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Antimicrobial and Phytochemical Study of Maerua
pseudopetalos Gilg & Bend.
A Thesis Submitted to the University of Khartoum in Fulfillment of
the Requirements of the Degree of Master of Science in Botany
By
Wisal Hassan Ali Mhjoub
B. Sc. Botany (University of El Nilein)
Supervisor: Dr. Sakina M.A. Yagi
Department of Botany
Faculty of Science
University of Khartoum
May 2012
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Dedication
To the Great mother ever, my dear mother
Gafera AbdElrahman.
To the crowns of my head, my brothers,
Emad, Slah, Safe and Esam.
To the sweet of my life, my Sisters,
Suaad, Rehab, Abeer, Safaa and Fatima.
To the person whom I love life for,
my son Huzaifa
To all of you ………………
The fruit of my efforts
All the love.
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Table of contents
Table of contents……………….…………………………………………………………...i
Acknowledgements……………………………………………………………………..….ii
Abstract ……………………………….…………………………………………………iii
List of Figures…………………... ……………..………………………………………..iv
List of Tables ………………………………………………………..……………………v
Introduction and literature review ............................................Error! Bookmark not defined.
1.1 General introduction................................................................ Error! Bookmark not defined.
1.2 Antimicrobial infection ........................................................... Error! Bookmark not defined.
1.2.1 Main-groups of antimicrobial agents ............................... Error! Bookmark not defined.
1.2.1.1 Penicillins .................................................... Error! Bookmark not defined.
1.2.1.2 Cephalosporins ............................................ Error! Bookmark not defined.
1.2.1.3 Other ß-lactams ........................................... Error! Bookmark not defined.
1.2.1.4 Amino glycosides ........................................ Error! Bookmark not defined.
1.2.1.5 Tetracyclines................................................ Error! Bookmark not defined.
1.1.1.6 Macrolides ................................................... Error! Bookmark not defined.
1.2.1.7 Quinolones ................................................... Error! Bookmark not defined.
1.2.1.8 Glycopeptides .............................................. Error! Bookmark not defined.
1.2.1..9 Other antimicrobial agents ......................... Error! Bookmark not defined.
1.2.1.10 Antifungal therapy ..................................... Error! Bookmark not defined.
1.2.2 Resistance to Antimicrobial Agent................................... Error! Bookmark not defined.
1.2.3 Factors contributing to the emergence of resistance ........ Error! Bookmark not defined.
1.2.4 Examples of some pathogenic bacteria ............................ Error! Bookmark not defined.
1.3 Protozoan Diseases.................................................................. Error! Bookmark not defined.
1.3.1 Flagellated Protozoa ......................................................... Error! Bookmark not defined.
1.3.1.1 Giardia ........................................................ Error! Bookmark not defined.
1.4 Plant under study ..................................................................... Error! Bookmark not defined.
1.4.1 Botanical description ........................................................ Error! Bookmark not defined.
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1.4.2 Distribution: ..................................................................... Error! Bookmark not defined.
1.4.3 Uses: ................................................................................. Error! Bookmark not defined.
1.4.4 Toxicity: ........................................................................... Error! Bookmark not defined.
1.4.5 Previous work on Maerua ................................................ Error! Bookmark not defined.
1.5 Research problem .................................................................... Error! Bookmark not defined.
1.6 Research hypothesis ................................................................ Error! Bookmark not defined.
1.7 Objective of the study ............................................................. Error! Bookmark not defined.
Materials and Methods .............................................................Error! Bookmark not defined.
2.1 Collection and preparation of plant material ........................... Error! Bookmark not defined.
2.2 Extraction ................................................................................ Error! Bookmark not defined.
2.3 Antimicrobial activity ............................................................. Error! Bookmark not defined.
2.3.1 Test organisms.................................................................. Error! Bookmark not defined.
2.3.2 Disc diffusion method ...................................................... Error! Bookmark not defined.
2.4 In vitro antigiardial activity ..................................................... Error! Bookmark not defined.
2.4.1 Parasite isolate .................................................................. Error! Bookmark not defined.
2.4.2 In vitro susceptibility assays............................................. Error! Bookmark not defined.
2.5 Phytochemistry ........................................................................ Error! Bookmark not defined.
2.5.1 Phytochemical screening .................................................. Error! Bookmark not defined.
2.5.1.1 Preparation of extracts ................................. Error! Bookmark not defined.
2.5.1.2 Phytochemical analysis ............................... Error! Bookmark not defined.
• Test for flavonoids ............................................ Error! Bookmark not defined.
• Test for tannins .................................................. Error! Bookmark not defined.
• Test for alkaloids ............................................... Error! Bookmark not defined.
• Test for saponins ............................................... Error! Bookmark not defined.
• Test for triterpenes and steroids (the Lieberman–Burchard)Error!
Bookmark
not defined.
• Test for cardiac glycosides (Keller–Killani test)Error! Bookmark not defined.
• Test for anthraquinones ..................................... Error! Bookmark not defined.
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2.5.2 Chromatographic Techniques........................................... Error! Bookmark not defined.
2.5.2.1 Preparation of thin layer chromatography plates (TLC)Error!
Bookmark
not defined.
2.5.2.2 Column chromatography (CC) .................... Error! Bookmark not defined.
2.5.2.3 Solvent systems ........................................... Error! Bookmark not defined.
2.5.2.4 Detection of spots on TLC .......................... Error! Bookmark not defined.
2.5.2.5 Preparation of the spray reagents ................ Error! Bookmark not defined.
2.5.2 Analysis of hexane extract ............................................... Error! Bookmark not defined.
2.6 Gas Chromatography\ Mass Spectroscopy analysis (GC/MS) Error! Bookmark not defined.
2.7 Column chromatography (CC) of chloroform extract ............. Error! Bookmark not defined.
Results and Discussion ...........................................................Error! Bookmark not defined.
3-1 Quantity of extracts ................................................................. Error! Bookmark not defined.
3-2 Antimicrobial activity ............................................................. Error! Bookmark not defined.
3-2-1 Antibacterial activity against Bacillus subtilis (Gram +ve)Error!
Bookmark
not
defined.
3-2-2 Antibacterial activity against Staphylococccus aureus (Gram +ve)Error!
Bookmark
not defined.
3-2-3 Antibacterial activity against Escherichia coli (Gram – ve)Error!
Bookmark
not
defined.
3-2-3 Antibacterial activity against Pseudomonas aeruginosa (Gram –ve)Error!
Bookmark
not defined.
3-2.4 Antibacterial activity against Salmonella typhi (Gram –ve)Error!
Bookmark
not
defined.
3.2.5 Antibacterial activity of Maerua pseudopetalosa roots at lower extracts concentrations
................................................................................................... Error! Bookmark not defined.
3.3 In vitro antigiardial activity against Giardia lamblia .............. Error! Bookmark not defined.
3-4 Phytochemistry ....................................................................... Error! Bookmark not defined.
3-4-1 Qualitative analysis of secondary metabolites ................. Error! Bookmark not defined.
3-3-2 Preliminary phytochemical screening by thin layer chromatography (TLC) .......... Error!
Bookmark not defined.
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3-4-3 GC/MS analysis of the hexane extract............................. Error! Bookmark not defined.
3.4.4 Bioassay guided fractionation of chloroform extract ....... Error! Bookmark not defined.
Conclusion ...............................................................................Error! Bookmark not defined.
Recommendations ..................................................................Error! Bookmark not defined.
References ...............................................................................Error! Bookmark not defined.
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Acknowledgements
All thanks to the almighty god for all the blessing he
offend me.
Then I present my appreciation and Gratitude to my teacher
Dr. Sakina Yagi which provided me with all possible support
to make this research.
Iam also thankful to Dr. Waleed Koko and Mohammed Ismail
mohammed Gharbi for conducting antigiardial activity at
their laboratory in MAPRI.
Iam also very much thankful to all of my colleagues and
friends in Botany Department.
My thanks to everyone provided me with assistance or
advice.
Last, but not least, I wish to express my sincere gratitude to
my beloved family, who supported me in every stage of my
life.
ii
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Abstract
The present study was carried
out on the roots of Maerua psedopetalosa (Family
Capparaceaeae). A plant used in Western Kordofan traditional medicine for the treatment of
cough and other pulmonary troubles, rheumatic pain and skin diseases. Different extracts
from the roots were assessed for their phytochemical constituents and in vitro antibacterial
and antigiardial activity.
Hexane, chloroform, ethyl acetate and methanol extracts from the roots were prepared and the
antibacterial activity at concentrations ranged between 20 mg/mL and 250 µg/mL was
determined against Bacillus subtilis, Staphylococccus aureus, Escherichia coli, Pseudomonas
aeruginosa and Salmonella typhi using the disc diffusion method.
Results showed that the response of the bacteria to the tested extracts varied among the strains
and are concentration dependent. The best antibacterial activity for all tested bacteria was
obtained from the hexane and chloroform extracts.The hexane extract at concentrations 20
and 10 mg/mL revealed high antibacterial activity against P. aeruginosa with inhibition zones
19 and 17 mm respectively. Whereas, the chloroform extract, at concentration 250 µg/mL,
displayed very high activity, higher than the standard antibiotic drug, against E. coli and S.
typhi with inhibition zones of 45 and 32 mm respectively.
Analysis of different classes of major secondary metabolites of the roots was carried out using
standard methods. Results revealed that, extracts contained saponins, alkaloids, triterpenes,
steroids and flavanoids. The hexane extract was subjected to GC/MS analysis and results
revealed the presence of 20 compounds. The major compounds were identified as 9,12
octadecenonic acid (28.83%); followed by 9- octadecenonic acid (24.86 %), 9octadecenamide (14.35 %) and hexadecanoic acid (11.64 %) respectively.
According to the antibacterial activity results, the chloroform extract was subjected to
bioassay guided fractionation using column chromatography technique. All subfractions at
concentration 5 mg/mL showed antibacterial activity lower than that obtained by the crude
extract. The decrease in potency of subfractions seems to indicate the loss of synergistic
action between the phytochemical constituents present in the chloroform extract due to its
subjection to the separation process.
The antigiardial activity test was performed for the different extracts of M. psudopetalosa
roots. The flagellated protozoa Gairdia lamblia used in this test were taken from patients at
Ibrahim Malik Hospital (Khartoum). Results showed that the ethyl acetate extract was the
iii
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most effective extract against G. lambelia with mortality of 70% higher than that obtained by
the positive control metrondizole.
In conclusion, the results of the present study provided scientific justification for the use of M.
psedopetalosa in traditional medicine and provide some information about its phytochemical
constituents. Moreover, this study paves the way for further attention and research to identify
active compounds for the development of new antimicrobial agents capable of decreasing the
burden of drug resistance and cost of management of diseases of clinical and public health
importance in the Sudan.
