This article was downloaded by: [Aston University] On: 26 August 2014, At: 01:32 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
GC–MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella a
Arslan Sarwar & Zakia Latif
a
a
Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan Published online: 22 Aug 2014.
To cite this article: Arslan Sarwar & Zakia Latif (2014): GC–MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947493 To link to this article: http://dx.doi.org/10.1080/14786419.2014.947493
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Aston University] at 01:32 26 August 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947493
SHORT COMMUNICATION GC –MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella Arslan Sarwar* and Zakia Latif Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan
Downloaded by [Aston University] at 01:32 26 August 2014
(Received 25 March 2014; final version received 19 July 2014) Salmonella resistance is becoming a worldwide serious health issue in these days; therefore, it is an urgent need to develop some alternative approaches to overcome this problem. Twenty bacterial strains were isolated and purified from different environmental sources and confirmed as Salmonella by morphological and biochemical analyses. Further confirmation was done by 16s rRNA sequencing. Antibiotic susceptibility test was performed by well diffusion assay against different concentrations of Ceftriaxone and Ciprofloxacin. The behaviour of both antibiotics was different against diverse strains of Salmonella. Salmonella strains resistant to both antibiotics were analysed for antibacterial activity of natural extracts of Nigella sativa (black seeds). N. sativa oil was found to be more effective against Salmonella species for which even Ceftriaxone and Ciprofloxacin were ineffective. Gas chromatography and mass spectrometry analysis of N. sativa oil was also accomplished, exhibiting 10 compounds including thymoquinone, p-cymene, cis-carveol, thymol, a-phellandrene, a-pinene, b-pinene, trans-anethole, a-longipinene and longifolene. Keywords: Salmonella; Nigella sativa oil; thymoquinone; Ceftriaxone; Ciprofloxacin
1. Introduction Salmonella is the leading and the most recurrent pathogen to cause food-borne diseases reported all over the world. Among the Salmonellas, typhoid fever or enteric fever is considered as the most prominent food-borne disease caused by Salmonella. Pakistan, India, Bangladesh, China, Indonesia, Nepal and Vietnam are considered as home to more than 80% typhoid fever cases recorded in the world (Crump et al. 2004). In the USA, 40,000 Salmonella cases were reported each year out of which 96% were caused by food (Mead et al. 1999). It is estimated that typhoid fever caused more than 21 million cases of morbidity and more than 0.2 million cases of mortality in the year 2000 all over the world and it is also estimated that 250 billion persons have suffered from typhoid fever since 1950 (Crump et al. 2004). Until now this disease remained a serious health problem in developing countries having incidence rates of 110/100,000 persons, but this situation is worse in India and Pakistan having 451/100,000 incidence cases (Kothari et al. 2008). The picture became more critical as WHO (WHO 2006) declared this disease as the fourth largest killer disease in Pakistan. Microorganisms resistant to antibiotics are emerging and spreading worldwide due to overprescription and misuse of traditional antibiotics. After frequent multiple drug-resistant Salmonella reported all over the world, fluoroquinolones and nalidixic acid became favourite choice of physicians to treat Salmonella infections (Parry et al. 2002). But unfortunately clinical abrupt use of fluoroquinolones resulted in 15 independent gyrA mutations which stimulated the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [Aston University] at 01:32 26 August 2014
2
A. Sarwar and Z. Latif
clonal expansion of Salmonella enterica serovar typhi in Asia and Africa. These resistant Salmonella strains enter into a carrier stage in which they pass from family to family without clinical signs and symptoms (Roumagnac et al. 2006) and establish many Salmonella cases. Other adverse side effects of uncontrolled and irrational use of antibiotics include allergic reactions, hypersensitivity and immuno-suppressions. Therefore, it is urgent to search for alternative antibacterial drugs from medicinal plants and other natural extracts for the treatment of Salmonella infections. Due to adverse effects associated with the use of antibiotics, Nigella sativa oil (also known as black seed or Kalvanji seeds) was selected. It has strong historical and religious background in Arabian and Indian civilisation and is used in food as well as medicine. The seeds were used as a flavouring agent, to improve digestion and produce warmth, especially in cold