Regional AMSA INA Scientific Poster

Invitro Test of Antibacterial Efficacy from Black Cumin Extract (Nigella

sativa Linn.) Against Streptococcus pneumoniae and Klebsiella pneumonia

Authors: Surya Wijaya, Alfi Fadillah

Sriwijaya Medical Faculty, Palembang, South Sumatera

Abstract
Background: There is the strain of standard antibiotic-resistant pneumonia bacteria, especially
Streptococcus pneumoniae and Klebsiella pneumoniae.1 In other side, black cumin (Nigella
sativa Linn.) is a traditional medicine which contains antibacterial compounds, such as
thymoquinone and thmohydroquinone.2,3,4 Based on above reasons, this study was conducted
suitable with the objective.

Objective: To test black cumin antibacterial effect against S. pneumoniae and K. pneumoniae, to
assess black cumin extract’s equivalent rate with standard antibiotic, and to know the potential of
black cumin for pneumonia treatment.

Method: The research was designed as experimental method. Black cumin seeds extract was
obtained by the soxhletation using multilevel extraction. The active antibacterial extract was
tested against S. pneumoniae ATCC 6303 isolate and K. pneumoniae isolate by agar diffusion
method using Kirby-Bauer disc in different concentration series. The result was analyzed by
SPSS version 16.5

Results: n-Hexane and ethyl acetate extract from black cumin seeds have inhibited the growth of
S. pneumoniae and K. pneumonia in 2 mg/ml. Equivalent rate of n-hexane and ethyl acetate
extract compared to ampicillin was 0.0008 μg/ml and 0.001 μg/ml S. pneumoniae and 4.25 μg/ml
and 5.61 μg/ml respectively against K. pneumoniae and compared to ciprofloxacin was 0.73
μg/ml and 0.93 μg/ml respectively against S. pneumoniae and 0.05 μg/ml and 0.06 μg/ml
respectively against K. pneumoniae.5

Conclusion: Ethyl acetate and n-hexane extract of black cumin seeds have inhibitory effect on
Streptococcus pneumoniae and Klebsiella pneumoniae so that extract of black cumin seeds is
potential for the complementary treatment of pneumonia.

Introduction
Background
Pneumonia is one of bacterial infectious disease which health of problem over world. It is
caused by pneumonia high mortality and morbidity rate in developing and developed country. It
is also noted as the major mortality and morbidity causes of children under five. More than two
million children suffer from pneumonia. World Health Organization notes about one fiftieth of
children mortality over world is caused by pneumonia, especially children in Africa and
Southeast Asia.1
In Indonesia, pneumonia has same impact. It is also noted main problem in children
under five. From the report of Indonesia Health Department, 477.429 children under five or
21.52% of Indonesia children suffer from pneumonia. In estimation, about two million Indonesia
children death is occurred because of pneumonia.1
The main cause of pneumonia is Streptococcus pneumoniae (Pneumococcus) infection.
Based on World Health Organization (WHO) and UNICEF, Streptococcus pneumoniae is 50%
etiology of pneumonia, and then followed with Haemophylus influenza type B (30%) and the
other infectious agent (include fungi and virus). Globally, over 1.6 million of children under five
deaths are caused by Streptococcus pneumoniae infection, especially in developing country.1
The other common pneumonia bacterium is Klebsiella pneumoniae, one of Gram
negative bacteria group. This bacterium is major causes of community-acquired pneumoniae in
adult. It is also noted as one of dangerous pathogen in the hospital and second major negative
bacteria primary bacteremia.1
The using of antibiotic has decreased the incidence of pneumonia significantly in recent
previous years. The drug of choice of pneumonia treatment is penicillin G or V. But, nowadays a
lot of bacteria is resistant of the standard antibiotic, include pneumonia bacteria, especially
Invasive Pneumonia Disease (pneumonia with Streptococcus pneumoniae as its etiology). In
other side, 80% of Klebsiella pneumoniae is resistant of beta-lactam antibiotic, such as penicillin
and ampicillin. Because of that, the research of potential antibacterial agent is needed for
antibiotic resistant-pneumonia bacteria strain treatment.1
The utilization of black cumin (Nigella sativa Linn.) as complementary treatment of
various diseases has done by millions of Asian, Mid-East, and African people over 2.000 years
ago.2 Black cumin’s herbs and oil is believed for respiratory disease, gastrointestinal disease, and
liver disease treatment. It is also used to improve quality of immune responses and eradicate
infectious agent. This pharmacological effect is appeared because of its pharmacological
properties.3 Those properties consist of several useful substances, such atsiri oil, melanthyne,
thymoquinone, thmohydroquinone, and dityhmoquinone (nigellon). It also has antibacterial and
antifungal. It shows antibacterial effect against Escherichia coli, Salmonella typhi, Pseudomonas
aeruginosa, Bacillus subtilis, Staphylococcus aureus, Micrococcus lysoideikticus, and Sarcina
lutea and antifungal effect against Candida Albicans.4 Based on above facts, the utilization of
black cumin extract is potential for pneumonia complementary treatment, especially antibiotic
resistant-pneumonia bacteria strain treatment.

