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May 2015
The National Ribat University
Faculty of Graduate studies & Scientific Research
Anti-Mycetoma, anti-oxidant and Phytochemical
Screening of Nigella sativa seeds
A Thesis Submitted for Fulfillment of the Requirements of Master Degree
in pharmacy ( pharmacognosy )
By: Rania Mubarak Awad Obaid
Supervisor: Prof. Yahia Mohamed Ahmed El-Imam
May 2015
كال ثعالى :
ذا ىوى﴿ ن ىو ۞وماينطق عن اليوى۞ماضل صاحبكم وماغوى۞ والنجم ا لاا وحي ا
﴾علمو شديد اللوى۞يوحى
صدق الله العظيم
"(5،4،3،2،1" ت)سورة النجم ،الآيا
عن خالد بن سعيد كال، خرجنا و معنا غالب بن أ بجر فمرض في الطريق،
فلدمنا المدينة و ىو مريض، فعاده ابن أ بي عتيق فلال لنا:
" عليكم بهذه الحبيبة السوداء فخذوا منها خمسا أ و س بعا فاسحلوىا، ثم اكطروا في أ هفو
ن عائشة أ م المؤمنين حدثتني أ نها بلطرات زيت في ىذا الجاهب و في ىذا الجاهب فا
:سمعت النبي صلى الله عليو و سلم يلول
ن ى لا السام، كلت و ما السام؟ كال: الموت) ا (ذه الحبة السوداء شفاء من كل داء ا
May 2015
أ خرجو البخاري
Dedication
I dedicate this research to
my family, my supervisor, friends
and all those who helped me to
complete this research………
إىى مه أحمو اسمه تنو إفتخاس )مثاسك عىض عثذ( مه عيمى اىعطاء تذون إوتظاس مه عيمى اىثقح
واىصثش إىى اىىىس اىزي ىش دسب اىىجاح
اىعزز.... إىى أت
مه مان دعائها سش وجاح (إىى معىى اىحة واىحىان واىتفاو إىى تسمح اىحاج) خضشج خيو اىسس
وحىاوها تيسم جشاح إىى اىشمعح اىت تحتشق ىتىش حات
إىى أم اىعززج.......
عثذ( إىى محة اىعيم واىتعيم عثمان وإىى مه إحتاسخ اىنيماخ مارا تقىه ىه وتأي إسم تىادح )عثذ
أتعث ىل مو اىشنش واىتقذش
إىى عم اىعزز.....
May 2015
TABLE OF CONTENTS
Chapter
No.
Particulars Page
No.
Acknowledgements i English abstract ii Arabic abstract iv
Chapter
one
INTRODUCTION & LITERATURE REVIEW 1.1 Introductions 1-2 1.2. Rational /Justification 3 1.3. Objective 3 1.4. literature review 4-60 1.4.1 Nigella sativa 4
1.4.1.1 History 4 1.4.1.2 Nigella sativa in Islam 5 1.4.1.3 Names &Etymology 5 1.4.1.4 Scientific classification 7 1.4.1.5 Origin 7 1.4.1.6 Characteristics 7 1.4.1.7 Species of N.sativa 8 1.4.1.8 Morphology and Description of N. Sativa 9 1.4.1.9 Description of oil of Nigella sativa 9 1.4.1.10 Nigella sativa at different stages of
growth 10-11
1.4.1.11 Chemical Constituents 12-13 1.4.1.12 Commercial Nigella oil 16 1.4.1.13 Adulterations 16 1.4.1.14 Roles of Nigella sativa constituents 17 1.4.1.14.1Thymoquinone 17 1.4.1.14.2 p- Cymene 17 1.4.1.15Traditional uses of N.sativa seeds 17-18 1.4.1.16 Antibacterial action of N.sativa 18 1.4.1.17 Antifungal action of N.sativa 19
1.4.1.18 Antimicrobial and antiparasitic effects 19-21
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1.4.1.19 Physiological effects 21-24 1.4.1.20 Anticancer effects 24-29
1.4.1.21 Anti inflammatory and immune
modulatory effects
29-30
1.4.1.22 Antioxidant and hepatoprotective effects 31-32 1.4.1.23 Toxicity of Nigella sativa 32-33 1.4.1.24 Actions on high risk groups 33
1.4.2Mycetoma (=Maduura Foot) 35 1.4.2.1Characteristics 35 1.4.2.2 Epidemiology 37-39 1.4.2.3 Etiology 40
1.4.2.3.1 Eumycetoma 40-41 1.4.2.3.2 Actinomycetoma 41-42
1.4.2.4 laboratory Diagnosis 43 1.4.2.4.1Specimen 43 1.4.2.4.2 Direct Microscopy 43-44 1.4.2.4.3 culture 45 1.4.2.4.4 Serology 45
1.4.2.5 Management 46-48 1.4.2.6 Detailed Descriptions for most common
microorganisms 49
1.4.2.6.1 Madurella mycetomatis 49 1.4.2.6.2 Pseudallescheria boydii (sexual state);
Scedosporium apiospermum(asexual state) 50
1.4.2.6.3 Nocardia spp. 52 1.4.2.6.4 Streptomyces spp. 53
1.4.2.6.5 Actinomadura spp 54 1.4.2.7 Detailed Descriptions for disease 56 1.4.2.7.1 Actinomycosis: 56 1.4.2.7.2 Mycetoma (Actinomycotic or
Eumycotic ) 57
1.4.2.7.3 Nocardiosis
59
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Chapter
two
MATERIALS & METHODOLOGY 2.1. Material 61-63 2.1.1. Nigella sativa Origin 61 2.1. 2.Other materials 62 2.1.3.Tested bacterial / fungal strains 63 2.2 Method 64-70 2.2.1Preparation of plant extracts 64 2.2.2Preparations of culture media 64 2.2.2.1 Preparations of Sabouraud Dextrose Agar: 64 2.2.2.2 Preparations of blood agar 64 2.2.3Preparation of Specimens before inoculated 64-65 2.2.2 Inoculation or culture of media 65 2.2.5 Phytochemical Screening 65 2.2.5.1 Test for Carbohydrates and / or Glycosides 65-66 2.2.5.2 Test for tannins 66 2.2.5.3 Test for alkaloids and / or nitrogenous bases 66 2.2.5.4 Test for flavonoids 67 2.2.5.5 Test for Saponins 67 2.2.5.6 Test for unsaturated sterols 68 2.2.5.7 Teste for Coumarins 68 2.2.5.8 Teste for Cardiac-glycoside 69 2.2.5.9 Test for Anthracenosides 69 2.2.6.Methods used for screenning anti-oxidant activity 70 2.4.6.1. DPPH radical scavenging assay 70 2.4.6.2. Iron chelating activity assay 70
Chapter
three
RESULTS & DISCUSSION 3.1. Results 71
3.1.1 Phytochemical investigation of the seeds of
N.sativa 71-74
3.1.2. Screening for antioxidant 74 3.1.3. Screening for antimicrobial activity 75-89 3.1.3.1. Subculture from Saudi Arabia examined after
7 days of incubation 75
3.1.3.2. Subculture from Saudi Arabia examined after
14 days of incubation 78
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3.1.3.3. Subculture from Saudi Arabia examined after
23 days of incubation 80
3.2. Discussion 90-92
Chapter
four
Conclusion & Recommendation 93
Chapter
fife
References 94-
113
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LIST OF TABLES
Table
No.
Table Name Page
No. 1.1 Some names of Nigella in comparison to local term 6 1.2 Chemical constituent of Nigella sativa seed 12-13 1.3 The nutritional value of Black Seeds 15 1.4 Biological activities of N.sativa 18 1.5 Methods of N.sativa application for treatment of diseases 34 1.6 Characteristics of the main species of the Actinomycetes 42 1.7 The color of the grains in mycetomas & related species 44
1.8 Differentiation of aerobic Actionmycetes 55
1.9 The main clinical differences between eumycotic and actinomycotic
mycetoma 60
2.1 Instruments 61 2.2 Media 61 2.3 Solvents 61 2.4 Tested fungal strains (Eumycetoma) 63 2.5 Tested bacterial strains (Actinomycetoma) 63 3.1 The percentage yield of the oil 71 3.2 The phytochemical screening of the methanolic extract 71-72 3.3 The phytochemical screening of the chloroformic extract 73-74 3.4 Screening of methanolic extract for antioxidant 74 3.5 Antifungal activity against (Pseudallescheria boydii and 4samples of
Madurella mycetomatis) after 8 days 82
3.6 Antifungal activity against (Pseudallescheria boydii and 4samples of
Madurella mycetomatis) after 30 days 82
3.7 Antibacterial activity against (Nocardia brasiliensis, N.asteroides and
Streptomyces samaliensis) after 8 days 86
3.8 Antibacterial activity against (Nocardia brasiliensis, N.asteroides and
Streptomyces samaliensis) after 30 days 86
3.9 Minimum inhibitory concentration of methanol oil extract against
mycetoma 89
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LIST OF FIGURES
Figure
No.
Figure Name Page
No. 1.1 Nigella sativa 8 2.2 Nigella damascene 8 1.3 Nigella arvensis 8 1.4 Oil of Nigella sativa: 9 1.5 Nigella plant with unripe seed pods 10 1.6 Unripe Nigella capsule (culinary) 10 1.7 Nigella plants at the end of their flowering period 10 1.8 Nigella seeds 11 1.9 T.S. of the N.sativa seed 11 1.10 Chemical Structures of some major compounds isolated from N.
sativa 14
1.11 Seeds of Argemone Mexicana 16 1.12 Onion (Allium cepa L) seeds 16 1.13 Mycetoma infections 36-37 1.14 Map showing the geographical distribution of some eumycetoma
agents 39
1.15 Map showing the geographical distribution of mycetoma in sudan 39
1.16 Madurella mycetomatis colonies 40 1.17 Pseudallescheria boydii colonies 41 1.18 Comparison between of Actinomycotic & Eumycotic grain 43 1.19 Actinomycete & Nocardia filaments 44 1.20 Actinomycete filaments from culture 45 1.21 Schematic Representation of the Balance between Amphotericin B-
Induced Nephrotoxicity, Patient Risk Factors, and Cost Effective
Therapy
47
1.22 Anti fungal drugs overall average cost in 1993 48 1.23 Microscopic morphology of Madurella mycetomatis 49 1.24 Microscopic morphology of Pseudallescheria boydii (sexual state);
Scedosporium apiospermum(asexual state) 51
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1.25 Microscopic morphology of Nocardia spp. 52 1.26 Microscopic morphology of Streptomyces spp. 53 1.27 Microscopic morphology of Actinomadura spp. 54 1.28 Organism morphology of Actinomycosis 56 1.29 Organism morphology of ( Actinomycotic or Eumycotic ) 58 1.30 Organism morphology of Nocardiosis 59 3.1 Madurella mycetoma(SA) subculture after 7 days 75 3.2 Madurella mycetoma(SU2) subculture after 7 days 75 3.3 Madurella mycetoma(pot) subculture after 7 days 76 3.4 Nocardia asteroides subculture after 7 days 76 3.5 Nocardia brasiliensis subculture after 7 days 76 3.6 Comparison of change in media colour between Culture tubes &
blank tube media 77
3.7 Madurella mycetoma(SA) subculture after 14 days 78 3.8 Madurella mycetoma(Su2) subculture after 14 days 78 3.9 Madurella mycetoma(pot) subculture after 14 days 79 3.10 Nocardia asteroides subculture after 14 days 79 3.11 Nocardia brasiliensis subculture after 14 days 79 3.12 Madurella mycetoma(SA) subculture after 23 days 80 3.13 Madurella mycetoma(SU1) subculture after 23 days 80 3.14 Madurella mycetoma(pot) subculture after 23 days 81 3.15 Nocardia asteroides subculture after 23 days 81 3.16 Nocardia brasiliensis subculture after 23 days 81 3.17 Culture of methanol extracts tested against Madurella
mycetomatis(SA) 84
3.18 Culture of methanol extracts tested against Madurella
mycetomatis(Pot) 84
3.19 Culture of methanol extracts tested against Madurella
mycetomatis(Su1) 84
3.20 Culture of methanol extracts tested against Madurella
mycetomatis(Su2) 84
3.21 Culture of methanol extracts tested against Pseudallescheria boydii 84 3.22 Culture of chloroform extracts tested against Madurella
mycetomatis(SA) 85
3.23 Culture of chloroform extracts tested against Madurella
mycetomatis(Pot) 85
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3.24 Culture of chloroform extracts tested against Madurella
mycetomatis(Su1) 85
3.25 Culture of chloroform extracts tested against Madurella
mycetomatis(Su2) 85
3.26 Culture of chloroform extracts tested against Pseudallescheria
boydii 85
3.27 Culture of methanol extracts tested against Nocardia brasiliensis 87 3.28 Culture of methanol extracts tested against Nocardia asteroides 87 3.29 Culture of methanol extracts tested against Streptomyces
samaliensis 87
3.30 Culture of chloroform extracts tested against Nocardia brasiliensis 88 3.31 Culture of chloroform extracts tested against Nocardia asteroides 88 3.32 Culture of chloroform extracts tested against Streptomyces
samaliensis 88
3.33 Minimum inhibition concentration of methanol oil extract against
mycetoma 89
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May 2015
Acknowledgments:
I thank almighty Allah for giving me health, strength and patience to perform this
study.
I would like to experess my sincere thanks to my supervisor Prof.Yahia M.Ahmed
El-Imam, Department of pharmacognosy, Faculty of pharmacy, Ribat National
University, for his continuous encouragement, advises and patience supervision of this
study.
Words are failed to express my grateful and thanks to Dr.Mohammed Chyad Al-
Noaemi, doctor of pathophysiology, faculty of medicine, Al-yarmok college, khartoum,
for his patience to help me with his knowledge, experience, direction, advising, with his
May 2015
fatherly care for me, his unlimited support and constant encouragement to complete this
study.
My special appreciation to Dr. Ahmad Mohamad Al-Barag, head of department of
Mycology laboratory in King Khalid hospital, faculty of medicine, King Saud University,
Saudi Arabia, Al-Riydh who was largely responsible for the improvement of this study
with his advise, supplement of valuable materials of the study and encouragement to
finish it.
My warmest heart thanks to Dr.Maawahib Abdel Moneim Ibrahim, member of
Mycology laboratory in Stak laboratory for her close constant help and support in
performing this study.
My special thanks for Nour , Def Allah, Melven and Roby (staff members of
Mycology laboratory in king Khalid hospital), for allowing me to use its facilitates and
their continuous help to do this work.
Sincere thanks and appreciation to my friends: Dr.Yasser Hamad Alnel & Dr.Mazen
Mohamad Mustafa for helping in writing & printing to complete this search.
Deep thanks to all who offered helps and encouraged me to complete this work.
Everlasting thanks to my father, mother, brothers and sisters, who show continuous
support and encouragement for me hoping to see this work finished, to see me in better
situations.
More and more everlasting thanks to ALLAH.
Abstract: Black cumin (Nigella sativa) is a spice native to Mediterranean region (family
Ranunculaceae). The seeds of black cumin have been used in traditional medicine by
many Asian, Middle Eastern and Far Eastern Countries to treat headache, coughs,
abdominal pain, diarrhea, asthma, rheumatism and other diseases. The oil extracts of the
seeds have been shown to possess antioxidant, anti-inflammatory, anticancer, analgesic
and antimicrobial activities.
In Sudan Madurella mycetomatis is the commonest causative organism causing
Eumycetoma and Streptomyces somaliensis is the commonest organism
causing actinomycetoma (Fahal et al, 2011).