climates. N. sativa oil seeds and oil have long been used to treat respiratory disorders, kidney, liver functions, circulatory system and as well as in cancer (Musa et al. 2004). Pharmaceutical properties exhibited by Kalvanji are anti-inflammatory, antihistaminic, antibiotic, anti-hypertensive, anti-microbial and antitumour activities. Many of these activities have been attributed to quinine and thymoquinone constituents from the essential oil of the black seeds (Islam et al. 2013). An authentic saying of the Prophet Muhammad (Peace Be Upon Him) about black seed is quoted in Al-Bukhari as: Abu Huraira (Allah be pleased from him) narrated that Allah’s Apostle (peace be upon him) said ‘Use the black seed, which is a healing for all diseases except “AsSam” and As-Sam is Death’ (Al-Bukhari 1976). It is also known as natural drug of Tib-e-Nabvi or medicine of the Prophet Muhammad (PBUH). Keeping in mind the importance of black seeds, this study was designed to use black seeds for the treatment of Salmonella infections. It also deals with the gas chromatography and mass spectrometry (GC – MS) analysis of antibacterial activity of black seeds against different strains of Salmonella genetically different determined by micro-satellite fingerprinting. 2. Results and discussion 2.1. Isolation and characterisation of bacterial strains The pure colonies on selected SS agar medium (Figure S1) with characteristic blackish spot in the centre were confirmed as Salmonella by microbiological and biochemical analyses. Seventeen out of 30 samples (56%) were collected from different environmental conditions contaminated with Salmonella. All strains were Gram negative: indole, Voges – Proskauer and urease tests were negative, whereas methyl red and citrate utilisation tests were positive (Sherman & Cappuccino 2007). 2.2. Molecular characterisation of bacterial isolates Six Salmonella strains based on their behaviour as resistant to both antibiotics and sensitive to N. sativa oil extract were selected for molecular characterisation by partial 16s rRNA sequencing. Four strains (As-1, AS-5, AS-18 and AS-38) were selected from samples collected from normal environment, whereas two strains (SG-1 and SR-1) were from clinical laboratory samples. Salmonella genera composed of two groups S. enterica and Salmonella bongori (CDC 2006), in this study all the isolated Salmonella strains belonged to S. enterica group but were different in serovar. Salmonella strain AS-1 identified as Salmonella paratyphi A with accession number KC820621 showed 98% resemblance with S. paratyphi A isolated from the USA with accession number NC_006511. Similarly Salmonella enteritidis (AS-5) isolated from rotten egg with accession number KC820622, showed 96% similarity with same strain isolated from the USA with accession number NC_011294. Likewise Salmonella typhimurium (AS-18) with accession number KC820623 showed 95% similarity with S. typhimurium with accession number
Natural Product Research
3
NC_003197. In the same manner Salmonella heidelberg with accession number JQ694356 exhibited 94% similarity with S. heidelberg (AS-38). Similarly strain SR-1 (Salmonella agona) was isolated from clinical samples. Previously, it was reported in Turkey meat (Rahimi et al. 2010). To our best knowledge, no reported evidence of S. agona from clinical sample was found in Pakistan. Phylogenetic analysis shows that S. agona strain having accession number JQ694195 showed 95% similarity with S. agona isolated from the USA (Figure S7). S. typhimurium was reported as the most common serovar reported worldwide, while S. enteritidis was reported as the second most common serovar all over the world, but high incidence rate reported in the USA (Hendriksen et al. 2011).
Downloaded by [Aston University] at 01:32 26 August 2014
2.3. Antibiotic susceptibility test It was observed by antibiotic susceptibility tests of Salmonella strains performed against 50 and 100 mg/mL Ciprofloxacin and Ceftriaxone by agar well diffusion assay that 15% and 30% strains exhibited complete resistance against both antibiotics, respectively (Figure S2). Overall 30% Salmonella strains were resistant to Ceftriaxone and 20% against Ciprofloxacin (Figure S2). Other Salmonella strains showed different behaviour against both antibiotics (Figure S3a and S3b). Similar research was conducted in the UK and it was reported that 23% Salmonella strains were resistant to Ciprofloxacin (Threlfall and Ward 2001). Ciprofloxacin is considered as an important medicine to treat Salmonella infections. In our research 15% Ciprofloxacinsusceptible Salmonella isolates were identified having # 20 mm zone of inhibition. These results are comparable with another reported research conducted in Bangladesh by Ahmed et al. (2006), that 11.7% Salmonella isolates showing Ciprofloxacin susceptibility are exhibiting inhibition zone diameter # 21 mm.