iv
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Δ
λϼΨϟ
΍
Maerua Δϟ΍ΩήϜϟ΍ΔΘΒϧϰϠϋΔγ΍έΪϟ΍ϩάϫΖϳήΟ΃
ϱΪϴϠϘΘϟ΍ΐτϟ΍ΕΎΗΎΒϧϯΪΣΎϛϡΪΨΘδΗ͓ϟ΍pseudopetalosa
ϡΰϴΗΎϣϭήϟ΍ϡϵ΍ˬέΪμϟ΍ΕΎΑ΍ήτο΍ΝϼόϟϥΎϓΩήϛΏήϏ̼
ΪϠͧ΍ν΍ήϣ΍ϭ
ΚϴΣϦϣέϭΰͧ΍ϦϣΕΎμϠΨΘδͪ΍ϒϠΘͮϢϴϴϘΗ͂΍Δγ΍έΪϟ΍ΖϓΪϫ
ΎϳΩέΎϘϟ΍ϭΎϳ͐ϜΒϠϟΩΎπͪ΍ΎϬσΎθϧϭΔϴ΋ΎϴϤϴϜϟ΍Ύ͡ΎϧϮϜϣ
͗δϜϴͫ΍κϠΨΘδϣέϭΰͧ΍ΕΎμϠΨΘδϣϦϣΪϳΪόϟ΍͖π̹͞
ethyl ΖϴΘϴγ΍ϞϳΎΜϳ΍ˬ chloroformϡέϮϓϭέϮϠϛˬHexane
͗ΑΎϣΡϭ΍͐Ηΰϴϛ΍ήΗ̼ methanolϝϮϧΎΜϴͪ΍ϭacetate
ΔϳϮμόϟ΍ΔϬΟ΍Ϯϣ̼Ϟϣϡ΍ήΟϭήϜϳΎϣϭϞϣϡ΍ήΠϠϣ
, ΔϴΒϫάϟ΍ΔϳΩϮϘϨόϟ΍ˬBacillus subtilisΔϘϴϗΪϟ΍
, ΔϴϧϮϟϮϘϟ΍ΔϴϜϳήηϻ΍ˬStaphylococccus aureus
, Pseudomonas ΔϳέΎͰΰϟ΍Δϔ΋΍ΰϟ΍Escherichia coli
ϚϟΫϭsalmonella typhi ϲϔϳΎΗϼϴϧϮͪΎδϟ΍ϭaeruginosa
ΔΑήθΘͪ΍ι΍ήϗϻ΍ΔϴΠϬϨϣϡ΍ΪΨΘγΎΑ
̹͓ϟ΍ΕΎμϠΨδϤϠϟΎϳ͐ϜΒϟ΍ΖΑΎΠΘγ΍ϥ΍Ξ΋ΎΘϨϟ΍ΕήϬχ΍
ΪϗϭˬκϠΨΘδͪ΍ΰϴϛήΗϰϠϋΪϤΘόΗϭΕϻϼδϟ΍͌ϋϒϠΘ͟ΎϫέΎΒΘΧ΍
Ύϳ͐ϜΒϟ΍Ϟϛ͗ΑϦϣΎϳ͐ϜΒϠϟΩΎπϣρΎθϧϞπϓ΍ϰϠϋϝϮμͨ΍̹
ϡέϮϓϭέϮϠϜϟ΍ϭHexane͗δϜϴͫ΍ϲμϠΨΘδϣϦϣΎϫέΎΒΘΧ΍̹͓ϟ΍
ϭΰϴϛήΗΪϨϋ͗δϜϴͫ΍κϠΨΘδϣήϬχ΍ΪϘϓchloroform
ΚϴΣΔϳέΎΒͰΰϟ΍Δϔ΋΍ΰϟ΍Ύϳ͐ϜΒϟΩΎπϣ͂ΎϋρΎθϧϞϣΞϣ
ήϬχ΍ΎϤϨϴΑˬ͂΍ϮΘϟ΍ϰϠϋϢϠϣϭςϴΒΜΘϟ΍ήτϗώϠΑ
˱
΍ΪΟ͂ΎϋρΎθϧϞϣΞϣΰϴϛήΗΪϨϋϡέϮϓϭέϮϠϜϟ΍κϠΨΘδϣ
ΔϴϜϳήηϻ΍ΔϬΟ΍Ϯϣ̼ϲγΎϴϘϟ΍ϱϮϴͨ΍ΩΎπͪ΍έΎϘϋϦϣϰϠϋ΍
ϭςϴΒΜΘϟ΍ήτϗώϠΑΚϴΣϲϔϳΎΗϼϴϧϮͪΎγϭΔϴϧϮϟϮϘϟ΍
͂΍ϮΘϟ΍ϰϠϋϢϠϣ
ΔϴόϴΒτϟ΍ΕΎΒϛήͪ΍ϦϋϒθϜϠϟϲ΋ΎϴϤϴϜϟ΍΢δͪ΍˯΍ήΟ΍̹
ΕήϬχ΍ΪϗϭΔϴγΎϴϗϕήσϡ΍ΪΨΘγΎΑϚϟΫϭέϭΰͧ΍̼ΔϳϮϧΎΜϟ΍
ˬsaponinsΕΎϴϧϮΑΎλϲϠϋϱϮΘ͞ΕΎμϠΨΘδͪ΍ϥ΍Ξ΋ΎΘϨϟ΍
ˬtriterpenesΕΎϨϴΑήΗϱ΍ήΗˬ alkaloids Ε΍ΪϳϮϟΎϜϟ΍
̹Ϊϗϭ flavanoidsΕ΍ΪϳϮϨϓϼϓϭ steroidsΕ΍Ϊϳϭ͐γ΍ϭ
ΔϴϨϘΗϡ΍ΪΨΘγΎΑϞϴϠΤΘϠϟ͗δϜϴͫ΍κϠΨΘδϣωΎπΧ΍
Ξ΋ΎΘϨϟ΍ΕήϬχ΃ΪϗϭΔϠΘϜϟ΍ϒϴσϊϣίΎϐϟ΍̼΍ήϏϮΗΎϣϭήϛ
ϚϳϮϧΎδϳΩΎΘϛϭϻ΍ξϣΎΣϲϫΎϬϨϣΔϴγΎγϷ΍˱
ΎΒϛήϣέϮϬχ
ξϣΎΣϪόΒΘϳ 9,12 octadecenonic acid 9- octadecenonic acidϚϳϮϨδϳΩΎΘϛϭϻ΍
9-octadecenamide ΪϳΎͳΎδϳΩΎΘϛϭϻ΍ξϣΎΣϭ
hexadecanoic acid ϚϳϮϧΎδϳΩΎδϜͫ΍ξϣΎΣϭ
͂΍ϮΘϟ΍ϰϠϋ
v
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κϠΨΘδϣξϳήόΗ̹Ύϳ͐ϜΒϠϟΩΎπͪ΍ρΎθϨϟ΍Ξ΋ΎΘϨϟ˱
ΎϘϓϭ
ϡ΍ΪΨΘγΎΑϚϟΫϭϲΟϮϟϮϳΎΒϟΎΑϪΟϮͪ΍͖δϜΘϠϟϡέϮϓϭέϮϠϜϟ΍
Ϊϗϭcolumn chromatographyϱΩϮϤόϟ΍̼΍ήΟϮΗΎϣϭήϜϟ΍ΔϴϨϘΗ
ϞϣΞϣΰϴϛήΗΪϨϋ͖δϜΘϟ΍ϦϣΔ͝ΎϨϟ΍ΕΎμϠΨΘδͪ΍ΕήϬχ΍
ΕΎμϠΨΘδͪ΍ϦϣΞΘϨͪ΍ϚϟΫϦϣϞϗ΍Ύϳ͐ϜΒϠϟΩΎπϣρΎθϧ
ΕΎϧϮϜͪ΍͗ΑϲϘϓ΍ϮΘϟ΍Ϟόϔϟ΍ϥ΍ΪϘϓ͂΍͖θϳ΍άϫϭΔϴγΎγϻ΍
ΎϬοήόΗΐΒδΑϚϟΫϭϡέϮϓϭέϮϠϜϟ΍κϠΨΘδϣ̼Δϴ΋ΎϴϤϴϜϟ΍
ϝΎμϔϧϻ΍ΔϴϠϤόϟ
ΕΎμϠΨΘδͪ΍ϲϠϋΎϳΩέΎϘϠϟΩΎπͪ΍ρΎθϨϟ΍έΎΒΘΧ΍˯΍ήΟ΍̹
Gairdia ΎϳΩέΎϘϟ΍ϞϴϔσάΧ΍̹ϭΕΎΒϨϟ΍έϭΰͧΔϔϠΘΨͪ΍
ϰϔθΘδϣϦϣϰοήϣϦϣέΎΒΘΧϻ΍΍άϫ̼ΔϣΪΨΘδͪ΍lamblia
κϠΨΘδϣϥ΍Ξ΋ΎΘϨϟ΍ΕήϬχ΍ΪϗϭϡϮσήͩΎΑϚϟΎϣϢϴϫ΍ήΑ΍
ΔϬΟ΍Ϯϣ̼ΔϴϟΎόϓΕΎμϠΨΘδͪ΍ήΜϛ΍ϦϣϥΎϛΖϴΘϴγ΍ϞϳΎΜϳϻ΍
ϦϣϪϴϠϋϞμΤΘͪ΍ϚϟΫϦϣϰϠϋ΍ϝΪό͛ΎϴϠΒϣϻΎϳΩέΎϘϟ΍
ϲγΎϴϘϟ΍ϝϭάϳΪϧ΍͐ϴϣϻ΍έΎϘϋ
ϡ΍ΪΨΘγϻ˱
ΎϴϤϠϋ˱
ΎϤϋΩϲτόΗΔγ΍έΪϟ΍ϩάϫΞ΋ΎΘϧϥ΍˱
ΎϣΎΘΧ
ξόΑϡΪϘΗΎϤϛϭϱΪϴϠϘΘϟ΍ΐτϟ΍̼Δϟ΍ΩήϜϟ΍ΕΎΒϧ
ϩάϫΪϬ͠ϚϟΫϰϠϋΓϭϼϋΔϴ΋ΎϤϴϜϟ΍Ύ͡ΎϧϮϜϣϝϮΣΕΎϣϮϠόͪ΍
ΔτθϨϟ΍ΕΎϧϮϜͪ΍ΪϳΪΤΘϟΚΤΒϟ΍ϦϣΪϳΰͪ΍˯΍ήΟϻΔγ΍έΪϟ΍
˯ΐϋϦϣϞϴϠϘΘϠϟΕΎΑϭήϜϴϤϠϟΓΩΎπϣΓΪϳΪΟήλΎϨϋήϳϮτΘϟ
ΎϤϴϓΔϴʹ΃Ε΍Ϋν΍ήϣ΍ϊϣϞϣΎόΘϟ΍ΔϔϠϜΗϭ͖ϗΎϘόϟ΍ΔϣϭΎϘϣ
ϥ΍ΩϮδϟ΍̼Δϳήϳήδϟ΍ϭΔϣΎόϟ΍ΔΤμϟΎΑϖϠόΘϳ
vi
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List of Figures
Fig.1: Life cycle of Giardia ……………………………………………………………..
(18)
Fig. 2: Maerua pseudopetalos leaves and flower………………………………………..
(20)
Fig. 3: A schematic representation of the procedure followed in the phytochemical
analysis of Maerua psedopetalosa roots……………………………………………….
(36)
Fig. 4: Antibacterial activity of Maerua pseudopetalosa roots extracts against Bacillus
subtilis……………………………………………………………………………………. (40)
Fig. 5: Antibacterial activity of
Maerua pseudopetalosa
roots extracts against
Staphylococccus aureus…………………………………………………………………
Fig. 6: Antibacterial activity of Maerua pseudopetalosa
roots extracts against
Escherichia coli…………………………………………………………………………..
Fig. 7: Antibacterial activity of
Maerua pseudopetalosa
(40)
(42)
roots extracts against
Pseudomonas aeruginosa………………………………………………………………..
(42)
Fig. 8: Antibacterial activity of Maerua pseudopetalosa roots extracts against
Salmonella typhi…………………………………………………………………………
(43)
Fig. 9: Antigiardial activity of roots of Maerua pseudopetalosa against Giardia lamblia
(50)
Fig.10: Thin layer chromatography of the hexane, CHCl3 and EtOAc extracts from
Maerua pseudopetalosa roots…………………………………………………………….
(52)
Fig.11: Thin layer chromatography of fractions of CHCl3 extract from Maerua
pseudopetalosa roots……………………………………………………………………..
vii
(59)
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List of Tables
Table 1: Major antifungal agents…………………………………………
(9)
Table 2: Column chromatography of CHCl3 extract of Maerua psedopetalosa roots
(35)
……………………………………………………..
Table3. Extractive value and organoleptic properties of plant extracts from Maerua
(38)
pseudopetalosa roots………………………………………
Table 4: Antibacterial activity of Maerua pseudopetalosa roots at lower extracts
(47)
concentrations……………………………………………………
Table
5:
Preliminary
phytochemical
screening
of
roots
of
Maerua
(53)
Table 6: GC/MS analysis of the hexane extract of Maerua pseudopetalosa
(55)
pseudopetalosa……………………………………………………………
roots……………………………………………………………………….
Table 7: Antibacterial activity of of fractions of CHCl3 extract from Maerua
roots………………………………………………………………….
viii
(60)
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CHAPTER ONE
Introduction and literature review
1
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Introduction and literature review
1.1 General introduction
Medicines form the second most essential need for mankind after water and
food. Plants have been used for medicinal purpose from the dawn of history
and for many centuries. The important vitality and marvel of plants have been
appreciated by man since ancient times and their medicinal significance has
been documented as early as 77 AD by Discorides who wrote the first Materia
medica in five volumes. Also, in the tradition and Hadith of Prophet
Mohammed (may God’s peace and blessing be upon him), the use of herbs
and plant was highly recommended as herbal drugs to cure different ailments
and to keep the health and fitness, even today plants are used by 80 % of the
world population as the only available source of medicine especially in
developing countries. In these countries people rely heavily on traditional
medicine and medicinal plants to meet primary health care needs. There are
considerable economic benefits in the development of indigenous medicines
and in the use of medicinal plants for the treatment of various diseases (Tyler,
1999).
In the last few decades, the study of medicinal plants and their traditional use
in different parts of the World has increased (Lev, 2006). Hundreds of plants
have been used as herbal remedies in indigenous medicine systems (Hussain
et al., 2008). While herbal medicines are assumed to be of great importance in
the primary healthcare of individuals in rural communities (Sheldon et al.,
1997; Tene et al., 2007), plant-based traditional knowledge coupled with the
high cost involved in the development of patentable chemicals and drugs
(Hack, 2006) are recognized as essential tools in search for new sources of
drugs and neutraceuticals (Sharma and Mujundar, 2003). Thus, antimicrobial
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activity of crude and semi-purified extracts of many plants has been widely
reported (Cos et al., 2002; Muschietti et al., 2005; Wannissorn et al., 2005;
Olajuyigbe et al., 2011). The increasing use of traditional therapies which the
laypeople considered as a part of their heritage now requires more
scientifically sound evidence for the principles behind plants’ therapeutic
effectiveness in complementary and alternative medicines (Patwardhan et al.,
2005).