In general, the current treatment for mycetoma is expensive and unsatisfactory. It needs a
long duration, and has many side effects. (Mahgoub ES.,1984) .
May 2015
If drugs not effective and bone is infected, amputate the limb or debride tissue and
continue treatment up to years.
In the present study, anti-mycetoma activity of the chloroform and methanol extracts of
Nigella sativa seed were studied in vitro against five isolates of fungi type
(Pseudallescheria boydii and 4samples of Madurella mycetomatis) and three isolates of
bacteria type ( Nocardia brasiliensis, N.asteroides and Streptomyces samaliensis ) of
mycetoma.
The results showed that the methanol and chloroform extracts had antimycetomal activity
against Nocardia brasiliensis and N.asteroides with a minimum inhibitory concentrations
of 4% and 8% respectively. The least anti-mycetomal activity was recorded against
Streptomyces somaliensis with methanol extract and MIC of 0.5% with chloroform
extract .
On the other hand chloroform extract showed activity against Madurella mycetomatis
with minimum inhibitory concentration 2% while in case of Pseudallescheria boydii the
minimum inhibitory concentration was 8% ,while methanol extract with MIC of 0.15%
with all fungal types.
The methanolic extract was also screened for its anti-oxidant activity. Iron chelating
recorded a high activity than DPPH radical scavenging assay.
Qualitative phytochemical screening of the methanol extract showed the presence of
tannins, alkaloids, quarternary bases & oxidized amines, saponins, carbohydrates,
reducing sugars, unsaturated sterols, coumarins, flavonoid, terpenoids, cardiac
glycosides, essential oil, fatty acids.
On other hand the qualitative phytochemical screening of the chloroform extract showed
the presence of alkaloids, quarternary bases & oxidized amines, saponins, unsaturated
sterols, coumarins, flavonoid, terpenoids and cardiac Glycosides.
اىخلاصح
) انؽثح انسداء )انعلا ساذافا( ي انراتم الأطهح ف يطقح انثؽش الأتغ انرسؾ انر ذؽذس ي ػائهح
رااخ قذ أسرخذيد تزس انك الأسد ف انطة انرقهذ ي قثم انؼذذ ي انثهذا ف انششق الاسؾ ( انؽ
. الإسال انشت انشياذضو غشا ي الأيشاعدل ششق أسا نؼلاض انظذاع انسؼال آلاو انثط
إيرلاك انفؼانح انؼادج نلأكسذ , انؼادج نلإنراتاخ , انؼادج قذ أشثد ا يسرخهض صد انؽثح انسداء
نهسشؽا , يسك شاؽاخ يؼادج نهكشتاخ.
انفطش نهاسريا انسثؽح انرسهسهح الأكصش ف انسدا انادسهح انفطشيح الأكصش شػا يسثث نهع
, ؽراض نفرشج غش اظػ , شػا يسثث نهع انثكرش نهسريا. تشكم ػاو انؼلاض انؽان نهاسريا تاع انص
ؽه نهؼلاض نذح ػذج أشاس ظاثح.
May 2015
نظاب أ الاسعح انظاتح يغ الإسرشاس ف ؼانح ػذو عاغ انؼلاض أطة انؼظى , رى انهعء نؽم ترش انؼؼ ا
ف أخز انؼلاض نذج سح تؼذ انؼهح.
نسرخهظ ي تزس ؼثح انثشك هاسريا انشاؽ انؼاد ن دساسح دذ ز انذساسح, ف
انهسكشح انكارتح ) انفطش ي انع أاع ػذ خسح ف انخرثش ذى الإخرثاس انرعشثانصال( انكهسفسو )
انكاسدح انثشاصهح, ) انثكرش عان يشلاز ااع ػذ انثذح استغ أاع ي انادسهح انفطشيح (
.اناسريا ي( انسثؽح انرسهسهحانكاسدح انعح,
انرائط ا انسرخهض انصان انسرخهض انكهسفسي نذى انرأشش انؼاد نهاسريا يغ أظشخ
% تانران. كا أقم ذأشش يؼاد 4% 2اقم ذشكض فؼال كا انكاسدح انعح انكاسدح انثشاصهح
% يغ انسرخهض 5.0فؼال كا نهسريا ػذ انسثؽح انرسهسهح يغ انسرخهض انصان أقم ذشكض
انكهسفسي .
أقم انادسهح انفطشيح ي اناؼح الأخش أظش انسرخهض انكهسفسي انرأشش انؼاد نهاسريا ػذ
%, نك كا أقم ذشكض 8كا أقم ذشكض فؼال انهسكشح انكارتح انثذح% ف إسرخذايا ػذ 2ذشكض فؼال كا
يغ انسرخهض انصان ػذ ظغ انع انفطش ي اناسريا. %0..5فؼال
ػذ ػم يسػ نهشاؽ انؼاد نلأكسذ نهسرخهض انصان تئخرثاس ذخهة أ ذكانة انؽذذ أظش شاؽ أػه ي
إقراص انشقق انؽشج تئسرخذاو يادج أخرثاس
. DPPH (1,1-diphenyl-2-picrylhydrazyl)
نذساسح انسػ انكائ انثاذ نهسرخهظ )انصال انكهسفسو ( ؼس أشثرد انذساسح ا ذاند ز ا
انسرخهض انصان ؽر ػه ياد دتاغح يركصفح, قهذاخ, انقاػذ انشتاػح, الأياخ انؤكسذج, طاتاخ,
ذ, ظلاكسذاخ قهثح, صخ ؽاس , انكشتذساخ, انسكشاخ انخرضنح, كيشاخ, فلافاخ, ذشت
أؼاع أيح.
ي اؼح أخش أشثرد انذساسح أ انسرخهض انكهسفسي ؽر ػه قهذاخ, انقاػذ انشتاػح, الأياخ
انؤكسذج, طاتاخ, كيشاخ, فلافاخ, ذشتذ, ظلاكسذاخ قهثح.
May 2015
1. Introductions:
Folk medicine is the mother of all other systems of medicine and even modern
medicine. The knowledge about certain herbs, which have curative and palliative effects
on disease conditions, has been transmitted from one generation to another by
experienced elders and also by tribal herbal specialists. Every culture throughout history
has used plants to treat medical problems. Originally, the specific utility of herbs was
assumes to be based on their shape or colour. With this primitive Doctrine of Signatures
approach, heart shaped leave were used against heart problems, plant with red flowers
were applied to treat bleeding disorders, and so on (Cassileth, 1998) Plant products have
been used for centuries as medicines. Today in most of the developing world, plant
remedies are the most prevalent treatments, with recipes handed down from generation to
generation. They are available, and there is generally a more culturally sensitive attitude
on the part of these practitioners (Spencer & Jacobs, 1999).
Nigella sativa, is one of the oldest medicinal plants which has been used for the
treatment of a broad range of illness. It has a wide range of, antibacterial (Toama et al,.
1974), (Halamova et al,. 2010), antifungal (Rogozhin et al,. 2011), antiviral (Kmen,.
2001), anti-arthritic (Tekeoglu et al ,2006), anti-neoplastic (Khan et al,. 2011),( Ali &
Blunden,. 2003), anti-diabetic (Kanter et al,. 2003) ,( Rchid et al,. 2004), (El-
Dakhakhny et al,. 2002), antioxidant (Khlife & Lupidi,. 2007),( Cemek et al,. 2006),
anti-inflammatory (El-Dakhakhny et al,. 2002),( Mansour & Tornhamre,. 2004), anti-
hypertensive (Ali & Blunden,. 2003) ,spasmolytic and bronchodilator (Gilani et al,.
2001), hypo-lipidemic effects (Dahri et al,. 2005) ,( Nader et al,. 2010), nephro and
hepatoprotective( Al-Kubaisy & Al-Noaemi,. 2007),( Nasim et al,. 2006).
May 2015
However we have a lot of diseases that have no treatment like Mycetoma (=Madura
foot ) which is endemic in tropical and subtropical regions {Sudan, Somalia, Senegal,
India, Yemen, Mexico, Venezuela, Columbia, Argentina and others (Fahal , 2011)}.
The African continent seems to have the highest prevalence especially in Sudan, which is
considered to be the (Mycetoma homeland), and Central Sudan is the most affected part
of the country (Fahal, 2011).
Rippon, 1988 reported that Mycetoma in Sudan appears to have the highest number of
cases per capita per year, which amounts to about 300 to 400 actual infections.
Mycetomas is a chronic progressive localized infection of the skin and subcutaneous
tissue which is believed to originate following the implantation of the causative agents
through minor trauma to the skin, usually from vegetative material such as thorns or
splinters( Fahal, 2004) , (Gumaa, 1994), (Mahgoub., 1994) ,(McGinnis,1996).
The disease frequently involves lower extremities of barefooted adult rural men involved
in agriculture or cattle related works.
Multiple nodules may appear on the same limb usually painless. These lesions rupture,
resulting in sinus tracts, and eventually if untreated, leads to destruction of deeper tissues
and bone, resulting in deformity and disability which may necessitate amputation,
(McElroy et al ,1992), (Ahmed et al,1999),( Sharma et al,2008).
Worldwide, 60% of mycetomas are bacterial (actinomycetoma) and 40% are fungal
(eumycetoma) ,(Kemper,2000),( Pang ,2004).
In Sudan Streptomyces somaliensis is the commonest organism causing actinomycetoma
and Madurella mycetomatis is the commonest causative organism causing Eumycetoma
(Fahal, 2011).
In general, the current treatment for mycetoma is expensive and unsatisfactory. It needs a
long duration, and has many side effects. As Nigella sativa was proved to have a broad
spectrum of antibacterial and antifungal activity, (Kemper, 2000),( Pang,,2004) the aim
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of the present study was to test its effects on mycetoma whether bacterial or fungal type
which could supplement the current therapeutic methods for mycetoma.
1.2 Rationale /Justification:
(ا ن ىذه الحبة السوداء شفاء من كل داء ا لا السام، كلت و ما السام؟ كال: الموت) :عائشة أ م المؤمنين أ نها سمعت النبي صلى الله عليو و سلم يلول عن
أ خرجو البخاري
Prophet Mohammed (peace be upon him) stated that ―The black seed heals all diseases
except death‖ (Sahih Bukhari 71:592).
Mycetoma disease is widely spread in sudan and it is considered as Mycetoma homeland.
Mycetoma caused by fungi are usually resistant to chemotherapy (Restrepo., 1994) and as
all anti fungal drugs induced nephrotoxicity, the current research was an attempt to find
an affordable and safer treatment for this disease.
1.3 Objectives:
Main objectives:
The aim of the present study is:
1- To investgate the chemical composition of N.sativa.
2- To screen the seed extracts for the antimicrobial and antioxidant activites.
3- To provide an effective antifungal drug that does not induce nephrotoxicity.
4- To avoid the social consequences of amputation.
5- To help in the eradication of Mycetoma, because it is a life-mutilating disease and
have a high morbidity and can be fatal and both surgical and medical treatments
are not satisfactory.
1.4 literature review:
1.4.1 Nigella sativa:
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Nigella sativa (Ns), an annual flowering plant belongs to the family Ranuncuaceae . Ns a
natural food additive, economically important and has a history of 2500 years which
makes it one of the safest plant extracts for human consumption. It is believed to be
indigenous in the dry temperate dimater of Mediterranean region but has been cultivated
into other parts of the world including the Arabian peninsula, northern Africa, Egypt,
Jammu-Kashmir, HimachalPradesh, Afghanistan, Baluchistan, Iran and probably western
Asia. The seeds, rich in essential oil, are consumed widely as condiment. In the
indigenous system of medicines, seeds and reregarded as stimulants and carminatives and
found to be useful in diarrhoea and dyspepsia. Also, this plant is used for culinary
purposes and for flavoring foods and beverages (Abdugniew et al., 1997).
1.4.1.1 History:
The earliest cultivation of N.sativa "is still scanty", but these is report that N.sativa seeds
have been found in several sites from ancient Egypt, including Tutan khamun's tomb.
Although its exact role in Egyptian culture is unknown, it is known that items entombed
with a pharaoh were carefully selected to assist him in the after life.
The earliest written reference to N. sativa is thought to be in the book of Isaiah in the Old
Testament where the reaping of nigella and wheat is contrasted
N. sativa "was another traditional condiment of the Old World during classical times; and
its black seeds were extensively used to flavor food.―
Although nigella is not mentioned in the common Bible translations, there is good
evidence that an obscure plant name mentioned in the Old Testament means nigella; if
true, this would indicate that nigella is cultivated since far more than two millennia
(www.ikitab.com/herbs/blackseed.html).
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1.4.1.2 Nigella sativa in Islam:
In Islam, it is regarded as one of the greatest forms of healing medicine available.
Prophet Muhammad once stated that the black seed can heal every disease -- except death
(Sahih Bukhari 71:592).
1.4.1.3 Names &Etymology:
Botanical Name: Nigella sativa
In English: it is called fennel flower, black caraway, nutmeg flower, Roman coriander, or
black onion seed. Etymology: Nearly all names of nigella contain an element meaning
black in reference to the unusually dark color of the seeds (Redgrave. et al, 1933).
There is a lot of confusion about the names of this spice: In some English sources,
Central Asia and Northern India, it is called black cumin, black caraway and black onion
seed but there is no botanical relation between Nigella sativa and any of these plants
(www.plantnames.unimelb.edu.au).
Table 1.1 Some names of Nigella in comparison to local term:
(www.plantnames.unimelb.edu.au).
Language Name Black German Schwarzkümmel Schwarz
Norwegian Svartkarve Svart
Swedish Svartkummin Svart
Latvian Melnsēklīte Melns
Lithuanian Juodgrūdė Juodas
Estonian Mustköömen Must
Finnish Mustakumina Musta
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Hungarian Feketeköméni Fekete
Latin Nigella Niger
Italian grano nero Nero
Spanish Niguilla Negro
Portuguese cominho-preto Preto
Romanian Negrillică Negru
Polish Czarnuszka Czarny
Ukrainian Chornushka Chornyj
Russian Chernushka Chyornyj
Czech černý kmín Černý
Slovak Černuška cern, cernoch
Slovenian vzhodna črnika Črn
Croatian crni kumin Crn
Serbian crno seme Crn
Greek Melanthion Melas
Arabic kamun aswad Aswad
Amharic tik'ur azmud tik'ur
Turkish kara çörek otu Kara
Turkish siyah kimyon Siyah
Farsi siah daneh Siah
Kurdish Siawasa Siawa
Sanskrit Krishnajira Krishna
Hindi Kalaunji Kala
Panjabi Kalonji Kala
Sinhala Kaladuru Kalu
Kannada kari jirige Karidu
Malayalam Karinjirakam Kari
Chinese hei zhong cao Hei
Thai thian-dam Dam
Indonesian jintan hitam Hitam
1.4.1.4 Scientific classification :
Kingdom: Plantae
Order: Ranunculales
Family: Ranunculaceae
Division: Magnoliophyta
Class : Magnoliopsida
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Genus: Nigella
Species: N .Sativa
(Redgrave. et al, 1933).
1.4.1.5 Origin:
Nigella sativa is an annual flowering plant. Is believed to be indigenous to the
Mediterranean region but has been cultivated into other parts of the world including the
Arabian Peninsula, northern Africa and Probably Western Asia. Today, the plant is
cultivated from Egypt to India (Redgrave.et al,. 1933).
