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ANTIBACTERIAL ACTIVITY TEST OF TOBACCO LEAF EXTRACT

(Nicotiana tabacum L.) AGAINST Mycobacterium Tuberculosis BACTERIA
Muh. Fitra Ilyas, Muh. Rusdi, Rudy Adyaksyah
Pharmacy Study Program, Faculty of Medicine and Health Sciences, Alauddin Makassar Islamic
University
Email: 70100114062@uin-alauddin.ac.id

ABSTRACT
Research on the extraction of antituberculosis from n-hexane extract, ethyl acetate, 96%
ethanol and water on tobacco leaves (Nicotiana tabacum L.). This study aims to determine the
inhibitory effect of n-hexane, ethyl acetate, ethanol 96% extract andwater tobacco leaf on
tuberculosis-causing bacteria and compounds that have the potential to be antituberculosis.
Extract n-hexan, ethyl acetate, ethanol 96% and water from the leaves of tobacco leaves
(Nicotiana tabacum L) has the potential to inhibit the growth of Mycobacterium tuberculosis
through tests performed by the method of MODS as well as classes of compounds contained in
extracts which have an inhibitory effect against Mycobacterium tuberculosis .
The sample was extracted by a multilevel extraction method using three solvents namely n-
hexane extract, ethyl acetate, 96% ethanol. Then proceed to the reflux method with water
solvent. Then the results of the extract were tested for inhibition of the growth ofbacteria
Mycobacterium tuberculosis H37Rvusing the MODS method. And the identification of compound
groups is carried out. The result of the inhibition of the growth of Mycobacteriumtuberculosis
showed that an extract of n-hexan, ethyl acetate, ethanol 96% and water from the leaves of
tobacco leaves (Nicotiana tabacum L) with each at a concentration of 2000 ppm showed activity
antituberculosis, evidenced from the number of cord fewer after being observed under a
microscope. which shows the inhibitory effect seen from the small number of cord that looks after
observed in the microscope, while the extract of n-hexan, ethyl acetate, ethanol 96% and water at
a concentration of 400 ppm and 800 ppm, 1200 ppm and 1600 ppm did not show any inhibition
against Mycobacteriumtuberculosis is seen from a large number of cords and approaches when
compared to negative controls. After that identification of compounds is carried out. The group of
compounds contained inextract tobacco leaf (Nicotiana tabacum L) after identification of groups
namely n-hexan extract containing flavanoid compounds, ethyl acetate extract containing steroid
and phenolic compounds, ethanol 96% containing alkaloids, flavonoids, terpenoids and phenolic
compounds , while the water extract contains alkaloids and phenolics.
Keywords : Tobacco Leaves, n-hexane Extract, Ethyl Acetate Extract, 96% Ethanol Extract, Water
Extract, Mycobacterium tuberculosis,MODEL
INTRODUCTION Millions of people are diagnosed and
Tuberculosis (TB) is a contagious successfully treated every year (53 million,
infectious disease that is a problem in the world. 2000 - 2016), but there are still many gaps in
Tuberculosis is the 9th cause of death detection and treatment (WHO, 2017).
worldwide and a major cause of infectious Tuberculosis sufferers who experience
agents. In 2016 an estimated 10.4 million people resistance become a new threat that continues.
suffered from tuberculosis, 90% of adults, 65% In 2016, there were around 600,000 new cases
of men, and 10% were accompanied by HIV. that had been resistant to Rifampicin, while this
Globally, the death rate falls 3% annually. drug was the most effective first-line drug. In
Tuberculosis sufferers fall by around 2% per addition there are 490,000 multidrug resistant
year and 16% die from tuberculosis. Most cases (MDR TB) (WHO, 2017).
deaths from tuberculosis can be prevented by
early diagnosis and appropriate treatment.
Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria
Mycobacterium Tuberculosis (Rudy Adyaksyah)
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Some of the reasons above encourage and stored for 2 x 24 hours in a place which is
researchers to test the compound group and test protected by direct sunlight while occasionally
the antituberculosis activity of tobacco leaf stirring, then filtered, separated from pulp and
extract (Nicotiana tabacum L.) against the filtrate. The pulp is macerated again with the
bacteria Mycobacterium tuberculosis same number of dancers, carried out 3 times.