The unique geographical position of Sudan and its interaction with different
cultures have undoubtedly left its influence on different aspects of the
Sudanese traditional medicine. Sudan has witnessed the fusion of Pharonic,
Christian and Islamic cultures with the local indigenous cultures. This ethnic
and cultural diversity led the country becoming a melting pot of African
cultures with respect to herbal medicine. The diversity is largely attributed to
immigration from the rest of the continent, in particular from the west. The
country is well-positioned to play a leading role in the area of medicinal
plants by virtue of its climate that varies from arid desert in the North to
tropical in the South. With this unique history and varied climate, terrain,
flora and fauna, the people of Sudan have developed their own traditional
medical culture. Medicinal and aromatic plants are not only used to meet
healthcare needs but also for cosmetics and perfumery purposes. Traditional
medicine is both popular and important as a medical system has been
integrated into the national healthcare schemes. There is vast experience in
the use of herbs in medical treatments. Many families specialize in herbal
medicines and this knowledge is conveyed from generation to generation.
Patients travel from urban to rural areas to consult herbalists, especially for
chronic diseases.
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There are more than 3,132 vascular plant species in Sudan. The Sudan Atlas
of Medicinal Plants has records of more than 2,000 medicinal plants collected
from different parts of the country. Several native plant species are in use in
traditional medicine. Moreover, the Medicinal and Aromatic Plants Research
Institute has trained a considerable number of specialists in the various fields
required for research in medicinal plants. Legislation is in force for the
registration of herbal preparations and herbal products (MAPRI, 1997).
1.2 Antimicrobial infection
Modren drugs used for antimicrobial chemotherapy affect characteristic
features of procaryotic cells that are not found in the eukaryotic cells of
human.
The recognition of bacteria as the cause of fever and infection was soon
followed by the search for substances that could destroy them. Chemicals
such as carbollic acid and iodine, known to kill bacteria cultures in the
laboratory. Other chemicals that destroy bacteria (e.g. Mercury) were used to
treat infections but invariably cause as much harm to the patient as to the
microbe. Ehrlich (a microbiologist) first perceived that what was required was
an agent that was selectively toxic to microbes. In 1904, he succeeded in
curing trypanosomiasis (sleeping sickness) with a dye called trypan. He
continued his work using a variety of compounds based on arsenic. Then, in
1935, Domagk found that streptococcal infections could be treated with a dye
called prontosil. This chemical was actually broken down in the body to form
the
effective
compounds,
sulphonilamides.
Other
antimicrobial
sulphonilamides were developed and widely used in the treatment of
infections. The ability to treat infections was revolutionized by the discovery
by Sir Alexander Fleming of the first naturally occurring antimicrobial
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substance. In 1928, Fleming noticed that colonies of Staphylococcus aureus
were “dissolved” where they occurred close to mould, Penicillium notatum,
which had inadvertently contaminated the plate. He then grew the same
fungus in a broth and found that the broth had marked inhibitory effect on
many types of bacteria. The antibacterial substances were difficult to purify
and were unstable, but after extensive work by Florey in the 1940s sufficient
penicillin could be made for the treatment of patients. Commercial production
of penicillin began during the Second World War and the search continued for
new antimicrobial agents from a range of micro-organisms living in natural
environments. In the 1940s streptomycin, chloramphenicol and tetracycline
were isolated from soil organisms, and cephalosporin from a fungus found in
sewage outlet. Erythromycin and rifampicin were discovered in the 1950s,
and gentamicin and fucidin in 1960s, all from soil organisms.
The term antibiotic was used to describe naturally occurring substances
produced by one microbial species and capable of inhibiting the growth of
another species.
In the laboratory, small alterations to the chemical structure of naturally
occurring antibiotics were found to alter the range of microbes against which
the drug was effective, the absorption by the body and the duration of action.
There are over 100 antimicrobial drugs available. Many are similar
compounds but, with minor modifications, affect different species of bacteria
(Cheesbrough, 2000). Some people use the term antibiotic to refer only to
naturally occurring drugs made by bacteria or fungi, and the term
antimicrobial agent to describe the whole range of antibacterial drugs now
available, many of which are modifications of naturally occurring substances
or are synthesized in the laboratory. Such a distinction is rather academic.
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Viruses are not affected by antibiotics, as they do not have the cell structures
targeted by these drugs (Wilson and breedon, 1990).
1.2.1 Main-groups of antimicrobial agents
1.2.1.1 Penicillins
The penicillins bind an enzyme involved in the productions of peptidoglycan
and as a result, prevent cell wall synthesis, they are bactericidal. The original
naturally occurring pencillins have a narrow spectrum of activity and are
mainly active against Gram positive bacteria. However, a large number of
different bencillin antibiotics have been synthesized, which have the same
basic ß- lactam ring structure but, by modification of the side chain,
Molecules, have a wider range of bacteria species. Penicillins are very useful
antibiotics, they can infiltrate most sites of infection and they are usually used
for initial treatment of infection until sensitivity testing indicates that an
alternative drug is necessary (Cheesbrough, 2000).
1.2.1.2 Cephalosporins
The Cephalosporins have a similar chemical structure to the penicillins. They
have a ß-lactam ring and additional side ring and different side chains. They
also kill bacterial cells by preventing cell wall synthesis. The first
cephalosporin, cephalothin, was obtained from a fungus in the mid 1960s.
This first generation of cephalosporin, had a similar spectrum to ampicillin.
Susceptibility to ß- lactamases limited their use until the second generation of
ß- lactamase resistant cephalosporins was introduced in 1970s. These drugs
are more potent and are active against wider range of bactria (Plumb, 1999).
1.2.1.3 Other ß-lactams
There are a variety of antibiotics that have a ß-lactam ring as part of their
structure. One group, the monobactams (e.g. imipenem) are particularly
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useful as they are very potent and effective against a range of Gram-positive
and Gram-negative bacteria. They also do not cause hypersensitivity reactions
in patients who are sensitive to penicillin. They have very broad spectrum,
active against most bactria including anaerobes, and are used to treat serious
infections caused by resistant bacteria (Plumb, 1999).
1.2.1.4 Amino glycosides
The amino glycoside antibiotics interfere with protein synthesis by binding to
bacterial ribosomes and preventing accurate reading of the messenger RNA.
They are bactericidal and active against many Grams negative, aerobic
bacteria and some Gram positive bacteria. They are often used in combination
with another antibiotic to provide activity against a broad range of organisms
(Wilson and Breedon, 1990).
1.2.1.5 Tetracyclines
The name tetracyclines is derived from their structure of four rings fused
together. They bind to bacterial ribosomes and block protein synthesis by
preventing transfer RNA from attaching to messenger RNA. The attachment
to the ribosomes is reversible and their effect is therefore bacteriostatic rather
than bactericidal. Within the group there are natural products such as
oxytetracycline, and semi-synthetic derivatives such as methacycline and
minocycline (Plumb, 1999).
1.1.1.6 Macrolides
These drugs inhibit bacterial protein synthesis by binding to the ribosome.
They are bacteriostatic as the attachment to the ribosome is reversible. The
first group of these drugs, erythromycin which was isolated from
Streptomyces spp .
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The naturally occurring macrolides are active against most Gram positive
organisms, neisseria, haemophilus and a range of anaerobes. They are also
effective against intracellular pathogens such as chlamydia and rickettsia.
Their activity against a number of emergent pathogens such as toxoplasma,
legionella and helicobacter has recently stimulated interest in the group, and a
range of synthetic macrolides is now being developed (e.g. clarithromycin)
(Wilson and Breedon, 1990).
1.2.1.7 Quinolones
These drugs are bactericidal. They inhibit the enzyme responsible for
supercoiling microbial DNA molecules. They are synthetic antibiotics, first
used in 1962 when naladixic acid was introduced. This drug is active against a
wide range of Gram negative bacteria, except Pseudomonas, and is used to
treat urinary tract infections, although resistance often develops during
treatment.
The addition of fluorine molecule into the compound was found to increase
both its potency and spectrum of activity and a new range of compounds, the
fluoroquinolones, was subsequently produced (e.g. ciprofloxacin, norfloxacin,
ofloxacin). These drugs are effective against Pseudomonas, Staphylococci
(including methicillin resistant strains) and intracellular pathogens such as
Chlamydia and Mycobacteria (Brown and Reeves, 1997).
1.2.1.8 Glycopeptides
These compounds were originally obtained from actinomyces found in soil.
Glycopeptides interfere with the synthesis of peptidoglycan, inhibiting the
formation of bacterial cell walls. Glycopeptides are active against Gram
positive organisms, in particular Staphylococcai (Wilson and Breedon, 1990).
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1.2.1..9 Other antimicrobial agents
Chloramphenicol.
Sulphonamides.
Trimethoprim.
Metronidazole.
Clindamycin.
Fusidic acid.
Rifampicin.
1.2.1.10 Antifungal therapy
Fungi may cause a variety of infections ranging from superficial infections of
the skin and mucous membranes to serious systemic infection which may be
fatal and are usually associated with immune suppression. The eucaryotic
cells of fungi are not susceptible to antibiotics, and specific antifungal agents
are required to treat the infections they cause (Table 1). Some of these agents
disrupt the cell membranes by altering their sterol content (e.g. amphotericin),
others affect cell formation (e.g. griseofulvin), disrupt protein synthesis by
substituting a nucleic acid (e.g. flucytosine), or interfere with cell wall
synthesis (e.g. imidazoles).
Table 1: Major antifungal agent.
Antifungal agent
Indication
Polyenes nystatin amphotericin B Superficial candidiasis, broad antifungal
spectrum, main form of treatment for
mycoses and nephrotoxic.
Flucytosine
Active against yeasts, used to treat
systemic infections.
Griseofulvin
Used for treatment of ringworm.
Allylamine terbinafine
New antifungal, active against
Candida and Aspergillus.
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1.2.2 Resistance to Antimicrobial Agent
Micro-organisms are not all intrinsically sensitive to all antibiotics. The terms
sensitive and resistant to antibiotics are used to distinguish between those
antibiotics that will or will not destroy a microorganism. On a simple level
bacteria can be described as sensitive to particular antibiotic if their growth is
inhibited or they are killed by concentration of the drug that could be
achieved by the usual dose regimen.
However in practice, it is not always possible to make clear distinctions
between sensitive and resistant strains, treatment with a particular antibiotic
may still be effective if given at higher dose. Determining the minimum
inhibitory concentration (MIC) of the antibiotic will assess the sensitivity of
particular Microorganism. If the MIC is high, the organism is resistant and
unlikely to be affected by treatment with the antibiotic, if it is low, then
treatment is likely to be effective provided the antibiotic is able to penetrate
the site of infection.
Bacteria may have natural resistance to certain antibiotics because the drug
cannot penetrate their cells or because they do not possess the portion to wish
the drug attaches. Some bacteria are naturally resistant to many antibiotics.
Resistance by selection occurs when one or two cells in population of bacteria
are naturally resistant to the antibiotic. On exposure to antibiotic these cells
are able to survive and multiply, eventually the sensitive cells are replaced by
resistant ones. This type of resistance develops rapidly when sulphonamides
are used and is also a problem with antituberculous drugs.
Acquired resistance to antibiotics has been evident since antibiotics were first
widely used, it may occur as a result of a mutation in the chromosome or the
acquisition of new DNA. Mutational resistance usually involves the
substitution of one or more amino acids in a target protein; for example,
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rifampicin resistance in Mycobacterium tuberculosis is due to a mutation in
RNA polymerase. This type of resistance may be induced during therapy.
The transfer of antimicrobial resistance frequently occurs on plasmids, small
molecules of DNA independent of the chromosome, which can be replicated
and transferred between cells by conjugation, transformation or transduction.
Plasmids provide a highly effective means of spreading resistance genes, both
within species and to another species.
Resistance genes may also be carried out on transposons. These are specific
sequences of DNA that can insert into both plasmids and chromosomes and
transfer or jump between them, they can carry genes encoding for resistance
to wide variety of antibiotics and play a key role in the dissemination of
antibiotic resistance, particularly where species develop resistance to more
than one drug. Multiple drug resistance is a term used to describe bacteria that
have developed resistance to several, unrelated antibiotics, for example Gram
negative bacilli that are resistant to both streptomycin and sulphoramides.
These multiresisant bacteria often have one plasmid or transposon that carries
several genes conferring resistance to several antibiotics.
The ability of microbes to acquire resistance to antibiotics was recognized
soon after the first drugs were introduced in the 1940. Initially, the steady
supply of new drugs was able to combat the problem. The cost of
development and control one usage to limit resistance affect the economic
viability of producing new antimicrobial drugs (Neu, 1992).
The development of vaccines has provided some solution for example the
vaccination of children against haemphilus influenza has helped to resolve the
problem of emerging resistance in this pathogen, however, vaccines are
unlikely to help combat resistances amongst commensal organisms such as
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enterococci, There is an increasing interest in bacteriophages, viruses that
destroy specific bacteria, although their use is associated with practical
problems such as selecting an appropriate phage and delivery to the site of
infection (Barrow and Soothill, 1997) .
1.2.3 Factors contributing to the emergence of resistance
Although much of the evidence is circumstantial, there are a number of
factors considered to play a key role in the development of antimicrobial
resistance;
-Unnecessary use of antimicrobial agent to treat trivial infections caused by
viruses.
-Use of antimicrobial agents as growth promoters or prophylactics in
agriculture.
- Uncontrolled sale of antimicrobial agent without prescription.
- Inappropriate use of antimicrobial agents (e.g. incorrect agent, dose,
duration).
