An Update On Antimicrobial Effects Of Nigella Sativa And Experience At King Faisal University, Damma
Auther:Muhammad Akram Randhawa
page: 37-44
ORIGINAL ARTICLES 
An Update On Antimicrobial Effects Of Nigella Sativa And Experience At King Faisal University, Dammam, Saudi Arabia
Muhammad Akram RandhawaDepartment of pharmacology, College of Medicine, King Faisal University, Dammam

Correspondence address: P.O. Box 2114, Dammam 31451, Kingdom of Saudi Arabia Tel: 00966.3.8577000/Ext. 2146 Fax: 00966.3.8571304E-Mail: mrakramsa@yahoo.co.uk  

Key words: Nigella sativa, thmoquinone, antimicrobial, antifungal

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Abstract

Nigella stiva (N. Sativa), one of the Ranunculaceae, commonly grows as a small shrub in Middle East, Middle Asia and neighboring countries. Since ancient times it is used as a folk medicine for the treatment of many ailments and as a flavoring agent in food. Many of the traditional uses of N. sativa have been confirmed via modern scientific techniques, such as analgesic, anti-inflammatory, immune stimulation, anti-cancer and anti-oxidant, etc. Antibacterial activity of N. sativa extracts and its active principles has also been investigated, and reported to be effective against many gram positive and gram negative bacteria, including resistant strains of Staphylococcus aureus and Pseudomonas aeroginosa. Similarly, antifungal activity has been reported against Candida albicans and dermatophytes isolated from animal infections. Moreover, N. sativa oil inhibited the virus titer in spleen and liver of mice infected with murine cytomegalovirus. Recently, some studies have been conducted at King Faisal University, Dammam to explore further the antifungal activities of N. sativa, considering the increasing occurrence of fungal infections and scarcity of effective remedies against them. The ether extract of N. sativa and its active principle, thymoquinone, were found to inhibit clinical isolates of eight strains of three important genera of dermatophytes: Trichophyton, Epidermophyton and Microsporum, with an MIC of 0.125 - 0.25 mg/ml for thymoquinone and 10 - 40 mg/ml for ether extract of N. sativa. Similarly, thymoquinone was shown to inhibit three opportunistic fungi: Aspergillus, Fusarium  and Scopulariopsis species, with an MIC of 0.1 - 1.0 mg/ml. We suggest N. sativa and its active principles as potential sources for the development of new antifungal drug. 

Introduction

N. sativa belongs to the botanical family of Ranunculaceae and commonly grows in Europe, Middle East, and Western Asia. In Arab countries it is commonly named as ‘Al-Habbah Al-Sawda’ and in English language is known as ‘black seed’1. The multiple uses of N. sativa in the folk medicine, especially in the Arab countries are probably related to the incentive from the authentic saying of the Prophet Mohammad (peace be upon him):قال رسول الله صلى الله عليه وسلم (عليكم بهذه الحبة السوداء فأن فيها شفاء من كل داء إلا السام) رواه أبو هريرة رضي الله عنه وأخرجه ألبحاري في صحيحهAbu Huraira (Allah Taala be pleased from him) narrated that Allah Taala’s Apostle (peace be upon him) said “Use the black seed which is a healing for all diseases except death”2. Ibne-Sina  in his famous book ‘Al-Qanoon fi el-Tibb’ has mentioned many medicinal uses of N. sativa including treatment of fever, common cold, headache, asthma, rheumatic diseases, scorpion and spider stings and bites of snake, cat and dog. Moreover, it is recommended for the treatment of microbial infections of skin (warts, collar-stud abscess and other abscesses, chronic fungal infections such as ring worm), eye and gastrointestinal tract as well as for the expulsion of worms from the intestines3. Multiple medicinal uses of N. sativa in the folk medicine encouraged many investigators to prepare various extracts of N. sativa, isolate active principles and to conduct experiments on laboratory animals and human beings using modern scientific techniques to demonstrate their pharmacological actions and therapeutic effects. These include, besides antimicrobial actions, immune stimulant1, anti-inflammatory4, 5; anti-cancer6, 7; anti-oxidant8, 9 hypoglycemic 10, 11, cardiovascular 12, 13, spasmolytic and bronchodilator effects14, 15. Numerous studies have been conducted to investigate the effectiveness of extracts of N. sativa and its active principles against bacteria, viruses, fungi and parasites. Some of the studies regarding anti-fungal activity against opportunistic fungi and clinical isolates of dermatophytes have been done at King Faisal University (KFU), Dammam. Considering the emerging incidence of microbial infections, particularly in immunocompromised individuals, studies conducted for the evaluation of antibacterial, antiviral and antifungal activities of N. sativa are being summarized below for the convenience of the interested readers and investigators. In the end some of the work done at KFU, Dammam, Saudi Arabia is also mentioned.