The liquid n-hexane extract obtained was then
RESEARCH METHODOLOGY
concentrated using a rotary evaporator until a
Tools and Materials
thick n-hexane extract was obtained. The pulp is
The tools used are autoclave
aerated to remove residual solvents. The pulp
(Hirayama), maceration vessel, chamber
was macerated again using an ethyl acetate
(Lamag), center glass, separating funnel,
solvent of 8000 ml, the same procedure was
incubator (memmert), beaker, watch glass,
carried out to obtain an ethyl acetate extract.
measuring cup, Laminan Air Flower (LAF)
Then macerated using 96% ethanol as much as
(ESCO), refrigerator (Modena), bowl,
8,000 ml, the same procedure was carried out
microscope, analytic balance, vortex mixer,
until 96% ethanol extract was obtained.
micro pipette (Socorex), 24 well plates, rotary
Furthermore, the refluxed sample
evaporator (Heidolph), iron spoons, analytical
Reflux Method
scales (Kern), and vials The
after the multilevel maceration process
material used is Distilled water
was completed, then the last method was reflux
(Aquadestillata), Aluminum Chloride, Pure
using distilled water solvent for 4 hours. After
culture (Mycobacterium tuberculosis), Tobacco
that the sample is filtered and separated from
leaves (Nicotiana tabacum L. ), DMSO,
the pulp and filtrate. The extract obtained is then
Ethanol, Ethyl Acetate, Filter paper,
evaporated. Each extract that has been dried is
Middlebrook 7H9, N-hexane, OADC Nutrient
used to be tested on Mycobacterium
(oxalid acid, albumin, destrosa, and catalase),
tuberculosis.
Nutrient PANTA, Reagent (Liebermann-Buc
RESULTS AND DISCUSSION
hard, FeCl3, Dragendroff)
Research Results
Work procedures
From the extraction of 800 g of tobacco
Preparation of Samples
leaf simplicia (Nicotiana tabacum L) using a
The research sample used was tobacco
multilevel maceration method with n-hexane,
leaf (Nicotiana tabacum L.). Sampling is done
ethyl acetate, 96% ethanol and water.
at 09.00 WITA. The leaves used are good
Table 1. Results of extraction oftobacco leaves
leaves, not damaged, not moldy and not yellow
Simplisia Weight %
or too old. The sample that has been taken is
800 g ofExtract HexRendame
then wet sorted to separate the sample from dirt
n
or other foreign material. Then the sample is
- 22.4 g 2.8%
washed with clean water to remove soil or other
impurities that are attached to the leaves. After Ethyl Acetate 21.4 g 2.67%
that the samples are chopped into small pieces,
then dried by aerating, protected from sunlight Ethanol 96% 39.5 g 4.94%
and then processed to become simplicia.
Extraction with Multilevel method Water 20.2 g 2.52%
Samples of tobacco plant leaves Test Results Leaf extract Tobacco (Nicotiana
(Nicotiana tabacum L) maserationthat have tabacum L) Againstbacteria Mycobacterium
been dried are weighed as much as 800 g for tuberculosis
multilevel maceration using 3 solvents, namely extracts for antibacterial activity test n-hexane,
n-hexane, ethyl acetate, ethanol 96%, samples ethyl acetate, and ethanol 96% and water and
inserted into a maceration vessel then the then tested on the bacterium Mycobacterium
sample soaked with solvent n-hexane as much tuberculosis strain H37RV.The purpose of this
as 8000 ml. The maceration container is closed
Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria
Mycobacterium Tuberculosis (Rudy Adyaksyah)
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screening test is to find out extracts activethat Table 4. Antituberculosis Test Results Ethanol
can inhibit microbial growth by observing Extract 96% Tobacco Leaf (Nicotiana tabacum
bacterial growth (cord) in the well in the plate L) against Bacteria Mycobacterium tuberculosis
with different concentrations, namely 400, 800, Esktrak
1200,1600 and 2000 ppm. Ethanol
Table 2. Test Results antituberculosis n-hexan Treatment Remarks
96%
Leaf Extract Tobacco(Nicotianatabacum L) 1 2
against the bacterium Mycobacterium Controls - Does not
tuberculosis - -
inhibit
extract Control + ++ ++ Inhibits
Treatment N-hexan Information Concentration Inhibits weak
1 2 + +
of 2000 ppm
Control - - - Not inhibit Concentration Does
Control + ++ ++ Inhibit - -
of 1600 ppm
concentration weak 1200 ppm not
+ +
of 2000 ppm Inhibiting - - inhibitDoes
Concentration Does not inhibit
- -
1600 ppm Concentration No inhibiting
1200 ppm not 800 ppm - -
Concentration - - inhibitDoes Concentration
not inhibit concentration Not hamper
- -
800 ppm Does not of 400 ppm
- -
Concentration inhibit
Concentration Does not Table 5. Test Results antituberculosis Water
- -
of 400 ppm inhibit Extract Tobacco leaves(Nicotianatabacum L)
Table 3. Antituberculosis Test Results Ethyl against the bacterium Mycobacterium
Acetate Extract Tobacco Leaf (Nicotiana tuberculosis
tabacum L) againstBacteria Mycobacterium extract
tuberculosis treatment Water Description
Esk 1 2
Controls- - - Not inhibit
Treatment EthylAcet Description Control + ++ ++ inhibit