1.4.1.6 Characteristics:
Taste of N. sativa flowers: Has a pungent bitter taste
Taste of N.sativa seeds: Is aromatic and slightly bitter; it is called pungent and smoky
and even compared to black pepper. There is, however, some pungency in unripe or not
yet dried seeds.
Odor of N. sativa flowers: A faint smell of strawberries. It is used primarily in candies.
Odor of N.sativa seeds: Have little odor, but when ground or chewed they develop a
vaguely oregano-like scent (Redgrave. et al ,1933).
1.4.1.7 Species of N.sativa:
There are three Species of Black Seeds (Redgrave et al ,1933):
1-Nigella sativa or small Fennel Flower.
2-Nigella damascene or Wild Fennel.
3-Nigella arvensis or Small Fennel.
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Fig 1.1
Fig 1.2
1.4.1.8 Morphology and Description of N. sativa:
The Stalk: It grows to 20–30 cm (7.9–12 in) tall, and the stalk is hard or stiff, ragged,
covered by soft hair, with finely divided.
The leaves: it blush green colored and feather crisscross and linear (but not thread-like) 1
to 2 inch long and are spear shaped.
Nigella
sativa
Nigella.
damascena
Nigella.
arvensis
Fig 1.3
May 2015
The flowers: are delicate, and usually colored pale blue and white or greenish blue, are
one inch in diameter with 5–10 petals. Flowers appear in early winters.
The fruit: is a large and inflated capsule composed of 3–7 united follicles, each
containing numerous seeds' ruts in winters. The mature fruit are round, triangular, black
colored, and aromatic, wrinkled and has many seeds in them.
The seed: black colored is used as a spice and pulp is white (Redgrave et al, 1933).
1.4.1.9 Description of oil of Nigella sativa:
The oil is reddish brown colour. They are also used as a stabilizing agent for edible
fats. Locally, oil is Anaesthetic (Redgrave et al, 1933).
Fig 1.4 Oil of Nigella sativa: (Redgrave et al, 1933).
1.4.1.10 Nigella sativa at different stages of growth:
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Fig 1.5 Nigella plant with unripe seed pods: (Redgrave et al, 1933)
Fig 1.6 Unripe Nigella capsule (culinary): (Redgrave. et al, 1933)
Fig 1.7 Nigella plants at the end of their flowering period: (Redgrave et al, 1933)
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Fig 1.8 Nigella seeds: (Redgrave et al, 1933)
Fig 1.9 T.S. of the N. seed:
(Redgrave et al, 1933)
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1.4.1.11 Chemical Constituents:
Table 1.2 Chemical constituent of Nigella sativa seed:
Main chemical
constituents
Names References
Essential oil
Fixed oil (Cheikh-Rouhou et al., 2007
and Houghton et al., 1995).
Thymoquinone (TQ) (Chopra et al.,1956 ,Adams,
2007 and Hajhashemi et al.,
2004)
P-cymene (Hajhashemi et al., 2004)
a-Pinene (Hajhashemi et al., 2004)
Dithymoquinone&
thymohydrquinon
(Vuorelaa et al., 2004,Adams,
2007 and Abou-Basha et al.,
1995)
Oligocondensation products
(nigellone)
(Adams, 2007 and tekeogluet
al., 2006).
Thymol (Hajhashemi et al., 2004 and
tekeogluet al., 2006).
Terpenoid Other terpene derivatves: Carvone ,
Limonene,4-terpineol, citronellol, t-
anethol, sesquiterpene
(Hajhashemi et al., 2004 and
Burits and Bucar, 2000)
Fatty oil :
1-Unsaturated fatty
acids:
Linoleic acid
(Adams, 2007 and
Hajhashemi et al., 2004)
Oleic acid (Adams, 2007 and
Hajhashemi et al., 2004)
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Eicodadienoic acid (Hajhashemi et al., 2004)
Dihomolinoleic acid (Hajhashemi et al., 2004)
2- Saturated fatty
acids:
Palmitic ,Stearic acid (Cheikh-Rouhou et al., 2007
and Adams, 2007).
Alkaloids:
1-Isoqinoline
alkaloide:
Nigellicimine & Nigellicimin-N-
Oxide
(tekeogluet al., 2006 and
Kruk et al., 2000),
2-Pyrazol alkaloids
or imdazole ring::
Nigellidine & Nigellicine (tekeogluet al., 2006 and
Kruk et al., 2000),
Saponins: Melanthin & Alpha-hederin
(nigelline)
(Adams, 2007 and Akhondian
etal., 2007)
Tannins (Adams, 2007).
sterol : sitosterol, stigmasterol (El-Mahmoudyet al., 2002).
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Fig 1.10 Chemical Structures of some major compounds isolated from N. sativa
Table 1.3 The nutritional value of Black Seeds:
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Nutrient
Average
References
Energy (kcal (MJ) (Haq et al., 1999)
Protein (g) (Haq et al., 1999 and Ghedira, 2006)
fat (Ghedira, 2006).
carbohydrates (Ghedira, 2006).
crude fiber (Ghedira, 2006) (Ali and Blunden, 2003).
carotene (vit.A) (Cheikh-Rouhouet al., 2007)
Thiamin (mg) (Benkaci-Ali etal, 2007)
Riboflavin (mg) (Benkaci-Ali etal, 2007).
Pyridoxine (mg) (Benkaci-Ali etal, 2007).
Niacin (mg) (Benkaci-Ali etal, 2007).
Calcium (mg) (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003).
Iron (mg) (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003 and Singh et al., 2005).
Copper (mg) (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003 and Singh et al., 2005).
Zinc (mg) (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003 and Singh et al., 2005).
Phosphorus (mg) (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003 and Singh et al., 2005).
Folacin (mg) (Benkaci-Ali etal, 2007)
Na (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003).
K (Benkaci-Ali etal, 2007) (Ali and Blunden, 2003).
1.4.1.12 Commercial Nigella oil:
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May also contain parts of the essential oil, mostly thymoquinone, by which it acquires
an aromatic flavor, trace elements, vitamins and enzymes
It contains 58% of essential fatty acids including omega 6 and omega 3
(www.caravanetresor.com/tiny-teasure.htm).
1.4.1.13 Adulterations:
The market samples of the seeds of N. sativa are often adulterated.
1- seeds of Argemone mexicana are often mixed with it :
Fig 1.11 seeds of Argemone mexicana
2-N. sativa seeds are also commonly confused with onion (Allium cepa L) seeds.
Fig 1.12 onion (Allium cepa L) seeds
(www.caravanetresor.com/tiny-teasure.htm)
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1.4.1.14 Roles of Nigella sativa constituents:
1.4.1.14.1Thymoquinone:
1- It is considered as anticancer specifically the rectum and colon cancer by gene
called (P53) (Salem and Hossain, 2000).
2- Prevent from the angiogenesis (Anti-tumer) (Salem and Hossain, 2000).
3- Antinflamatary and important in treatment of romatoide problem
(Ali and Blunden, 2003 and Ramadan, 2007).
4- In treatment of the respiratory distress syndrome acute (Gilani et al., 2001).
5- Hypoglycemic effect (Zaoui et al., 2002a).
1.4.1.14.2 p- Cymene:
It is considered bactericidal, specifically against E.coli and prevent the
deterioration of apple juice(El-Fatatry, 1975).
1.4.1.15 Traditional uses of black seed:
1- Black Seed is used in perfumery, especially in soaps, and as a spice in cakes,
breads, pastries, confectionery, sauces, cheese, etc. Also used as flavouring
material.
3- The seeds of N. savita are considered carminative, stimulant, diuretic,
Emmenoagogue, Galactaogogue, and are used in the treatment of mild cases of
puerperal fever.
4-They are externally applied for eruptions of skin. Alcoholic extracts of the seeds
show antibacterial activity against Micrococcus pyrogenes var. aureus and
Escherichia coli.
May 2015
5- Preliminary clinical trials indicate its possible therapeutic use in some
conditions of cough and bronchial asthma. Clinical trials also indicate its use for
control of Blood Sugar & Chololesterol.
6- It is used for different treatment hair fall, head ache, rhinitis, piles, pain,
tastelessness, and indigestion.
7-The seeds are also used in the treatment of hydrophobia, tertiary fever, and
paralysis.
Table 1.4 Biological activities of N.sativa :
Antibacterial Antitumor Hypotensive
Anti yeast Antitoxic Diuretic
Antioxidants Antispasmodic Hypoglycemic
Antihyper cholesterolemic Hemostatic Hypolipaermic,
Bronchodilator
Analgesic Antifungal activities
1.4.1.16 Antibacterial action of N.sativa:
The crude extracts of N. Sativa were reported to have promising effect on multi-
organism as anti-bacterial ,anti-fungal , anti-yeast ,anti-protozoal ,anti -helmintic
action.
El-Fatatry, 1975 showed that Bacillus subtilis cannot grow in the diet containing
Nigella sativa because it stops the growth, that indicates the seed of N.sativa
contain anti-bacterial material .
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The crystal material isolated from oil solidification of Nigella sativa which is
known as thymoquinone has a large effect on bacteria (El-Fatatry, 1975).
The volatile oil that was extracted from the Nigella sativa showed an effect on
Gm+ve bacteria as: Staphylococcus aurous inhibition and the Gm-ve bacteria as:
E.colli likes effect of Streptomycin (Hanafii et al., 1991).
1.4.1.17 Antifungal action of N.sativa:
Nigella sativa was found to have antifungal activity against Candida albicans on
rats (Khan ., 2003) ( Manohar Et al., 2001).
The oil of Nigella sativa has a large effect on the tapeworm and threadworm
(Mahmoud et al., 2002).
Akhtar & Riffat., 1991 reported that Nigella sativa seeds has the same effect on on
tapeworm as that of neioklzumid .
Some of the other therapeutic properties attributed to N.sativa oil are anticestodic,
antinematodic, hepatoproctative (Mahmoud et al., 2002).
Antioxidant and antiviral effect against murine cytomegalo virus infection (Salem
and Hossain, 2000).
The crude extract of N.stiva seed exhibited spasmolytic and bronchiodialatory
activities (Gilani et al., 2001) .
1.4.1.18 Antimicrobial and antiparasitic effects :
Extracts of black cumin oil (BCO) have shown promising effects against bacteria, fungi,
parasites and worms. The purified compound thymoquinone (TQ) from black cumin oil
was found to have high antimicrobial effect against Gram positive microorganisms
(Manohar et al., 2001). The seed extracts of black cumin were found to inhibit the growth
May 2015
of Escherichia coli, Bacillus subtilis and Streptococcus feacalis (Mansour et al., 2002).
The antimicrobial activity of black cumin seed extract was further established against
several species of pathogenic bacteria and yeast (Manohar et al., 2001). The filter paper
discs impregnated with the diethyl ether extract of black cumin seeds caused
concentration dependent inhibition of Gram positive Staphylococcus aureus and Gram
negative Pseudomonas aerginosa and E.coli and a pathogenic yeast Candida albicans.
The extract showed antibacterial synergism with streptomycin and gentamicin and
showed an additive antibacterial synergism with streptomycin and showed an additive
antibacterial action with spectinomycin, erythromycin, tobramycin, doxycycline,
chloramphenicol, nalidixic acid, ampicillin, lincomycin and sulfamethoxyzole-
trimethoprim combination (Manohar et al., 2001). Interestingly, the extract of black
cumin seed successfully eradicated a non-fatal subcutaneous staphylococcal infection in
mice when injected at the site of infection (Mansour and Tornhamre, 2004). Recently,
crude extracts of black cumin seeds showed promising antimicrobial effects against
bacterial isolates with multiple resistances against antibiotics (Morsi, 2000). The most
effective extract were the crude alkaloid and water extracts. The antiparasitic actions of
BCO have been well documented (El-Mahdy et al., 2005 and Mahmoud et al., 2002).
The antihelminthic activities of BCO were studied by Ghedira (2006) who reported that
the essential oil from the black cumin seeds showed pronounced activity even in 1:100
dilutions against tapeworms and earthworms. Anticestodal effects of black cumin seeds
were studied in children infected naturally with the respective worms. A single oral
administration of 40 mg/kg of black cumin ethanolic extract reduced the percentage of
the fecal eggs without producing any adverse side effects in the doses tested (Akhtar and
Riffat, 1991). When given orally to Schistosoma mansoni-infected mice, a 2- week
treatment with BCO reduced the number of S.mansoni worms in the liver and decreased
the total number of ova deposited in both the liver and the intestine ( Mahmoud et al.,
2002). When BCO was administered in combination with praziquantel, the drug of choice
May 2015
for the treatment of schistosomiasis, the most prominent effects was a further lowering of
the dead ova number over that produced by praziquantel alone. These changes were
correlated mainly with the ability of BCO to improve liver function and the
immunological system of infected mice and partly to its antioxidant effects (Mahmoud et
al., 2002). The protection is also due to the ability of BCO and TQ to reduce the
cytogenetic damage induced by schistosomiasis infection (Aboul-Ela, 2002). The
antiviral effect of BCO was investigated using murine cytomegalovirus as a model
(Salem and Hossain, 2000). Intraperitoneal administration of BCO to mice strikingly
inhibited the virus titers in spleen and liver on day 3 of infection. The antiviral effects of
BCO were more potent than the action of Chinese traditional herbal medicine hochuekki
against murine cytomegalovirus (Hossain et al., 1999).
1.4.1.19 Physiological effects of TQ:
The oil extract of black cumin seeds has been reported to exert effects on various systems
including the respiratory, cardiovascular, gastric and uterine and smooth muscle (Shah
and Ray, 2003). The effects of intravenous administration of volatile oil and of TQ were
investigated on the respiratory system of the rat (Pagola et al., 2004). TQ was found to
increase the intratacheal pressure in the dose range of 4-32 ml/kg and 1.6-6.4mg/kg,
respectively. Although black cumin (N. sativa L.) oil (BCO) increased significantly the
respiratory rate in rats, it has any effect. The effects of BCO were antagonized
significantly by the treatment of the animals with atropine and reserpine, which may
mean that the oil-induced respiratory effects can be mediated through the release of
histamine and the indirect activation of muscarinic and cholinergic mechanisms (Pagola
et al., 2004).This also suggested that the removal of TQ from black cumin seed oil might
provide a potential centrally acting respiratory stimulant (Pagola et al., 2004).It was also,
demonstrated that the intravenous administration of black cumin oil (BCO) (4-32 ml/kg)
or TQ (0.2-1.6 mg/kg) to rats decreased the arterial blood pressure and the heart rate in a
May 2015
dose-dependent manner (Ghoisheh et al., 1999), hinting that the oil may possess
antihypertensive effects. The cardiovascular depressant effects of the oil were
significantly antagonized by atropine and cyproheptadine, which may mean that these
effects were mediated mainly centrally via indirect and direct mechanisms that involved
both 5-hydroxy tryptaminergic mechanisms (Ghoisheh et al., 1999). BCO has also been
shown to increase bile secretion in dogs and uric acid in rats as well as protect rats
against histamine-induced bronchospasm (El-Dakhakhany,. et al 2002a). The fatty and
petroleum extracts shortened bleeding time and inhibited fibrinolytic activity in rabbits
(Tekeoglu et al., 2006).In a recent study, the crude extract of black cumin seeds was
found to exhibit spasmolytic and bronchodilator activities mediated possibly through
calcium channel blockade and this activity was concentrated in the organic fraction of the
extract (Gilani et al., 2001).