Antibacterial effects

The antibacterial effect of the phenolic fraction of N. sativa oil was reported first16. Later thymohydroquinone was isolated from the volatile oil of N. sativa and found to have high activity against gram-positive microorganisms17. Long after that, diethyl-ether extract of N. sativa was reported to possess concentration dependent inhibition of gram-positive bacteria (represented by Staphylococcus aureus) and gram-negative bacteria (represented by Pseudomonas aerogenosa and Escherichia coli). It also showed synergistic effect with streptomycin and gentamycin and additive effect with spectinomycin, erythromycin, tobramycin, doxycycline, chloramphenicol, nalidixic acid, ampicillin, lincomycin and co-trimoxazole18. That grew more interest in N. sativa and soon after that was reported to possess anti-bacterial activity against Listeria monocytogenes19, reduce bacterial counts in cheese20 and promote wound healing in farm animals21.Antimicrobial resistance is increasing worldwide due to multiple factors. The microorganisms tend to develop resistance to virtually every antibiotic used, though the time and extent of resistance can vary22. Therefore, it is mandatory to develop new remedies against pathogenic microbes. Realizing the importance of this issue some researchers investigated the activity of N. sativa extracts against resistant microorganisms and reported to have promising effect against multi-antibiotic resistant gram positive and gram negative bacteria23. Similarly, ether extract of N. sativa was found to be effective against resistant Staphylococcus aureus and Pseudomonas aeroginosa24, 25. Recently, combination of garlic and black cumin extracts were reported to be more effective against Staphylococcus aureus and E. coli as compared to the individual extracts26.
Antiviral effects
Immune deficiency (e.g. due to AIDs) predisposes to infections including viral infections; and bolstering the immune system may achieve virology control even without antiviral drugs27. In view of the multiple uses of N. sativa it was suspected that black seed may have some stimulating effect on the immune system of the human body and was found to enhance helper T cell (T4) and suppressor T cell (T8) ratio and increased natural killer (NK) cell activity in healthy volunteers 1. Besides improvement in immunity, black seed extract had some inhibitory effect on the human immune deficiency virus (HIV) protease but the active principle(s) responsible for this activity was not identified28. Moreover, N. sativa oil when given intra-peritoneally to mice infected with murine cytomegalovirus for 10 days the virus was undetectable in the liver and spleen, while it was still detectable in the control mice. This action was possibly related to increase in number and function of M-phi and CD4 (+) T cells and increased production of INF-gamma29.  
Antifungal effects
Assessment of N. sativa for antifungal activity has been reported rarely. Initially, inhibitory effect of the diethyl-ether extract of N. sativa extract was reported against Candida albicans18. Later, the ether extract of N. sativa was found to inhibit dermatophytes isolated from sheep skin infection30
Experience at KFU, Dammam, Saudi Arabia:
Considering the scarcity of studies regarding the activity of N. sativa against fungi and the growing need for the development of new anti fungal drugs, ether extract of N. sativa and its derivative, thymoquinone, were investigated for their in vitro activity against three opportunistic fungi: Aspergillus niger, Fusarium solani, Scopulariopsis brevicaulis; and eight species of three important genera of dermatophytes: Trichophyton, Epidemophyton and Microsporum, isolated from the clinical cases reporting in the Department of Dermatology, King Fahd Hospital of the University, Al-Khober.The fungi were sub-cultured in dermasel agar with supplement of chlorhexidine and chloramphenicol. Then 5 mm disc of 7-10 