2.4. Antibacterial activity of black seed crude extract and oil Salmonella strains subjected to well plate assay against black seed crude extract gave promising results. Salmonella strains showing complete resistance against both antibiotics were sensitive against crude extract of black seeds (data not shown). The results of antibacterial activity of black seeds oil clearly showed that it has significant effect on all strains collected from different conditions (Figures S4 and S5). These results to some extent are comparable with those of Trigonella foenum-graecum seed oil (Ritu et al. 2010). Overall 90% Salmonella strains were sensitive against black seed oil including strains AS-5, AS-38 and SR-1 which were highly resistant against Ciprofloxacin and Ceftriaxone (Figure S5). In case of other strains, zone of inhibition was variable but greater in diameter as compared to antibiotics. The remaining black seed solid material did not show any promising antibacterial activity.
2.5. Minimum inhibitory concentration determination of N. sativa crude extract and N. sativa oil The bacteriostatic activity of N. sativa crude extract and N. sativa oil was determined by broth dilution method. The results showed that minimum inhibitory concentration (MIC) of N. sativa crude extract is 100 times higher than N. sativa oil, which shows the strong concentration of bacteriostatic compounds present in the N. sativa oil rather than in its crude extract. S. typhimurium and S. agona were least susceptible towards N. sativa oil and N. sativa crude extract as well having MIC as low as 200 and 400 mL/mL, respectively. On the other hand, Salmonella typhi and S. paratyphi A were less susceptible against N. sativa crude extract and N. sativa oil (Table S1).
Downloaded by [Aston University] at 01:32 26 August 2014
4
A. Sarwar and Z. Latif
2.6. GC –MS analysis of black seed Oil In GC – MS analysis, total ion current was kept at 1.00. The chromatogram was calibrated at 26 min retention time because all the required and major compounds of Kalvanji oil were found within that retention time. After obtaining overall chromatogram, the compounds representative of high peak area (Figure S6) were identified by archives of chemical compounds (Rose 1994), based on their geometrical symmetry, molecular weight and retention time (Paarakh 2010). Thymoquinone and p-cymene were identified at 15 and 8.5 min with highest peak area, respectively. In addition, eight other compounds were identified from the oil extract of N. sativa oil including a-phellandrene, a-pinene, b-pinen, cis-carveol, trans-anethole, thymol, alongipinene and longifolene, (Figure S6). Their empirical formula, molecular weight and retention time are summarised in Table S1. In this study, the GC –MS evaluation of N. sativa oil indicated that Thymoquinone and pcymene are compounds found in the oil with highest peak area as compared with the other compounds with the retention time 15.1 and 8.4 min with 164 and 134 molecular weight, respectively (Solatia et al. 2012). Thymoquinone retains not only antibacterial activity, but also anti-inflammatory and anti-cancer activities(Wooa et al. 2012). And 75% Salmonella strains showed results with diameter between 15 and 30 mm zones of inhibition with N. sativa oil. Strain MC showed highest zone of inhibition diameter 30 mm. Previously, Sheikh et al. (2010) have reported approximately 14 mm zone of inhibition with N. sativa oil against S. typhi. This deviation in results may be due to the quality of N. sativa oil, because it is also reported that the antibacterial activity of N. sativa oil is purely dependent upon the quantity of Thymoquinone and Thymohydroquinone compounds, and the percentage of these active compounds depends upon the method of oil extraction from black seeds (Halawani 2009). 3. Conclusion New antibiotic formulations have long been dependent upon isolation of bioactive compounds from natural extracts. With emerging bacterial resistance, it is the need to discover new bioactive compounds having lesser side effects and greater beneficial effects to the human body. To achieve this, further research and investigation is required to exploit their therapeutic properties to conflict the disease. We have analysed the effects of N. sativa oil on Salmonella. In future, we can also determine the fruitful effects of N. sativa oil against a variety of diseases. It is concluded that contemporary drugs can be formulated after comprehensive research on new bioactive compounds, their therapeutic action and after clinical trial. Supplementary material Experimental details relating to this paper are available online, alongside Figures S1 –S7 and Tables S1 –S2. References Ahmed D, D’Costa LT, Alam K, Balakrish Nair G, Hossain MA. 2006. Multidrug-resistant Salmonella enterica serovar typhi isolates with high-level resistance to Ciprofloxacin in Dhaka, Bangladesh. Antimicrob Agents Chemother. 50:3516–3517. Al-Bukhari 1976. Division (71) on medicine. In: Sahi Al-Bukhari, editor, The collection of authentic sayings of Prophet Mohammad (peace be upon him). 2nd ed. Turkey: Hilal Yayinlari, Ankara. CDC. 2006. Salmonella surveillance: annual summary, 2006. Atlanta, GA: US Department of Health and Human Services, CDC, 2008. Crump JA, Luby PS, Mintz ED. 2004. The global burden of typhoid fever. Bull World Health Organ. 5:82. Halawani E. 2009. Antibacterial activity of thymoquinone and thymohydroquinone of Nigella sativa oil L. and their interaction with some antibiotics. Adv Biol Res. 3:148–152.