1.2.4 Examples of some pathogenic bacteria
• Staphylococcus aureus
Staphylococcus aureus is a facultatively anaerobic, Gram-positive coccus. It
is a spherical bacterium, frequently part of the skin flora found in the nose and
on skin. S. aureus can cause a range of illnesses from minor skin infections to
life-threatening diseases such as pneumonia, meningitis, osteomyelitis,
endocarditis, toxic shock syndrome, and septicemia. Its incidence is from
skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It
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is still one of the most common causes of nosocomial infections, often
causing postsurgical wound infections.
• Bacillus subtilis
Bacillus subtilis is a Gram-positive, catalase-positive bacterium commonly
found in soil. B. subtilis is rod-shaped, and has the ability to form a tough,
protective
endospore,
allowing
the
organism
to
tolerate
extreme
environmental conditions. B. subtilis is not considered as human pathogen; it
may contaminate food but rarely causes food poisoning (Joseph and Palmer,
1989).
• Salmonella typhi
Salmonella typhi are an enterobacteria, strictly human pathogens. Gram–
negative, facultative aerobic rod with flagellate motility. They cause enstric
fever (typhoid and paratyphoid) (Joseph and Palmer, 1989).
• Escherichia coli
Escherichia coli is a Gram negative bacterium, facultative anaerobic and nonsporulating, that is one of several types of bacteria that normally inhabit the
intestine of humans and animals (commensal organism). Most E. coli strains
are harmless, virulent strains of E. coli can cause gastroenteritis, urinary tract
infections and neonatal meningitis (David, 2001).
• Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium
with unipolar motility. An opportunistic human pathogen. It uses a wide range
of organic material for food; in animals, the versatility enables the organism
to infect damaged tissues or people with reduced immunity. The symptoms of
such infections are generalised inflammation and sepsis. If such colonisations
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occur in critical body organs such as the lungs, the urinary tract, and kidneys,
the results can be fatal.
1.3 Protozoan Diseases
Diarrhea is a very common illness, especially in the developing world, and is
frequently experienced by travelers. Cryptosporidium, Cyclospora, Isospora,
Giardia, amoeba, and Sarcocystis are pathogenic protozoan parasites that can
cause these gastrointestinal illnesses. Commensal parasites are also relatively
common in developing countries and less frequently identified in the
developed world. Worldwide, Giardia is the most common protozoan
infection in the gastrointestinal tract of humans. It was probably first seen by
Anton van Leuwenhoek in the late seventeenth century. In Tennessee,
Giardia cysts have been identified in human feces from about 600 BC.
Cryptosporidium and Giardia have also been reported from samples 500 to
3000 years old from the Andean regions in Peru, and from 4300- to 1100year-old samples from the coastal regions of Peru. Cryptosporidium became
much more relevant to public health in the early 1980s with the emergence of
the AIDS epidemic. Opportunistic and emerging parasitic infections also
include Isospora, particularly in HIV and AIDS patients. Cyclospora has been
observed in certain regions of the developing world; however, globalization
of the food supply and increase in international travel has revealed that
parasitic infections can also cause epidemics in the developed world.
1.3.1 Flagellated Protozoa
1.3.1.1 Giardia
Giardia intestinalis (also known as G. lamblia) is a flagellated protozoan that
inhabits the small intestine of man and other animals, including monkeys,
rodents, dogs, cats, horses, goats, cattle, birds, reptiles, and fish (Newman et
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al., 2001). Like many other protozoa, G. intestinalis has a trophozoite and a
cyst stage. The trophozoite is oblong, pear-, or kidney-shaped, rounded
anteriorly, and pointed posteriorly. The trophozoite is flattened laterally,
being convex dorsally and concave ventrally; much of the ventral surface
comprises the sucking disk that the organism uses to attach firmly to the
intestinal mucosa. The trophozoite is microscopic in size, averaging 10 to 20
µm long by 5 to 15 µm in breadth; a prominent pair of nuclei on each side of
the organism near the anterior end gives a facelike appearance. There are four
pairs of flagella, one pair arising near the anterior and posterior end,
respectively, and two pairs arising near mid-body. Rapid movement of the
flagella allows the trophozoite to move from place to place. Trophozoites
divide by a complicated process of longitudinal binary fission that results in
two daughter trophozoites. Transmission from one host to another is
accomplished by viable cysts (Fig. 1). As trophozoites transit down the colon,
they prepare for encystation by retracting their flagella. The cytoplasm
becomes condensed, and a thin, tough hyaline membrane (cyst wall) is
secreted. The cysts are oval in shape and measure 8 to 12 µm in length by 7 to
10 µm in breadth. Mature cysts have four nuclei located at one end of the
cyst. As the cyst matures, internal structures and the sucking disk are doubled.
When excystation occurs in a new host, division results in two identical
trophozoites, which grow flagella and initiate infection.
Diagnosis of infection is typically by microscopic detection of cysts in freshly
collected stool (or trophozoites in diarrheic stools). Organisms can
occasionally be seen in direct exams, but a concentration procedure is
recommended. Because of their distinctive shape, appearance of the nuclei,
and other features, the diagnosis can often be made on wet, unstained
samples. However, staining may enhance detection and confirmation of
infection. In addition to direct or stained specimens, commercial direct
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fluorescent antibody (DFA) assays are available and often used as the gold
standard for diagnosis. Enzyme-linked immunosorbent assay (ELISA)
formatted tests are commercially available and are extremely useful for
screening large numbers of samples.
Infection with Giardia in an appreciable number of cases results in irritation
of the duodenum with excess secretion of mucus and dehydration,
accompanied by epigastric pain, flatulence, and chronic diarrhea with
steatorrheic-type stool containing a large amount of mucus and fat but
typically with no blood. It is recognized that giardiasis can cause stunting and
interference with growth, particularly in children in developing countries
where repeated infections are the norm.
Metronidazole or tinidazole is the recommended drug of choice for treating
giardiasis. Nitazoxanide, furazolidone, and paromomycin are alternatives.
Paromomycin is not absorbed from the gastrointestinal tract and is often used
during pregnancy but it is less efficacious than the other agents.
Transmission of Giardia is by viable cysts that are swallowed (Fig.1).
Contaminated food and water are the most common source of exposure
although intimate contact with an infected individual may represent a
common mechanism. Giardiasis is typically more common in children than in
adults, especially in a crowded setting such as day care centers. However, in
the United States and other developed countries, outbreaks of giardiasis are
also observed in adults. These are often linked to contaminated food or drink,
or are associated with recreational water venues. Cats and dogs are also
recognized to harbor Giardia. Despite the morphologic similarity of the
organisms infecting humans and animals, molecular analysis has shown
distinct clades or assemblages that seem to suggest some degree of host
specificity, with certain assemblages being more restricted in their host
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preference than others. Further differences in virulence between isolates have
also been proposed, but evidence to date has been inconsistent. Giardia cysts
have a relatively high resistance to routine water treatment procedures,
including chlorination, which has led to numerous waterborne outbreaks.
Surface water can be widely contaminated, and as a result, giardiasis is one of
the most common intestinal parasitic infections. This implies that to provide
potable water, surface water should be treated by flocculation, sedimentation,
filtration, and finally, chlorination. Use of chlorine alone at levels normally
used in municipal treatment facilities does not rapidly inactivate cysts,
especially at lower water temperatures, so other measures must be in place.
Purifying water for use when camping or traveling overseas can include
boiling, filtration through filters with pore size of less than 1 µm, or treatment
with chlorine or iodine preparations (some recommend iodine preparations to
be more effective than chlorine preparations).
Giardiasis can occur year-round in all settings, temperate as well as tropical.
However, there is strong evidence that some seasonality occurs in temperate
regions with increased incidence in the summer months, peaking in early fall
(Hill and Nash, 2006).
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(www.dpd.cdc.gov/DPDx/HTML/ImageLibrary/Giardiasis_il.htm).
Fig.1: Life cycle of Giardia
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1.4 Plant under study
Maerua pseudopetalos Gilg & Bend
Kingdom: Planate – plant
Subkingdom: Tracheobionate – vascular
Super division: Spermatophyte – seed plants
Division: Mangoliophyta –flowering plants
Class: Magnoliopsida - dicotyledons
Subclass: Dilleniidae
Order: Capparales
Family: Capparaceaeae
Genus: Maerua
Species: pseudopetalosa
Synonyms: Courbonia virgata Brongn; C. pseudopetalosa Gilg. & Bened.
Vernacular names: (Ar) Kordala, Karkadan, Kurdan.
1.4.1 Botanical description:
Glabrous shrub up to 6 m high with tuberous roots and twiggy leafy
branches. Leaves narrowly elliptic or anceolate, 12-5 x 3-13 mm, coriaceous;
petioles 0.5-1.5 mm long. Inflorescence axillary, solitary in the upper leafaxils; pedicals 8-20 mm long; sepals 4, elliptic, 10-12 mm long; petals absent;
stamens many, 2-2.5 cm long. Fruit globose or ovoid, cylindrical, 2 cm
across, 1-seeded, yellow turning dark brown, beaked (Elamin, 1990) (Fig. 2).
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Leaves
Flower
Fig. 2: Maerua pseudopetalos leaves and flower.
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1.4.2 Distribution:
Widespread, occurring from Senegal to Nigeria and to Sudan, Ethiopia,
Somalia and South wards to Uganda (http:// www.aluka.org / action / show
metadata).
Distribution in Sudan:
In deciduous bush land and grassland in the North (Berber), Red Sea hills,
Kordofan and Darfour. (Elamin, 1990).
1.4.3 Uses:
The fruit is eaten in Sudan to make one strong. The root, when chewed, is at
first bitter, then sweetness follows, and Sudanese use the root to make sweet
drinks and as milk substitutes.
The root is said to be an efficient precipitant of suspensions in water and is
used in Sudan as water-purifiers and storage in rural areas. The roots are
chopped up and thrown into the water.
Fruit and roots are used in topical application to the chest for cough and other
pulmonary troubles and for treatment of rheumatic pain and skin diseases.
1.4.4 Toxicity:
The tuberous root has been found to contain toxic principle. An amount of the
root containing about 0.25 gm of tetramine has proved fetal within an hour
when swallowed by human beings. The subcutaneous lethal dose, in mouse is
0.5 to 1 mg /25 g/ body weight the symptoms being convulsive spasm,
collapse, and death within 30 min (Watt and Breyer-Brandwijk, 1962).
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1.4.5 Previous work on Maerua
Literature survey revealed that no phytochemical work or estimation of the
biological activity M. pseudopetalos was carried out. However, the antiinflammatory activities of aqueous methanolic extract of M. angolensis stem
bark were evaluated by Adamu et al. (2007), using carrageenan – induced
hind paw oedema and cotton pellet granuloma models in rats. The aqueous
methanolic extract dose-dependently inhibited carrageenan-induced oedema
in rats. In the granuloma pouch, the extract exhibited a 52.25% reduction in
granuloma weight at the dose of 500 mg/kg. These activities were comparable
to that of diclofenac sodium (5 mg/kg), the standard agent used in the study.
The oral median lethal dose (LD50) value of the extract in rats, found to be
greater than 5000 mg/kg, suggests that it is non-toxic at the anti-inflammatory
doses used in the study.
Mavura et al. (2008) attempted to explain the mechanism of sediments
settling as aided by M. subcordata juice. The study included: chemical
composition of the juice such as mineral contents, protein and polysaccharide
contents. These tests were carried out both in the juice extracts and in the
“flocs”. While there were no significant amounts of minerals found in the
juice or in the flocs, there were significant amounts of polysaccharides in the
juice as well as in the flocs. However, it was found that there were
insignificant amounts of proteins in the settled flocs. This was indication that
polysaccharides in the juice were precipitated along with the sediments
present in water. Further biochemical experiments on the identity of
polysaccharides which apparently aided flocculation indicated that they were
of branched type, amylopectin.
Eight compounds have been isolated from M. arenaria namely, ß-sitosterol,
ursolic acid, 4-hydroxybenzoic acid, methyl grevillate, glycerol 1,321
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didodecanoate,
1-? -coumaroylglycerol
and
ß-sitosterol
3-? -
ß-D-
glucopyranoside (Ali et al., 2008).
Moreover, The hexane fraction obtained from the total alcohol extract of the
aerial parts of M. crassifolia Forssk grown in Egypt upon repeated column
chromatography afforded one new compound identified as 1, 23 dimethoxy
tricosa-6-one, along with six known compounds identified as long chain
hydrocarbon (triacontane), ceryl alcohol, lupeol palmitate, ß-sitosterol
palmitate, lupeol acetate, and a-amyrin (Ibraheim et al., 2008).
1.5 Research problem
A large sector of the Sudanese population use traditional medicine to meet
their primary healthcare needs. In addition to being accessible and affordable,
it is part of their belief systems. Often, traditional medicine provides the only
available healthcare service to the population in many parts of the country.
The research problem of the present work was to determine the antimicrobial
potential of M. pseudopetalos. This selection was guided in the first place by
ethnobotanical claim in traditional medicine suggestive of their antimicrobial
activity that associated with coughs and other pulmonary troubles as well as
skin diseases and secondly by lack of information in literature on their
chemical constituents and biological activity.