Traditionally black cumin plant has been in use in the Middle East as a natural remedy
for diabetes. Significant reductions in blood glucose and cholesterol levels in humans
following the use of the plant were reported by Bamosa et al. (1997). The oil of this plant
has a great potential in the treatment of diabetic animals because of its combined
hypoglycemic (Zaoui et al., 2002a) and immune-potentiating properties (Haq et al.,
1999). An extract mixture of black cumin, myrrh, gum Olibanum. gum asafetida and aloe
plants was found to lower blood glucose in streptozotocin diabetic rats (Mansour and
Tornhamre, 2004). It was found that the plant extracts significantly decreased hepatic
gluconeogenesis, suggesting that it may prove to be useful therapeutic agent in the
treatment of non-insulin-dependent diabetes mellitus. Similar insulinotropic effects of
BCO were recently observed in streptozotocin plus nicotinamide-induced diabetes
mellitus in hamsters (a model of type 2 diabetes) orally fed with the oil (Fararhet al.,
2002). The ability of BCO to lower blood giucose concentrations was later confirmed in
streptozotocin diabetic rats following 2, 4 or 6 weeks of treatment (El-Dakhakhany et al.,
2002b).
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In many Islamic countries black cumin and its derived products are consumed for
traditional treatment of blood homeostasis abnormalities and as a treatment for
dyslipidemia (Zaoui et al., 2002a). Several studies support the use of BCO extract for the
treatment of thrombosis and dyslipidemia (Labhal et al., 1997, Enomoto et al., 2001 and
zaoui et al., 2002a). In addition an aqueous suspension of black cumin seeds was found
to decrease the serum total lipids and body weight in Psammomys obesus sand rat
(Labhal et al., 1997). Analogous results, accompanied by decreases in serum lipid levels
have also been observed in rats chronically treated with black cumin fixed oil (Zaoui et
al., 2002b). When animals were treated with an oral dose of 1 ml/kg body weight of the
black cumin seed fixed oil on daily basis for 12 weeks, the serum cholesterol,
triglycerides and the count of leukocytes and platelets decreased significantly as
compared to the control values (Zaoui et al., 2002a).
Black cumin is also used in Islamic medicine as a diuretic and hypotensive plant. In an
attempt to experimentally support the above traditional uses of the plant, a study was
conducted on the diuretic and hypotensive effects of the extract of black cumin seeds in
the spontaneously hypertensive rat (Zaoui et al., 2000). An oral dose of black cumin
extract significantly increased diuresis, after 15 days of treatment. The urinary
execretions of CI-, Na
+, K
+ and urea were increased after 15days of treatment. Evidence
indicates that BCO has a protective role against gastric ulcers (El-Dakhakhany et al.,
2000b). Oral administration of BCO for 2 weeks in rats produced a significant increase in
gastric mucin content and glutathione level and a significant decrease in gastric mucosal
histamine content without significant changes in free acidity and peptic activity of the
gastric juice (El-Dakhakhany et al., 2000b). Ethanol administration, however, produced
100% ulcer induction accompanied by a reduction in free acidity, mucin content and
glutathione level without any significant changes in peptic activity. When animals were
pretreated with BCO before ulcer induction by ethanol, a protection ratio of 53.56% was
noted as compared to the ethanol group (El-Dakhakhany et al., 2000b).
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1.4.1.20 Anticancer effects:
The avtive constituents of BCO have been found to exert antineoplastic effects both in
vitro and vivo using various models of carcinogenesis. Black cumin seed preprations (TQ
and TQ2) have been demonstrated to have significant antineoplastic activity against
various tumor cells in vitro (Swamy and Tan, 2000). The active principles of black cumin
showed 50% cytotoxicity against Ehrlich ascites carcinoma, Daltons lymphoma ascites
and Sarcoma-180 cells at a concentration of 1.5, 3 and 1.5mg, respectively, with little
activity against lymphocytes (Ali and Blunden, 2003). In vitro cytotoxicity was also
demonstrated against human pancreatic adenocacinoma, uterine sarcoma and leukemic
cell lines (Gali-Muhtasib et al., 2004). The growth inhibitory was found to be related to
the extracts ability to inhibit DNA synthesis as measured by the incorporation of tritiated
thymidine into cells. These findings were later confirmed by Worthen et al. (1998) who
assayed the in vitro cytotoxicity of a crude gum, a fixed oil and two purified components
of black cumin seed, TQ and TQ2, on several parental and multidrug resistant human
tumor cell lines. Although as much as 1% w/v of the gum or oil was devoid of
cytotoxicity, both TQ and TQ2 were cytotoxic for all of the tested cell lines. The ethyl
acetate fraction of black cumin seeds was later found to exhibit significant growth of
normal human endothelial cells (Swamy and Tan, 2000). Badary and Gamal El-Din
(2001) also showed that TQ inhibited the survival of fibrosarcoma cells with IC50 of
15mM by inhibiting the incorporation of 3H thymidine into cells. The cellular
mechanism of antineoplastic activity of TQ was only recently investigated (Shoieb et al.,
2003). In this study, the cellular mechanisms of TQ-induced cytotoxicity in parental and
cisplatin-resistant osteosarcoma human breast adenocarcinoma, human ovarian
adenocarcinoma and Madin-Darby canine cell lines have been examined. The cisplatin
resistant cells were the most sesetive to TQ treatment, while the canine cell lines were the
least sensitive. A dose of 25 mM of TQ induced apoptosis of osteosarcoma cells 6h after
May 2015
treatment. This dose also decreased the number of cells in S-phase and increased cells in
G1-phase, indicating cell cycle arrest at G1. These results suggest that TQ induces cell
cycle arrest and apoptosis in cancer cells (Shoieb et al., 2003). The growth- inhibitory
effects of TQ against colon cancer cells were found to be mainly due to the ability of this
compound to induce G1 cell cycle arrest and apoptosis. The apoptotic effects of TQ are
modulated by Bcl-2 protein and are linked to and dependent on p53 (Gali-Muhtasib et al.,
2004).
Several studies have shown that BCO and TQ retard the carcinogenic process in animals.
The active principles of black cumin seeds containing fatty acids were found to
completely inhibit the Ehrlich ascites carcinoma in mice (Mansour and Tornhamre,
2004). A dose of 100mg/kg body weight (b.w.) of black cumin extract delayed the onset
of papilloma formation and reduced the mean number of papillomas per mouse (Ali and
Blunden, 2003). Interperitoneal administration of black cumin (10mg/kg b.w.) 30 days
after subcutaneous administration of 20-methylcholanthrene-induced soft tissue sarcoma
restricted tumer incidence to 33.3% compared to 100% in methylcholanthrene-treated
controls (Ali and Blunden, 2003). In vivo Ehrich ascites carcinoma tumer development
was completely inhibited by the active principle at the dose of 2mg per mouse per day for
10 days (Shoieb et al., 2003). Furthermore, BCO was reported to possess a protective
effect on chemical-induced carcinogenesis in hamster cheek pouch (Wortthen et al.,
1998). In another study, the administration of a dose of 1mg of TQ twice weekly for 4
weeks demonstrated powerful chemo-preventive effects against benzo (α) pyrene (BP)-
induced for stomach tumors (Badary et al., 1999). TQ inhibited both BP-induced for
stomach tumor incidence and multiplicity by 70% and 67%, respectively. More recently,
this same group (Badary and Gamal El-Din, 2001) demonstrated that the administration
of 0.01% of TQ in drinking water 1 week before and after 20-methyl-cholanthrene
treatment significantly inhibited fibrosarcoma tumor incidence and tumor burden by 43%
and 34%, respectively. Moreover, TQ delayed the onset of methylcholanthrene-induced
May 2015
fibrosarcoma tumors that appeared at 12 weeks and produced less methylcholanthrene-
induced mortality. The possible modes of anticarcinogenic actions of TQ in the above
two studies were suggested to be through its antioxidant and anti-inflammatory activites,
coupled with enhancement of detoxification processes.
In a stady, the effect of CC-5 (ethyl acetate fraction of BCO) was evaluated for its in vivo
antitumor activity against intraperitoneally implanted murine P388 leukemia and
subcutaneously implanted Lewis lung carcinoma cells in BDF1 mice (Kumara and Huat,
2001). At doses of 200 and 400 mg/kg b.w., the fraction prolonged the life span of these
mice by 153% compared to DMSO-treated control mice. The antitumor activity of a 21-
day treatment CC-5 against subcutaneously implanted LL/2 was tested and found to
produce a 60 - 70 % tumor inhibition rate. A triterpene saponin was isolated from the
CC-5 fraction and identified to be α-hederin. This compound was found to exert more
potent anticancer effects compared to the commonly used anticancer drug, cyclo-
phosphamide. When α-hederin was given at doses of 5 and 10 mg/kg b.w. to mice with
formed tumors, it produced significant dose-dependent tumor inhibition rate values of
50% and 71%, respectively, on day 15, compared to 42% on day 15 in the
cyclophosphamide (CP)-treated group. The underlying mechanism(s) of antitumor
activity of α-hederin is not defined yet (Kumara and Huat, 2001). The protective effect of
black cumin seeds on carcinogenesis induced by methylintrosourea in Spargue Dawley
rats was recently investigated (Mabrouk et al., 2002). When given orally (0.2 g ground
black cumin seeds) alone or with honey, a 6-month treatment MNU-induced colon
adenocarcinomas by 80% ((Mabrouk et al., 2002). There was an associated elevation of
malondialehyde and nitric oxide in sera obtained from methylnitrosourea-treated animals,
which was lowered by ingestion of black cumin seeds. Interestingly, combined oral
treatment of honey and black cumin seeds protected 100% against methylnitrosourea-
induced oxidative stress, carcinogenesis and abolished the nitric oxide and
malondialdhyde elevations shown in sera of animals that did not receive these nutrients
May 2015
(Mabrouk et al., 2002). TQ has also been shown to improve the therapeutic index of
several anticancer agents and to protect non-tumor tissues from chemotherapy-induced
damage. TQ protected against ifosfamide-induced Fanconi syndrome in rats and
enhanced its antitumor activity in Ehrlich ascites carcinoma-bearing mice (Badary, 1999).
The disease Fanconi syndrome is characterized by wasting off glucose, electrolytes and
organic acids along with elevated serum creatinine and urea as well as decreased
creatinine clearance rate (Mansour and Tornhamre, 2004). It also corrected the damage
induced by ifosfamide on phosphorus, glucose, serum creatinine and urea levels and
significantly normalized creatinine clearance rate. This effective dose of TQ was found to
be very safe (Badary et al., 1998). TQ protected the kidney against ifosfamide-induced
damage through an antioxidant mechanism, since it significantly prevented ifosfamide-
induced renal glutathione depletion and lipid peroxide accumulation. Further more, mice
treated with ifosfamide in combination with TQ showed less body weight loss and
mortality rate compared to ifosfamide single therapy. Moreover, investigations by Nagi
and Mansour (2000) showed that oral administration of TQ (10mg/kg/day) with drinking
water starting 5 days before a single injection of DOX (15mg/kg) and containing during
the experimental period ameliorate the DOX-induced cardiotoxicity in rats. TQ also
protected against the nephropathy in rats associated with hypoalbuminemia,
hypoproteinemia, elevated serum urea, hyperlipidemia and a high urinary excretion of
protein and albumin. The nephropathy observed in this model resembles histologically
and clinically the focal and segmental glomerulosclerosis that occurs in humans (Zima et
al., 1997). Treatment of rats with TQ (10mg/kg per day) supplemented with the drinking
water for 5 days before (DOX), and daily thereafter significantly lowered serum urea,
serum and kidney levels of triglycerides and total cholesterol and suppressed DOX-
induced proteinuria and albuminuria (Badary et al., 2000). TQs protective effects against
DOX damage to the heart and kidney was found to be mainly due to its superoxide
scavenging and antilipid peroxidation effects.
May 2015
1.4.1.21 Anti inflammatory and immune modulatory effects:
Black cumin seeds and its derived products have been traditionally used as treatments for
rheumatism, liver diseases and related inflammatory disorders. The effects of black
cumin seed on the immune system has been investigated by several researchers
(Houghton et al., 1995, Hag et al.,1995, El-Dakhakhny et al., 2000a). all studies have
shown that the oil and its most abundant component, TQ, inhibit many inflammatory
mediators, and, thus may be useful in ameliorating inflammatory and autoimmune
conditions. Ali and Blunden (2003) reported that the black cumin-derived nigellone, the
carbonyl polymer of TQ, was very effective at low concentrations in inhibiting histamine
release from rat peritoneal mast cells in vitro. He suggested that the mechanism of action
is mainly due to the ability of TQ to decrease intracellular calcium by inhibiting protein
kinase C and partly due to its ability to inhibit oxidative energy metabolism.
Several studies pointed to the effect of black cumin on the human immune system (Ali
and Blunden, 2003 and Ramadan, 2007). The seeds of black cumin were found to
produce an increase in the ratio of helper to suppressor T cells and enhance natural killer
cell activity in normal volunteers (Tekeoglu et al., 2006). In vitro studies showed that the
crude fixed oil and pure TQ were potent inhibitors of eicosanoid generation, namely
thromboxane B2 and leucotriene B4, by inhibiting both cyclooxygenase and
lipoxygenase, respectively (Houghton et al., 1995). Thromboxane B2 has been implicated
in the mechanism of hepatocyte plasma membrane bleb formation, which is an early
event in hepatocyte injury when exposed to oxidative stress (Ali and Blunden, 2003). In
another study, black cumin seeds enhanced the production of IL-3 by human
lymphocytes and had a stimulatory effect on macrophages (Haq et al., 1995). Besides, the
immune-modulatory effect of black cumin purified proteins was found in mixed
lymphocyte cultures and caused increased secrection in the levels of the cytokines IL-Ib
and IL-8 (Haq et al., 1999). Moreover, the fixed oil of black cumin increased the release
May 2015
of PGE2, inhibited the release of leukotrienes and histamine from normal and sensitized
rat‘s lungs. Other pieces of evidence include the inhibition of TNF-a production in
murine septic peritonitis by TQ (El-Dakhakhny et al., 2000) and the unique
immunomodulatory properties of ethyl acetate (CC-5) fraction of black cumin at non-
cytotoxic doses (Swamy and Tan, 2000). The ability of TQ to modulate cytokines and
enhance the immune system has been implicated as the main reason for its protective
effect against schistosome egg infection in the liver (Mahmoud et al., 2002).
In an attempt to determine the immunomodulatory role of TQ, the effect of this
compound on the production of nitric oxide (NO) by rat peritoneal macrophages was
investigated. It was found that it reduced production of NO in supernatants of
lipopolysaccharide- stimulated macrophages without affecting the cell viability. The
protein and mRNA levels of inducible nitric oxide synthase in peritoneal macrophages
were also decreased by TQ. Immunofluorescence staining of inducible nitric oxide
synthase in macrophages showed decreased immune-reactivity for inducible nitric oxide
synthase after TQ treatment. The anti-inflammatory effects of black cumin have been
found to be comparable to that of 100mg/kg aspirin (El-Mahmoudy et al., 2002).
1.4.1.22 Antioxidant and hepatoprotective effects:
Health food stores sell black cumin seeds as natural remedy for a variety of complaints
including liver diseases (Kruk et al., 2000). The hepatoprotective effects of TQ have been
well documented and have been found to be related to its strong antioxidant potentials. In
fact, the antioxidant and free radical scavenging properties of many plants have been
found to play an important role in their hepatoprtective activity (Thabrew et al., 1995).
Oxidant stress can increase the susceptibility to irreversible injury by free radicals that
can result in lipid peroxidation, protein oxidation, protein inactivation, disturbance in
calcium homeostasis and consequent loss of cell viability (Kruk et al., 2000).
