ANTITUBERCULOSIS ACTIVITY OF ETHANOLIC EXTRACT OF MIMBA (Azadirachta indica JUSS.) ON TUBERCULOSIS PATIENTS BY Cut Fatimah, ErfanWahyudi, and Ernawati

1. Indonesian Journal of Cancer Chemoprevention, 2015, 6(2): 59-63
ISSN: 2088 – 0197
e-ISSN: 2355-8989
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ANTITUBERCULOSIS ACTIVITY OF ETHANOLIC
EXTRACT OF MIMBA (Azadirachta indica JUSS.) ON
TUBERCULOSIS PATIENTS
Cut Fatimah1*, ErfanWahyudi1, and Ernawati2
1
Faculty of Agriculture, Universitas Tjut Nyak Dhien, Medan, Sumatera Utara
2
Faculty of Pharmacy, Local Health Laboratorium, Sumatera Utara
Abstract
WHO has identified more people with tuberculosis disease. Indonesia is the third country
with tuberculosis patients. Mycobacterium tuberculosis as a cause of tuberculosis has been resistant
to antituberculosis drugs. In previous research proved that the ethanol extract of mimba cortex can
inhibit growth of Mycobacterium tuberculosis based on in vivo toxicity test on guinea pigs infected by
Mycobacterium tuberculosis H 37 Rv. The previous results of toxicity test is mild toxic category. This
study aimed to determine the antituberculosis potency of ethanolic extract of mimba cortex (EEM)
on tuberculosis patients. As many as 30 patients were divided into 3 groups, each consisting of 10
patients. Group I patients were given by FDCs for tuberculosis treatment category I for intensive
phase. Group II patients were given by FDCs for tuberculosis treatment category I for intensive phase
and 2 capsules of EEM 100 mg each day. Group III patients were given by FDCs for tuberculosis
treatment category I for intensive phase and 3 capsules of EEM 100 mg each day. Antituberculosis
assay was done by taking patients’ sputum speciments every two weeks and tested using Lowenstein-
Jensen method. After two months of treatment for stage I, the treatment will be continued by
advanced FDCs, and evaluated every two weeks for 4 months. The result showed that FDCs for
tuberculosis treatment in combination with 3 capsules of EEM in each day showed negative result of
Mycobacterium tuberculosis in week six, meanwhile the combination of FDCs for tuberculosis
treatment with 2 capsules EEM each day showed similar result with the treatment of FDCs only to
gain negative result of Mycobacterium tuberculosis in week eight, and the treatment with advanced
FDCs showed negative result of Mycobacterium tuberculosis after advanced four weeks.
Keyword : mimba cortex, antituberculosis, tuberculosis patients.
INTRODUCTION
Tuberculosis (TB) is still the main health
problem in the world. Everyone tried to prevent
TB. Even Bill Gates and George Soros funding
organization which called GF ATM (Global Fund
against AIDS, TB and Malaria) gave fund to
Indonesian tuberculosis prevention to get in
charge in tuberculosis prevention (DepKes RI,
2008). Various improvements in tuberculosis
prevention programs have been reached, such as
Directly Observed Treatment (DOTS) program.
Indonesia almost reached DOTS program target
of 70/85, which means at least 70% of TB patients
in Indonesia have been found and at least 85% of
them have been cured. Indonesia was also
introduced to some programs, such as HDL
(Hospital DOTS Linkage) that holds DOTS
program in hospitals, PPP (Public Private
Partnership) or PPM (Public Private Mix) which
involving private sectors on TB prevention.
DOTS program also be held to prevent MDR
(Multi Drug Resistance) on TB (DepKes RI,
2008).
As technology to prevent TB develops,
TB bacteria also “develops”. There are MDR
cases on therapy of TB where TB bacteria is
resistant to first-line drugs, especially rifampicin
and INH. In this case, the treatment cost and risks
to side effects are higher. There are many MDR
cases in Indonesia. Various possible second-line
drugs have been used to prevent MDR TB. MDR
TB therapy consists of sensitive primary anti TB
drugs, plus one of flouroquinolon, injected anti-
TB drugs (such as amikacin) and two or more oral
secondary anti TB drugs (thioacetazone, PAS,
ethionamide, cycloserine).
*Corresponding author email: cutmah57@gmail.com

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Higher cost on MDR-TB therapy and risks of TB
therapy failure make pressuring MDR TB cases
by therapy benefit enhancement through DOTS
program becomes the best solution. In fact,
bacteria which are resistant to rifampicin and
INH, can also resistant to all first-line drugs.
