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Jurnal Farmasi Lampung

ETHANOL AND N-HEXAN OF MANILA PALM SEEDS (Veitchia merrillii)
AS ANTIBACTERIAL AGAINST Staphylococcus aureus
AND Escherichia coli BY BIOAUTOGRAPHY

Siti Nurjanah, Akhmad Rokiban, Mutiara Puspita

Department of Pharmacy Mathematics and Natural Sciences faculty,
Tulang Bawang University, Lampung

Email : [email protected]
085840315713

Abstract
Manila palm seeds are one of the plants that have antibacterial properties. Compounds that
act as antibacterial were tannins, flavonoids, and alkaloids. The purpose of this study was to
prove the antibacterial activity of ethanol and n-hexane fractions of manila palm seeds
(Veitchia merrillii). The extraction process of manila palm seeds was carried out by
maceration methode using 70% ethanol. The extract was continued with the fractionation
process with ethanol, n-hexane and chloroform solvents. Antibacterial activity testing using
the cup mate methode with concentrations of 10%, 30%, 50%, 70%, and 100%, positive
control of tetracycline and aquadest as negative control. The compound content test used
the Thin Layer Chromatography (TLC) methode with the stationary phase using silica plate
G60 F254 and comparison of the mobile phase of chloroform: methanol: water (2: 5: 3) (v /
v). Bioautography test using the contact methode, namely the elution TLC plate, was placed
on the NA medium containing a bacterial suspension for 3 hours. Antibacterial test results of
the largest female palm seed ethanol fraction were at a concentration of 100% with a
diameter of inhibitory zone of 13.59mm on S. aureus while 11.56mm in E. coli. The results of
TLC showed the presence of tannin with a price of Rf 0.84, a flavonoid compound with a
price of Rf 0.47 and an alkaloid compound at a price of Rf 0.54. Bioautography results
showed a inhibition zone with a price of Rf 0.84 in S. aureus and E. coli bacteria which are
tannin compounds.

Keywords: Adonidia merrillii (Becc.), Veitchia merrillii (Becc.). Staphylococcus aureus,

Escherichia coli, KLT, and Bioautografi

INTRODUCTION Based on this, then sought other

alternatives in treating infections that is by
Infections in Indonesia today are still
using ingredients from nature, with the
a serious problem, because one of the
hope of minimizing undesirable side
most common causes of infectious
effects such as those that occur in
diseases is caused by bacteria [1]. How to
treatment with antibiotics or other active
deal with bacterial infections is often the
substances [4]. Biodiversity can be used
use of antibiotics that are still prescribed in
as a source for obtaining new anti-
recent decades as a solution to deal with
bacterial compounds [5].
infections [2]. The use of antibiotics has
One of the biodiversity that has the
undesirable side effects, one of which is to
potential to be developed as a medicine is
cause resistance if the use is inappropriate
manila palm plant (Veitchia merrillii) [6].
[3].
Manila palm (Veitchia merrillii) is a type of