May 2015
The oil of black cumin and TQ are known to possess strong antioxidant activities (Nagi
and Mansour, 2000, Meral et al., 2001, El-Dakhakhny et al., 2002a and Mohmoud et al.,
2002), TQ has been shown to inhibit non-enzymatic peroxidation in ox brain
phospholipid liposomes (Houghton et al., 1995) with a potency that is 10 times higher
than BCO. Using TLC screening methods, Burits and Bucar (2000) showed that TQ and
BCO components, namely, carvacol, t-anethole and 4- terpineol possess strong radical-
scavenging properties. Moreover, TQ showed extremely high superoxide anion radical-
scavenging abilities in pure chemical systems (Nagi and Mansour,2000). This high
scavenging power of TQ was responsible for its protective effects against DOX induced
cardiotoxicity in rats (Nagi and Mansour, 2000).TQ was observed to be metabolized by
liver DT diaphorase to dihydrothymoquinone, a phenolic metabolite that acts as a radical
scavenger and inhibits lipid peroxidation in vitro (Mansour et al., 2002). The most
comprehensive evidence on the antioxidant effects of BCO and its components came
from the studies conducted that TOH, TQ and TQ2 exhibit antioxidant properties and
acted as scavengers of various reactive oxygen species. TOH, for example, acted as 1O2
quencher, while TQ and TQ2 showed superoxide dismutase- like activity removing O.
El-Dakhakhny et al.(2002a) also showed that BCO as well as nigellone and TQ exert
inhibitory actions on the production of leukotriene- type mediators of inflammation in
vitro. The high antioxidative action of BCO and its components suggests their importance
for the treatment of various diseases occurring with participation of reactive oxygen
species. Hepatoprotective effects of TQ were also documented against carbon
tetrachloride- induced toxicity (Mansour et al,. 2004). Similar hepatoprotective effects in
the same system were obtained following a 4-weeks oral intake of BCO in male albino
rats (El-Dakhakhny, 2003). It was shown that black cumin seeds given orally every day
for 2 months decreased the lipid peroxidation, increased the antioxidant defense system
and prevented the lipid peroxidation- induced liver damage in experimentally induced
May 2015
diabetic rabbits (Meral et al., 2001), suggesting that the seed may be used in diabetic
patients to prevent lipid peroxidation.
1.4.1.23 Toxicity of Nigella sativa:
The toxicity properties of TQ and THQ were investigated in male rats whereby the drugs
were dissolved in propylene glycol, injected into 30 male rats and LD50 determined
(Vuorelaa et al., 2004). The oral administration of aqueous extracts of the seeds of black
cumin for 14 days has been shown to cause no toxicity symptoms in male Sprague-
Dawley rats (El-Mahady et al., 2005). The safety of consuming black cumin seeds was
also reported by Al-Homidan et al. (2002) whereby the seeds did not affect the growth of
7-day-old Hibro broiler chicks when fed black cumin seeds at 20 and 100 g/kg of the diet
for 7 weeks.
Although several studies have reported the safety of consuming black cumin seeds, a
recent comprehensive investigation has shown that the plant is relatively unsafe if
consumed for prolonged periods of time (Zaoui et al., 2002b). Treatment of animals with
a daily oral dose of 1ml/kg b.w. of BCO for 12 weeks resulted in significant slowdown of
the body weight in black cumin- treated animals compared to untreated control animals.
Changes in key hepatic enzymes levels and histopathological modifications (heart, liver,
kidneys and pancreas) were not observed in rats treated with black cumin after 12 weeks.
However, the serum cholesterol, triglyceride and glucose levels and the count of
leukocytes and platelets decreased significantly, compared to the control values, while
Hematocrit and Hemoglobin levels increased significantly. The decrease in body weight
in black cumin treated rats was thought to be related to the decreased in serum lipids and
glucose levels as a consequence of a possible reduction in food intake by BCO
administration (Zaoui et al., 2002b).
1.4.1.24 Actions on high risk groups :
The seed generally safe in pregnancy, lactation & for children but its volatile oil advised
to be avoided in pregnancy (Gilani et al., 2001).
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Table 1.5 Methods of N.sativa application for treatment of diseases:
Disease and
condition
Methods of application
Acne Half a teaspoon of oil in bowl of hot water, vapour bath with a
towel over head.
Asthma & cough Rub the back and chest with oil, drink one tsp after weab a
towel 3 times a day.
Cold & flu 1tsp of oil, three times a day. Drink hot lemon with honey.
Lethargy 1tsp of oil with orange juice for 10 days.
Nervous tension ½ tsp oil with herbal tea like lemon balm, clary sage, passion
flower, st.Johns Wort.
Healthy
complexion
Rub ½ tsp oil all over face; wash with cold water.
Tired leg,
muscles,etc
Massage on affected area.
Backache,
arthritis, bruises
and rheumatism
Heat oil slightly and massage intensely. Drink 1tsp oil with 1
tsp olive oil 3 times a day.
High blood
pressure
Drink 1tsp in any hot drink; take 2 lobes of garlic before
breakfast.
Stomach
complaint
Drink mint tea with lemon and take 1tsp oil three times a day
or until relieved.
Diarrhea 1tsp oil with a cup of yoghurt. Take 2 times daily. Also eat
boiled rice with yogurt.
Hair loss Stroke the scalpe thoroughly with lemon, leave for 15 minutes
May 2015
then wash and dry, apply ½ -1 tsp oil.
Headache Rub the forehead and sides of the head and the part of the face
near the ear with the oil. Drink ½ tsp of oil after a meal 3 times
aday.
Earache Mix ½ tsp oil with ½ tsp olive oil, warm and then drip drops
into the ear and cover the ear with a woolen shawl or hat.
Intestinal
parasites
Take one tsp oil with ―warm wood‖ capsules. Eat plenty of
onions and garlic.
Colic (babies) Warm oil in hand, massage the whole abdomen with it,
stroking clockwise.
Sinusitis Inhale through nose with vapour bath, take 1tsp daily in
chronic cases, 3 times daily in acute cases.
Skin fungus Affected area with cider vinegar, then apply oil, repeat if
necessary.
1.4.2Mycetoma (=Madura Foot):
1.4.2.1Characteristics:
Chronic localized subcutaneous infection that involves underlying bone later in the
disease course.
- The lesions are multiple abscesses.
- Main symptoms/signs are cold swelling of the affected site (tumefaction),
formation of sinuses that drain pus to the surface of the skin, and presence of
grains.
- Grains are granules (small colonies), about 1-2 mm diameter, of the etiologic
agent with different color.
- The commonly affected site is the foot; however, it can be in leg, thigh, arm,
shoulder, or head.
- Infection is acquired following trauma to the skin by plant material from trees,
shrubs, or vegetation debris. Thus more seen in rural areas (in farmers,
Sheppard‘s, walking bare-foot in agricultural land or city parks)
- ―Madura foot‖ referring to the first case seen in ―Madura‖ region of India which
was in the foot of that patient.
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- Infection is very chronic takes months to be fully established and years to deal
with. It is not contagious. More seen in tropics and subtropics.
- Etiologies are Fungi which cause eumycotic mycetoma (Eumycetoma) or
actinomycetes which cause actinomycotic mycetoma ( actionmycetoma ). Their
natural habitats are plant materials (Rippon .,1988).
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May 2015
Fig 1.13 Mycetoma infections
1.4.2.2 Epidemiology:
Mycetoma has a worldwide distribution, which is, however, extremely uneven.
Mycetoma is endemic in tropical and subtropical regions and the African continent seems
to be the area of the highest prevalence (Figure 1.14). Worldwide, mycetoma prevails in
the mycetoma belt that stretches between the latitudes of 15° South and 30° North
(Boiron et al 1998 and Magana,.1984). The belt includes Sudan, Somalia, Senegal, India,
Yemen, Mexico, Venezuela, Columbia, Argentina and other countries (Magana, 1984,
Mahgoub,. 1973, Mariat, 1963). In Africa, mycetoma is most frequently seen in Sudan,
Senegal, Mauritania, Kenya, Niger, Nigeria, Ethiopia, Chad, Cameroon, Djibouti and
Somalia
(Abbott,. 1956, Agarwal., and Mathur 1985, Basset et al 1965, Cameron et al., 1973,
Develoux et al., 1988, Develoux et al., 1995, Lynch, 1964, and Singh, 1978). Lynch in
1964 gave an estimation of 300 - 400 new cases per year in Sudan.
It has been extensively reported in India (Bocarro, 1909, Singh, 1978). There were
reports on mycetoma from the United States, Ceylon, Germany, Egypt, Turkey,
Philippines, Japan, Lebanon, Thailand, Iran, The Netherlands and Saudi Arabia (Corray,
1962, de Hoog,et al., 1993 , El Mofti et al., 1965., Gammel, 1927, Erbakan et al., 19730,
Tight and Bartlett 1981).
Areas where mycetoma prevails are relatively arid with a short rainy season of 4-6
months. Rainfall is50 to 1000 mm per year, with a relative humidity of 60-80% and fairly
constant temperatures of 30- 3rC for 24 hours a day. This is followed by a dry season of
6-8 months with a relative humidity of 12- 18%, day temperatures rising to 45-60°C.
Temperatures may fall to 15-18°C during the night (Mahgoub and Murray 1973). This
alteration in extreme weather conditions may be a prerequisite to the survival of the
causative organism in its natural niche.
May 2015
Geographical distribution of the mycetoma causative agents shows considerable
variations, which could be explained on these and other environmental factors, especially
the rainfall (Mariat, 1963). Many microorganisms are capable of causing mycetoma. The
most prevalent etiological agent of eumycetoma worldwide is Madurel/a mycetomatis
(McGinnis, 1996). Most of the mycetoma cases in Africa are eumycetoma caused by M.
mycetomatis. In some parts of central Africa, including Sudan, M. mycetomatis causes
more than 70% of all mycetoma infections (Gumaa, 1994).
Fig 1.14 Map showing the geographical distribution of some eumycetoma agents
(Gumaa, 1994).
May 2015
Fig 1.15 Map showing the geographical distribution of mycetoma in sudan (Gumaa,
1994).
1.4.2.3Etiology:
1.4.2.3.1Eumycetoma:
- It is caused by several mold fungi.
- The color of grains in this type of mycetoma is black or white.
- Fungi include: Madurella, Pseudallescheria (Scedosporium), Pyrenochaeta
(Pycnidia producer), Acremonium, and the ascomycetes Leptosphaeria and
Neoteatudina, others (larone, 2002).
- The common etiologies in Saudi Arabia and neighboring
countries are :
o Madurella mycetomatis causes the majority of the cases with black
grains. It is imperfect dematiaceous mold with brown colonies and
diffused hony – colored pigment. Produces phialoconidia from phialides,
and chlamydospores.
May 2015
Fig 1.16 Madurella mycetomatis colonies
o Madurella grisea :Another species of Madurella, similar to M.mycet. but
with grey colonies.
o Pseudallescheria boydii – causes white grain mycetoma. It is
Ascomycete mold forming cleistothecia and ascospores. The imperfect of
it is the moniliaceous mold: Scedosporium apiospermum which forms
annelloconidia from annelids.
Fig 1.17 Pseudallescheria boydii colonies
1.4.2.3.2Actinomycetoma:
Caused by about 10 species of aerobic actionmycetes.
o Color of grains yellow, white, yellowish-brown. Pinkish-red.
o Actinomycetes are filamentous higher bacteria. The filaments (very thin
about 1.0µm wide) appear as long branching, beaded, or as long rods.
They are Gram-positive (Rippon, 1988).
o Main etiologies:
May 2015
Streptomyces somaliensis – causes the majority of the cases –
color of grains yellow to yellow - brown.
Actinomadura madurae – white or yellow grains.
Actinomadura pelletieri – pinkish-red grains.
Nocardia brasiliensis - white grains.
N.asteroides, N.caviae, N.coeliaca –white or yellow grains.
Latter species of Nocardia usually cause Nocardiosis (which is
subcutaneous, pulmonary, or brain abscess infection).
Nocardia is acid-fast to partially acid fast when stained by Ziehl-
Nelsen stain (ZN) while Streptomyces and Actinomadura are
nonacid-fast.
o These actinomycetes are differentiated by their decomposition pattern of
gelatin. Also by few other biochemical tests and colony morphology
(They have adherent dry colonies). See Table of characteristics.
o The anaerobic actinomycete ;Actinomyces israelii causes the infection:
―Actinomycosis‖ which is subcutaneous, cervicofacial, pulmonary,
abdominal, uterine, or brain abscess infection. It also causes dental caries.
The organism is filamentous and will have yellow grains ( sulfer
granules) (McNeil & Brown, 1994).
Table 1.6 Characteristics of the main species of the Actinomycetes:
(McNeil & Brown, 1994).
Organism Ca Ty Xa Hx Urea Growth in
0.4X salt
Gelatin Colony
S.somaliensis + + - - - - + Brown
A.madurae + + - + - + + Brown
A.pelletieri + + - + - + Brown
May 2015
N.brasiliensis + + - + + + Orange
N.asteroides - - - - + - White
Decomposition of Ca=Casien, Ty=Tyrosine, Xa=Xanthine, Hx=Hypoxanthine
1.4.2.4laboratory Diagnosis:
1.4.2.4.1Specimen:
Visible grains, Biopsy tissue (not skin pinch), curetting of sinuses, pus, blood for
serology.
- First determine color of grains – it helps identify etiology and initiate treatment.
- Make histologic section, or grinde tissue and crush grains and make smears –
stain by: Hematoxylin –Eosin, Gram, ZN; if fungi do 20% KOH or periodic
acid Schiff stain.
- Extract serum for serology (Isenberg , 2004).
1.4.2.4.2Direct Microscopy:
- Will reveal grains in tissue ;
o Homogenous texture = actinomycetes.
o Heterogenous texture = fungi.
May 2015
Fig 1.18 Comparison between Actinomycotic & Eumycotic grain
- Actinomycete grains = will not reveal filaments easily.
- Fungal grains = will contain easily seen hyphae and chlamydospores.
- Grain will have different morphology and color (White, black, yellow,
pink….etc) depending on etiology.
Table 1.7 The color of the grains in mycetomas & related
species(Mahgoub, 1973):
Color of grains Species
Eumycetoma
black grains
M. mycetomatis, M. grisea, Leptospheria senegalensis,
Exophiala jeanselmei, P.romeroi, C.lunata, Phialophora
verrucosa, P. parasitica
Eumycetoma
pale grains
P.boydii, Aspergillus nidularis, A. flavus, Fusarium Sp,
Acrimonium Sp, Neotestudina rosatii, dermatophytes
Actinomycetoma
red grains
Actinomadura pelleitieri
Actinomycetoma
yellow grains
streptomyces somalinsis
Actinomycetoma
pale grains
N. brasiliensis, N. cavae, N. asteroids, Actinomadura
madurae
- Direct microscopy of specimen from Nocardiosis and actinomycosis will show
branching thin filaments by silver stain (Isenberg, 2004). .
May 2015
Fig 1.19 Actinomycete & Nocardia filaments
1.4.2.4.3 Culture:
On sabouroud dexterose agar(SDA), Neutral- Sabouroud dexterose agar,Brain
Haert Infusion-Agar(BHI-A), Blood agar and incubate at 37˚C and at 25-30˚C
aerobically.
- For actinomycosis also culture on cooked meat medium and incubate
anaerobically at 37˚C.
- The organisms will grow slowly and may be contaminated with skin flora –
purify – identify.
