycemia arises because the absorption of

glucose into cells and inhibited metabolism

disturbed while Diabetic Mellitus is a chronic

disorder that is specifically related to glucose

metabolism in the body, but the metabolism of

fats and proteins are also affected.

Chloroform extract testing is done to

prove the pharmacological effects of plant leave

M. umbellata

(Houtt.) Stapf var. degrabrata K.

as a drug that can treat diabetic situation and

determine the optimum dose. This study used a

chloroform extract of the plant with a

concentration of 2%, 4%, and 6% w/v.

Na-CMC suspension 1% w/v was used as a

negative control and a positive control used

glibenclamide 0.0195 mg/ml.

Antihyperglycemic

effect

was

determined using the glucose tolerance and the

treatment condition was kept uniform with the

aim to reduce the factors that may affect the

outcome of the experiment.

Animals used in this study were mice

(

Mus musculus

) male sex in a healthy condition.

Female mice were not used because of their

hormonal system is unstable compared with

male

mice.

Nevertheless, the biological

variation factors of the test animals can not be

removed so it can affect research results.

Before treatment, the mice were fasted

beforehand to avoid the influence of food at the

time of measurement of blood glucose levels

and to increase the speed of drug absorption.

Measurement of blood glucose levels in

mice were performed for 3 hours with 1 hour

intervals. It is based on literature indicating that

the absorption of glucose in the body takes

about 30-60 minutes and will decline after 2-3

hours, then to see a decrease in glucose levels

more obvious use for a period of 3 hours after

administration of the test preparation (Syahrul

2010 ).

After the measurement of blood

glucose levels for 3 hours, from the diagram it

appears that the higher the concentration of

chloroform extract

M. umbellata

(Houtt.) Stapf

var. degrabrata K. the rate of decrease in blood

glucose levels also increase. The biggest

influence is shown by a comparison giving

chloroform extract

M. umbellata

(Houtt.) Stapf

var. degrabrata K. at a concentration of 6% w/v.

CONCLUSION

compound having 25 carbon atoms.

Chloroform extracts of the leaves of

plants Melochia umbellata (Houtt.) Stapf var.

K. degrabrata can lower blood glucose levels,

which is consistent with the use of this plant in

ethnobotany.

ACKNOWLEDGEMENTS

Thank you so much to DIKTI that has

supported fund this research through PKM-P

and also to Prof. Dr. Nunuk Hariani S., MS. as

the main supervisor and Prof. Dr. Hanapi H.

Usman, MS. as the first supervisor.

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Penentuan Struktur Molekul

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degrabrata

K.

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Hasanuddin

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