These bacteria are called super strain, which have
been found in Indonesia. XDR, extreme drug
resistance or extensive drug resistance, is the most
feared TB case where MDR bacteria are resistant
to three or more second-line drugs. It is the most
concerned possibility on TB case. In September
2006, 52 of 53 XDR patients in Africa –which
also have HIV +- were reported died in 25 days
and practically there was no medicine. Other
XDR datas are 1) at least 2 of 52 patients who
were health care professionals died from XDR TB
in 25 days in South Africa, 2) as many as 18,000
samples were collected by CDC and WHO, 20%
are MDR and 2% are XDR, 3) as many as 4%
MDR TB cases in America are XDR, 4) as many
as 19% MDR TB cases in Latvia are XDR, and 5)
as many as 15% MDR TB cases (WHO, 2008).
There are 425.000 new cases of MDR in
the world every year. Now XDR only exists in
some countries. It was also reported that TB
bacteria strain in Beijing easily mutated into
MDR, even XDR. TB disease and bacteria
develop as the world develops. Further
knowledge is needed in order to handle the
development of this disease (WHO, 2008).
Tuberculosis is a new threat for
Indonesia. In 2004 there are around 250.000 case
and each year around 140.000 people die. Most
Tuberculosis patients are citizen at the productive
age (15-55 YO), this disease is the third most
deadly disease after CHF and Pulmonary Disease
(Depkes RI, 2005).
One of the factor to increase the number
of TB patient is patient’s adherence in the
consumption of long term of TB therapy as the
treatment of TB takes 6 months for treatment.
Other factors are the case of drugs poly-resistant,
lack of host immunity, resistance of drugs and
economy factor of the society (Depkes RI, 2005).
TB bacteria resistance and slow rate of
the development of new TB drugs cause urgency
to explore new alternative solution by using
natural compunds as TB co-therapy. Empirically,
mimba (Azadiracta indica Juss) has been used to
cure various diseases. Based on Dalimarta (2000),
mimba heartwood in boiled water is used to treat
cough. Recent study showed that ethanolic extract
of mimba cortex inhibited the growth of
Mycobacterium tuberculosis bactreria in vitro
(Fatimah, 2012) and ethanolic extract of mimba
cortex can cure tuberculosis on animal that has
been infected with Mycobacterium tuberculosis H
37 Rv, and reduce toxicity on toxicity assay
(Fatimah et al., 2015). Based on study of mimba
heartwood potentials and its usage empirically,
mimba heartwood possibly has potency to be
natural TB co-therapy agent.
In this study, ethanolic extract of mimba
cortex (EEM) in capsules were given to TB
patient in order to explore its antituberculosis
activity. Capsules were given as TB co-therapy
with main therapy of FDCs for tuberculosis
treatment.
MATERIALS AND METHODS
Preparation of EEM capsules
Mimba cortex was dried and powdered.
The extraction was done by percolation using
96% ethanol. The extract then was capsuled to be
consumed more easily.
Antituberculosis Potency Test on Patients
This study was demonstrated on TB type
1 patients. TB type 1 patients are the patient which
have the characteristics 1) posistive new acid
resistant bacteria (BTA) pulmonary TB patient, 2)
negative new acid resistant bacteria pulmonary
TB patient with positive thorax rontgen, 3) extra
pulmonary TB patient. The patients were briefed
to understand the benefits of the drug to be given
to them, and they have made a memorandum of
understanding stated that they were willing and
obedient.
The whole sputum specimens from
patients were taken for examination using
Lowenstein-Jensen media culture (CV. Varka
Bayak Medan). The patients were invited to have
sputum checked at Health Laboratory in Medan.
As many as 30 patients were divided into
3 groups, each consisting of 10 patients. Group I
patients were given by FDCs for tuberculosis
treatment category I for intensive phase
(combination of isoniazide, pirazinamide, and
ethambutol for each day). Group II patients were
given by FDCs for tuberculosis treatment
category I for intensive phase and 2 capsules of
EEM 100 mg each day. Group III patients were
given by FDCs for tuberculosis treatment
category I for intensive phase and 3 capsules of
EEM 100 mg each day. The treatment were done
in 2 month. During the treatment, the patients

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were continuously controlled and maintained
consumption of nutritious foods, also got helped
to buy milk. Every two weeks in 2 months,
sputum specimens were taken to be examined in
the presence of Mycobacterium tuberculosis
culture using Lowenstein-Jensen method to
observed the presence of bacteria from each group
of patients. Furthermore, patients who are still
positive for tuberculosis, continously treated with
FDCs for tuberculosis treatment and capsules of
EEM 100 mg based on the appropriate dose group
above for the next 4 months or until a negative
response on cultivation testing of Mycobacterium
tuberculosis using Lowenstein-Jensen method.