Downloaded by [Aston University] at 01:32 26 August 2014
Natural Product Research
5
Hendriksen RS, Vieira AR, S, Lo Fo Wong DMA, Jensen AB, Wegener Henrik C, Aarestrup FM. 2011. Global monitoring of Salmonella serovar distribution from the World Health Organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog Dis. 8:887–900. Islam MH, Ahmad IZ, Salman MT. 2013. Antibacterial activity of Nigella sativa oil seed in various germination phases on clinical bacterial strains isolated from human patients. E3 J Biotech Pharma Res. 4:8–13. Kothari A, Pruthi A, Chugh TD. 2008. The burden of enteric fever. J Infect Dev Ctries. 4:253–259. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. 1999. Food-related illness and death in the United States. Emerg Infect Dis. 5:607–625. Musa D, Dilsiz N, Ulakoglu G, Ulakoglu G, Bitiren M. 2004. Antitumor activity of an ethanol extract of Nigella sativa oil seeds. Biologia. 59:735–740. Paarakh MP. 2010. Nigella sativa oil Linn. – a comprehensive review. IJNPR. 1:409–429. Parry CM, Hien TT, Dougan G, White NJ, Farrara JJ. 2002. Typhoid fever. N Engl J Med. 347:1770–1782. Rahimi E, Ameri M, Kazemeini HR, Elbagi M. 2010. Prevalence and antimicrobial resistance of Salmonella isolated from retail raw turkey, ostrich and partridge meat in Iran. BJVM. 13:23–30. Ritu K, Atanu C, Pal SB, Ashutosh G, Kalyan S. 2010. Antimicrobial activity of ethanolic extract of Trigonella foenumgraecum Linn. IRJP. 1:181–183. Rose ME. 1994. Interpretation of mass spectra, 4th edition F.W. McLafferty and F. Turecˇek Published by University Science Books, Mill Valley, 1993 ISBN 0-935702-25-3, xiii þ371 pp. Rapid Commun Mass Spectrom. 8:501. Roumagnac P, Weill FX, Dolecek C, Baker S, Brisse S, Chinh NT, Le TA, Acosta CJ, Farrar J, Dougan G, et al. 2006. Evolutionary history of Salmonella typhi. Science. 314:1301–1304. Epub 2006/11/25. Sheikh MI, Soriful I, Atikur R, Mostafizur R, Mashiur R, Mizanur R, Abdur R, Firoz A. 2010. Control of some human pathogenic bacteria by seed extracts of cumin. Agric Conspec Sci. 75:39–44. Sherman N, Cappuccino JG. 2007. Microbiology – a laboratory manual. Upper Saddle River, NJ: Pearson Education Inc. Solatia Z, Baharinb BS, Bagheri H. 2012. Supercritical carbon dioxide (SC-CO2) extraction of Nigella sativa oil L. oil using full factorial design. Ind Crop Prod. 36:519–523. Threlfall EJ, Ward LR. 2001. Decreased susceptibility to ciprofloxacin in Salmonella enterica serotype typhi, UK. Emerg Infect Dis. 7:448–450. WHO. 2006. 6th International conference on typhoid fever and other Salmonellosis. Geneva: WHO. Wooa CC, Kumara AP, Sethia G, Tan KHB. 2012. Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem Pharm. 83:443–451.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
GC–MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella a
Arslan Sarwar & Zakia Latif
a
a
Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan Published online: 22 Aug 2014.