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palm plant that is well known by the wider (NA) and Nutrient Broth (NB), aquadest
community and even the whole world. This (H2O), 70% ethanol (C2H6O), ethanol
plant is often used as plant decoration and 96% ( C2H6O), chloroform (CHCl3), n-
function as a road rectifier. Putri palem is a Hexane (CH3 (CH2) 4CH3), Ammonia
cosmopolitan ornamental plant, found in (NH3), Liebermann-Burchard, iron (III)
tropical and subtropical regions [7]. chloride (FeCl3) and Bouchardate
Putri palms belong to the Arecacea reagents.
tribe and this tribal plant is known as a
medicinal plant, which has long been used Manufacture of Manila Palm Seed
by the community in overcoming Extract and Faction
flatulence, curing myopic eyes and is great Simplisia as much as 500 g of
for mothers who have given birth. The manila palm seeds are put in a dark
results mentioned that areca nut seeds container and then macerated with 70%
(araceae) can reduce blood sugar levels in ethanol extraction liquid until the simplicia
mice. According to plant chemotaxonomy, is completely submerged in solvent 7
plants belonging to one family have the times maceration. The maserat is then
same secondary metabolite compounds evaporated using a rotary evaporator until
and physiological activity [7]. a liquid extract is obtained. The extract
Based on previous research that has obtained was fractionated by adding
been carried out states that the methanol ethanol and n-hexane in a ratio of 1: 1 to
extract of female palm seeds has obtain ethanol fraction and n-hexane
secondary metabolite compounds namely fraction. Then the ethanol fraction is
flavonoids, alkaloids, terpenoids, tannins, fractionated again with the addition of
and saponins [8]. This is supported by chloroform in a ratio of 1: 1 to obtain
microbiological research which states that ethanol and chloroform fractions. Then the
the nanoparticle extract of ethanol extract ethanol fraction obtained is evaporated
of manila palm seeds (Adonidia merrillii with a rotary evaporator until a liquid
(Becc.) Becc.) With a concentration of 2% fraction is obtained.
has antibacterial activity against
Stapylococcus aureus and Escherichia Anti-Bacterial Power Test
coli, amounting to 2.076 ± 0.083 mm and Prepare 24 sterile petri dishes. Then
3.11 ± 0.107 mm. Tetracycline as a 12 petri dishes were poured 100 µL of
positive control produces inhibition zones each suspension of E. coli bacteria into NA
of 8.793 ± 0.361 mm and 7.736 ± 0.346 media and 12 petri dishes were each
mm [9]. poured 100 µL of S. aureus bacterial
The purpose of this study aims to suspension. Then it is shaken so that it is
prove the antibacterial active compounds homogeneous, NA is poured as much as
of ethanol and n-hexane fractions of 10 ml into each petri dish then let it
manila palm seeds (Veitchia merrillii) that condense, after which a pit is made using
can inhibit the growth of S. aureus and E. tipmikropipet. Then enter the ethanol
coli bacteria. fraction test solution of manila palm seeds
with a concentration of 10% (Ket1), 30%
(Ket2), 50% (Ket3), 70% (Ket4), and 100%
RESEARCH METHODS (Ket5), tetracycline as positive control (K
Tools and Materials +) and aquadest as negative control (K-)
Tools used are glass tools, rotary and n-hexane fraction test solution for
evaporators. Materials used are manila manila palm seeds with a concentration of
palm seeds, tetracycline, E. coli bacteria, 100% (Kkl), tetracycline as positive control
S. aureus bacteria, agar nutrient media (K +) and aquadest as negative control
(K-) into the holes using these holes
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micropipette. All petri dishes were calculate its Rf. Then compare the results
incubated for 24 hours at 37ºC. of the Rf value obtained on TLC
Furthermore, observations and Bioautography with the results of the Rf
measurements of the inhibition zone value on the TLC plate.
formed around the wellbore are made
using a calipers. Data Analysis
The data obtained were then
analyzed by the Analysis of Variance
TLC Test and Bioautography (ANOVA) method with an advanced tukey
The TLC plate to be used is test to determine the ethanol fraction and
activated by heating it in an oven at 100ºC the n-hexane fraction of female palm
for 30 minutes. The ethanol fraction of seeds against E. coli and S.aureus
manila palm seeds and the fraction of n- bacteria.
hexane of manila palm seeds were each
bottled 3 times on 1 cm x 7 cm TLC plate
using capillary tubes, allowed to sit for RESULTS AND DISCUSSION
several minutes until dry and put into a
Manila Palm Seed Extract and Fraction
saturated chromatography chamber. with
Simplisia as much as 500 grams of
chloroform: methanol: water eluting liquid
manila palm seeds was extracted by
with a ratio (2: 3: 5) (v / v / v) for ethanol
maceration using 70% ethanol solvent.
fraction and chloroform: methanol eluting
The maceration method was chosen
fluid with a ratio (9: 1) (v / v). The plates
because it has its own advantages such
are left eluted until the mobile phase
as the process is quite simple,
reaches the desired limit. The plates are
inexpensive, easy to do and does not use
removed from the vessel, then the TLC
high temperatures which can damage the
Plate is removed and dried, the stain that
chemical compounds contained in the
appears on the chromatogram is then
extract [12]. Maserat in a rotary evaporator
observed in UV light with wavelengths of
so that you get 500 ml of liquid extract.
254 nm and 366 nm. The spots are
Then fractionation of extracts with various
detected by Bouchardat spray reagents for
types of solvents was carried out with
alkaloids and will show brown, ammonia
various levels of polarity, namely n-hexane
for flavonoids showing yellow, green,
(non-polar), chloroform (semipolar), and
brown or pink, FeCl3 for tannins will
ethanol (polar). N-hexane solvents are
produce a strong green, red, purple, blue
used to attract compounds that are non-
or black color, and Lieberman-Burchard for
polar, chloroform solvents to attract
saponin [10]. FeCl3 for tannins will
compounds that are semipolar and ethanol
produce a strong green, red, purple, blue
solvents to attract polar compounds. The
or black color, and Liberman-Buchard for
use of these three solvents is to obtain
terpenoids shows red orange or purple
compounds that are truly pure. The
[11]. Then calculate the Rf obtained.
fractions used in this study were ethanol
The active compound which has
fraction and n-hexane fraction. The
antibacterial activity is then detected using
obtained fraction was concentrated using
a bioautographic method by means of an
a hotplate and produced 60 ml ethanol
eluted plate affixed to the surface of the
fraction and 50 ml n-hexane fraction.
media so that in the petri each has been
inoculated with a suspension of E. coli and
S. aureus. After 3 hours the plate was
removed and removed and then incubated
Escherichia coli Bacteria
at 37ºC for 24 hours then observed the
zone of inhibition formed and then
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The result of antibacterial power test concentration with inhibition zone diameter
of ethanol fraction of manila palm seeds 11.56mm. Whereas in the fraction of n-
against E. coli bacteria showed a zone of hexane manila palm seeds did not
inhibition, marked by the presence of a produce inhibition zone diameters against
clear zone around the wellbore at each E. coli bacteria. This is proven by the
concentration (10%, 30%, 50%, 70%, and absence of a clear zone around the well
100%) and in the positive control, but in with a concentration of 100% fraction.
the n-hexane fraction of manila palm
seeds and the negative control showed no Staphylococcus aureus bacteria
clear zone. The results can be seen in The results of the antibacterial power
Figures 1 and 2. test of the ethanol fraction of manila palm
seeds against S. aureus bacteria showed
a zone of inhibition, marked by the
50% K+ presence of a clear zone around the
100% wellbore at each concentration (10%,
10% 70 % 30%, 50%, 70%, 100%) and at positive
control, but in the fraction of n-hexane
K- manila palm seeds and negative control
30 % showed no clear zone. The results can be
seen in Figures 3 and 4
(A) (B) 50 %
Figure 1 Test results of antibacterial power k-
70% 100%
of ethanol fraction of manila palm seed
10%
against E. coli A. Concentration of 10%,
30%, 50%, 70%, B. Concentration of
100%, positive control (+) tetracycline and K+
negative control (-) aquades . 30%