After the completion of the entire course of
treatment, patients got medical checkup, to see the
neem bark’s potency to inhibit bacteria growth
and also the effects to human health.
Analysis of Antituberculosis potency of
EEM
Sputum specimens from patients were
identificated and cultivated for Mycobacterium
tuberculosis by transfering the sputtum into a test
tube and added by phosphate buffer pH 7 and
homogenized. As many as 0.1 ml was pippeted
and inoculated on two tubes containing
Lowenstein-Jensen medium, leveled to the entire
surface of the medium, incubated at 37o
C for 6
weeks and observed the growth in each week with
this analysis criteria:
(-) : No growth
(+1) : Slightly in yellow colonies (1-200
colonies)
(+2) : ½ of the media covered by the yellow
color colonies (200-500 colonies)
(+3) : ¾ of the media covered by the yellow
color colonies (500-2000 colonies)
(+4) : The media covered entirely by a colony
of yellow (more than 2000 colonies).
RESULTS AND DISCUSSIONS
EEM showed Antituberculosis Potency
Test on Patients
Mimba (Azadirachta indica JUSS) is
Indian plant that also available in the side road
of Indonesia as shades. Almost all parts of
mimba can be used, from its leaf, cortex,
flower, root even the seed oil. Mimba tree can
reach 9 meter in heights for only six years and
20 m after decades. It can grow rapidly in less
mineral ground and dry places. That makes
mimba very suitable for reforestation in
Indonesia. It contains nimbin and nimbidin that
have antimicrobial activity as well as antivirus,
bactericide and fungiside (Kardinan and
Taryono, 2003). People in India have used
mimba for various purposes, that it is called
village pharmacy.
More than 40 drugs compounds, either
single or combination from mimba tree have
been identified. Mimba leaves can be used to
heal wound, antibacteria, antifungi, antiviral,
antiacne, anti helmintic, diabetes, ulcus peptic,
antivomit, antitumor, anticancer, asthma,
hypertention, gout, antiinflammation, sore,
antifever, cardiovascular disease and appetite
enhancer. Mimba heartwood in boiled water is
used as antimalaria, arthritis, sore,
hemorrhoids, antitumor, antiobesity, tonicum,
nose bleeding, and cough (Dalimarta, 1999).
Numerous studies have reported that
mimba seeds can inhibit the growth of Salmonella
thyposa, Streptococcusaureus (Ambarwati,
2007), Streptococcus mutant and Streptococcus
faecalis (Almas, 1999). Mimba oils can also
inhibit the growth of E. coli and Kleibsiella
pneumonia (Sai Ram et al., 2000). Besides, the
etanolic extract of mimba cortex can inhibit the
growth of Mycobacterium tuberculosis (Fatimah,
2012).
Table 1. The result of treatment with FDCs for
tuberculosis treatment category I for intensive phase
without capsules of EEM
Patient
code
number
Category of sputum analysis result
Replication
I II III IV
I.1 3+ 2+ 2+ 1+
I.2 3+ 2+ - -
I.3 3+ 2+ 2+ 1+
I.4 3+ 2+ 2+ 1+
I.5 3+ 1+ 1+ -
I.6 3+ 1+ - -
I.7 3+ 2+ - -
I.8 3+ 2+ 1+ -
I.9 3+ 2+ 1+ -
I.10 3+ 2+ 1+ -
According to Table 1, the data showed
that after the treatment with FDCs for

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tuberculosis treatment category I for intensive
phase without capsules of EEM, as many as
three of ten patients were still categorized as +1
after 8 weeks of treatment.
As seen in Table 2, after the treatment
of FDCs for tuberculosis treatment category I
for intensive phase added with 2 capsules of
EEM showed that from ten patients, three
patients still categorized as +1 on eighth week.
Table 2. The result of treatment with FDCs for
tuberculosis treatment category I for intensive phase
added with 2 capsules of EEM
Patient
code
number
Category of sputum analysis result
Replication
I II III IV
II.1 3+ 2+ 2+ 1+
II.2 3+ 2+ 1+ -
II.3 3+ 2+ - -
II.4 3+ 2+ 2+ 1+
II.5 3+ 2+ - -
II.6 3+ 1+ 1+ -
II.7 3+ 2+ 2+ 1+
II.8 3+ 2+ - -
II.9 3+ 2+ 1+ -
II.10 3+ 1+ 1+ -
Meanwhile from Table 3, it could been
seen that after six weeks treatment of FDCs for
tuberculosis treatment category I for intensive
phase added with 3 capsules of EEM, no one
patient were detected as +1.