1.6 Research hypothesis
The roots of M. pseudopetalos possess antibacterial activity and other
interesting activity like antigiardial activity.
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1.7 Objective of the study
The present study was conducted to:
§ Investigate in vitro antibacterial activity of crude extracts of roots of M.
pseudopetalos.
§ Investigate in vitro antigiardial activity of crude extracts of roots.
§ Determine the secondary metabolites present in the roots.
§ Select roots extracts with higher activity for more detailed
phytochemical and antibacterial activity tests.
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Matrials and methods
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Materials and Methods
2.1 Collection and preparation of plant material
Roots of Maerua pseudopetalosa were collected in September 2009 from
Western Kordofan. Voucher specimen was deposited at the Herbarium of
Botany Department, University of Khartoum. Roots were cut into small
pieces, dried under shade and ground into a soft powder with a grinding mill.
The powdered roots (1 kg) were stored in glass storage containers in the dark
in the research laboratory.
Chemicals:
All chemicals used were of Analar grade.
2.2 Extraction
The dried powder (500 g) was extracted sequentially using hexane (3 X 3 L),
chloroform (CHCl3) (3 X 3 L), ethyl acetate (EtOAc) (3 X 3 L) and methanol
(MeOH) (3 X 3 L). Extracts were filtered, concentrated under reduced
pressure, weighed and kept in a desicator.
2.3 Antimicrobial activity
2.3.1 Test organisms
The bacteria used were of the American type culture collection (ATCC). They
were obtained from the stock culture of National Sanitary Laboratory and
Microbiology Laboratory, Department of Botany, Faculty of Science,
University of Khartoum. Strains maintained for tests were Escherichia coli
ATCC25922, Staphylococcus aureus ATCC25923, Pseudomonas aeruginosa
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ATCC27853, Bacillus subtilis (NCTC8236) and Salmonella typhi ATCC
6539.
2.3.2 Disc diffusion method
The microbial growth inhibitory potential of the extracts was determined by
using the agar disc diffusion method as described by Mbavenge et al. (2008).
Muller Hinton agar (38 g) was suspended in one litre of distilled water, heated
on a boiling water bath until dissolved and then sterilized by autoclaving at
121° C for 15 minutes.
The test organism was maintained by bi-weekly transfer on agar slants of
nutrient agar medium. Growth was washed from slants with sterilized 3 mL of
normal saline. This suspension was used to inoculate in wide based flask
containing 200 mL of the same medium supplemented with 10 g of agar per
Litre. The flask was incubated for 24 hours at 37° C. Growth was harvested
by washing with 15 mL of normal sterilized saline. Usually 0.08 to 0.1 mL of
the concentrated suspension was used to inoculate 100 mL of agar medium.
A loopful of isolated colonies was inoculated into 4 mL peptone water and
incubated at 37 °C for 4 h. The turbidity of actively growing bacterial
suspension was adjusted to match the turbidity standard of 0.5 McFarland
units prepared by mixing 0.5 mL of 1.75% (w/v) barium chloride dehydrate
with 99.5 ml L% (v/v) sulphuric acid. This turbidity was equivalent to 106 –
108 colony forming units per millilitre (CFU/mL) (McFarland, 1907). 100
microlitres of inocula of all tested microorganisms were inoculated on Muller
Hinton Agar medium for bacteria. The medium solution was placed into Petri
dishes (17 mL/dish). Then, the agar plates were kept in refrigerator for
overnight. Whaterman filter paper discs of 6 mm diameter were impregnated
with 10 µL of the solution of crude extract (equivalent to 20, 10, 5 and 1
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mg/mL and 500, 250 and 125 µg/mL of the dried extract) or fraction (at 5
mg/mL) dissolved in dimethyl sulfoxzide (DMSO). The paper discs were
dried and placed on the surface of the inoculated agar plates. Standard disc of
ampicilin (10 µg/disc) was used as positive control, while DMSO was used as
a negative control. The Petri dishes were inverted and incubated for 24 hours
at 37º C. Three replicates of the test extracts were made. Clear inhibition
zones around the discs indicated the presence of antimicrobial activity.
2.4 In vitro antigiardial activity
2.4.1 Parasite isolate
Gairdia lamblia used in all experiments were taken from patients of Ibrahim
Malik Hospital (Khartoum). All positive samples were examined by wet
mount preparation. Then the positive sample was transported to the laboratory
in nutrient broth medium. Trophozoites of G lamblia were maintained in
RPMI 1640 medium containing 5% bovine serum at 37 ± 1°C. The
trophozoites were maintained for the assays and were employed in the log
phase of growth.
2.4.2 In vitro susceptibility assays
In vitro susceptibility assay using the sub- culture method of Cedilla et al.
(2002) was adopted, This method being described as highly stringent and
sensitive for assessing the anti-protozoal effects (gold standard) particularly in
Entamoba histolytica, Gairdia intestinalis and Trichomonas vaginalis
(Arguello et al., 2004). Five mg from each extract was dissolved in 50 µL of
(DMSO) at Eppendorf tube containing 950 µL distilled water in order to reach
concentration of 5 mg/mL (5000 ppm). The concentrates were stored at -20°C
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for further analysis. Sterile 96-well microtite plate was used for different plant
extracts, positive control and negative control.
Three out of 8 columns of microtitre plate wells (8 columns × 12 rows) were
chosen for each extract, 40 µL of an extract solution (5 mg/mL) were added to
the first column wells C-1: On the other hand, 20 µL of complete RPMI
medium were added to the other wells the second column and third column (
C-2 and C-3). Serial dilutions of the extract were obtained by taking 20 µL of
extract to the second column wells and taking 20 µL out of the complete
solution in C-2 wells to C-3 wells and discarding 20 µL from the total
solution of C-3 to the remaining 20 µL serial solutions in the successive
columns. 80 µL of culture medium was complemented with parasite and
added to all wells. The final volume in the wells was 100 µL.
In each test metronidazole (a trichomonocide) pure compound [(1-(2hydroxyethl)-2-methyl-5 nitroimidazole], was used as a positive control in
concentration 312.5 µg/mL, whereas untreated cells were used as a negative
controls (culture medium plus trophozoites). For counting, the samples were
mixed with Trypan blue in equal volume. The final number of parasites was
determined with haemocytometer three times for counting after 0, 24, 48 and
72 h. The mortality % of parasite for each extracts activity was carried out
according to the following formula:
Mortality of parasite (%) = (Control negative – tested sample) × 100
Control negative
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2.5 Phytochemistry
2.5.1 Phytochemical screening
2.5.1.1 Preparation of extracts
Two types of extracts were prepared; for the MeOH extract the powdered root
(25 g) was extracted with (200 mL) of MeOH with stirring at an interval of 4
h for 24 h. After filtration, the solvent was evaporated under reduced pressure
in a rotatory evaporator at 45 °C to afford the MeOH crude extract.
For the aqueous extract the powdered root (25 g) was extracted with (200 mL)
of distilled water with stirring at an interval of 4 h for 24 h. After filtration,
the water extract was freeze dried.
The root extracts (both MeOH and aqueous extracts) were subjected to
phytochemical screening for the identification of major groups of chemical
constituents using standard procedures (Harborne (1973), Trease and Evans
(1989)). The phytochemical components analysed were alkaloids, saponins,
flavonoids, tannins, anthraquinones and cardic glycosides.
2.5.1.2 Phytochemical analysis
• Test for flavonoids
Plant sample (0.5 g) was suspended in 5 mL of water and 2.5 mL of methanol
added to it. After filtration 1 mL of NaOH 10% was added to 1 mL of the
filtrate. The appearance of a yellow precipitate indicated the presence of
flavonoids.
• Test for tannins
Water (7.5 mL) was added to plant extract (1 g) and heated in a water bath. It
was then filtered upon cooling. Few drops of iron III chloride (FeCl3) 0.5%
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were added to 2 mL of the filtrate. The appearance of a green or dark-blue
precipitate indicated the presence of tannins.
• Test for alkaloids
Sample (2 g) was heated in a test tube containing 25 mL of HCl (1%) for 15
min in a boiling water bath. The suspension was then filtered and 5 drops of
Meyer's reagent (potassium tetraiodomecurate) were added into the filtrate (1
mL). The formation of a precipitate indicated the presence of alkaloids.
• Test for saponins
A quantity of 0.5 g of extract was introduced into a test tube containing 7.5
mL of distilled water and the mixture heated for 5 min in a boiling water bath.
The solution was then filtered and cooled to room temperature. Five milliliters
of the filtrate was introduced into a test tube and agitated for 10 seconds. The
formation of persistent foam indicated the presence of saponins.
• Test for triterpenes and steroids (the Lieberman–Burchard)
Sample (0.5 g) was dissolved in chloroform (3 mL) and a few drops of acetic
anhydride and concentrated H2SO4 were added. A purple coloration indicated
the presence of triterpenes while bluish-green coloration indicated the
presence of steroids. The formation of two layers upon addition of H2SO4 is
characteristic of the presence of both triterpenes and steroids.
• Test for cardiac glycosides (Keller–Killani test)
The sample (0.2 g) was suspended in 5 mL of water in a test tube and treated
with 2 mL of glacial acetic acid containing one drop of FeCl3 solution. Then 1
mL of concentrated H2SO4 was added gradually along the wall of the test
tube. The formation of a brown ring at the interface indicated the presence of
deoxysugars, characteristics of cardenolides.
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• Test for anthraquinones
The sample (0.5 g) was boiled with 1 mL of 10% H2SO4 and filtered. 2.5 mL
of benzene was added to the filtrate and shaken. The benzene layer was
transferred with a pipette to a test-tube and then 2 mL of 10% ammonia
solution was added. The presence of a pink or red –violet colour in the lower
ammonia phase indicated the presence of anthraquinones.
2.5.2 Chromatographic Techniques
Chromatographic separations were carried out using handmade or
precoated silica gel GF245 TLC plates (Merck). Silica gel (Merck, type 60 &
70-230 mesh) were used for column chromatography,
2.5.2.1 Preparation of thin layer chromatography plates (TLC)
The coating materials were usually applied as aqueous slurries. Slurry was
made by mixing 30 g of silica gel G with 60 mL of distilled water in a motor
until it was of uniform consistency and free of air bubbles. The slurry was
spread using a DESGA spreader at 0.25 mm thick layer on five glass plates
(20 x 20 cm). The coated plates were dried at room temperature, then placed
vertically in an oven and activated by heating to 110 °C for 30 minutes.
2.5.2.2 Column chromatography (CC)
Column chromatography was performed on a glass column packed with
silica gel. Extract was chromatographed after being absorbed onto a small
amount of packing material then applied to the top of column.
2.5.2.3 Solvent systems
Twenty mL solvent always freshly prepared mixtures were introduced
into the tank one hour prior the chromatography. The tanks were lined with
filter paper and were closed tightly by greasing the lid. This was used to
assure the saturation of the atmosphere with the solvent vapors. Samples were
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applied on a starting line about (1-2 cm) from the bottom of the plate, (1-1.5
cm) apart using a tipped dropper.
The chromatographs were developed by the ascending method at room
temperature. The most commonly used solvent systems in the present work
were:
Chloroform: Hexane (8:2 v/v)
Chloroform: Ethylacetate (8:2 v/v)
Ethylacetate: Hexane (8:2 v/v)
Chloroform: Methanol (17:3 v/v)
Hexane: Chloroform (8:2 v/v)
2.5.2.4 Detection of spots on TLC
TLC plates were viewed under UV light at 254 and 366 nm for
fluorescence or quenching spots. Then sprayed with the appropriate reagent.
Rf values were calculated as follows:
Rf value = Distance moved by the solute
Distance moved by the solvent
2.5.2.5 Preparation of the spray reagents
• Vanillin/H 2SO4
Six grams of vanillin mixed in 250 mL ethanol and then 2.5 mL concentrated
sulphuric acid was added.
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• Dragndorff’s reagent:
(A) Bismitintrate (0.6 g) dissolved in 2 mL concentrated HCl. 10 mL of water
was added.
(B) Potassium iodide (6 g) dissolved in 10 mL water.
Solution (A) mixed with (B). Seven mL concentrated HCl were added to the
mixure, then diluted with 400 mL water and was used to detect the presence
of alkaloids.
2.5.2 Analysis of hexane extract
Oily components of the hexane extract were first transformed into their
methyl esters. Methylene chloride (100 µL) and 1 mL 0.5 M NaOH in
methanol were added to oil extract in a test-tube and heated in a water bath at
90 °C for 10 min. The test tubes were removed from the water bath and
allowed to cool before the addition of 1 mL 14% BF3 in methanol. The test
tubes were heated again in a water bath for 90 °C for 10 min, and cooled to
room temperature. One mL of distilled water and 200 µL hexane were added
to the test tubes and then fatty acid methyl esters were extracted by vigorous
shaking for one minute. After centrifugation, the top layer which is the fatty
acid methyl esters was collected and transferred into a sample bottle for
analysis.