To cite this article: Arslan Sarwar & Zakia Latif (2014): GC–MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.947493 To link to this article: http://dx.doi.org/10.1080/14786419.2014.947493
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [Aston University] at 01:32 26 August 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 http://dx.doi.org/10.1080/14786419.2014.947493
SHORT COMMUNICATION GC –MS characterisation and antibacterial activity evaluation of Nigella sativa oil against diverse strains of Salmonella Arslan Sarwar* and Zakia Latif Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan
Downloaded by [Aston University] at 01:32 26 August 2014
(Received 25 March 2014; final version received 19 July 2014) Salmonella resistance is becoming a worldwide serious health issue in these days; therefore, it is an urgent need to develop some alternative approaches to overcome this problem. Twenty bacterial strains were isolated and purified from different environmental sources and confirmed as Salmonella by morphological and biochemical analyses. Further confirmation was done by 16s rRNA sequencing. Antibiotic susceptibility test was performed by well diffusion assay against different concentrations of Ceftriaxone and Ciprofloxacin. The behaviour of both antibiotics was different against diverse strains of Salmonella. Salmonella strains resistant to both antibiotics were analysed for antibacterial activity of natural extracts of Nigella sativa (black seeds). N. sativa oil was found to be more effective against Salmonella species for which even Ceftriaxone and Ciprofloxacin were ineffective. Gas chromatography and mass spectrometry analysis of N. sativa oil was also accomplished, exhibiting 10 compounds including thymoquinone, p-cymene, cis-carveol, thymol, a-phellandrene, a-pinene, b-pinene, trans-anethole, a-longipinene and longifolene. Keywords: Salmonella; Nigella sativa oil; thymoquinone; Ceftriaxone; Ciprofloxacin
1. Introduction Salmonella is the leading and the most recurrent pathogen to cause food-borne diseases reported all over the world. Among the Salmonellas, typhoid fever or enteric fever is considered as the most prominent food-borne disease caused by Salmonella. Pakistan, India, Bangladesh, China, Indonesia, Nepal and Vietnam are considered as home to more than 80% typhoid fever cases recorded in the world (Crump et al. 2004). In the USA, 40,000 Salmonella cases were reported each year out of which 96% were caused by food (Mead et al. 1999). It is estimated that typhoid fever caused more than 21 million cases of morbidity and more than 0.2 million cases of mortality in the year 2000 all over the world and it is also estimated that 250 billion persons have suffered from typhoid fever since 1950 (Crump et al. 2004). Until now this disease remained a serious health problem in developing countries having incidence rates of 110/100,000 persons, but this situation is worse in India and Pakistan having 451/100,000 incidence cases (Kothari et al. 2008). The picture became more critical as WHO (WHO 2006) declared this disease as the fourth largest killer disease in Pakistan. Microorganisms resistant to antibiotics are emerging and spreading worldwide due to overprescription and misuse of traditional antibiotics. After frequent multiple drug-resistant Salmonella reported all over the world, fluoroquinolones and nalidixic acid became favourite choice of physicians to treat Salmonella infections (Parry et al. 2002). But unfortunately clinical abrupt use of fluoroquinolones resulted in 15 independent gyrA mutations which stimulated the
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
Downloaded by [Aston University] at 01:32 26 August 2014
2
A. Sarwar and Z. Latif
clonal expansion of Salmonella enterica serovar typhi in Asia and Africa. These resistant Salmonella strains enter into a carrier stage in which they pass from family to family without clinical signs and symptoms (Roumagnac et al. 2006) and establish many Salmonella cases. Other adverse side effects of uncontrolled and irrational use of antibiotics include allergic reactions, hypersensitivity and immuno-suppressions. Therefore, it is urgent to search for alternative antibacterial drugs from medicinal plants and other natural extracts for the treatment of Salmonella infections. Due to adverse effects associated with the use of antibiotics, Nigella sativa oil (also known as black seed or Kalvanji seeds) was selected. It has strong historical and religious background in Arabian and Indian civilisation and is used in food as well as medicine. The seeds were used as a flavouring agent, to improve digestion and produce warmth, especially in cold climates. N. sativa oil seeds and oil have long been used to treat respiratory disorders, kidney, liver