Download Isolation of Chemical Compounds Fraction 8 in Ethyl Acetate Phase

Isolation of Chemical Compounds Fraction 8
in Ethyl Acetate Phase from Metanol Extract in Binahong Leaves
(Anredera cordifolia (Ten) Steenis)
Ratna Djamil, Henri Marjuandi
Faculty of Pharmacy Pancasila University, Jakarta 12640, Indonesia
[email protected]
ABSTRACT
Binahong is one type of plants whose leaves are used as medicine. From previous research, it is
reported that ethyl acetate phase from methanol extract in binahong leaves contain secondary
metabolites such as saponin, steroids, triterpenoids, and flavonoids. Ethyl acetate phase from
methanol extract of binahong leaves also have bioactivity toward the larvae of Artemia Salina
Leach. This research aims to determine the content of chemical compounds fraction 8 in ethyl
acetate phase from methanol extract in binahong leaves that is obtained through vacuum liquid
chromatography using dichloromethane-isopropanol and methanol as the eluent. It is obtained
nine fractions (fraction 1 – fraction 9) which then fraction 8 is refractionated by column
chromatography using methanol as eluent that acquires five fractions (fraction 8.1 – fraction 8.5).
Later on, through preparative high performance liquid chromatography using methanol-water as
eluent, fraction 8 obtains three peaks. The first peak is identified by UV-VIS, FTIR, and GC-MS.
The result exhibits the maximum isolate absorption at 264 nm from UV-VIS, bears the group of
C=C and NH in FTIR, and according to GC-MS analysis the isolate of fraction 8.4.1 contains
adenine with 97% similarity.
Keywords: Binahong (Anredera Cordifolia (Ten.) Steenis), Amino acid, Adenine.
INTRODUCTION
Anredera is one species of the Basellaceae has been recognized to have many benefits for
treating various diseases. Familia Basellaceae have various species such as Anredera baselloides
(Kunth) Baill, Anredera cordifolia (Ten.) Steenis, Anredera diffusa (Moq.), Anredera
The International Biopolymeric Micro/Nanoparticles for Drug an Protein Delivery , Faculty of Pharmacy, Pancasila
th
University, October 26 ,2013
leptostachys (Moq.) Steenis, Anredera spicata, Anredera vesicaria, Anredera cumingii, and
others.
Binahong is the Indonesian names for Anredera cordifolia, From previous researches,
binahong was identified to have several secondary metabolites such as flavonoids, saponins,
steroids, triterpenoids, and coumarins that have various bioactivity for human benefits.
This research will be delivered as a discovery of fraction 8 in ethyl acetate phase from
methanol extract in binahong leaves. The molecular structure of compounds is determined based
on UV-VIS, FTIR, and GC-MS.
METHODS
General Experimental Procedure
Ethyl acetate crude extract from the binahong leaves is fractionated by vacuum liquid
chromatography, column chromatography, and thin layer chromatography. The result is isolated
furthermore by preparative high performance liquid chromatography. The obtained isolate is
identified by UV-VIS, Fourier Transform Infra-Red (FTIR), and Gas Chromatography – Mass
Spectrophotometer.
Extraction and Isolation
Prior to fractionation of ethyl acetate phase from methanol extract of the leaves, thin layer
chromatography analysis is conducted with dichloromethane-isopropanol and methanol as
eluent.
Ethyl acetate fraction is refractionated by Vacuum Liquid Chromatography (VLC) using silica
gel H60 and dichloromethane-isopropanol and methanol as gradient. The produced fractions are
thickened by rotary evaporator vacuum until it forms crude.
Fraction 8 from VLC procedure is refractionated by sephadex column chromatography to
obtained a more simplified fraction. The result with similar chromatogram is combined together.
The purification of isolate is performed by High Performance Liquid Chromatography
(HPLC) at 254 nm. Sample is diluted in methanol, centrifuged and injected to HPLC until certain
peaks are acquired.
The final isolate is evaporated from methanol by rotary evaporator vacuum and is frozen by
freeze dryer for 48 hour.
The dried powder of isolate from fraction 8 is identified with UV-VIS, FTIR, and GC-MS to
determine the chemical compounds molecular structure.