Objective:
a. To test black cumin antibacterial effect against pneumonia causes, S. pneumoniae and K.
pneumoniae
b. To assess black cumin extract’s equivalent rate with standard antibiotic
c. To know the potential of black cumin for pneumonia treatment, especially antibiotic resistant-
pneumonia bacteria strain treatment.
Benefits:
a. Provide scientific base and information for scientist and society about the advantages of black
cumin (Nigella sativa Linn.) as antibacterial which can be used as complementary pneumonia
treatment.
b. Complete research data of black cumin (Nigella sativa Linn.) antibacterial effect.

c. Provide reference and information data for further research.

Method
The research was designed as experimental with Post-test only Control Group Design. This
research was held in Balai Besar Laboratorium Palembang (BBLK) and Indralaya Science
Faculty’s Laboratory, with Streptococcus pneumoniae (Gram positive) and Klebsiella
pneumoniae (Gram negative) as the research triplo sample. Black cumin seeds extract was
obtained by the soxhletation using multilevel extraction (extraction with n-hexane, ethyl acetate,
and methanol). Then, the active antibacterial extract was tested against S. pneumoniae ATCC
6303 isolate and K. pneumoniae isolate by agar diffusion method using Kirby-Bauer disc in six
different concentration series (10%, 8%, 6%, 4%, 2%, 1%). After that, the result is tested by
equivalent test to find the regression formula which is equivalent weight with standard antibiotic.
The result was statistically analyzed by SPSS version 16 with ANOVA test (to analyze normal
distribution data and its homogeny variant), Post-Hoc Test (to know the difference between
control and tested group), and Pearson test (to test hypothesis and conclude the research).4

Black cumin seeds (Nigella sativa Linn.) Extraction with n-heksana

Extraction with ethyl acetate

The Soxhletation using-Multilevel
Extraction with three solvent Extraction with ethanol

Antibacterial activity test Agar Diffusion method

with three solvent using Kirby-Bauer disc

Extract with potential solvents

Minimal Inhibitory Concentration 6 concentration gradients

(MIC) Test 10%, 8%, 6%, 4%, 2%, 1%
Standard Antibiotic Regression Formula

Equivalent Weight Test

Nigella sativa-Ampicillin and Equivalent weight Nigella sativa-
Siprofloksasin
Ampicillin and Siprofloksasin

Results
The result of each extract antibacterial activity test has shown ethyl acetate and n-hexane
extract of black cumin seeds have inhibitory effect against Streptococcus pneumoniae (Gram
positive) and Klebsiella pneumoniae (Gram negative). Whereas, methanol extract of black cumin
seeds only have inhibitory effect against Streptococcus pneumoniae (Gram positive). Ethyl
acetate extract of black cumin seeds has higher antibacterial activity than n-hexane extract of
black cumin seeds in inhibiting Streptococcus pneumoniae and Klebsiella pneumoniae growth.5
(shown in table 1).
Table 1. The Result of n-Hexane, Ethyl Acetate, and Methanol with 10% Concentration Against
Streptococcus Pneumoniae (Gram Positive) and Klebsiella Pneumoniae (Gram Negative)
Minimal Inhibitory Concentration (mm)
No
Extract Streptococcus pneumoniae Klebsiella pneumoniae
1 n-Hexane 15.83 7.33
2 Ethyl Asetate 24.67 9.33
3 Methanol 11 -

n-Hexane and ethyl acetate extract from black cumin seeds have inhibited the growth of
S. pneumoniae and K. pneumonia and the value of MIC of both extract was 2 mg/ml.5 (shown in
table 2, table 3, table 4, and table 5).

Table 2. The Result of Minimum Inhibitory Zone’s Diameter n-Hexane Black Cumin Extract
against Streptococcus pneumoniae and Klebsiella pneumoniae
Extract Minimum Inhibitory Zone’s Diameter Mean (mm)
No Concentration
(mg/ml) Streptococcus pneumoniae Klebsiella pneumoniae
1 10 8.78 ± 1,57 6.83 ± 1,30
2 8 7.44± 1,50 4.94 ± 4,02
3 6 5.89 ± 1,02 4.44 ± 3,50
4 4 4.44± 1,39 3.61 ± 2,82
5 2 2.55± 0,50 1.67 ± 1,53
6 1 - -

Table 3. The Result of Minimum Inhibitory Zone’s Diameter Ethyl Acetate Black Cumin Extract
against Streptococcus pneumoniae and Klebsiella pneumoniae
Extract Minimum Inhibitory Zone’s Diameter Mean (mm)
No Concentration
Streptococcus pneumoniae Klebsiella pneumoniae
(mg/ml)
1 10 13.55 ± 2,51 9 ± 2,85
2 8 9.33 ± 2,31 7.67 ± 2,03
3 6 7.89 ± 0,19 4.33 ± 1,20
4 4 6.33 ± 1,15 3.22 ± 1,83
5 2 3.89 ± 0,19 2.33 ± 1,20
6 1 - -