days fresh fungal coloni was inoculated on dermasel agar plates containing ether extract of N. sativa, thymoquinone, griseofulvin, clotrimazole or amphotericin B. These plates were incubated at 30º C for 4 days to 2 weeks, according to the type of fungus. Diameter of the fungal growth was measured and the % inhibition of the growth of fungi calculated, taking controls as 100 %. Minimal inhibitory concentration (MIC) was determined as the concentration of test drug showing 90 % inhibition of the fungal growth31. Asprgillus species are common opportunistic pathogens, can cause topical and systemic infections. Amphotetricin B is effective but azoles (e.g. fluconazole) are not very effective to treat Aspergillus infections. In our pilot study at KFU, thymoquinone inhibited Aspergillus niger, obtained from the laboratory contamination, with an MIC of 1mg/ml32. Later, another study was carried out to compare its activity with amphotericin B against standard strains of Aspergillus niger obtained from UK and was found to possess similar activity to apmhotericin B33. Fusarium species are also opportunistic pathogens and resistant to most anti-fungal drugs, even new azoles (e.g. voriconazole). Amphotericin B is currently used but not very successful. Results of our initial experiments revealed an inhibitory effect of thymoquinone against Fusarium solani giving an MIC of 1mg/ml34 Scopulariopsis species are rare pathogens and resistant to many antifungal drugs. According to our observations, thymoquinone was more effective than Amphotericin B and griseofulvin. There was 100% inhibition of the growth of Scopulariopsis brevicaulis with thymoquinone 1mg/ml, while amphotericin B 1mg/ml inhibited only 70 % growth. However, clotrimazole was much more effective than the above mentioned drugs. Its 0.03mg/ml inhibited 100% growth of Scopulariopsis brevicaulis35. Dermatophytosis is common worldwide, can infect skin, hair and nail. Some strains are becoming resistant to commonly available antifungal drugs. In our study, thymoquinone had moderate activity against dermatophytes, while griseofulvin was more effective. Ether extract of N. sativa was also effective but in relatively higher concentrations. MIC of thymoquinone against various dermatophytes was mostly 0.25mg/ml with a range of 0.125 - 0.25 mg/ml, while that of griseofulvin was mostly 7.75 mg/ml with a range of 0.95 - 15.5 mg/ml. 40 mg/ml of N. sativa extract inhibited 80-100% of the growth of most dermatophytes. Proportionately greater effect of thymoquinone than N. sativa extract points out that antifungal activity of N. sativa is primarily due to thymoquinone and related active principles36. We presume that due to the well known analgesic and anti-inflammatory activities of N. sativa 4, 5, its extracts and thymoquinone would have additional benefit, i.e. control of symptoms of inflammation in clinical use. Our studies disclosed the potentiality of the employment of N. sativa as a natural source for the development of new antifungal drugs. Although currently there are several antifungal drugs, the prolonged duration of treatment, drug toxicity, interactions, fungal resistance and high costs are the encountered difficulties37, 38. Additionally, the advent of HIV infection and induction of immune suppression e.g. for organ transplants or by cancer chemotherapy increased the predisposition to invasive fungal infections39.
Acknowledgements
The author is thankful to other participants in various studies carried out at the King Faisal University, Dammam, particularly Professor Dr. Saleh Aljabre, and Associate Professor Dr. Omar Alaklobi from the Department of Dermatology for the collection of samples and identification of the fungi. Technical support from Mr. Lari Bartholomew and Mr. Abdulrehamn Alfiki and the financial support from the Deanship of the King Faisal University are also gratefully acknowledged. 


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