The International Biopolymeric Micro/Nanoparticles for Drug an Protein Delivery , Faculty of Pharmacy, Pancasila
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University, October 26 ,2013
RESULTS AND DISCUSSION
From the extracted binahong leaves (Anredera cordifolia (Ten.) Steenis) with ethyl acetate
adenine is found in the compound. This compound is obtained through several stages ofisolation
include partitioning,fractionationand variouschromatographic techniques.
Fractination by VLC obtains nine fractions and fraction 8 is chosen to be isolated furthermore
by column chromatography. In column chromatography, 350 fractions are earned and then
combined based on their same chromatogram. Afterwards, the simplified fractions go through
refractination by HPLC process. In HPLC process fraction 8.4 is indicated to have the dominant
absorbent peaks.
Fraction 8.4 in preparative HPLC obtains 3 peaks. Fraction 8.4.1 at Rt 4.8 minutes, fraction
8.4.2 at Rt 8.9 minutes, and fraction 8.4.3 at Rt 15.8 minutes (figure 1).
8.9
[Au]
1.4
15.8
C:\Clarity\D.Binahong Ratna\Data\FRAKSI 8.5 11_31 26-Jan-2011
1.2
1.0
0.6
23.0
23.5
25.4
20.7
22.1
19.8
17.2
13.7
14.4
14.8
12.7
10.5
7.1
2.8
4.0
1.6
1.8
2.0
0.2
18.8
0.4
18.0
4.8
voltage
0.8
0.0
0
5
10
15
Time
20
25
30
[min.]
Figure 1
Figure 2
Figure 1
Fraction 8.4.1 is identified by UV-VIS spectrophotometer at wave length 600nm-200nm and
exhibits spectrum with maximum absorbent 0.820 at 264.0 nm wave length (figure 2).
According to FTIR identification indicates functional groups stretched C=C aliphatic,
aromatic, and C=N at wave 1660 cm-1 , also the presence of amine group (NH) at wave 3640 cm1
(figure 3).
Figure 3
Figure 1
The International Biopolymeric Micro/Nanoparticles for Drug an Protein Delivery , Faculty of Pharmacy, Pancasila
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University, October 26 ,2013
Abundance
TIC: BINAHONG F1.D
5.77
5.80
5.92
140000
5.98
12.05
130000
120000
6.23
110000
9.98
100000
90000
80000
70000
60000
50000
40000
30000
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
Time-->
Figure 4
Figure 1
A bundanc e
S c a n 3 2 2 ( 5 . 7 7 7 m in ) : B I N A H O N G F 1 . D
135
9000
8000
7000
6000
5000
4000
108
3000
2000
54
1000
66
81
92
119
0
40
60
80
100
147
120
140
207
163
160
180
281
200
220
240
260
280
200
220
240
260
280
m / z -->
A bundanc e
# 3 0 0 5 8 : A d e n in e
135
9000
8000
7000
6000
5000
4000
108
3000
54
2000
1000
66
43
81
92
0
40
60
80
119
100
120
140
160
180
m / z -->
Figure 5
Figure 1
Identification with gas chromatography is aimed to determine molar mass from chemical
compound and its similarity by Willey database. The chromatogram result from fraction 8.4.1
exhibits that the isolate is pure. The indicated chemical compounds content in fraction 8.4.1 can
be seen in (figure 4).
Adenine has the molecular formula C5H5N5 with 135 as its molar mass. It is identified as
adenine because it has similar Rt with adenine which are 5.77 minutes, 5.81 minutes, 5.92
minutes, 5.98 minutes and 6.23 minutes, also it’s calculated that fraction 8.4.1 has 97% similarity
with adenine. Fraction 8.4.1 isolate at 5.7 minutes compared with adenine with library Willey is
identified with mass spectrum which result can be seen on figure 5.
structure of adenine
Figure 1
The International Biopolymeric Micro/Nanoparticles for Drug an Protein Delivery , Faculty of Pharmacy, Pancasila
th
University, October 26 ,2013
CONCLUSION
Ina study ofleafbinahong(Anredera cordifolia) have successfullyisolatedanadenincompound
from fraction 8.4.1 and exhibits molecular structure through various spectrophotometric and
chromatography procedure of UV-VIS, FTIR, GCMS, and preparative HPLC.
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The International Biopolymeric Micro/Nanoparticles for Drug an Protein Delivery , Faculty of Pharmacy, Pancasila
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University, October 26 ,2013