functions, circulatory system and as well as in cancer (Musa et al. 2004). Pharmaceutical properties exhibited by Kalvanji are anti-inflammatory, antihistaminic, antibiotic, anti-hypertensive, anti-microbial and antitumour activities. Many of these activities have been attributed to quinine and thymoquinone constituents from the essential oil of the black seeds (Islam et al. 2013). An authentic saying of the Prophet Muhammad (Peace Be Upon Him) about black seed is quoted in Al-Bukhari as: Abu Huraira (Allah be pleased from him) narrated that Allah’s Apostle (peace be upon him) said ‘Use the black seed, which is a healing for all diseases except “AsSam” and As-Sam is Death’ (Al-Bukhari 1976). It is also known as natural drug of Tib-e-Nabvi or medicine of the Prophet Muhammad (PBUH). Keeping in mind the importance of black seeds, this study was designed to use black seeds for the treatment of Salmonella infections. It also deals with the gas chromatography and mass spectrometry (GC – MS) analysis of antibacterial activity of black seeds against different strains of Salmonella genetically different determined by micro-satellite fingerprinting. 2. Results and discussion 2.1. Isolation and characterisation of bacterial strains The pure colonies on selected SS agar medium (Figure S1) with characteristic blackish spot in the centre were confirmed as Salmonella by microbiological and biochemical analyses. Seventeen out of 30 samples (56%) were collected from different environmental conditions contaminated with Salmonella. All strains were Gram negative: indole, Voges – Proskauer and urease tests were negative, whereas methyl red and citrate utilisation tests were positive (Sherman & Cappuccino 2007). 2.2. Molecular characterisation of bacterial isolates Six Salmonella strains based on their behaviour as resistant to both antibiotics and sensitive to N. sativa oil extract were selected for molecular characterisation by partial 16s rRNA sequencing. Four strains (As-1, AS-5, AS-18 and AS-38) were selected from samples collected from normal environment, whereas two strains (SG-1 and SR-1) were from clinical laboratory samples. Salmonella genera composed of two groups S. enterica and Salmonella bongori (CDC 2006), in this study all the isolated Salmonella strains belonged to S. enterica group but were different in serovar. Salmonella strain AS-1 identified as Salmonella paratyphi A with accession number KC820621 showed 98% resemblance with S. paratyphi A isolated from the USA with accession number NC_006511. Similarly Salmonella enteritidis (AS-5) isolated from rotten egg with accession number KC820622, showed 96% similarity with same strain isolated from the USA with accession number NC_011294. Likewise Salmonella typhimurium (AS-18) with accession number KC820623 showed 95% similarity with S. typhimurium with accession number
Natural Product Research
3
NC_003197. In the same manner Salmonella heidelberg with accession number JQ694356 exhibited 94% similarity with S. heidelberg (AS-38). Similarly strain SR-1 (Salmonella agona) was isolated from clinical samples. Previously, it was reported in Turkey meat (Rahimi et al. 2010). To our best knowledge, no reported evidence of S. agona from clinical sample was found in Pakistan. Phylogenetic analysis shows that S. agona strain having accession number JQ694195 showed 95% similarity with S. agona isolated from the USA (Figure S7). S. typhimurium was reported as the most common serovar reported worldwide, while S. enteritidis was reported as the second most common serovar all over the world, but high incidence rate reported in the USA (Hendriksen et al. 2011).
Downloaded by [Aston University] at 01:32 26 August 2014
2.3. Antibiotic susceptibility test It was observed by antibiotic susceptibility tests of Salmonella strains performed against 50 and 100 mg/mL Ciprofloxacin and Ceftriaxone by agar well diffusion assay that 15% and 30% strains exhibited complete resistance against both antibiotics, respectively (Figure S2). Overall 30% Salmonella strains were resistant to Ceftriaxone and 20% against Ciprofloxacin (Figure S2). Other Salmonella strains showed different behaviour against both antibiotics (Figure S3a and S3b). Similar research was conducted in the UK and it was reported that 23% Salmonella strains were resistant to Ciprofloxacin (Threlfall and Ward 2001). Ciprofloxacin is considered as an important medicine to treat Salmonella infections. In our research 15% Ciprofloxacinsusceptible Salmonella isolates were identified having # 20 mm zone of inhibition. These results are comparable with another reported research conducted in Bangladesh by Ahmed et al. (2006), that 11.7% Salmonella isolates showing Ciprofloxacin susceptibility are exhibiting inhibition zone diameter # 21 mm.
