ISOLATION AND IDENTIFICATION OF CHEMICAL

COMPOUNDS IN n-BUTHANOL EXTRACT FROM

KELADI TIKUS LEAVES (Typhonium flagelliforme
(Lodd.) Blume, Araceae.

Yunahara Farida
1
, Lamsari
1
, Wahyudi P.S
2
, Wahono S
3

1
Faculty of Pharmacy, Pancasila University, Jakarta 12640, Indonesia
2
Department of Chemistry, FMIPA, University of Indonesia, Depok 1624, Indonesia
3
Agency for the Assessment and Apllication of Technology, Indonesia (BPPT)

Abstract
Typhonium flagelliforme (Lodd.) Blume), also known as Keladi tikus,
familia Araceae, is one of the restorative plant for anti cancer. This study was
isolated and identification of chemical compounds from n-buthanol extract
of the plant. This research is continued from previous research which is done
by phytochemical screening, n-buthanol extract was isolated using column
chromatography. The fractions were analyzed by TLC and the toxicity test
using BSLT (Brine Shrimp Lethality Test). The fraction with the smallest LC
50

value was identified by UV-VIS Spectrophotometer, FTIR Spectrophotometer
and Gas Chromatography-Mass Spectrometer. The results showed the
B.4.3.1 isolate is the most active fraction with LC
50
209.89 ppm. Based on
the result of GCMS, in the n-buthanol extract of Typhonium flagelliforme
(Lodd.) Blume) is guess contains flavonoid compounds as dihidroflavonol
groups, flavon, isoflavon and auron

Key words: isolation, Typhonium flagelliforme (Lodd). Blume, n-buthanol
extract, BSLT, identification



Introduction
Traditional herbal is a part of Indonesian
biodiversity that need to be conserved
especially for human welfare. Plants
used as medicine in the healing process
and prevention of disease. Socialization
of the movement "back to nature"
through the public media and health
seminar increasingly encouraged so
that many people are turning to
traditional materials as medicine, which
have proven efficacy and traditional
medicine are considered safety and side
effects than chemical-based medicine.
Keladi tikus (Typhonium flgelliforme
(Lodd) Blume, is a native Indonesian
plant that grows wild and unknown
widely. Based on research and empirical
evidence, this plant has efficacy in
treating some diseases such as ulcers,
wounds, cancer and neutralizing toxicity
of medicine. Therefore many studies
done on T. flagelliforme to know its
activity or to know both of activity and
the content of compounds. Several
chemical constituents had been
identified from T.flagelliforme. The
hexane extract was reported to contain
saturated hydrocarbons and aliphatic
acids (Choo, et.al, 2001); the ethyl
acetate extract was found to contain
aromatic fatty acids (Chen, et.al, 1997);
the root of this plant were reported to
contain phenylpropanoid glycosides,
sterols and a cerebroside which has
antihepatotoxic (Huang, et,al, 2004).
Therefore, further research will be
conducted. The study was conducted in
three phases, there are n-hexane, ethyl
acetate and n-buthanol representing a
different polarity to isolate and identified
active chemical compounds. The focus
of this study are isolation and
identification of n-buthanol extract from
Typhonium leaves. The research include
of screening phytochemicals,
fractionation, isolation by TLC, Column
Chromatography and BSLT assay, and
the identification using spectroscopy
methods.
Methodology
Materials
n-buthanol phase of methanol
extract from T. flagelliforme leaves, silica
gel, methanol, ethyl acetate, chloroform
and cerium sulfate. Equipments: UV-Vis
spectrophotometer FTIR spectrometer,
GC-MS, Buchi Rotavapor and Column
Chromatography
Methods
Phytochemical Screening.
Phytochemical screening to identify
compounds such as flavonoids by the
reduction test (Mg-HCl/amylalcohol),
saponins by the foam formation test,
tannins by the FeCl
3
reagent, quinones
by the NaOH reagents, steroids
/triterpenoids by the Liebermann-
Burchards reagent, coumarins by the
fluorescence test with ammonia and
essential oils by the odor test, based on
the method of Farnsworth
Fractionation. A total of 10.84 g
of the extract was fractionated by column
chromatography using eluent system of
ethyl acetate and methanol (guided by
TLC and BSLT).
Isolation and identification.
Isolation is done by TLC preparative.
Further identification by UV-Vis
spectrophotometer, FT-IR spectrometer
and gas chromatography- mass
spectrometer.