( Koneman and Roberts,1985)
Fig 1.20 Actinomycete filaments from culture
1.4.2.4.4 Serology:
- Test for antibody using known antigen from each etiologic agent.
- Methods used immunodiffusion (I.D), and/or
counterimmunoelectrophoresis ( C.I.E). (Anaissie et al., 2003).
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1.4.2.5 Management:
- Usually actinomycetoma respond better to treatment than eumycetoma.
- Generally if bone is infected the response to treatment is poor (Mahgoub,
1994).
Actinomycetoma:
- Trimethoprim – Sulfametoxazol (Cotrimoxazole/septrin) + Streptomycin
sulfate (Mahgoub,1972) .
- Or : Dapsone + Streptomycin sulfate.
- Or : Doxycycline + Cefachlor.
- For Actinomycosis : Penicillin G
Actinomycetoma is amenable to medical treatment with antibiotics and other
chemotherapeutic agents. Combined drug therapy is always preferred to a single
drug to avoid drug resistance and to eradicate residual infection. The common
drugs in use include combination of:
a. Streptomycin sulphate (14 mg/kg daily), Diaminodiphenyl sulphone
(Dapsone) (1.5 mg/kg twice daily).
b. If there is no response for few months or if there is persistent side effect then
dapsone is replaced by Co-trimoxazole (14 mg/kg twice daily).
c. Amikacin sulphate alone or in combination with Co-trimoxazole
d. In resistant cases other drugs such as Rifampicin, Sulfadoxine-
pyrimethamine (Fansidar) and Sulphonamides
(Mahgoub, 1976) (Mahgoub, 1994) (Mahgoub, 1972) (Welsh et al, 1987) .
May 2015
All drug of antifungal Induced Nephrotoxicity and the most common factors that
have been identified in retrospective studies of nephrotoxicity include:
a) Baseline renal dysfunction.
b) Previous or concurrent administration of nephrotoxic agents (i.e.
Cyclosporin, Aminoglycosides)
c) Prolonged therapy with high dosages of (i.e. treatment for invasive fungal
infection).
d) Underlying disease state associated with renal dysfunction (i.e. diabetes,
sepsis).
) Luber et al,.1999) , ) Wingard et al,.1999)
Fig 1.21 Schematic Representation of the Balance between Amphotericin B-
Induced Nephrotoxicity, Patient Risk Factors, and Cost Effective Therapy(
Luber et al,.1999, Wingard et al,.1999):
Eumycetoma:
- Ketoconazole(Nizoral) tablets or Itraconazol or voriconazole
(Mahgoub, 1984) .
May 2015
If drugs not effective and bone is infected = Amputate the limb or
debride tissue and continue treatment.
Treatment duration is long – up to many years.
All drugs have two costs. In addition to their acquisition costs, cost of
monitoring and treatment of side effects must also be considered. These
secondary costs are especially important with antifungal agents. Those invasive
fungal infections are also one of the most expensive complications that can be
encountered in hospitalized patients. Until recently, studies examining the
pharmacoeconomics of antifungal therapy were a low priority, as few antifungal
therapies were available and costs related to antifungal acquisition and
administration appeared (Rentz et al., 1998)
Figure 1.22 Antifungal drugs overall average cost in 1993
(Rentz et al., 1998):
1.4.2.6 Detailed Descriptions for most common microorganisms:
May 2015
1.4.2.6.1Madurella mycetomatis
Pathogenicity: Causes black grain mycetoma (larone, 2002) .
Rate of
growth:
Moderate at 37˚C; mature in 10 day. Grows much more slowly
at 25˚C.
Colony
morphology:
Varies greatly; may be smooth or folded and glabrous or
powdery and ranges in color from white to yellowish brown.
There is usually a brown diffusible pigment in the agar . reverse
is dark brown.
Microscopic
morphology:
On sabouroud dexterose agar forms only septate hyphae (1-6µm
in diameter ) with numerous chlamydoconidium –like enlargwd
cells. On cornmeal agar , some strains produce phialides that
bear round or oval conidia at their tips.may form large, black
masses of modified hyphae ( sclerotia) in old cultures.
M.mycetomatis differs biochemically from Madurella grisea in
assimilating lactose but not sucrose.
Fig 1.23 Microscopic morphology of Madurella mycetomatis
1.4.2.6.2 Pseudallescheria boydii (sexual state); Scedosporium
apiospermum(asexual state)
May 2015
Pathogenicity : Has been known for many years to cause mycetoma .The
infection occurs most often in the feet or hands but may
occur on any exposed parts of the body.this organisem is
now also recognized as an agent of phaeohyphomycosis. It
can infect the subcutaneous tissue, bones, brain, eye, lungs,
sinuses, meninges, and other body sites. Disseminated
infection has been reported in immunocompromised patients
(larone, 2002).
Rate of growth: Moderately rapid; mature in 7 days. The sexual stage
(P.bodydii) is inhibited by cyclohexamide, the asexual stage
(S.apiospermum) is not inhibited.
Colony
morphology:
Surface has a spreading, white, cottony aerial mycelium
which later turns gray or brown. Reverse is at first white but
usually becomes gray or black.
Microscopic
morphology:
Hyphae are septate (2-4 µm in diameter ), with simple long or
short conidiophores bearing coninda singly or in small
groups ( may resemble mould phase of Blastomyces
dermatitidis, the conidia (4-7 x 5-12µm) are unicellular and
oval, with the larger end to ward the apex, and appear cut
off at the apex, and appear cut off at beas (i.e.,truncate);they
become dark with age, the grapbium type of a sexual
conidiation is seen occasionally; it is characterized by long,
erect, narrow conidophores that are cemented together,
diverge at the apex, and bear clusters of oval, truncate
conidia (2-3 x 5-7µm). in the sexual stage, large brown
cleistotheca (50-250µm in dimeter) are formed and release
May 2015
elliptical ascospores when ruptured. The sexual stage may
sometimes be induced by culturing on corn-meal agar or
potato dextrose agar; the cleistothecia are most likely to
form in the center of the colony .
Fig 1.24 Microscopic morphology of Pseudallescheria boydii (sexual state);
Scedosporium apiospermum(asexual state)
1.4.2.6.3Nocardia spp.:
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Pathogenicity : Cause nocardiosis, which symptomatically may be similar to
tuberculosis or actinomycosis. Disease may begin as a
pulmonary infection and later involve the central nervous
system, kidneys, and other organs. Skin lesions or
subcutaneouse abscesses may be the only manifestation of
infection; occasionally mycetomas develop in the extremities.
on rare occasions, the eye has been infected. The organisms
are ubiquitous in nature and may therefore be encountered as
contaminants or colonizers.
Rate of growth: Moderately rapid; mature in 7-9day. Optimal growth is at 35-
37c. Grow on sabouraud dexterose agar (SDA) without
antibiotics and also on Lowenstein –Jensen (LJ ) and
middlebrook 7H11 media ( frequently survive
decontamination procedures used for isolation of acid- fast
bacilli). Excellent recovery has been reported on nonselective
buffered charchoal yeast extract (BCYE) agar and also on
Thayer-Martin agar (especially useful for inhibiting other
organisms in mixed cultures) (larone, 2002).
Colony
morphology:
Grow aerobically on SDA without antibiotics, forming raised,
irregular, folded colonies varying from white to orange,
depending on the species. May be glabrous or develop a white
chalky coating.
Microscopic Delicate, branching, often beaded, intertwining filaments that
May 2015
morphology: fragment into bacillary and coccoid forms; best exhibited on
slide culture using a minimal medium, such as cornmeal-
Tween 80agar. They are gram positive and often, but not
always, partially acid fast (use a modified kinyoun method).
Young primary cultures are usually the most acid fast; acid
fastness may be enhanced on middlebrook 7H11 medium or
by growing cultures for 3-4 weeks in a proteinaceous medium,
such as litmus milk or bromocresol purple milk.
Fig 1.25 Microscopic morphology of Nocardia spp.
1.4.2.6.4Streptomyces spp.
May 2015
Pathogenicity: Most streptomyces spp. Are considered nonpathogenic
contaminants. Other species, such as S.somaliensis, cause
mycetomas and occasionally appears to be an etiologic agent of
infection.
Rate of
growth:
Rapid or moderate; mature in 4-10days. Optimum growth
occurs at 30c.
Colony
morphology:
Surface is slightly folded, hard, and leathery; may develop a
fine chalky or powdery aerial mycelium. Many strains have
various pigments of gray, orange, rose, red, or occasionally
green. Culture often produces the characteristic odor of freshly
tilled soil (larone, 2002).
Microscopic
morphology:
Hyphae are long, thin (1µm or less in diameter), and abundantly
branching, with filaments which may be straight, wavy, or
spiral. Small oblong conidia are produced at distinct points on
the filament; this is best observed on slide culture. Some
species do not form conidia readily.
Fig 1.26 Microscopic morphology of Streptomyces spp.
1.4.2.6.5Actinomadura spp.
May 2015
Pathogenicity : A frequent cause of mycetoma. They have also been isolated
from sputum, wounds, blood, and other sites, suggesting the
ability to colonize or infect some patients.
Rate of growth: Usually rapid on Lowenstein-Jensen (LJ) medium; slower on
Sabouraud dexterose agar (SDA). Optimum growth is at 35-
37c.
Colony
morphology:
Waxy, folded, membranous, or mucoid. May be white, tan,
pink, orange, or red. White aerial hyphae may develop after 2
weeks of incubation; best seen on LJ medium(larone, 2002).
Microscopic
morphology:
Narrow abundantly branched filaments (0.5-1µm in diameter )
that are gram positive, non-acid fast, and nonfragmenting.
Short chains of round conidia may be produced from limited
portions of the aerial hyphae; this is best observed on slide
culture.
Fig 1.27 Microscopic morphology of Actinomadura spp.
Table 1.8 Differentiation of aerobic Actionmycetes: (larone, 2002).
May 2015
1.4.2.7 Detailed Descriptions for disease:
May 2015
1.4.2.7.1Actinomycosis:
Etiologic
agents:
Actinomyces israelii (in human), Actionmyces bovis (in animals),
and other Actinomyces spp.; occasionally Arabnia, Rotbia, and
Eubacterium spp. (larone, 2002)
Sites of
Infection :
Neck and face area, lung, thoracic cavity, abdomen, pelvis,
multiple systemic sites.
Tissue
reaction :
Typically suppuration with abscesses containing granules
composed of the bacterial filaments. The innermost portion of the
wall of the abscess sometimes contains foamy macrophages.
Palisading epithelioid macrophages and giant cells often surround
the abscess and may be encapsulated by fibrosing granulation
tissue. Splendore-Hoeppli material usually radiates around the
abcesses. Sinus tracts connecting the absesses or opening to the
body surface are common.
Morphology
of
organism:
Granules from an abscess or draining sinus tract are 30 - 3000µm
or more in dimeter and are commonly called ―sulfur
granules‖.when crushed, the granules appear microscopically as
opaque masses with peripheral, gelatinous, club-shaped bodies.
The granules are composed of numerous delicate (less than 1µm
in diameter) bacterial filaments that branch and may exhibit
beading. In some instances, only small groups of branching
filaments form instead of the characteristic granules. The
organisms are gram positive and nonacid fast; they stain well with
GMS and Giemsa stains but not with H&E, PAS, and Gridley
fungus. Other anaerobic bacteria may also be present. Branching
filaments of Nocardia are partially acid fast.
May 2015
Fig 1.28 organism morphology of Actinomycosis
1.4.2.7.2Mycetoma (Actinomycotic or Eumycotic ):
Etiologic
agents :
Actinomycotic: Nocardia spp, Actinomadura spp, Streptomyces spp.
Eumycotic:Pseudallescberia boydii, Madurella spp, Exophiala
jeanselmei, Acremonium spp, Fusarium spp, Curvularia spp, and
occasionally other moulds (larone, 2002).
Sites of
infection :
Subcutaneous tissue and skin; long-standing infections may ivolve
muscle, fascia, and bone. The infection is most commonly on lower
leg or foot, rarely disseminated.
Tissue
reaction :
Similar reaction are seen with all mycetomas, i.e., both
actinomycotic and eumycotic. Multiple draining sinus tracts with
neurophilic abscesses containing granules and necrotic debris are
characteristic. The abscesses and sinus tracts are surrounded by
chronic inflammation consisting of palisading epothelioid
macrophages, multinucleated giant cells. Lymphocytes, and plasma
cells. Between the abscesses is granulation tissue that is usually
vascular and contains many inflammatory cells. Splendore-Hoeppli
May 2015
material very often surrounds the granules. Fibrosis may occur in
long-standing infections.
Morphology
of
organism :
The etiologic agent typically organizes into aggregates in infected
tissue to form granules ranging in size from 25µm to 5µm.
-Actinomycotic Mycetoma: Granules (White, yellow, or red) are
composed of narrow (1µm or less in diameter) interwined filaments
that are radially oriented and most numerous at the edge of the
granule. Nocardia spp. Are usually at least partially acid fast, while
Actinomadura and Streptomyces spp. Are not acid fast.All are gram
positive and stain well with GMS and Giemsa, but not with H&E,
PAS, or Gridley fungus.
-Eumycotic Mycetoma: Granules (white, yellow, browen, or black)
contain septate, variously shaped, somewhat distorted hyphae (2-
6µm in diameter) that are often accompanied by numerous
chlamydoconidia and swollen cells; the fungal forms are most
commonly visible at the periphery of the granule.
Fig 1.29 organism morphology of ( Actinomycotic or Eumycotic )
May 2015
1.4.2.7.3Nocardiosis
May 2015
Etiologic
agents:
Nocardia asteroids (most common), Nocardia brasiliensis,
Nocardia otitidiscaviarum (formally Nocardia caviae), and
rarely other Nocardia spp. (larone , 2002).
Sites of
infection:
Lung, central nervous system, skin and subcutaneous tissue,
multiple systemic sites.
Tissue
Reaction:
Generally, the reaction is suppurative, sometimes
necrotizing. Poorly defined, variably encapsulated abscesses
may form. Occasionally, granulomatous tissue is seen in
chronic infections. The Splendore-Hoeppli phenomenon is
rare in Nocardia infections.
Morphology
of organism:
Delicate, narrow (0.5-1.0µm in diameter) filaments that tend
to branch at right angles; coccobacillary elements may also
form branched that they have been described as resembling
‖Chinese letters‖. They are partially acid fast when stained
with the modified Kinyoun stain or other acid fast stain
using a weak acid solution for decolorization. The
organisms are gram positive and stain well with GMS,
especially when the staining time in the silver nitrate
solution is increased; they do not reliably stain with H&F,
PAS, or Gridley fungus.
Fig 1.30 organism morphology of Nocardiosis
May 2015
Table 1.9 The main clinical differences between eumycotic and actinomycotic
mycetoma: (Mahgoub, 1994).