2.4. Antibacterial activity of black seed crude extract and oil Salmonella strains subjected to well plate assay against black seed crude extract gave promising results. Salmonella strains showing complete resistance against both antibiotics were sensitive against crude extract of black seeds (data not shown). The results of antibacterial activity of black seeds oil clearly showed that it has significant effect on all strains collected from different conditions (Figures S4 and S5). These results to some extent are comparable with those of Trigonella foenum-graecum seed oil (Ritu et al. 2010). Overall 90% Salmonella strains were sensitive against black seed oil including strains AS-5, AS-38 and SR-1 which were highly resistant against Ciprofloxacin and Ceftriaxone (Figure S5). In case of other strains, zone of inhibition was variable but greater in diameter as compared to antibiotics. The remaining black seed solid material did not show any promising antibacterial activity.
2.5. Minimum inhibitory concentration determination of N. sativa crude extract and N. sativa oil The bacteriostatic activity of N. sativa crude extract and N. sativa oil was determined by broth dilution method. The results showed that minimum inhibitory concentration (MIC) of N. sativa crude extract is 100 times higher than N. sativa oil, which shows the strong concentration of bacteriostatic compounds present in the N. sativa oil rather than in its crude extract. S. typhimurium and S. agona were least susceptible towards N. sativa oil and N. sativa crude extract as well having MIC as low as 200 and 400 mL/mL, respectively. On the other hand, Salmonella typhi and S. paratyphi A were less susceptible against N. sativa crude extract and N. sativa oil (Table S1).
Downloaded by [Aston University] at 01:32 26 August 2014
4
A. Sarwar and Z. Latif
2.6. GC –MS analysis of black seed Oil In GC – MS analysis, total ion current was kept at 1.00. The chromatogram was calibrated at 26 min retention time because all the required and major compounds of Kalvanji oil were found within that retention time. After obtaining overall chromatogram, the compounds representative of high peak area (Figure S6) were identified by archives of chemical compounds (Rose 1994), based on their geometrical symmetry, molecular weight and retention time (Paarakh 2010). Thymoquinone and p-cymene were identified at 15 and 8.5 min with highest peak area, respectively. In addition, eight other compounds were identified from the oil extract of N. sativa oil including a-phellandrene, a-pinene, b-pinen, cis-carveol, trans-anethole, thymol, alongipinene and longifolene, (Figure S6). Their empirical formula, molecular weight and retention time are summarised in Table S1. In this study, the GC –MS evaluation of N. sativa oil indicated that Thymoquinone and pcymene are compounds found in the oil with highest peak area as compared with the other compounds with the retention time 15.1 and 8.4 min with 164 and 134 molecular weight, respectively (Solatia et al. 2012). Thymoquinone retains not only antibacterial activity, but also anti-inflammatory and anti-cancer activities(Wooa et al. 2012). And 75% Salmonella strains showed results with diameter between 15 and 30 mm zones of inhibition with N. sativa oil. Strain MC showed highest zone of inhibition diameter 30 mm. Previously, Sheikh et al. (2010) have reported approximately 14 mm zone of inhibition with N. sativa oil against S. typhi. This deviation in results may be due to the quality of N. sativa oil, because it is also reported that the antibacterial activity of N. sativa oil is purely dependent upon the quantity of Thymoquinone and Thymohydroquinone compounds, and the percentage of these active compounds depends upon the method of oil extraction from black seeds (Halawani 2009). 3. Conclusion New antibiotic formulations have long been dependent upon isolation of bioactive compounds from natural extracts. With emerging bacterial resistance, it is the need to discover new bioactive compounds having lesser side effects and greater beneficial effects to the human body. To achieve this, further research and investigation is required to exploit their therapeutic properties to conflict the disease. We have analysed the effects of N. sativa oil on Salmonella. In future, we can also determine the fruitful effects of N. sativa oil against a variety of diseases. It is concluded that contemporary drugs can be formulated after comprehensive research on new bioactive compounds, their therapeutic action and after clinical trial. Supplementary material Experimental details relating to this paper are available online, alongside Figures S1 –S7 and Tables S1 –S2. References Ahmed D, D’Costa LT, Alam K, Balakrish Nair G, Hossain MA. 2006. Multidrug-resistant Salmonella enterica serovar typhi isolates with high-level resistance to Ciprofloxacin in Dhaka, Bangladesh. Antimicrob Agents Chemother. 50:3516–3517. Al-Bukhari 1976. Division (71) on medicine. In: Sahi Al-Bukhari, editor, The collection of authentic sayings of Prophet Mohammad (peace be upon him). 2nd ed. Turkey: Hilal Yayinlari, Ankara. CDC. 2006. Salmonella surveillance: annual summary, 2006. Atlanta, GA: US Department of Health and Human Services, CDC, 2008. Crump JA, Luby PS, Mintz ED. 2004. The global burden of typhoid fever. Bull World Health Organ. 5:82. Halawani E. 2009. Antibacterial activity of thymoquinone and thymohydroquinone of Nigella sativa oil L. and their interaction with some antibiotics. Adv Biol Res. 3:148–152.