2.6 Gas Chromatography\ Mass Spectroscopy analysis (GC/MS)
The sample was analyzed using QP 20 -10 Shimadzu GC-MS equipment
(Japan). Supelco equity 1 column with a film thickness of 30 cm x 0.25
microns was used. The total flow rate was 50 mL/min and column flow rate
was 1.69 ml/min. Ultra high purity Helium was used as the carrier gas with
injector split ratio of 1.0. The ion source and interphase temperatures were
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200 ºC and 260 ºC respectively. The solvent cut time was 2.50 min and
detector gain was 0.30 kv. A Wiley 229 library search was conducted on
major peaks of the sample in order to identify the components of the sample.
The relative percentage of each compound was determined.
2.7 Column chromatography (CC) of chloroform extract
The chloroform crude extract of the roots of M. psedopetalosa was subjected
to CC. Silica gel (60 g) was mixed with hexane and packed to a height of 25
cm in 4.5 cm diameter glass column. Chloroform extract (3.3 g) was
dissolved in small volume of hexane, mixed with 5 g of silica gel, allowed to
dry and loaded on top of the packed column. Initially the column was eluted
with 100% hexane and subsequently, the polarity of the eluting solvent was
sequentially increased with EtOAc and MeOH respectively. Essentially, a
volume of 200 mL of 100% hexane was initially used, followed by increasing
mixtures of hexane: EtOAc and finally the column was eluted with EtOAc:
MeOH mixture. Fractions of 100 mL portions were collected and
concentrated under reduced pressure. Finally, 7 fractions were obtained on
combining the eluates according to their similarity in behaviour on TLC
(Table 2, Fig. 3). Each fraction was transferred to preweighed glass vials to
dry completely at room temperature.
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Table 2: Column chromatography of CHCl3 extract of Maerua
psedopetalosa roots.
Fraction No
Eluent
1-3
Hexane 100%
4
Hexane: EtOAc (9.5:0.5 v/v)
5
(9: 1 v/v)
6-8
(8: 2 v/v)
9
(7: 3 v/v)
10-11
(5: 5 v/v)
12-13
(4: 6 v/v)
14-17
(EtOAc 100% )
18-20
EtOAc: MeOH (9: 1 v/v)
21-22
(1: 1 v/v)
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Fig. 3: A schematic representation of the procedure followed in the
phytochemical analysis of Maerua psedopetalosa roots.
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ResulTs and discussion
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Results and Discussion
3-1 Quantity of extracts
Among all the plant extracts, methanol (1.3%) extract was found to have
maximum extractive yield followed by EtOAc with 0.5%, CHCl3 with 0.33%
and hexane (0.30%) extracting the lowest quantity (Table 3). Harbone (1973)
reported that the less polar solvents (such as chloroform) are particular useful
for the extraction of less polar flavonoid aglycones such as flavanone,
dihydroflavonles, flavones and flavonols, which are highly methylated while
the more polar plant metabolites are generally, isolated from plant materials
by extraction with ethyl acetate, alcohol and water. The colour, texture and
odour of the plant extracts in different solvents were also characterized (Table
1). Most dried extracts were generally appeared brown in colour and vary in
texture from oily (hexane), sticky, resinous (CHCl3 and EtOAc) to waxy in
MeOH extract. The hexane was best organic solvent that retaining the natural
fragrances of the roots in the extract. This may be due to the non-polar
characteristic of fragrant components present in the roots.
Table 3. Extractive value and organoleptic properties of plant extracts
from Maerua pseudopetalosa roots.
Plant extract
Yield (%)
Colour
Texture
Odor
Hexane
0.30
Brown
Oily
Unpleasant
Chloroform
0.33
Dark black
Sticky, resinous
Agreable
Ethyl acetate
0.50
Brownish black
Sticky, resinous
Agreable
Methanol
1.30
Brown
Waxy, thick
Caramel-like
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3-2 Antimicrobial activity
The antimicrobial activity of different extracts obtained from M.
pseudopetalosa roots at concentrations 20, 10 and 5 mg/mL was determined
against Bacillus subtilis, Staphylococccus aureus, Escherichia coli,
Pseudomonas aeruginosa and Salmonella typhi using the disc diffusion
method. The inhibition diameters can be classified into four categories
(Monks et al., 2002); No activity (diameter of inhibition < 7 mm); weak
activity (diameter of inhibition between 7 mm and 10 mm), moderate activity
(diameter of inhibition between 11 mm and 16 mm) and good or higher
activity (diameter of inhibition between > 16 mm). Results are presented in
Figures 4-8.
3-2-1 Antibacterial activity against Bacillus subtilis (Gram +ve)
The CHCl3 extract showed antibacterial activity against B. subtilis at the
concentrations 20 and 10 mg/mL with inhibition zone of 28 and 12 mm
respectively. The EtOAc extract showed moderate antibacterial against B.
subtilis at concentration 20 mg/mL with inhibition zone of 11 mm and weak
activity at concentration of 10 mg/mL with inhibition zone of 9 mm. No
antibacterial activity was observed for the hexane and MeOH extracts. The
results obtained suggested that the antibacterial principals against B. subtilis
were lies mainly at the CHCl3 and EtOAc extracts (Fig. 4).
3-2-2 Antibacterial activity against Staphylococccus aureus (Gram +ve)
High antibacterial activity against S. aureus was obtained at concentration 20
mg/mL for both the hexane and CHCl3 extracts with inhibition zones of 18
and 17 mm respectively. Other concentrations as well as the EtOAc and
40
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MeOH extracts showed more or less the same susceptibility with inhibition
zones ranging from 12 to 15 mm indicative of moderate activity (Fig. 5).
A- Bacillus subtilis
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Fig. 4: Antibacterial activity of Maerua pseudopetalosa roots extracts
against Bacillus subtilis.
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Fig. 5: Antibacterial activity of Maerua pseudopetalosa roots extracts
against Staphylococccus aureus.
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3-2-3 Antibacterial activity against Escherichia coli (Gram – ve)
The hexane extract at all tested concentrations showed potent antibacterial
activity against E. coli with inhibition zones ranged between 25 and 20 mm.
The susceptibility of CHCl3 extract towards E. coli revealed highest
antibacterial activity at concentration 20 mg/mL with an inhibition zone of 45
mm; whereas; at concentration 10 mg/mL it gave moderate activity with an
inhibition zone of 12 mm. No antibacterial activity (inhibition zone 5 mm)
was obtained at 5 mg/mL. However, the EtOAc and MeOH extracts showed
moderate antibacterial activity with inhibition zones ranged from 15 to 11 mm
and weak activity (inhibition zone 10 mm) at 5 mg/mL for the MeOH extract
(Fig. 6).
3-2-3 Antibacterial activity against Pseudomonas aeruginosa (Gram –ve)
Only the hexane extract at concentrations 20 and 10 mg/mL revealed high
antibacterial activity against P. aeruginosa with inhibition zones 19 and 17
mm respectively. Other extracts were either displayed weak activity or not
active at all (Fig. 7).
3-2.4 Antibacterial activity against Salmonella typhi (Gram –ve)
At a concentration 20 mg/mL, the CHCl3 extract displayed very high
antibacterial activity against S. typhi with inhibition zone of 32 mm. The
susceptibility was less for the EtOAc extract at same concentrations
(inhibition zone 16 mm). The MeOH extract displayed moderate antibacterial
activity whereas the hexane extract was inactive at all tested concentrations
(Fig. 8).
42
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C- Escherichia coli
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Fig. 6: Antibacterial activity of Maerua pseudopetalosa roots extracts
against Escherichia coli.
D- Pseudomonas aeruginosa
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Fig. 7: Antibacterial activity of Maerua pseudopetalosa roots extracts
against Pseudomonas aeruginosa.
43
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E- Salmonella typhi
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Fig. 8: Antibacterial activity of Maerua pseudopetalosa roots extracts
against Salmonella typhi.
In this study, The results showed that the response of the bacteria to the tested
extract varied among the strains and are concentration dependent. The
differences in susceptibility may be due to the differences in cell wall
composition and/or genetic content of their plasmids (Karaman et al., 2003).
In addition, the differences between the susceptibility of the Gram positive
and Gram negative bacteria may be attributed to the differences in their cell
wall components and thicknesses (Yao, 1995). However, the fact that Gram
negative bacteria were more susceptible to the extracts of M. pseudopetalosa
roots is significant as Gram positive bacteria are usually reported as being
more affected by plant extracts (Yani et al., 2005; Sofidiya et al., 2009;
Afolayan et al., 2009). In the Gram negative bacteria, the extracts of M.
pseudopetalosa roots were able to overcome the permeability barrier provided
by the cell wall and the membrane accumulated resistance mechanisms
(Adwan and Abu-Hasan, 1998).
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3.2.5 Antibacterial activity of Maerua pseudopetalosa roots at lower
extracts concentrations
The antimicrobial activity of different extracts obtained from M.
pseudopetalosa roots at concentrations 1000, 500, and 250 µg/mL were also
determined and results are represented in Table 4. The hexane extract devoid
of antibacterial activity against all the tested bacteria. Interestingly, the active
extracts did not show susceptibility against the tested bacteria in a
concentration dependant manner. The CHCl3 extract showed antibacterial
activity against B. subtilis at the three concentration used. The highest activity
was observed at concentration 250 µg/mL with inhibition zone of 28 mm,
followed at concentrations 1000 and 500 µg/mL with inhibition zones of 12
and 5 mm respectively. The EtOAc extract showed moderate antibacterial
activity against B. subtilis at concentration 250 µg/mL with inhibition zone of
11 mm and weak activity at concentration of 1000 µg/mL with inhibition
zone of 9 mm. No antibacterial activity was observed at 500 µg/mL. The
MeOH extract exhibited no antibacterial activity.
High antibacterial activity against S. aureus was obtained at concentration
250 µg/mL for the CHCl3 extract with inhibition zone of 17 mm. Other
concentrations as well as the EtOAc and MeOH extracts showed more or less
the same susceptibility with inhibition zones ranging from 12 to 15 mm.
The susceptibility of CHCl3 extract towards E. coli revealed highest
antibacterial activity at concentration 250 µg/mL with an inhibition zone of
45 mm; whereas; at concentration 1000 µg/mL it gave moderate activity with
an inhibition zone of 12 mm. No antibacterial activity (inhibition zone 5 mm)
was obtained at 500 µg/mL. However, the MeOH extract showed moderate
antibacterial activity with inhibition zones of 11 and 15 mm at concentrations
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1000; 500 µg/mL respectively and weak activity (inhibition zone 10 mm) at
250 µg/mL.
The CHCl3 extract displayed very good antibacterial activity against S. typhi
at concentration 250 µg/mL with inhibition zone of 32 mm. The susceptibility
was less at concentrations 500 and 1000 µg/mL. The EtOAc and MeOH
extracts inhibited the bacteria in all tested concentrations with inhibition zone
ranging from 12 to 16 mm indicative of moderate activity.
These differences in susceptibility could be due to the nature and level of the
antimicrobial agents present in the extracts and their mode of action on
different test microorganisms (Barbour et al., 2004).
From the above results it was clear that the best antibacterial activity for all
tested bacteria was obtained from the CHCl3 and at concentration 250 µg/mL.
Moreover; very high inhibition zone, higher than standard antibiotic drug, was
observed against E. coli (inhibition zone of 45 mm) and S. typhi (inhibition
zone of 32 mm).
46
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Table 4: Antibacterial activity of Maerua pseudopetalosa roots at lower
extracts concentrations.
Extract/
Inhibition zone* (mm)
Concentration µg/mL B. subtilis
S. aureus
Hexane
1000
0
0
500
0
0
250
0
0
Chloroform
1000
12
13
500
5
13
250
17
28
Ethyl acetate
1000
9
12
500
0
11
250
11
15
Methanol
1000
4
12
500
0
14
250
0
12
Ampicillin
5
17
16
* Values are representative of three independent determinations.
47
E. coli
S. typhi
0
0
0
0
0
0
12
5
45
10
7
32
11
13
11
16
13
13
11
15
10
12
13
12
25
22
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3.3 In vitro antigiardial activity against Giardia lamblia
According to the results obtained from the antibacterial activity test, where
the extracts showed high susceptibility against bacteria (E. coli and S. typhi)
responsible of diarrhea, antigiardial activity test was performed for the
different extracts of M. psudopetalosa roots. In fact, G. lamblia is one of the
most common intestinal pathogenic protozoan parasites (Newman et al.,
2001). It is becoming increasingly important among HIV/AIDS patients.
There are reports that some cases of acute and chronic diarrhea in AIDS
patients may be associated with giardial infection (Merchant and Shroff,
1996). However, metronidazole, the common drug of choice, can cause
mutagenicity in bacteria (Legator et al., 1975) and is carcinogenic in rodents
(Rustia and Shubik, 1972). It also possesses undesirable side effects and
treatment failures have been reported (Llibre et al., 1989).
The activity of different extracts of M. psudopetalosa roots against G.
lambelia was investigated using three different concentrations and results are
presented on Fig. 9. Extract is considered active with mortality value = 50% .
The hexane extract showed mortality of 50.7% after 48 h at concentration 250
µg/mL. All other concentrations were considered inactive as they showed
weak mortality (? 50%) of G. lambelia (Fig. 9-a).
At concentration 500 µg/mL the CHCl3 extract showed an increase of number
of the parasite (- 57.4%) after 24 h then a mortality of 56.7% and 46.8% of G.
lambelia was observed after 48 and 72 h respectively. Antigiardial activity
was also obtained at concentrations 250 µg/mL (58.7%) and 125 ppm (50%)
after 72 h (Fig. 9-b).