Eumycetoma Actinomycetoma
Causative agents Fungi Bacteria
Lesion
characteristics
Well-encapsulated with a clear
margin
Diffuse, no clear margin
Sinuses Relatively few Many
Color of grains Different colors, but mostly
white or black
Different colors, other than black
Course of
infection
Slowly progressive More inflammatory and rapid
progression
Bone invasion After a long time Rapid
Cavities in X-ray Small in number, but large in
size with clear margins
Numerous, small in size (except
in case of Actinomadura
madurae) with unclear margins
Management
Drug of choice Ketoconazole
ltraconazole
Dapsone + streptomycin
Rifampicin or fansidar
Amikacin + cotrimoxazole
Management of
choice
Both medical and surgical
intervention required
Only medical treatment is
sufficient (in some advanced
cases in combination with
surgery)
Surgery only May cure small, well-
encapsulated lesions
Up to 90% recurrence in
most cases
Not indicated
Medical
treatment only Partial cure or
improvement
Recommended before
surgery to prevent local
spread
Needed after surgery to
prevent recurrence
Useful in most of cases
Most successful
outcome
Early diagnosis followed by
adequate antifungal treatment
in combination with surgery
Early diagnosis followed by
medical treatment for a
sufficiently long period
May 2015
2.1 Materials:
2.1.1 Nigella sativa Origin:
The seeds were purchased from Qasiem garden and authenticated by (Roby marry
in King Khalid hospital lab)
Table 2.1 Instruments:
Instrument Model No Serial No Company Country
Incubator lib- 080M 07083144 prime company khartom
/sudan vertical Autoclave - 7421st.JF.190 Medica
instrumentMFG.Co
Autoclave H86613AC1 2521993-101-
01
Hot air oven LDO-080N 07083131 prime company khartom /
sudan Sensitive Balance kern 440-33N - prime company khartom /
sudan Sensitive Balance TE601&Sec:BL 23603172 Sartorius
Colony counter - - prime company khartom /
sudan Soxhelt apparatus - - Quick Fit England
Rotatary vapour - - Buchi Switzerland
star dust - - Japan Japan
Table 2.2 Media:
Type Qty in Grams/litre of medium Code Packing
Nutrient Agar 28.00 M001-500G 500gm
Sabouraud Dextrose
Agar 65.00 M063-500G 500gm
Columbia CNA agar
base
42.50 M002-500G 500gm
Table 2.3 Solvents:-
Name Chemical
Symbol M.Wt Company Country
Distilled water - - - - Chloroform CHCL3 119.38 British drug house England
Methanol CH3OH E.Merck Germany
May 2015
2.1.2 Other materials:-
Bunsen burner.
Petri dishes.
Wire loops.
Glass ware.
Test tube.
Tips.
Cork borer (No2).
Bottles.
Cylinder.
Pipette.
Physiological saline.
Sterile swab.
Forceps.
Antipapal.
Slop holder.
Tube holder.
Para film.
Electronic pipette.
Cornal tube.
Micro filter.
Centrifuge.
Glass pit.
Electronic chaker.
Betridish.
Mixing machen.
Basintgater.
May 2015
2.1.3 Tested bacterial / fungal strains:-
Standard strains were obtained from natural culture type collection (NCTC) , England
and American type culture collection (ATCC), USA as well as specimens were collected
from patients in Sudan (SU1), (SU2) and Saudi Arbia(SA), (Pot).
The test organisms used were:
Table 2.4 Tested fungal strains (Eumycetoma):
m/o Abbrev. Code number
Madurella mycetomatis M.m(SU1) ATCC 6380
Madurella mycetomatis M.m(SU2) From pat.
Madurella mycetomatis M.m(Pot) 586/587
23/3/11
Madurella mycetomatis M.m(SA) 24/12/13
Pseudallescheria boydii
(Scedosporium)
Pseudo. ATCC27853
Madurellagrisea M.g M ¾
Table 2.5 Tested bacterial strains (Actinomycetoma):
Streptomyces
somaliensis
S.s MO7/1109
26/8/07
Streptomyces
somaliensis
S.s 442
16/11/12
Actinomaduramadurae A.m 690
5/5/97
Actinomaduramadurae A.m MO/991
21/7/5
Nocardiabrasiliensis N.b 7/3/4
24/12/13
Nocardiaasteroides N.a 16/6/98
24/12/13
(SA) Saudi Arabia sample in Sabroud dexterose agar. (Pot) Saudi Arabia sample in Potato desterose
agar. (SU1) and (SU2) Sample from Sudan in SDA.
May 2015
2.2 Methods:-
2.2.1Preparation of plant extracts:- 100g of N.sativa powder was extracted by maceration using chloroform as a solvent.
Another 100g was extracted by the same method using methanol.
The solvent was evaporated with a rotary evaporator and the oil was collected.
The oil obtained from both extracts was serially diluted (0.05%, 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%) using chloroform and methanol respectively.
2.2.2 Preparations of culture media:
2.2.2.1 Preparations of Sabouraud Dextrose Agar:
1000 ml of distilled water and 1ml of anti-bubbles was added to 65g of Sabouraud
Dextrose Agar powder in glass bottle,
And was sterilized for 3 hours at 121Coin an Autoclave, then after 3 hours the media was
removed from the autoclave and was cooled for 15 minutes, 100mg of chloramphenicol
in 6ml of ethanol I was added to the media to prevent bacterial contamination without
affecting the growth of mycetoma.
15 ml of the prepared agar was transferred into sterile plastic tubes and petrie dishes and
the agar in the tubes was allowed to set in slope position.
2.2.2.2 Preparations of blood agar:
500 ml of blood agar was prepared by adding 21.25 g of Cloumbia CNA agar base to 500
ml distilled water and 3 drops of anti-bubbles was added and autoclaved for 1 hr. and 45
min., 50 ml of blood was added. 15 ml of the prepared agar was transferred into sterile
plastic tubes and petrie dishes and the agar in the tubes was allowed to set in slope
position (McGinnis, 1980).
2.2.3 Preparation of Specimens:
Specimens of exudates, pus, and drainage should be examined for granules by using a
dissecting microscope. If granules are present, the colour is noted, and then a portion of
the specimen is teased apart gently, crushed between two glass slides, and examined
microscopically; the remainder is washed several times in sterile distilled water, crushed
with a sterile glass rod, and inoculated onto appropriate media (granules may be bacterial
and should be plated accordingly). If no granules are present, the specimen is examined
microscopically for hyphae and other fungal elements and directly inoculated onto
isolation medium (larone, 2002)..
2.2.4 Inoculation or culture of media:
May 2015
Inoculation was performed in a sterile advanced safety cabinet using Sabouroud
dexterose agar and blood agar media.
The plates were inverted and the tubes were put in test tube rack and incubated
aerobically at 35-37˚C for 7 days insure the growth of mycetoma was begin and clearly
change was shown up to 21 -23 days to reach the complete growth (McGinnis, 1980).
2.2.5 Phytochemical Screening:
The methanol extracts from the defatted vegetal products may contain important groups
of natural constituents, as for example:
- Polyphenols (tannins, etc.),
- Reducing compound,
- Alkoloid salts,
- Polyphenolic glycosides (anthracenosides, coumarins, flavonosides),
- Sterol glycosides (cardiotonics, saponosiders),
- Triterpene glycosides,
- Anthocyanosides.
Very good results may be also obtained by extraction with alcohol (70-80 per cent).
2.2.5.1 Test for Carbohydrates and / or Glycosides:
The dried powdered seed (5g) was boiled with 100ml of distilled water; the aqueous
solution was filtered through a cloth of muslin and the liquid obtained was tested as
follows:
- Molishs test:
An aliquot of the filtrate (2ml) was mixed with 0.2 ml of ethanolic-naphthol
(20%) followed by 2ml of sulphuric acid (98%) poured carefully on the side of the
test tube to form two layers. A violet zone at the junction of the two layers
indicated the presence of carbohydrates.The filtrate (5ml) was heated with 5ml
fehlings solution, and a red precipitate indicated the presence of reducing sugars
(Gonalez, 1962).
2.2.5.2 Test for tannins:
May 2015
The dried powdered plant (1gm) was extracted with aqueous ethanol (50%) and tested for
tannins by the following tests:
- Addition of 1ml of ferric chloride reagent, a green blackish colour indicated the
presence of complexes Gallic tannins.
- To about 5ml of an aqueous extract 0.5g of NaH2PO4 , was added , warmed, cooled
and filtered . to the filtrate, 2ml of 2% solution of phenazone was added. Formation
of a precipitate or turbidity indicates the presence of tannins.
- Vanillin in hydrochloric acid (2ml) was added to 5ml of ethanol extract. A crimson
red colour obtained indicated the presence of condensed tannins.
- A match stick was dipped in a few ml of aqueous extract and the stick was left to
dry, then dipped again in hydrochloric acid, removed quickly and dried near the
flame , a red colour indicated the presence of condensed tannins(Gonalez,
1962)(Clauss; 1961).
2.2.5.3 Test for alkaloids and / or nitrogenous bases:
The powdered seed (10g) was extracted with acidified water and the acidic filtrate was
rendered alkaline by the addition of ammonium hydroxide solution, and the mixture
extracted with chloroform. The chloroform extract was evaporated to dryness, and the
residue was dissolved in 2ml of dilute HCL and tested with the following reagents:
Mayers, modified Dragendorffs and iodoplatinate modified reagent. A white, orange
precipitate and violet colour occurred, respectively, with the reagents, indicating the
presence of alkaloids and / or nitrogenous bases (Wall et al., 1954).
2.2.5.4 Test for flavonoids:
- Shinoda test:
May 2015
A 1ml of 10% ethanol extract of each plant was treated with 0.5ml of 10%
hydrochloric acid, then magnesium metal strips was added. A red colour developed
indicating the presence of flavonoids.
- Amyl alcohol test (for free and combined flavonoids):
A 5ml aliquot of 1% hydrochloric acid extract of the plant was shaken with 5ml of
amyl alcohol. A yellow colour in the amyl alcoholic layer indicated the presence of
free flavonoid aglycones.The aqueous layer was boiled with 10ml of concentrated
hydrochloric acid for two minutes. The acidic solution was cooled and extracted
with amyl alcohol. A brownish yellow colour indicated the presence of flavonoid
glycosides ( Fuluon, 1932)(Gessman , 1962).
2.2.5.5 Test for Saponins:
- Froth test:
The plant sample (1gm) was boiled in 10ml water for a few minutes ,filtered and
shaked. A persistent froth indicated the presence of saponins.
- Haemolysis test:
The alcohol extract of the plant (10ml) was evaporated at low temperature and the
residue dissolved in 10 ml of normal saline. The solution (10ml) was added to 10
ml of a suspension of red blood corpuscles in normal salin 1:40. Haemolysis
occurred indicating the
presence of saponins ( Harper,1939) (Habrone, 1973).
2.2.5.6 Test for unsaturated sterols:
May 2015
The dried powdered plant (1g) was extracted with few ml of ethanol , filtered and
evaporated to dryness, the residue was dissolved into two equal portions and tested as
follows:
- Libermann-burchards test:
To the first portion, 1ml of acetic anhydride was added and 2ml of concentrated
sulphuric acid along the side of the tube. a reddish violet colour at the junction of
the two layers indicated the presence of unsaturated sterols and/or triterpenes.
- Salkowiskis test:
To the second portion an equal volume of concentrated sulphuric acid was added.A
red colour was produced indicating the presence of unsaturated sterols and/or
triterpenes( Stahl .1969).
2.2.5.7 Test for Coumarins:
A small amount of moistened plant sample was placed in a test tube , the tube was
covered with filter paper moistened with dilute sodium hydroxide solution.
The covered test tube was then placed in boiling water bath for several minutes. The
paper was removed and exposed to ultraviolet light. Ayellow – green colour appearing
within a few minutes indicating the presence of coumarins (Franwarth. 1966).
2.2.5.8 Teste for Cardiac-glycoside
- (Legal's test)
May 2015
1.0 ml extract was dissolve in 5.0 ml pyridine, 2 drops 2% Sodium Nitroprusside and 2
drops 20% NaOH were added. A deep red color faded to brown indicates presence of
cardenolide.
- Kedde's test
1.0 ml extract was mixed with 1.0 ml 2 % 3,5- dinitrobenzoic acid in methanol and 1.0
ml aqueous NaOH. A violet color precipitate indicates lactone ring present in
cardenolide.
2.2.5.9 Test for Anthracenosides:
4 ml extract was concentrated to 2 ml, then ammonia solution (25 %,1-2 ml) was added
by shaking. A cherish-red colour of the alkaline solution indicates the presence of emodol
(aglycones of anthracenosides) in an oxidized form (Borntragers reaction).
2.2.6 -Methods used for screenning anti-oxidant activity:
2.2.6.1. DPPH radical scavenging assay:
May 2015
The DPPH radical scavenging was determined through use In 96-wells plate, the test
samples were allowed to react with 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl stable
free radical (DPPH) for half an hour at 37ºC. The concentration of DPPH was kept as
(300μM). The test samples were dissolved in DMSO while DPPH was prepared in
ethanol. After incubation, decrease in absorbance was measured at 517nm using
multiplate reader spectrophotometer. Percentage radical scavenging activity by samples
was determined in comparison with a DMSO treated control group. All tests and analysis
were run in triplicate (Shimada et al., 1992).
2.2.6.2. Iron chelating activity assay:
The ferrous ( Fe+2
) were monitored by measuring the formation of ferrous ion-ferrozine
complex. The experiment was carried out in 96 microtiter plate. The plant extracts were
mixed with Ferrous Sulphate(FeSO4). The reaction was initiated by adding 5 mM
ferrozine. The mixture was shaken and left at room temperature for 10 min. the
absorbance was measured at 562 nm. EDTA was used as standard, and DMSO as control.
All tests and analysis were run in triplicate (Dinis et al., 1994).
3. Results:-
3.1 Phytochemical investigation of the seeds of Nigella sativa:
May 2015
100gm of the seeds were extracted with chloroform by maceration. Another 100gm of the
seeds were extracted with methanol by the same method and the percentage yield of the
oil was recorded in table (3.1).
Table 3.1 The percentage yield of oil extract:
Solvents Yield%v/w
Chloroform 30
Methanol 15
The methanolic extract was subjected to qualitative chemical tests and the results are
presented in table (3.2).
Table 3.2 The phytochemical screening of the methanolic extract:
Constituents Test/Reagent Observations Result Hydrolysable tannins
1- Ferric chloride Greenish brown
colour
+Ve
2- 2%solution of
phenazone
Turbidity +ve
Condensed tannins
1- Ferric chloride Greenish brown
colour
+Ve
2- Vanillin in
hydrochloric
acid
A crimson red
colour
+ve
3- Match stick Red colour +ve
Alkaloids (salt)
Mayers reagent White
precipitate
+Ve
Dragendorffs reagent Orange
precipitate
+Ve
Iodoplatinate
modified reagent
Violet colour +Ve
Quarternary bases &
oxidized amines
Mayers reagent Yellow
preciptate
+Ve
Saponins
Froth test Standing
foaming
+Ve
Haemolysis test Haemolysis
occurred
+Ve
May 2015
Carbohydrates Molishs test Violet zone +Ve
Reducing sugar Fehlig solution (|I+II) No Red
precipitates
+Ve
unsaturated sterols Liebermann
Burchards reaction
Reddish violet
colour at the
junction of the
two layers
+Ve
Salkowiskis test Red colour +Ve
Anthracenosides Borntragers reaction No colour
change
-Ve
Coumarins Ammonia +UV light Flouresence or
Ayellow – green
colour
+Ve
Flavonoid Shinoda test Red colour +Ve
Free flavonoids
(flavonoid aglycones)
Amyl alcohol test Yellow colour +Ve
Combined flavonoids
(flavonoid glycosides)
Amyl alcohol test Brownish
yellow colour
+Ve
Terpenoids Salkowiskis test No change +Ve
Cardiac Glycosides
(Cardenolide)
Keddees test violet color
precipitate
+Ve
Cardiac Glycosides
(Cardenolide)
Legal's test deep red color
faded to brown
+Ve
Chlorophyll Visual test No green colour -Ve
Essential oil Visual test On filter paper +Ve
Fatty Acids Sudan red 1 Red +Ve
The chloroformic extract was subjected to qualitative chemical tests and the results are
presented in table (3.3).