ate concentration Inhibits weak
1 2 + +
of 2000 ppm
Controls - Does not Concentration Does
- - - -
inhibit of 1600 ppm
Control + ++ ++ Inhibits Concentration not
Concentration Inhibits weak of 1200 ppm - - inhibitDoes
+ +
of 2000 ppm not inhibit
Concentration Does Concentration Does not
- - - -
of 1600 ppm of 800 ppm inhibit
1200 ppm not Concentration Does not
- -
- - inhibitDoes 400 ppm inhibit
not inhibit
Concentration Does not
ofConcentrati - - inhibit
on of 800 ppm
Concentration Not inhibiting
- -
of 400 ppm
Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria
Mycobacterium Tuberculosis (Rudy Adyaksyah)
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Description: Table 6. phytochemical screening test Tobacco

- : There is growth of Mycobacterium leaf extract(Nicotianatabacum L)
tuberculosis (many)
+ : There is growth (Few) Extract Compoun Reagent results
++ : No growth of Mycobacterium d
tuberculosis N- Alkaloids Dragendorf Yellow
Control + : Isoniazid hexane (-)
Control - :bacteria Mycobacterium Terpenoids LB Yellow
tuberculosis / Steroids (-)
Phenolic FeCl3 Orange
As for h The results obtained after
(-)
incubation for 7 days showed that the negative
control (-) with the treatment of + 50 µl DMSO Flavanoid AlCl3 Yellow
+ 950 µl media + Mycobacterium tuberculosis (+)
contained bacterial growth which was mostly Ethyl Alkaloids Dragendorf Green (-
seen in the microscope observation, for positive acetate )
control (+) with treatment + 50 µl ddH2O + drug / Steroids LB Green
isoniazid + 950 mL of media + Mycobacterium (+)
tuberculosis there is no bacterial growth at all. Steroids
The extract which can inhibit the growth of Phenolic FeCl3 Black(+
Mycobacterium tuberculosis is the four extracts, )
namely n-hexane extract, ethyl acetate, and 96% Flavanoid AlCl3 Clear (-
ethanol and water each at a concentration of )
2000 ppm. The extract showed a strong enough Ethanol Alkaloid Dragendorf Orange(
inhibition on the growth of Mycobacterium 96% +)
tuberculosis seen from the appearance of the Terpenoid / LB Brown
microscope with the results that the inhibition of Steroids (+)
bacterial growth was indicated by the number of Phenolic FeCl3 Black
cords which was less than the four extracts (+)
namely n-hexane extract, ethyl acetate and 96% Flavanoid AlCl3 Yellow
ethanol and water at the concentration of 400 (+)
ppm, 800 ppm, 1200 ppm and 1600 ppm did not Water Alkaloid Dragendorf Orange
have any inhibition at all against the bacteria (+)
Mycobacterium tuberculosis so that the best Terpenoid / LB Yellow
inhibition of growth of Mycobacterium Steroid (-)
tuberculosis was found in each extract at a Phenolic FeCl3 Black
concentration of 2000 ppm. The inhibition (+)
power is seen in the presence of cord growth. Flavanoid AlCl3 Brown
(-)
Phytochemical Screening Test Results
Result of identification of compound
Based on phytochemical screening tests
compounds extracts of tobacco leaves (
on tobacco leaf extract, the results obtained can
Nicotiana tabacum L) at 2000 ppm
be seen in the table as follows.
concentration those with inhibitory activity
against the bacteria Mycobacterium tuberculosis
contain compounds as follows, n-hexan extract
contains flavanoid compounds, ethyl acetate
extracts contain steroid and phenolic
compounds, ethanol extract 96% contains
alkaloid compounds, flavanoids, terpenoids, and

Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria
Mycobacterium Tuberculosis (Rudy Adyaksyah)
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phenolics, while water extracts contain alkaloids
and phenolics.
CONCLUSION
The extract n-hexan, ethyl acetate, ethanol
96% and water from the leaves of tobacco
leaves (Nicotiana tabacum L) has the potential
to inhibit the growth of Mycobacterium
tuberculosis through tests performed by the
method of MODS.
Extract n-hexan, ethyl acetate, ethanol 96%
and water from the leaves of tobacco leaves
(Nicotiana tabacum L) with each at a
concentration of 2000 ppm showed activity
antituberculosis almost the same in the test on
the inhibition of Mycobacteriumtuberculosis,is
not evident from the number of cords more little
after being observed under a microscope. While
at concentrations of 400 ppm, 800 ppm 1200
ppm and 1600 ppm in each extract there was no
inhibition of the bacteria Mycobacterium
tuberculosis.
The group of compounds contained inextract
tobacco leaf (Nicotiana tabacum L) after
identification of groups namely n-hexan extract
containing flavanoid compounds, ethyl acetate
extract containing steroid and phenolic
compounds, ethanol 96% containing alkaloids,
flavonoids, terpenoids and phenolic compounds
, while the water extract contains alkaloids and
phenolics.
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Antibacterial Activity Test of Tobacco Leaf Extract (Nicotiana Tabacum L.) AgainstBacteria
Mycobacterium Tuberculosis (Rudy Adyaksyah)