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 FadillahSriwijaya Medical Faculty, Palembang, South Sumatera

AbstractBackground: 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.

IntroductionBackground

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.

pneumoniaeb. 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.

MethodThe 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

ResultsThe 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)

No Minimal Inhibitory Concentration (mm)

6 concentration gradients10%, 8%, 6%, 4%, 2%, 1%

Black cumin seeds (Nigella sativa Linn.)

The Soxhletation using-Multilevel Extraction with three solvent

Antibacterial activity test with three solvent

Extract with potential solvents

Minimal Inhibitory Concentration (MIC) Test

Agar Diffusion method using Kirby-Bauer disc

Extraction with n-heksana

Extraction with ethyl acetate

Extraction with ethanol

Equivalent Weight TestNigella sativa-Ampicillin and

Siprofloksasin

Standard Antibiotic Regression Formula

Equivalent weight Nigella sativa-

Ampicillin and Siprofloksasin

Extract Streptococcus pneumoniae Klebsiella pneumoniae1 n-Hexane 15.83 7.332 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

No Extract Concentration

(mg/ml)

Minimum Inhibitory Zone’s Diameter Mean (mm)

Streptococcus pneumoniae Klebsiella pneumoniae

1 10 8.78 ± 1,57 6.83 ± 1,302 8 7.44± 1,50 4.94 ± 4,023 6 5.89 ± 1,02 4.44 ± 3,504 4 4.44± 1,39 3.61 ± 2,825 2 2.55± 0,50 1.67 ± 1,536 1 - -

Table 3. The Result of Minimum Inhibitory Zone’s Diameter Ethyl Acetate Black Cumin Extract against Streptococcus pneumoniae and Klebsiella pneumoniae

NoExtract

Concentration (mg/ml)

Minimum Inhibitory Zone’s Diameter Mean (mm)

Streptococcus pneumoniae Klebsiella pneumoniae

1 10 13.55 ± 2,51 9 ± 2,852 8 9.33 ± 2,31 7.67 ± 2,033 6 7.89 ± 0,19 4.33 ± 1,204 4 6.33 ± 1,15 3.22 ± 1,835 2 3.89 ± 0,19 2.33 ± 1,206 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 AmpicillinBlack Cumin

Extract Ampicillin (µg/ml)

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 CuminExtract

Ciprofloxacin (µg/ml)

Streptococcus pneumoniae Klebsiella pneumoniae

1 mg/ml n-hexane 0.73 0.05

1 mg/ml Ethyl acetate 0.93 0.06

DiscussionEthyl 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.

ConclusionEthyl 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.

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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).