K- Figure 3 Results of antibacterial power test

of ethanolic fraction of manila palm seed
100% against S. Aureus A. Concentration of
10%, 30%, 50%, 70%, B. 100%
K+ concentration, positive control (+)
tetracycline and negative control (-)
aquades .
Figure 2 Test results of antibacterial power
of n-hexane fraction of manila palm seed k-
against E. coli bacteria 100%
concentration, positive control (+) 100%
tetracycline and negative control (-)
aquades.
The results of the antibacterial power
test against E. coli showed that the K+
ethanol fraction of the smallest female
inhibition zone at 10% concentration with
the diameter of inhibition zone 6.32mm Figure 4 Test results of antibacterial power
and the largest inhibitory zone at 100% of n-hexane fraction of manila palm seed
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against S. aureus bacteria 100% compounds, tannins, saponins, and
concentration, positive control (+) alkaloids which are as antibacterial [13].
tetracycline and negative control (-)
aquades. TLC Testing
The results of the antibacterial test TLC is a method used to separate
on S. aureus showed that the ethanol physical-chemical compounds based on
fraction of manila palm seeds had the the stationary phase and the mobile phase
smallest inhibition zone at a concentration components. TLC analysis aims to
of 10% with a diameter of inhibition zone determine the compound content of the
of 7.34 mm and the largest inhibitory zone ethanol fraction and n-hexane for manila
at a concentration of 100% with a diameter palm seeds. Before bottling the sample,
of inhibition zone of 13.59 mm. Whereas in the stationary phase to be used is first
the fraction of n-hexane manila palm activated in the oven for 30 minutes at a
seeds did not produce inhibition zone temperature of 100º C. Then the chamber
diameters against S. aureus bacteria. The to be used is saturated first using the best
average results of the inhibition zone mobile phase chloroform: methanol: water
diameter of the ethanol and n-hexane (2: 5: 3) (v / v / v) for ethanol and n-hexane
fractions against S. aureus and E. coli fractions: chloroform (7: 3) (v / v) [14]. TLC
bacteria can be seen in Table 1. plates that were eluted with the mobile
phase were then observed for spots at UV
Table 1. Average diameter of inhibition 254 and at UV 366 nm. Next the blotches
zones of ethanol and n-hexane fractions of with Bouchardat spray reagents for
manila palm seeds against S. aureus and alkaloids and will show brown, ammonia
E. coli bacteria for flavonoids show yellow, green, brown
Treatment Obstacles zone (mm) or pink, FeCl3 for tannins will produce
fraksi green, red, purple, blue or strong black,
etanol (et) and Lieberman-Burchard for saponins
dan S.aureus E.coli [10]. Liberman-Buchard for terpenoids
kloroform shows red orange or purple [11]. The
(kl results of cross section of thin layer
K- 0,00±0,00a 0,00±0,00a chromatography can be seen in Figures 5
and 6.
Ket1 7.34±0,69c 6.32±0,34c
Ket2 8.47±0,42cd 7.26±0,38cd
Ket3 10.00±0,34e 8.27±0,21de
Ket4 12.16±0,34f 9.26±0,34ef
Ket5 13.59±0,60g 11.56±0,58f
K+ 18.56±0,46b 17.51±0,47b (A) (B) (C) (D) (E) (F) (G)