Brine Shrimp Lethality assay

The assay was carried out according to
McLaughlin et. al (1998) and Meyer et al.
(1982) using nauplii of Artemia salina
that are generally considered as a bench
top assay aiming at discovery of
cytotoxic compounds. Sample was
prepared in 3 concentrations 10; 100;
and 1000 ug/ml by dissolving them in
DMSO (not more than 50 l in 5 ml
solution) plus sea water to attain
concentrations. Ten nauplii were added
to each vial. All vials were covered and
stored at room temperature for 24 hours
under the light. Observe the result after
24 hours, count the mortality number of
Artemia salina Leach from each
concentration. Furthermore, calculated
mortality rates or mortality (%) by
comparing the total number of Artemia
salina Leach who dies with the total
number of Artemia salina Leach tested.
LC
50
value was calculated using
regression equations with log
concentration as X (axis) and Y (ordinat)
values as the probit LC
50
value obtained
by calculating the value X from the
equations obtained.
Results and Discussions
The results of phytochemical
screening on n-buthanol extract from
T.flagelliforme leaves contains
flavonoids, tanins and coumarin. The
result of fractionation by column
chromatography I obtained 7 fractions.
The LC
50
values of the brine shrimp
obtained from 7 fractions shown in
Table 1.
Table 1. LC
50
values against Brine Shrimp of
column chromatography I
Fraction Weight (g) LC
50
(ppm)
B.1 0,23 247,34
B.2 0,87 66,39
B.3 1,62 314,89
B.4 2,10 33,67
B.5 2,83 78,70
B.6 1,54 621,76
B.7 1,16 2364,28

Based on BSLT tests obtained the most
active fraction is the 4
th
fraction (B.4)
with LC
50
value was 33.67 ppm, and a
HR
f
value of 29.8.

The most active results of the B.4
fraction is fractionated by column
chromatography using ethyl acetate-
methanol (5:1), obtained 4 fractions.
Test results of toxicity test using BSLT
method that the most active fraction was
the B.4.3 fraction with LC
50
value was
74.00 ppm, and a HR
f
value of 50. The
results shown in Table 2.




Table 2. LC
50
values against Brine Shrimp of
column chromatography II

Fraction Weight (mg) LC
50
(ppm)
B.4.1 60 130,32
B.4.2 80 463,62
B.4.3 260,7 74,00
B.4.4 1300 215,70

The results of the most active fraction
(B.4.3) was fractionated by column
chromatography using a mobile phase
chloroform-methanol (10:1), obtained 2
fractions. The most active fraction is the
2
nd
fraction with LC
50
value was 209,89
ppm with a HRf 60,86 The results shown
in Table 3.
Table 3. LC
50
values against Brine Shrimp of
column chromatography III

Fraction Weight (mg) LC
50
(ppm)
B.4.3.1 15 209,89
B.4.3.2 10 474,80
.
Isolation The B.4.3.1 isolate was
performed using TLC preparative (silica
gel GF
254
), mobile phase chloroform-
methanol (1:5). A blue-ribbon
fluorescence was formed at 366 nm.
The result of TLC preparative shown in
Figure 1.

Figure 1. Result of TLC Preparative of
the B.4.3.1 isolate

Identification. The isolate of B.4.3.1
was identified by UV-Vis
spectrophotometer in methanol. The UV-
Vis spectrum of B.4.3.1 isolate at 230.5
nm and 281.0 nm shown in Figure 2.


Figure 2. The UV-Vis Spectrum of the
B.4.3.1. isolate

Test results two maximum wavelengths
that are characteristic of flavonoids is
supported by the results of
phytochemical screening analysis, the
flavonoids are thought to be flavonoid
groups of flavanon and dihydroflavonol.
The FTIR spectrum of the B.4.3.1 isolate
showed absorption peaks at 3355.91cm
-
, 2923.88 cm
-
, 2852.52 cm
-
,1537,16cm
-1
, 779.19 cm
-
indicates the
presence of OH group, CH stretching,
stretch C = C, C = CH bending, CH
aliphatic (Figure 3).

Figure 3. The FTIR spectrum of the
B.4.3.1 isolate


Identification of the B.4.3.1 isolate by
GCMS, was produced several peaks
shown in Figure 4.


Figure 4. The GCMS spectrum of the
B.4.3.1 isolate

The results of GCMS analysis showed
the retention time 28.53 and 9.68
obtained compounds with [M
+
] at m/z
316 and 237. These results indicate
conformity with the literature data
(Markham) is shown with the core
flavonoid compounds dihidroflavonol,
flavones, isoflavones and auron with the
additional of methyl groups.

Figure 5. The spectrum fragmentation of
the B.4.3.1 isolate

Conclusion
n-buthanol extract from T.flagelliforme
leaves indicate the presence of
flavonoids, tannins and coumarin
compounds. Based on the analysis of
data from UV-Vis spectrophotometer
obtained maximum wavelength at 230.5
nm and 281.0 nm which is typical of
flavonoid compounds; flavanon and
dihydroflavonol group. Based on
analysis by GCMS with retention time
28.53 and 9.68 at m/z 316 and 237 and
data supports (phytochemical screening,
UV-Vis spectrophotometer and FT-IR
spectrophotometer) are suspected the
flavonoids group, they are
dihydroflavonol, flavones, isoflavones
and auron.

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