Downloaded by [Aston University] at 01:32 26 August 2014
Natural Product Research
5
Hendriksen RS, Vieira AR, S, Lo Fo Wong DMA, Jensen AB, Wegener Henrik C, Aarestrup FM. 2011. Global monitoring of Salmonella serovar distribution from the World Health Organization global foodborne infections network country data bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog Dis. 8:887–900. Islam MH, Ahmad IZ, Salman MT. 2013. Antibacterial activity of Nigella sativa oil seed in various germination phases on clinical bacterial strains isolated from human patients. E3 J Biotech Pharma Res. 4:8–13. Kothari A, Pruthi A, Chugh TD. 2008. The burden of enteric fever. J Infect Dev Ctries. 4:253–259. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffin PM, Tauxe RV. 1999. Food-related illness and death in the United States. Emerg Infect Dis. 5:607–625. Musa D, Dilsiz N, Ulakoglu G, Ulakoglu G, Bitiren M. 2004. Antitumor activity of an ethanol extract of Nigella sativa oil seeds. Biologia. 59:735–740. Paarakh MP. 2010. Nigella sativa oil Linn. – a comprehensive review. IJNPR. 1:409–429. Parry CM, Hien TT, Dougan G, White NJ, Farrara JJ. 2002. Typhoid fever. N Engl J Med. 347:1770–1782. Rahimi E, Ameri M, Kazemeini HR, Elbagi M. 2010. Prevalence and antimicrobial resistance of Salmonella isolated from retail raw turkey, ostrich and partridge meat in Iran. BJVM. 13:23–30. Ritu K, Atanu C, Pal SB, Ashutosh G, Kalyan S. 2010. Antimicrobial activity of ethanolic extract of Trigonella foenumgraecum Linn. IRJP. 1:181–183. Rose ME. 1994. Interpretation of mass spectra, 4th edition F.W. McLafferty and F. Turecˇek Published by University Science Books, Mill Valley, 1993 ISBN 0-935702-25-3, xiii þ371 pp. Rapid Commun Mass Spectrom. 8:501. Roumagnac P, Weill FX, Dolecek C, Baker S, Brisse S, Chinh NT, Le TA, Acosta CJ, Farrar J, Dougan G, et al. 2006. Evolutionary history of Salmonella typhi. Science. 314:1301–1304. Epub 2006/11/25. Sheikh MI, Soriful I, Atikur R, Mostafizur R, Mashiur R, Mizanur R, Abdur R, Firoz A. 2010. Control of some human pathogenic bacteria by seed extracts of cumin. Agric Conspec Sci. 75:39–44. Sherman N, Cappuccino JG. 2007. Microbiology – a laboratory manual. Upper Saddle River, NJ: Pearson Education Inc. Solatia Z, Baharinb BS, Bagheri H. 2012. Supercritical carbon dioxide (SC-CO2) extraction of Nigella sativa oil L. oil using full factorial design. Ind Crop Prod. 36:519–523. Threlfall EJ, Ward LR. 2001. Decreased susceptibility to ciprofloxacin in Salmonella enterica serotype typhi, UK. Emerg Infect Dis. 7:448–450. WHO. 2006. 6th International conference on typhoid fever and other Salmonellosis. Geneva: WHO. Wooa CC, Kumara AP, Sethia G, Tan KHB. 2012. Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem Pharm. 83:443–451.