Figure 5. Results of Spots of Thin Layer

The results of this study indicate that Chromatography Test for Ethanol Fraction
the ethanol fraction of female palm seeds of manila Palm Seed with Chloroform
has antibacterial inhibition against E. coli Motion Phase: Methanol: Water (2: 5: 3).
and S. aureus bacteria. This is because in A: visible light, B: UV 366, C: UV 254, D:
manila palm seeds contain flavonoid FeCl3, E: Bourchardat F. Ammonia G:
liberman burchard
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The results of the cross section of

thin layer chromatography showed positive
results containing tannin compounds after
spraying with FeCl3 which was marked by
the formation of a strong black color on the
plate. The results of cross section of thin
(A) (B) (C) (D) layer chromatography showed positive
results containing alkaloid compounds
Figure 6. Spotting results of Thin Layer after being sprayed with bouchardate with
Chromatography Test for N-Hexane the formation of a brown color on the
Fraction of manila Palm Seed with N- plate. The results of thin layer
Hexane Motion Phase: Chloroform (7: 3). chromatography showed positive results
A: Visible Light, B: UV 254, C: UV 366, D: containing phalvonoid after spraying using
Liberman Burchard ammonia with the formation of a yellow
The results of the cross section of color on the plate, the results of the
thin layer chromatography test can be negative thin layer chromatography
seen in table 2. contained saponins after being sprayed
Table 2. Spots cross section results with Libermann-Burchard because there
was no purple formation on the plate and
Compo Detec Positif Research
und tion Result Result
Inf the negative cross-section results
Flavon Amo Yellow Brownish + contained terpenoid compounds because
yellow they did not color formation changes in the
oid niak orange
plate.
and
Stain spots on the ethanol fraction of
green
manila palm seeds by looking at the
Alkaloid Bouc Brown Brown + results of the spray test found no good
hard spot stains on the TLC plate after elution
at with the mobile phase of chloroform:
methanol: water (2: 5: 3) so that the Rf
Saponi Liber Purple Didn't - value is unknown due to the mismatch of
n man experie the mobile phase used. Previous studies
n- nce of female palm seed extract showed the
burc change presence of the same compounds tannins,
hard flavonoids, and alkaloids [8].
Tanin FeCl3 Green Deep + TLC-Bioautographic Testing
red, black Antibacterial activity test results
purple showed that the ethanol fraction of manila
blue palm seeds has antibacterial activity, so it
or was continued to the bioautographic test.
deep Bioautography is a method for detecting
black spots on chromatograms from TLC results
terpeno Liber Red Didn't - that have antibacterial activity by attaching
id man orange experie TLC plates to the surface of the agar
n- or nce media that has been planted with
burc purple change microorganisms. The location of the active
hard compound will appear as a clear zone with
a murky background.

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In this research the contact 2.Ethanol fraction of manila palm
bioautography method is used because it seeds has been proven to contain
is easier, simpler and most often used. alkaloids, flavonoids and tannins, but
those that provide antibacterial effects
are tannins with an Rf value of 0.84
0,84 0,84
B. Suggestions
Suggestions for further research
need further isolation and identification of
the antibacterial compounds contained in
the extracts or fractions of manila palm
seeds that have antibacterial activity.

Acknowledgement
Thank you to Ms. Siti Nurjanah,
M.Sc, as the supervisor lecturer 1. Mr.
Akhmad Rokiban, AP., S.Si, Apt, as the
supervisor lecturer 2. Ms. Yuli Wahyu
(A) (B) Trimulyani M.Sc., as the examiner lecturer,
Figure 7. Bioautographic results of the all lecturers and Laboratory staff University
ethanol fraction of manila palm seeds with of Tulang Bawang Microbiology and
the mobile phase of chloroform: methanol: University of Lampung Botanical
water (2: 5: 3) (v / v) against bacteria (A) Chemistry Laboratory. Thank you also to
S. aureus and (B) E.coli. the parents, father and mother, sister,
The clear zone formed in Figure 7 brother and friends who have provided
shows the presence of antibacterial support to the author so that he can
activity in both plates at an Rf value of complete this final project.
0.84. strong suspected to be a tannin
compound, but the Rf value at the time of References
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1.Ethanol fraction of manila palm
seeds has antibacterial activity against
S. aureus and E.coli, while the n-
hexane fraction does not have
antibacterial activity.
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8