The highest effective concentration of M. psudopetalosa roots against G.
lambilia was obtained from EtOAc extract at 250 µg/mL with mortality of
48
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70.5% after 48 h and 66.17% after 72 h. Moreover, the EtOAc extract showed
mortality higher than the positive control (metrondizole). At concentration
125 µg/mL the EtOAc extract reveald antigiardial activity with mortality of
61.67% after 72 h (Fig. 9-c).
The MeOH extract only in the lowest concentration 125 µg/mL showed
antigiardial activity with mortality of 56% after 72 h (Fig. 9-d).
Thus, from the above results it was found that the EtOAc extract was the most
effective extract against G. lambelia with mortality of 70% higher than that
obtained by the positive control metrondizole. In this study an interesting
observation was noted; an increase in number of parasite was observed
mainly after 24 h in the hexane, CHCl3 and EtOAc extracts which might
reflect the presence of nutritive ingredients for the parasite in these extracts as
well. In general, no much work was carried out on the Sudanese medicinal
plants for their antigiardial activity. Hassan et al. (2011) investigated the
antigiardial activity of Citrullus lanatus var. citroides extracts and
cucurbitacins isolated compounds. They found that all extracts and isolated
compounds showed high antigiardial activity.
49
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b- Chloroform extract
a- Hexane extract
ϴϬ
ϴϬ
ϲϬ
ϲϬ
ϰϬ
ϰϬ
)
%
(
y
ti
l ϮϬ
a
t
r
o
M
) ϮϬ
%
(
y
ti
l Ϭ
a
t
r
o
M-ϮϬ
Ϭ
ϱϬϬ
ђŐͬŵ> ϮϱϬ
ђŐͬŵ> ϭϮϱ
ђŐͬŵ> ctrl -ve
ctrl +ve
-ϮϬ
ϱϬϬ
ђŐͬŵ> ϮϱϬ
ђŐͬŵ> ϭϮϱ
ђŐͬŵ>
ctrl -ve
ctrl +ve
ϱϬϬ
ђŐͬŵ> ϮϱϬ
ђŐͬŵ> ϭϮϱ
ђŐͬŵ> ctrl -ve
ctrl +ve
-ϰϬ
-ϲϬ
-ϰϬ
-ϴϬ
c- Ethyl acetate extract
D- Methanol extract
ϭϬϬ
ϳϬ
ϴϬ
ϲϬ
ϲϬ
)
%
(
y
itl
a
t
r
o
M
ϱϬ
ϰϬ
ϰϬ
ϮϬ
)
%
(
y ϯϬ
ti
l
a
t
r ϮϬ
o
M
Ϭ
-ϮϬ
-ϰϬ
-ϲϬ
-ϴϬ
ϱϬϬ
ђŐͬŵ> ϮϱϬ
µg/mL
ϭϮϱ
ctrl -ve
µg/mL
ctrl +ve
ϭϬ
zϭ
zϮ
Ϭ
zϯ
-ϭϬ
-ϮϬ
Fig. 9: Antigiardial activity of roots of Maerua psedopetalosa against Giardia lamblia.
50
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3-4 Phytochemistry
3-4-1 Qualitative analysis of secondary metabolites
Analysis of different classes of major secondary metabolites present in water and
MeOH extracts of the roots of M. pseudopetalosa was carried out using the
method described by Harbone (1973). Results are presented in Table 5. It was
clear that both extracts revealed the same classes of secondary metabolites but
not necessarily the same compounds. Both extracts contained saponins,
alkaloids, triterpenes, steroids and flavanoids. Tannins, cardiac glycosides and
anthraquinones were not detected on both extracts. Phytochemical screening of
the extract from M. angolensis stem bark was carried out by Adamu et al.
(2007). They revealed the presence of tannins, flavonoids and saponins. The
observed antimicrobial properties in this study could be due to constituents
belonging to the groups of phytochemicals that were identified in the crude
extract. Antimicrobial properties of alkaloids, triterpenes, steroids and
flavonoids for examples are well documented (Cowan, 1999; Navarro and
Delgado, 1999).
3-3-2 Preliminary phytochemical screening by thin layer chromatography
(TLC)
Preliminary phytochemical screening of the hexane, CHCl3, EtOAc and MeOH
extracts from roots of M. pseudopetalosa was carried out using TLC technique.
TLC plates of all extracts were developed using vanillin/H2SO4 reagent and
results are presented in Figure 10. Several spots with different Rf values were
observed in the three extracts indicating that the root was rich in secondary
metabolites. The hexane extract exhibited many dark violet- coloured spots with
51
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different polarities indicating the presence of significant number of non-polar
compounds. Also, the CHCl3 extract revealed dark violet-coloured spots with
different degree of polarities with less number of spots as compared with the
hexane extract (Fig. 10). The EtOAc extract gave fewer spots but having the
same colour reaction as in the hexane and EtOAc extracts (Fig. 10). Several
solvent systems were tried for the MeOH extract but no good resolution was
obtained suggesting that other chromatographical media like cellulose or
polyamide should be used. Thus, the observed antimicrobial activity of extracts
may be due to the presence of these components. Okwute (1992) reported that
terpenes, alkaloids, saponins, phenolic compounds and cardiac glycosides are
known to possess antimicrobial and antiplasmodial activity and pesticide
properties.
Rf
Ϭ
͘
ϴ
Ϭ
͘
ϰ
Hexane extract
CH3Cl extract
EtOAc extract
Fig.10: Thin layer chromatography of the hexane, CHCl3 and EtOAc
extracts from Maerua pseudopetalosa roots.
Solvent system: hexane extract, Toluene: EtOAc (97:3 v/v); CHCl3 extract, hexane: EtOAc
(7:3 v/v); EtOAc extract, toluene: EtOAc: formic acid (5:4:1 v/v); spray reagent:
vanillin/H2SO4
52
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Table 5: Preliminary phytochemical screening of roots of Maerua pseudopetalosa.
Class of compounds
Name of the test or test reagent
Observations
Results
EtOH extract
H2O extract
Alkaloids
Meyer's reagent
Formation of a precipitate
++
++
Flavonoids
10% NaOH
Yellow precipitate
+
+
Tannins
0.5% FeCl3
Green or dark-blue precipitate
-
-
Saponins
Shaking of aqueous solution
Formation of persistent foam
+++
+++
Cardiac glycosides
Keller–Killani test
Brown ring at the interface
-
-
Anthraquinones
10% ammonia
Pink colour in ammonia phase
-
-
Triterpenes and steroids
Lieberman–Burchard
Purple colour for triterpenes
++
++
Bluish-green colour for steroides
++
++
53
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3-4-3 GC/MS analysis of the hexane extract
The hexane extract was subjected to GC/MS analysis and results are
presented in Table 6. Results revealed the presence of 20 compounds. The
major compounds were identified as 9,12 octadecenonic acid (28.83%);
followed by 9- octadecenonic acid (24.86 %), 9-octadecenamide (14.35 %)
and hexadecanoic acid (11.64 %) respectively. The hexane extract possessed
high antibacterial activity at concentrations 20, 10 and 5 g/mL against E. coli,
S. aureus, and P. aeruginosa. This antimicrobial activity might be attributed
to the presence of saturated and unsaturated fatty acids. Fatty acids are known
to have antibacterial and antifungal properties (Agoramoorthy et al., 2007).
Lauric, palmitic, linolenic, linoleic, oleic, stearic and myristic acids are
known to have potential antibacterial and antifungal agents (McGaw et al.,
2002; Seidel and Taylor, 2004). Moreover, a previous study of liphophylic
extracts derived from 15 different plant parts of Pistacia vera showed activity
against E. coli, Enterococcus faecalis and S. aureus (Özçelik et al., 2005).
Similarly, linoleic acid isolated from Schotia brachypetala displayed
antibacterial activity against B. subtilis, S. aureus, Klebsiella pneumoniae and
E. coli (McGaw et al., 2002).
54
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Table 6:: GC/MS analysis of the hexane extract of Maerua psedopetalosa
roots.
3H
D
N
1R
&RPSRXQG
1D
PH
5
7L
PH $U
H
D
1 1,4- Benzenedicarboxylic acid
9,45
3,18
2 1,3- Benzenedicarboxylic acid
9,624
0,64
3 Nonanedioic acid
10,0
10,043
0,39
4 3- hexanon
hexanon, 2,5 – dimethyl
10,446
0,08
5 Methyl tetradecanoate
12,916
0,52
6 11- octadecenonic acid
17,944
0,42
7 Hexadecanoic acid
18,588
11,64
8 Heptadecanoic acid
20,982
0,9
55
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9 9,12- octadecenonic acid
22,481
28,83
10 9- octadecenonic acid
22,642
24,86
11 11- octadecenonic acid
22,721
2,84
12 octadecenonic acid
23,192
4,26
13 Decane
Decane,1- Iodo –
24,507
0,27
14 Heptadecane
Heptadecane, 2,6,10,15 - tetra methyl
26,412
0,33
15 Hexadecanoic acid
27,069
0,27
16 Tetratriacontane
28,2
0,56
17 9- octadecenamide,(z) –
28,674
14,35
56
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18 Tetradecane
29,899
0,31
19 1,2- Benzenedicarboxylic acid, disooctyl ester
30,527
5
20 7U
L
GH
F
D
QRL
F
D
F
L
G
33,634
0,26
57
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3.4.4 Bioassay guided fractionation of chloroform extract
According to the antibacterial activity results the CHCl3 extract was
subjected to phytochemical analysis using column chromatography technique.
The CHCL3 extract was fractionated on silica gel column and eluted with
increasing mixture of hexane: EtOAc and EtOAc: MeOH respectively. The
fractions were analyzed by TLC and similar fractions were pooled to give 7
subfractions Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6 and Fr.7 (Fig. 11-a). Spraying the
plate with Dragndorff’s reagent revealed the presence of alkaloids in Frs. 4
and 5 (Fig. 11-b).
All subfractions at concentration 5 mg/mL were tested for antibacterial
activity by disc diffusion assay. Fr.1 showed weak antibacterial activity
against E. coli and S. typhi with inhibition zones 7 and 8 mm respectively.
Fr.2 showed weak antibacterial activity against all tested bacteria. Except
towards B. subtilis, the same observation was displayed by Fr.3. It showed
moderate antibacterial activity against B. subtilis with inhibition zone 13 mm.
Fractions 4, 5 and 6 showed only weak antibacterial activity against E. coli, P.
aeruginosa and S. typhi. Fr.7 showed weak antibacterial activity against B.
subtilis, E. coli, P. aeruginosa and S. typhi (Table 7). The decrease in potency
of fractions obtained from the CHCl3 extract when compared to the crude
CHCl3 extract seems to indicate the loss of synergistic action between any of
the phytochemical constituents present in the CHCl3 extract due to its
subjection to the separation process. In other words, the results indicated that,
the active components in the crude CHCl3 extract may be acting
synergistically to produce good antimicrobial effects.
58
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Rf
a
Ϭ͘
ϳ
Ϭ͘
ϱ
ϭ
Rf
Ϯ
ϯ
ϰ
ϱ
ϲ
ϳ
ϯ
ϰ
ϱ
ϲ
ϳ
b
Ϭ͘
ϳ
Ϭ͘
ϲ
ϭ
Ϯ
Fig.11: Thin layer chromatography of fractions of CHCl3 extract from
Maerua pseudopetalosa roots.
Solvent system: Toluene: EtOAc: formic acid (5:4:1 v/v); spray reagent: plate a;
vanillin/H2SO4 and plate b; Dragndorff’s reagent.
59
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Table 7: Antibacterial activity of of fractions of CHCl3 extract from
Maerua pseudopetalosa roots.
Inhibition zone* (mm)
Fraction
(5 mg/mL)
Fr 1
Fr 2
Fr3
Fr 4
Fr 5
Fr 6
Fr 7
Ampicillin
(5 µg/mL)
B. subtilis
S. aureus
E. coli
P. aeruginosa S. typhi
0
0.8
1.3
0
0
0
0.8
0
0.7
0.8
0
0
0
0
0.8
0.7
0.8
0.7
0.8
0.8
0.8
0
0.8
0.7
0.8
0.7
0.7
0.7
0.7
0.7
0.9
0.8
0.8
0.7
0.8
17
16
25
15
20
*Values are mean representative of three independent determinations.
60
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Conclusion
In this study, results showed that the response of the bacteria to the tested
extract varied among the strains. The best antibacterial activity for all tested
bacteria was obtained from the hexane and CHCl3 extracts. At concentration
250 µg/mL the CHCl3 extract displayed very high inhibition zones, higher
than that obtained from standard antibiotic drug, against E. coli (inhibition
zone of 45 mm) and S. typhi (inhibition zone of 32 mm).
The EtOAc extract was the most effective extract against G. lambelia with
mortality of 70% higher than that obtained by the positive control
metrondizole.
Only, the hexane extract at concentrations 20 and 10 mg/mL revealed highest
antibacterial activity against P. aeruginosa with inhibition zones 19 and 17
mm respectively.
Water and methanol extracts contained saponins, alkaloids, triterpenes,
steroids and flavanoids.