May 2015
Table 3.3 The phytochemical screening of the chloroformic extract:
Constituents Test/Reagent Observations Result Hydrolysable tannins
1- Ferric
chloride
Greenish brown
colour
-Ve
2- 2%solution of
phenazone
Turbidity -ve
Condensed tannins
1- Ferric
chloride
Greenish brown
colour
-Ve
2- Vanillin in
hydrochloric
acid
A crimson red
colour
-Ve
3- A match stick Red colour -Ve
Alkaloids
Mayers reagent White precipitate +Ve
Dragendorffs
reagent
Orange
precipitate
+Ve
iodoplatinate
modified reagent
Violet colour +Ve
Quarternary bases &
oxidized amines
Mayers reagent Yellow preciptate +Ve
Saponins
Froth test Standing foaming +Ve
Haemolysis test Haemolysis
occurred
+Ve
Carbohydrates Molishs test Violet zone -Ve
Reducing sugar Fehlig solution
(|I+II)
No Red
precipitates
-Ve
unsaturated sterols Libermann
Burchards reaction
Reddish violet
colour at the
junction of the
two layers
+Ve
Salkowiskis test Red colour +Ve
Anthracenosides Borntragers reaction No colour change -Ve
Coumarins Ammonia +UV
light
Flouresence or
Ayellow – green
colour
+Ve
Flavonoid Shinoda test Red colour +Ve
Free flavonoids
(flavonoid aglycones)
Amyl alcohol test Yellow colour +Ve
Combined flavonoids Amyl alcohol test Brownish yellow +Ve
May 2015
(flavonoid glycosides) colour
Terpenoids Salkowiskis test Red colour +Ve
Cardiac Glycosides
(Cardenolide)
Keddees test Violet color
precipitate
+Ve
Cardiac Glycosides
(Cardenolide)
Legal's test Deep red color
faded to brown
+Ve
3.2 Screening for antioxidant activity:
Table 3.4 Screening of methanolic extract for antioxidant:
Plant extract %RSA±STD
(DPPH)
Iron ch %±STD
(Iron chelating)
Nigella sativa 07±0.03 16±0.01
3.3 Screening for antimicrobial activity:
May 2015
Different concentrations of chloroformic and methanolic oils were screened for their
antimicrobial activity against Actinomycetoma( Nocardia brasiliensis, N.asteroides and
Streptomyces samaliensis ) and Eumycetoma ( Pseudallescheria boydii and 4 samples of
Madurella mycetomatis) using SDA and blood agar as culture media.
The tubes and plates were examined for inhibition of the growth of the above species
from three subcultures for each organism and the results obtained are shown on the
following tables and figures.
3.3.1Subculture from Saudi Arabia examined after 7 days of incubation:
Fig 3.1 Madurella mycetoma(SA) subculture after 7 days
Fig 3.2 Madurella mycetoma(SU2) subculture after 7 days
May 2015
Fig 3.3 Madurella mycetoma(Pot) subculture after 7 days
Fig 3.4 Nocardia asteroides Figures 3.5 Nocardia brasiliensis
subculture after 7 days subculture after 7 days
(SA) Saudi Arabia sample in Sabroud dexterose agar.
(Pot) Saudi Arabia sample in Potato desterose agar.
(SU1) and (SU2) Sample from Sudan in SDA.
May 2015
Fig 3.6 Comparison of change in media colour between
Culture tubes & blank tube media:
3.3.2Subculture from Saudi Arabia examined after 14 days of incubation:
Normal colour
Colour
change to
darker
from
growth
blank tube media
3Type of Cultures tubes
May 2015
Fig 3.7 Madurella mycetoma(SA) subculture after 14 days
Fig 3.8 Madurella mycetoma(SU2) subculture after 14 days
May 2015
Fig 3.9 Madurella mycetoma(Pot) subculture after 14 days
Fig 3.10 Nocardia asteroides Fig 3.11 Nocardia brasiliensis
subculture after 14 days subculture after 14 days
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3.3.3Subculture from Saudi Arabia examined after 23 days of incubation:
Fig 3.12 Madurella mycetoma(SA) subculture after 23 days
Fig 3.13 Madurella mycetoma(SU1) subculture after 23 days
May 2015
Fig 3.14 Madurella mycetoma(Pot) subculture after 23 days
Fig 3.15 Nocardia asteroides Fig 3.16 Nocardia brasiliensis
subculture after 23 days subculture after 23 days
May 2015
The chloroform and methanol extracts were tested separately against ((Pseudallescheria
boydii and 4 samples of Madurella mycetomatis)) and the results are shown on (Tables
3.5-3.6) and (Figures 3.17 – 3.26).
Table 3.5 Antifungal activity against (Pseudallescheria boydii and 4 samples of
Madurella mycetomatis) after 8 days:
Organesim Oil type Concentration of oil
0.05% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% Control
M.m(SA1) Methanol - - - - - - - - - - - +
M.m(Pot) - - - - - - - - - - - +
M.m(su2) - - - - - - - - - - - +
M.m(Su1) - - - - - - - - - - -
Psudo - - - - - - - - - - - +
M.m(SA1) Chloroform + - - - - - - - - - - +
M.m(Pot) + - - - - - - - - - - +
M.m(Su2) - - - - - - - - - - - +
M.m(Su1) - - - - - - - - - - - +
Psudo + + + + + - - - - - - +
Table 3.6 Antifungal activity against (Pseudallescheria boydii and 4 samples of
Madurella mycetomatis) after 30 days:
Organesim Oil type Concentration of oil
0.05% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% Control
M.m(SA) Methanol - - - - - - - - - - - +
M.m(Pot) - - - - - - - - - - - +
M.m(Su2) - - - - - - - - - - - +
M.m(Su1) - - - - - - - - - - - +
Psudo - - - - - - - - - - - +
M.m(SA) Chloroform + + - - - - - - - - - +
M.m(Pot) + + - - - - - - - - - +
M.m(Su2) + + - - - - - - - - - +
M.m(Su1) - - - - - - - - - - -
Psudo ++ ++ + + + + + + - - - +
Interpretation of the results:
May 2015
On the eighth day of incubation the control showed growth of the organism. While
cultures of four samples of Madurella mycetomatis and Pseudallescheria boydii treated
with methanolic extract showed total inhibition of the growth with all concentrations.
On other hand cultures of Madurella mycetomatis treated with chloroformic extracts
showed inhibition of growth with all oil concentrations (2% and above)
While in case of cultures of Pseudallescheria boydii inhibition of growth was recorded
only with concentration 8% and above.
May 2015
Fig 3.17 Culture of methanol extracts tested against Madurella mycetomatis(SA)
Fig 3.18 Culture of methanol extracts tested against Madurella mycetomatis(Pot)
Fig 3.19 Culture of methanol extracts tested against Madurella mycetomatis(Su1)
Fig 3.20 Culture of methanol extracts tested against Madurella mycetomatis(Su2)
Fig 3.21 Culture of methanol extracts tested against Pseudallescheria boydii
May 2015
Fig 3.22 Culture of chloroform extracts tested against Madurella mycetomatis(SA)
Fig 3.23 Culture of chloroform extracts tested against Madurella mycetomatis(Pot)
Fig 3.24 Culture of chloroform extracts tested against Madurella mycetomatis(Su1)
Fig 3.25 Culture of chloroform extracts tested against Madurella mycetomatis(Su2)
Fig 3.26 Culture of chloroform extracts tested against Pseudallescheria boy
May 2015
The chloroform and methanol extracts were tested separately against (Nocardia
brasiliensis, N.asteroides and Streptomyces samaliensis) and the results are shown on
(Tables 3.7 - 3.8) and (Figures 3.27 – 3.32).
Table 3.7 Antibacterial activity against (Nocardia brasiliensis, N.asteroides and
Streptomyces samaliensis) after 8 days:
Organesim Oil type Concentration of oil
0.05% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% Control
N.a Methanol + + - - - - - - - - - +
N.b + + - - - - - - - - - +
S.s - - - - - - - - - - - +
N.a Chloroform + + + + + + + + - - - +
N.b + + + + + + + + - - - +
S.s - - - - - - - - - - - +
Table 3.8 Antibacterial activity against (Nocardia brasiliensis, N.asteroides and
Streptomyces samaliensis) after 30 days:
Organesim Oil type Concentration of oil
0.05% 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% Control
N.a Methanol + + + + - - - - - - - +
N.b + + + + - - - - - - - +
S.s + + + + + + + + + + + +
N.a Chloroform + + + + + + + + - - - +
N.b + + + + + + + + - - - +
S.s - - - - - - - - - - - +
Interpretation of the results:
On the eighth day of incubation the control and methanolic extract showed growth of the
organism. While cultures treated with chloroformic extracts showed total inhibition of the
growth with Streptomyces somaliensis in all concentrations but on the other hand
cultures of Nocardia brasiliensis and N.asteroides treated with methanolic and
chloroformic extract showed inhibition of growth with oil concentrations of 4% and 8%
respectively.
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Fig 3.27 Culture of methanol extracts tested against Nocardia brasiliensis.
Fig 3.28 Culture of methanol extracts tested against Nocardia asteroides .
Fig 3.29 Culture of methanol extracts tested against Streptomyces samaliensis .
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Fig 3.30 Culture of chloroform extracts tested against Nocardia brasiliensis.
Fig 3.31 Culture of chloroform extracts tested against Nocardia asteroides .
Fig 3.32 Culture of chloroform extracts tested against Streptomyces samaliensis .
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Table 3.9 Minimum inhibitory concentration of methanol oil extract
against mycetoma:
Organisms Concentration of methanol oil dilution Control
2.5% 1.25% 0.625% 0.31% 0.15% 0.078% 0.039% M.m(SA) - - - - + ++ +++ +
M.m(Pot) - ++ ++ ++ ++ ++ ++ + M.m(Su2) - + + ++ +++ +++ ++++ +
Fig 3.33 Culture of Minimum inhibitory concentration of methanol oil extract against mycetoma
3.2. Discussion:
M.m(SA)
M.m(Su2)
M.m(Pot)
May 2015
Nigella sativa seeds were subjected to extraction by maceration using methanol. A new
batch of the plant material was extracted by the same method using chloroform as a
solvent.
Qualitative phytochemical screening tests of the methanolic extract of Nigella sativa
seeds revealed the presence of steroids, tannins, alkaloids, quarternary bases & oxidized
amines, carbohydrates, reducing sugars, terpenoids, essential oils, fatty acids, flavonoids,
coumarins, cardiac glycosides, and saponins.
The extract was devoid of anthracenosides..
These results agreed with was previously reported by Lewis, 2006 and Adekunle &
Adekunle 2009 that N. sativa seeds contain alkaloids, flavonoids, tannins and terpenoids ,
while Eloff, 1998 and Hashem and El-Kiey, 1982 detected tannins only in the methanolic
extract. Our results were also in agreement with Chaouche et al 2011 who reported
flavonoids, tannins, coumarines, saponins and sterols in the methanolic extracts and
disagreed with them in that they did not detected alkaloids in the methanolic extracts.
While the qualitative phytochemical screening tests of the chloroform extract of Nigella
sativa seeds showed the presence of alkaloids, saponins, steroids, terpenoids, flavonoids,
coumarins and cardiac glycosides.
These results agreed with those previously reported by Morsi, 2000.
These differences might be due to age of the plant, climatic factors and different methods
used.
The oil of nigella sativa seeds was subjected to preliminary screening against three
isolates of mycetoma bacteria types [Actinomycetoma: Streptomyces somaliensis –
(causes the majority of the cases) Nocardia brasiliensis and N.asteroides] and five
isolates of fungi types [Eumycetoma: four samples of Madurella mycetomatis (causes
the majority of the cases) and one sample of Pseudallescheria boydii].
May 2015
The chloroform extract exhibited activity against all bacterial types with a minimum
inhibitory concentration (MIC) of 8% with Nocardia brasiliensis and N.asteroides and
MIC of 0.5% with Streptomyces somaliensis. It also showed activity against the fungi
types with a MIC of 2%. And showed MIC 8% with Pseudallescheria boydii. This
antimicrobial activity is due to the presence of steroids, terpenoids, flavonoids, coumarins
and cardiac glycosides in the chloroform extract as reported by Morsi, 2000.
The methanol extract exhibited activity against the fungal types with a minimum
inhibitory concentration of 0.15% with the four samples of Madurella mycetomatis and
MIC of 8% with Pseudallescheria boydii. It also showed activity against bacterial types
with MIC 4% with Nocardia brasiliensis and N.asteroides and no inhibition was noticed
with Streptomyces somaliensis. This antimicrobial activity against all strain, is due to the
presence of the essential oils as reported by Enomoto et al., 2001 and Karapinar & Aktug,
1987. Mason & Wasserman, 1987 attributed the activity to essential oils due to enzyme
inhibition by the oxidized compounds, possibly through nonspecific interactions with the
proteins. Also antimicrobial activity was found to be due to presence of tannins as
reported by Eloff, 1998 and Hashem and El-Kiey, 1982 who explain that tannins forms
complexes with proteins through forces such as hydrophobic effects, hydrogen bonding
and covalent bond formation, thus, tannins act as antibacterial agent by inactivating
microbial adhesins, enzymes, and cell envelope transport proteins.
Flavonoids have been found to be effective antimicrobial substances against a wide range
of microorganisms. This activity is probably due to their ability to form complexes with
soluble proteins and also with bacterial cell walls, as well as disruption of membrane
particularly with lipophilic flavonoids.
Triterpenes and many saponins were also known to have antimicrobial activity.
The antimicrobial activity of this plants has been studied previsouly by other researchers
against other types of bacteria different from what we are reporting in this study
(Escherichia coli, Bacillus subtilis, Streptococcus feacalis, Staphylococcus aureus,
May 2015
Pseudomonas aerginosa, Proteus vulgaris, Salmonella typhi, and Klebsiella.pneumonia)
and different types of fungi (Candida albicans, A.fumigatous, A.flavus and A.niger) ,
(Manohar et al., 2001, Mansour et al., 2002, Hafez, 1991, El-Fatatry, 1975, Hanafii &
Hatim, 1991, Khan, 2003)
So this is the first attempt to investigate the effect of Nigella sativa seeds oil against
mycetoma.
The results obtained in this study indicated that the antimicrobial activities were found in
two different extracts suggesting existence of different antimicrobial principles.
Phytochemical screening results explain the correlation between the biological activity
(antimicrobial) exhibited by this plant and detected constituents.
The methanolic extract was also screened for its anti-oxidant activity with Iron chelating
recorded a high activity than DPPH radical scavenging assay. These results were
similar to that reported by Nagi and Mansour, 2000. The anti-oxidants activity was
probably due to phenolic compounds, (tannins and flavonoids).
4.1. Conclusion:
-This study showed that oil of Nigella sativa has antimycetoma activity against both
fungal and bacterial types. And it has been used for a long time as food without any
May 2015
serious toxicity reported, it can be considered as a safe and affordable treatment for
mycetoma.
4.2. Recommendations:
1- Further studies in Minimum Inhibitory concentration (MIC) recommended to be done
for N.sativa as pure seed.
2- Further studies using other models to confirm the anti-mycetoma activity of the plant.
3- Toxicological studies to ensure the safety of the extract.
4- To be formulated in a suitable dosage form and to be tested in vivo.
5- Suitable dose must be adjusted to prevent from toxicity or over dose effects.
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