Equivalent rate of n-hexane and ethyl acetate extract compared to ciprofloxacin was 0,73
μg/ml and 0,93 μg/ml respectively against S. pneumoniae and 0,05 μg/ml and 0,06 μg/ml
respectively against K. pneumoniae.5

Table 4. The Result of Equivalent Test n-Hexane and Ethyl Acetate with Ampicillin
Black Cumin Ampicillin (µg/ml)
Extract Streptococcus pneumoniae Klebsiella pneumoniae
 1 mg/ml n-hexane 0.0008 4.25
1 mg/ml Ethyl acetate 0.001 5.61

Table 5. The Result of Equivalent Test n-Hexane and Ethyl Acetate with Ciprofloxacin
Black Cumin Ciprofloxacin (µg/ml)
Extract Streptococcus pneumoniae Klebsiella pneumoniae
1 mg/ml n-hexane 0.73 0.05
1 mg/ml Ethyl acetate 0.93 0.06

Discussion
Ethyl acetate and n-hexane extract of black cumin seeds have inhibitory effect against
Streptococcus pneumoniae (Gram positive) and Klebsiella pneumoniae (Gram negative) in 2
mg/ml.5 This pharmacological effect has appeared because of thymoquinone substance. This
substance is reported as useful active compound which has a lot of pharmacological effects, as
mentioned antimicrobial active compound.4
From this report, we also can see that ethyl acetate extract of black cumin seeds has
higher antibacterial activity than n-hexane extract of black cumin seeds in inhibiting
Streptococcus pneumoniae and Klebsiella pneumoniae growth. This condition may caused by
better ethyl acetate solvent ability. Ethyl acetate dissolves more active compounds in black
cumin (Nigella sativa Linn.) than n-hexane.5
Gram positive bacterium (Streptococcus pneumoniae) is more sensitive to ethyl acetate
and n-hexane extract of black cumin seeds than Gram negative bacterium (Klebsiella
pneumoniae). It may caused by the extract preparation of antibacterial compound. 5 Nevertheless
it is in extract preparation, ethyl acetate and n-hexane extract of black cumin seeds have shown
positive and effective antibacterial effect in inhibiting Streptococcus pneumoniae (Gram
positive) and Klebsiella pneumoniae (Gram negative) growth.
n-Hexane and ethyl acetate extract from black cumin seeds have inhibited the growth of
S. pneumoniae and K. pneumonia and the value of MIC of both extract was 2 mg/ml. Equivalent
rate of n-hexane and ethyl acetate extract compared to ampicillin was 0.0008 μg/ml and 0.001
μg/ml S. pneumoniae and 4.25 μg/ml and 5.61 μg/ml respectively against K. pneumoniae and
compared to ciprofloxacin was 0.73 μg/ml and 0.93 μg/ml respectively against S. pneumoniae
and 0.05 μg/ml and 0.06 μg/ml respectively against K. pneumoniae.5 It states equivalent rate of
black cumin n-hexane and ethyl acetate extract is very small. It means black cumin n-hexane and
ethyl acetate extract fulfill the standard complementary therapy of pneumonia.
We also state several recommendations because of the limitation of the research. We
recommend for further research to find the best active compound of black cumin (Nigella sativa
Linn.) isolation method. Another in vivo animal research and human clinical study also must be
conducted to get therapeutic effect of black cumin (Nigella sativa Linn.) data, especially its
antibacterial pharmacological effect. We also recommend another research to find the most
effective preparation of black cumin for pneumonia treatment.
Conclusion
Ethyl acetate and n-hexane extract of black cumin seeds have inhibitory effect on bacteria
that cause pneumonia diseases, such as Streptococcus pneumoniae (Gram positive) and
Klebsiella pneumoniae (Gram negative) so that extract of black cumin seeds is potential for the
complementary treatment of pneumonia.

Reference:
1. Ginting, Susi.(2009). Pneumonia, Penyebab Kematian Balita Nomor Satu. Biz, 6 Januari
2009.

2. Diratphagar. (2009). Mengenal Manfaat Jintan Hitam. Available at

http://dijenbun.deptan.go.id/rempahbun/ [Accessed 23 March 2011]

3. Holetz, Barberi, G.L. Pessini, NN. R. Sanchez, Cortez G., C.V. Nakamura, B.P.D. Filho.
(2002). Screening of Some Plant Used in The Brazilian Folk Medicine for The Treatment of
Infectious. Journal of Bioline International. Available at http://www.bioline.org.br/request?
oc0229. [Accessed 23 March 2011]

4. Abu Al-basal.(2009). In vitro and In vivo Anti-Microbial Effect of Nigella Sativa Linn.
Seed Extract against Clinical Isolate from Skin Wound Infections. American Journal 6 (8):
1440-1447, 35(2):15-50

5. Nugroho, Ilham. (2010). In Vitro Test of Antibacterial Efficacy from Black Cumin
Extract (Nigella Sativa Linn.) and Determination of Its Minimal Inhibitory Concentration
(MIC) Against Streptococcus pneumoniae and Klebsiella pneumoniae. Skripsi Sarjana
Kedokteran Fakultas Kedokteran UNSRI (unpublished).