The hexane extract revealed the presence of 20 compounds. The major
compounds were identified as 9,12 octadecenonic acid (28.83%); followed by
9- octadecenonic acid (24.86 %), 9-octadecenamide (14.35 %) and
hexadecanoic acid (11.64 %) respectively.
In conclusion, the results provide promising baseline information for the
potential use of the crude extracts from M. pseudopetalosa roots in the
treatment of bacterial and giardial infections.
61
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Recommendations
Since plants produce a diverse range of bioactive molecules making them rich
sources of different types of medicines, further pharmacological and
toxicological studies will be necessary to establish their safety as
antimicrobial agents in the traditional medicine.
Also, the Isolation and identification of pure compounds and evaluation of
their biological activity as new source of natural drugs are needed.
62
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References
Abdalla, A. A.and Nour, H. O. A., (2001). The agricultural potential of
Sudan–Executive intelligence review, Ministry of Agriculture and
Forestry, Sudan, pp.37-45.
Adamu, A., Abdurahman, E.M., Ibrahim, H., Abubakar, M.S., Magaji,
M.G.and Yaro, A.H. (2007). Effect of aqueous methanolic stem
bark extract of Maerua angolensis DC. on acute and sub-acute
inflammations. Nig. Journ. Pharm. Sci., Vol. 6 No. 2, P. 1 – 6.
Adwan, K.and Abu-Hasan, N. (1998). Gentamicin resistance in clinical
strains of Enterobacteriaceae associated with reduced gentamicin
uptake. Folia Microbiol. 43: 438–440.
Afolayan, A.J.and Ashafa, A.O.T. (2009). Chemical composition and
antimicrobial activity of the essential oil from Chrysocoma ciliate
L. leaves. J. Med. Plants Res., 3: 390–394.
Agoramoorthy, G., Chandrasekaran, M., Venkatesalu, V.and Hsu., M.J.
(2007). Antibacterial and antifungal activities of fatty acid methyl
esters of the Blind-your-eye mangrove from India. Brazilian Journal
of Microbiology 38: 739-742.
Ali, Y., Riaz, N., Afza, N., Kajhoro, M. A., Malik, A., (2008).
Phytochemical studies on Maerua arenaria. J. Chem. Soc. Pak., 30,
103-105.
Amooti-Kyomya, S., 1994, Traditional health systems and the
conventional system in Uganda, In: Islam A., Wiltshire R.
(Eds.),Traditional Health Systems and Public
Policy, International
63
Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark.
Development Research Centre, Ottawa, Canada Anonymous, 1994,
Development plan.
Arguello GR, Cruz M, Romero ML, Ortega PG (2004). Variability and
variation in drug susceptibility among Giardia duodenalis isolates
and clones exposed to 5-nitromidazoles and benzimidazoles in
vitro. J. Antimicrob. Chemotharpy, 54: 711-721.
Barbour, E.K.; Sharif, M.A.; Sagherian, V.K.; Habre, A.N.; Talhouk, R.S.
and Talhouk, S.N. (2004). Screening of selected indigenous plants
of Lebanon for antimicrobial activity. J. Ethnopharmacol., 93,1-7.).
Barrow, p. and, Soothill, J. (1997). Bacteriophage therapy and
prophylaxis; rediscovery and reviewed assessment of potential.
Trend Microbial, 5: 268-71.
Brown, E.M. and Reeves, D.S. (1997). Quinolones in anti biotic and
chemotherapy .anti infective agents and there use in therapy ,7th
edn, ch.32. (FO,Grady,HP lampert,RG finich,eds). Churchill
livingstone, London.
Cedillo, R., Chave, B., Gonzalez, A.,Tapia, A., Yepez, L. (2002) In vitro
effect of nitazoxanide against Entamoba histolytica, Gairdia
lamblia and Trichomonas vaginalis trophozoites. The Journal of
Eukaryotic Microbiology 49, 201-208.
Cheesbrough, M., (2000) District laboratory practice in Tropical countries.
Part II, Cambridge, London, pp, 62-70.
Cos, P., Hermans, N., De Bruyne, T., Apers, S., Sindambiwe, J.B.,
Berghe,
D.V.,
Pieters, L.and Vlietinkck, A.J.( 2002). Further
evaluation of Rwandan medicinal plant extracts for their
64
Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark.
antimicrobial and antiviral activities. J. Ethnopharmacol.79: 155–
163.
Cowan, M.M., 1999. Plant products as antimicrobial agents. Clinical
Microbiol Rev 12, 564–582.
David, G.W.(2001). Pharmaceutical Analysis. School of Pharmacy,
University of Strathclyde, Glasgow, UK.
Elamin, H.M. (1990). Trees & shrubs of the Sudan. Ithaca press. 484p.
Elsir, L .A. H ., Koko, S.W., El-Badri E. O., Mahmoud, M. D., and
Mohd, H. S. 2011. In vitro antigiardial activity of Citrullus lanatus
Var. citroides extracts and cucurbitacins isolated compounds.
Journal of Medicinal Plants Research Vol. 5(15), pp. 3338-3346.
Hack, S.K. (2006). Do not put too much value on conventional medicine.
J. Ethnopharmacol. 100: 37–39.
Harborne JB (1973). Phytochemical methods, London. Chapman and
Hall, Ltd. pp. 49-188.
Hill, D.R., Nash, T.E. (2006). Intestinal flagellate and ciliate infections. In:
Guerrant RL, Walker DH, and Weller PF (eds.) Tropical Infectious
Diseases: Principles, Pathogens, and Practice, 2nd edn., pp. 984–
1002. Philadelphia, PA: Elsevier.
Hussain, K.; Shahazad, A.; Hussain, S.Z. (2008). An ethnobotanical
survey of important wild medicinalplants of Hattar district Haripur,
Pakistan. Ethnobot. Leafl. 12: 29–35.
65
Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark.
Ibraheim, Z.Z., Amany, S. A. and Ramadan, M.R. (2008). Lipids and
triterpenes from Maerua crassifolia growing in Egypt. Saudi
Pharmaceutical Journal 16(1).
Joseph, C.A. and Palmer, S.R.(1989). Outbreak of Salmonella infection in
hospital in England and Wales 1978-87. BMJ, 298:1161-4.
Karaman, I., Sahin, F., Gulluce, M.; Ogutcu, H., Sngul, M. and
Adiguzel,
A. ( 2003).
Antimicrobial activity of aqueous and
methanol extracts of Juniperus oxycedrus L. J. Ethnopharmacol. 85:
231–235.
Legator, M.S., Connor, T.H. and Stoeckel, M. (1975). Detection of
mutagenic activity of metronidazole and nitridazole in body fluids
of humans and mice. Sci., 188: 1118-1119.
Lev, E. ( 2006). Ethno-diversity within current ethno-pharmacology as
part of Israeli traditional medicine-A review. J. Ethnobiol.
Ethnomed. 2: 4.
Llibre, J.M., Tor, J., Manterola, J.M., Carbonell, C. and Foz , M. (1989).
Blastocystis hominis chronic diarrhoea in AIDS patients. Lancet, 1:
221.
MAPRI. (1997). Review of Trade in Wildlife Medicinal Plant in
Khartoum, Medicinal and Aromatic Plant Research Institute,
Khartoum. Sudan.
Mavura W.J., M.C. Chemelil, W.W. Saenyi and H.K. Mavura., 2008.
Investigation of chemical and biochemical properties of Maerua
subcordata plant extract: a local water clarification agent. Bull.
Chem. Soc. Ethiop. 22(1), 143-148.
66
Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark.
Mbaveng A.T., Ngameni B., Kuete V., Simo I.K., Ambassa P., Roy R.,
Bezabih M., Etoa F.X., Ngadjui B.T., Abegaz B.M., Meyer J.J.,
Lall N. and Beng V.P., (2008). Antimicrobial activity of the crude
extracts and five flavonoids from the twigs of Dorstenia barteri
(Moraceae). J Ethnopharmacology, 116(3): 483- 9.
McFarland, J. 1907. Nephelometer; JAMA 14:1176-1178.
McGaw, L.J.; Jäger, A.K. and Van Staden, J. (2002). Isolation of
antibacterial fatty acids from Schotia brachypetala. Fitoter., 73,431433.
Merchant, R.H. and Shroff, R.C. (1996). The prevalence of HIV infection
in children with extensive tuberculosis and chronic diarrhoea. AIDS
Asia, 3: 21-23.
Monks, N.R., C. Lerner, A.T. Henriques, F.M. Farias, D.D.S.Schapoval,
E.S. Suyenaga, A.B. Da Rocha, G. Schwarsmann, and B. Mothes.
2002. Anticancer, antichemotactic and antimicrobial activities of
marine sponges collected off the coast of Santa Catarina, southern
Brazil. Journal of Experimental Marine Biology and Ecology
281:1–12.
Muschietti, L.; Derita, M.; Sülsen, V.; Muñoz, J.D. ; Ferraro, G.;
Zacchino; S. and Martino, V.( 2005). In vitro antifungal assay of
traditional Argentine medicinal plants. J. Ethnopharmacol.102:
233–238.
Navarro, V. and Delgado, G. (1999). Two antimicrobial alkaloids from
Bocconia arborea. J. of Ethnopharmacol. 66, 223–226.
67
Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark.
Neu, H.C., (1992). The crisis in anti biotic resistance – science, 257:106473.
Newman, R.D., Moore, S.R., Lima, A .A., Nataro, J.P., Guerrant, R.L.
and Sears, C.L. (2001). A longitudinal study of Giardia lamblia
infection in North-east Brazilian children. Trop. Med. Int. Health, 6:
624-634.
Okwute SK (1992). Plant derived pesticidal and antimicrobial agents
foruse in agriculture. A review of phytochemical and biological
studies on some Nigerian plants. J. Agric. Sci. Technol. 2 (1): 6270.
Olajuyigbe, O.O.; Babalola,
A.E. and
Afolayan,
A.J.( 2011).
Antibacterial and phytochemical screening of crude ethanolic
extracts of Waltheria indica Linn. Afr. J. Microbiol. Res. 5: 3760–
3764.
Özçelik, B.; Aslan, M., Orhan, I. and Karaoglu, T. (2005). Antibacterial,
antifungal and antiviral activities of the lipophylic extracts of
Pistacia vera. Microbiol. Res., 160, 159-164.
Patwardhan, B., Warude, D., Pushpangadan, P. and Bhatt, N. (2005).
Ayurveda and traditional Chinese medicine: A comparative
overview. Evid.-Based Compl. Alt. 2: 465–473.
Plumb, D.C., (1999). Veterinary drug handbook. 3rd ed. Low State
University Press/ Ames.
Rustia, M. and Shubik,
P.
(1972). Induction of lung tumors and
malignant lymphomas in mice by metronidazole. J. Natl. Cancer
Inst., 48: 721- 729.
68
Please purchase PDFcamp Printer on http://www.verypdf.com/ to remove this watermark.
Seidel, V. and Taylor, P.W. (2004). In vitro activity of extracts and
constituents of Pelagonium against rapidly growing mycobacteria.
Int. J. Antimicrob. Agen., 23, 613-619.
Sharma, P.P. and Mujundar , A.M.( 2003). Traditional knowledge on
plants from Toranmal Plateau of Maharastra. Indian J. Tradit.
Know 2: 292–296.
Sheldon, J.W.; Balick, M.J. and Laird, S.A.( 1997). Medicinal plants: can
utilization and conservation coexist Advances in Economic Botany.
Econ. Bot. 12: 1–104.
Sofidiya, M.O.; Odukoya, O.A.; Afolayan, A.J. and Familoni, O.B.(
2009). Phenolic contents, antioxidant and antibacterial activities of
Hymenocardia acida. Nat. Prod. Res., 23: 168–177.
Tyler, V. E. (1999). Herbs of choice. The Therapeutic use of
phytomedicinals. New York: Pharmaceutical Products Press.
Tene, V.; Malag, O.; Finzi, P.V.; Vidari, G.; Armijos, C. and
Zaragoza, T. (2007) . An ethnobotanical survey of medicinal plants
used in Loja and Zamora-Chinchipe, Ecuador. J. Ethnopharmacol.
111: 63–81.
Wannissorn, B.; Jarikasem, S.; Siriwangchai, T. and Thubthimthed, S.
(2005). Antibacterial properties of essential oils from Thai
medicinal plants. Fitoterapia. 76: 233–236.
Watt, J.M. and Breyer-Brandwijk, M.G. (1962). The Medicinal and
poisonous plants of Southern and Eastern Africa, 2nd ed.
Livingstone, London.
69
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Wilson, J. and Breedon, P. (1990). Universal precautions, nursing times,
86(37):67-70.
Yani, V.V., Oyedeji, O.A., Grierson, D.S. and Afolayan, A.J. (2005).
Chemical analysis and antimicrobial activity of essential oil
extracted from Helichrysum aureonitens. S. Afr. J. Bot. 71: 239–
241.
Yao, J.; Moellering, R., Murray, P., Baron, E., Pfaller, M., Tenover,
F. and Yolken, R., Eds.; .( 1995). Antibacterial agents. In: Manual
of Clinical Microbiology; ASM Press: Washington, DC, USA,; pp.
1281–1290.
(http://WWW.aluka.org/action/showmetadata).
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