Table 3. The result treatment with FDCs for
tuberculosis treatment category I for intensive phase
added with 3 capsules of EEM
Patient
code
number
Category of sputum analysis result
Replication
I II III IV
III.1 3+ 1+ - -
III.2 3+ 1+ - -
III.3 3+ 1+ - -
III.4 3+ 1+ - -
III.5 3+ 1+ - -
III.6 3+ 1+ - -
III.7 3+ 1+ - -
III.8 3+ 1+ - -
III.9 3+ 1+ - -
III.10 3+ 1+ - -
Table 4. Result of advanced treatment with FDCs for
tuberculosis treatment category I for intensive phase
without capsules of EEM
Patient
code
number
Category of sputum
analysis result
Replication
I II III
I.1 1+ 1+ -
I.3 1+ - -
I.4 1+ - -
For patients who still detected as +1
after the treatment with FDCs for tuberculosis
treatment category I for intensive phase without
capsules of EEM, they were treated again with
FDCs 3 times a week, and their sputum were
examined every 2 weeks, given the results as
stated on Table 4. Based on Table 4, among 3
patients who got the treatment, there were 1
patient who still catogorized as +1 through four
weeks, but in sixth week, all the tests were
given negative.
For patients of Group II who still
detected positive Mycobacterium tuberculosis
after 8 weeks treatment of FDCs for
tuberculosis treatment category I for intensive
phase added with 2 capsules of EEM, also
continued treatment with the same drugs, and
the results were shown in Table 5.
Table 5. Results of treatment with advanced OAD-KDT
category 1 added with extract of mimba cortex a dose
of 2 capsules daily 1 times
Patient
code
number
Category of sputum
analysis result
Replication
I II
II.1 - -
II.4 1+ -
II.7 3+ -
According to Table 5, it could be seen
in Table 5, among 3 patients treated, there were
1 patient who still categorized as +1 through
four weeks, but in sixth week, all the tests
remained negative.

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CONCLUSION
Based on this study, the treatment of
EEM capsule as co-chemotherapy for FDCs for
tuberculosis treatment showed good potential as
antituberculosis on TB patients. The data showed
that 1) the treatment by the combination of 3
capsules of EEM with FDCs category I for
intensive phase in each day could change the
condition of +3 categorized patients to be
categorized as negative after 6 weeks of
treatment. 2) The treatment by using 2 capsules of
EEM with FDCs category I for intensive phase in
each day showed that from the patients detected
as +3, as many as three of ten patients was still
catogorized as +1 patients after 8 weeks of
treatment. 3) From the medical checked up after
the treatment with the capsule of EEM showed
that there were no problem after the treatment.
ACKNOWLEDGEMENT
We acknowledge DP2M DIKTI who
grant this study through Competitive Grants
Research 2014, the head of Lung Disease Clinic,
Sumatera Utara, the head of Local Health
Laboratory, Sumatera Utara, and also the head
and staff of Phytochemical Laboratory, Faculty of
Pharmacy Universitas Tjut Nyak Dhien – Medan.
REFERENCES
Dalimatha, Setiawan, 2000, Atlas Tanaman Obat
Indonesia, Trubus Agriwidya, I(I), Jakarta.
Depkes RI, 1995, Materia Medika Indonesia, 4th
edition, Departemen Kesehatan
Republik Indonesia, Jakarta, 297-
307,322-339.
DepKes RI, 2008, Pedoman Nasional
Penanggulangan Tuberkulosis,
Departemen Kesehatan Republik
Indonesia, Jakarta
Kardinan, A. dan Taryono, 2003, Tanaman obat
penggempur kanker, PT. Agromedia
Pustaka, Jakarta.
Misnadiarly, 2006, Tuberkulosis dan
Mikobakterium Atipik., PT. Dian Rakyat,
Jakarta, 98-100.
Rembold, T., 1989, Azadirachtin, their structure and
mode of action. Federal Republic of
Germany Press. 15, 1639-1643,
Germany.
Fatimah, 2012, Uji Aktivitas Antituberkulosis
Ekstrak Etanol Kulit Batang Mimba
(Azadirachta indika Juss.) terhadap
Mycobacterium tuberculosis, Universitas
Tjut Nyak Dhien Medan, Sumatera
Utara.
Fatimah, C., Wahyudi, F., Ernawati, 2015,
Antituberculosis and Toxicity Assay of
ethanolic extract of mimba cortex
(Azadirachta indica JUSS.)., Indon.J. Canc.
Chemo., 6(1), 30-33.
Ganiswara, 1995, Farmakologi dan terapi, Gaya
Baru, Jakarta.
World Health Organization, 2008, Guideline for
the programmatic management of
drugresistant tuberculosis. Emergency
Update. Accesed from
http://www.who.int/gtb Why DOTS-Plus
for MDR-TB (cited 14 april 2008).
/publication/busdocs/index.html.