Antibacterial and antifungal activities of different parts of Tribulus terrestris L. growing in Iraq

Antibacterial and antifungal activities of different parts of Tribulus terrestris L. growing in Iraq first-author †E-mail:firas.bayati@gmail.com †E-mail:firas.bayati@gmail.com firas.bayati@gmail.com Department of Biology, College of Education, University of Mosul, Mosul-00964, Iraq Department of Biology, College of Education, University of Mosul, Mosul-00964, Iraq Antimicrobial activity of organic and aqueous extracts from fruits, leaves and roots of Tribulus terrestris L., an Iraqi medicinal plant used as urinary anti-infective in folk medicine, was examined against 11 species of pathogenic and non-pathogenic microorganisms: Staphylococcus aureus , Bacillus subtilis , Bacillus cereus , Corynebacterium diphtheriae , Escherichia coli , Proteus vulgaris , Serratia marcescens , Salmonella typhimurium , Klebsiella pneumoniae , Pseudomonas aeruginosa and Candida albicans using microdilution method in 96 multiwell microtiter plates. All the extracts from the different parts of the plant showed antimicrobial activity against most tested microorganisms. The most active extract against both Gram-negative and Gram-positive bacteria was ethanol extract from the fruits with a minimal inhibitory concentration (MIC) value of 0.15 mg/ml against B . subtilis , B . cereus , P . vulgaris and C . diphtheriae . In addition, the same extract from the same plant part demonstrated the strongest antifungal activity against C . albicans with an MIC value of 0.15 mg/ml. Antimicrobial activity Tribulus terrestris Urinary tract infections INTRODUCTION 1 Traditional medicine has been practiced for many centuries by a substantial proportion of the population of Iraq. The interest in the study of medicinal plants as a source of pharmacologically active compounds has increased worldwide. It is recognized that in some developing countries, plants are the main medicinal source to treat infectious diseases. Plant extracts represent a continuous effort to find new compounds with the potential to act against multi-resistant bacteria. Approximately 20% of the plants found in the world have been submitted to pharmacological or biological test, and a substantial number of new antibiotics introduced on the market are obtained from natural or semi-synthetic resources (Mothana and Lindequist, 2005 2005 ). Tribulus terrestris (Puncture Vine, Caltrop, Yellow Vine and Goathead) is a flowering plant of the Zygophyllaceae family, native to warm temperature and tropical regions of the old world in Southern Europe, Southern Asia, Africa and Northern Australia. It can thrive even in desert climates and poor soil (Abeywickrama and Bean, 1991 1991 ). In Iraq T . terrestris is used in folk medicine as tonic, aphrodisiac, analgesic, astringent, stomachic, anti-hypertensive, diuretic, lithon-triptic and urinary anti-infectives (Majeed and Mahmood, 1988 1988 ; Saad Aldein, 1986 1986 ). Different parts of Turkish and Iranian T . terrestris have been reported to have antibacterial activity (Abbasoglu and Tosun, 1994 1994 ; Kianbakht and Jahaniani, 2003 2003 ); however, the antimicrobial activity of Iraqi T . terrestris has not been studied. In the current study, we evaluated in vitro antimicrobial activity of different parts of T . terrestris growing in Iraq using different extracts. MATERIALS AND METHODS 1 Plant materials 2 Fruits, leaves and roots of T . terrestris used in this study were collected at the end of November 2006 from Mosul countryside, Nineveh Province, Iraq, with assistance of local traditional healers, and authenticated by Dr. Abdulazeez Shekho, Department of Biology, University of Mosul, Iraq. Voucher specimen of the plant (No. 62) was dried and deposited at the herbarium of Department of Biology, University of Mosul, Iraq. Preparation of the extracts 2 1. Aqueous extracts 3 The air dried fine powdered plant fruits, leaves and roots (100 g) were infused in distilled water until complete exhaustion. The extract was then filtered using Whatman No. 1 filter paper and the filtrate was evaporated in vacuo and dried using either a rotary evaporator at 60 °C or a freeze drier (Kandil et al., 1994 1994 ). The final dried samples were stored in labeled sterile bottles and kept at −20 °C. 2. Ethanol extracts 3 Ethanol extracts were accomplished according to established protocols (le Grand et al., 1988 1988 ). Each dried plant sample was ground and extracted in a percolator with 95% ethanol. About 10 ml of ethanol per gram of plant sample was used. The ethanol extract was dried under a reduced pressure at 40 °C. The dried extract was stored in sterile bottles until further use. 3. Chloroform extracts 3 Powdered samples (100 g) from each plant part were extracted with chloroform using a soxhlet extractor for continuously 10 h or until the used solvent turned pure and colorless (Chhabra et al., 1982 1982 ). The solvent was removed using a rotary vacuum evaporator at 40 °C to give a concentrated extract, which was then frozen and freeze-dried until use. Microbial cultures 2 Ten strains of bacteria and one yeast were used as test microorganisms. The bacterial strains included Gram-positive Staphylococcus aureus , Bacillus subtilis , Bacillus cereus and Corynebacterium diphtheriae ; Gram-negative Escherichia coli , Proteus vulgaris , Serratia marcescens , Salmonella typhimurium , Klebsiella pneumoniae and Pseudomonas aeruginosa ; and the yeast Candida albicans . All microorganisms were clinical isolates, obtained from the Microbiology Laboratory at Department of Biology, University of Mosul, Iraq, and very carefully identified using standard microbiological methods. Inoculum preparation 2 Mueller-Hinton broth and Sabouraud dextrose agar (SDA) were applied for growing and diluting the microorganism suspensions. Bacterial strains were grown to exponential phase in Mueller-Hinton broth at 37 °C for 18 h and adjusted to a final density of 10 8 CFU/ml by diluting fresh cultures and comparing with McFarland density. C . albicans was aseptically inoculated on Petri dishes containing autoclaved, cooled and settled SDA medium. The Petri dishes were incubated at 31 °C for 48 h to give white round colonies against a yellowish background. These were aseptically subcultured on SDA slants. The yeast colonies from SDA slants were suspended in sterilized 0.9% sodium chloride solution (normal saline), which was compared with McFarland solution. One ml yeast suspension in normal saline was added to 74 ml of sterile medium, kept at 45 °C, to give concentration of 2×10 7 cells/ml (according to the manufacturer’s instructions). Antimicrobial activity 2 Extracts were tested against the strains for their inhibitory activity, using a common broth microdilution method in 96 multiwell microtiter plates, in duplicate, as reported by Koneman ( 1995 1995 ) and recommended by the National Committee for Clinical Laboratory Standard (NCCLS, 2001 2001 ). For susceptibility testing, 50 µl of Mueller-Hinton broth was distributed from the second to the twelfth test tubes. Dry extracts from each plant part were initially dissolved in 100 µl of dimethyl sulfoxide (DMSO) and then in Mueller-Hinton broth, to reach a final concentration of 10 mg/ml; 100 µl of these suspensions were added to the first test well of each microtiter line, and then 50 µl of scalar dilutions were transferred from the second to the ninth well. The 10th well was considered as growth control, since no extracts solutions were added. Then, 50 µl of the bacterial suspensions were added to each well. The final concentrations of the extracts adopted to evaluate the antibacterial activity were 0.01 to 5.00 mg/ml. Plates were incubated for 24 h at 37 °C. Maxipime (Hemofarm) at the concentration range of 0.01~5.00 mg/ml was prepared in Mueller-Hinton broth and used as standard drug for positive control. As an indicator of bacterial growth, 40 µl p -iodonitrotetrazolium (INT) violet dissolved in water was added to the wells and incubated at 37 °C for 30 min (Buwa and van Staden, 2006 2006 ). The lowest concentration of each extract showing no growth was taken as its minimal inhibitory concentration (MIC) and confirmed by plating 5 µl samples from clear wells on Mueller-Hinton agar medium. The colorless tetrazolium salt acts as an electron acceptor and is reduced to a red-colored formazan product by biologically active organisms (Eloff, 1998 1998 ). Where bacterial growth was inhibited, the solution in the well remained clear after incubation with INT. As for C . albicans a simple turbidity test was used to determine the MIC values of T . terrestris extracts by adding 0.1 ml of each extract (0.01~5.00 mg/ml) from each plant part into tubes containing 9.8 ml sterile Mueller-Hinton broth, and tubes were then inoculated with 0.1 ml of yeast suspension and incubated at 31 °C for 48 h. Amphotericin B (0.01~5.00 mg/ml) was used as positive control. The optical density was determined using a Spectro SC spectrophotometer (LaboMed Inc., USA) at 630 nm. The MIC values were taken as the lowest concentration of the extracts that showed no growth after 48 h of incubation by comparing with the control tube that included 9.8 ml Mueller-Hinton broth and 0.1 ml of yeast suspension in addition to 0.1 ml of each extract in different concentration (un-incubated). RESULTS 1 Three different extracts from fruits, leaves and roots of Iraqi T . terrestris were tested at various concentrations (0.01~5.00 mg/ml), and the evaluated MIC values are reported in Table 1 1 . All the plant parts showed antibacterial activity against most tested bacteria. Aqueous extract from T . terrestris fruits showed good activity against the tested bacteria and the strongest activity was seen against C . diphtheriae ( MIC =0.62 mg/ml), which was similar to what was achieved by the standard drug Maxipime; meanwhile, S . typhimurium was inhibited using the highest extract concentration ( MIC =5.00 mg/ml). In addition, S . marcescens and P . aeruginosa resisted all aqueous extracts of various concentrations. Ethanol and chloroform extracts of T . terrestris fruits demonstrated very close activities against all reference bacteria. Very strong activity was seen against S . aureus , B . subtilis , B . cereus , C . diphtheriae , E . coli and P . vulgaris using both extracts. The highest antibacterial activity was seen against B . subtilis , B . cereus , C . diphtheriae and P . vulgaris in the ethanol extract ( MIC =0.15 mg/ml), while B . subtilis , B . cereus and C . diphtheriae were the most sensitive bacteria to the chloroform extract ( MIC =0.31 mg/ml). S . marcescens , S . typhimurium , K . pneumoniae and P . aeruginosa were inhibited by high concentrations of ethanol and chloroform extracts ( MIC =2.50 and 1.25 mg/ml). A: Aqueous extract; E: Ethanol extract; C: Chloroform extract; M: Maxipime; AB: Amphotericin B; NT: Not tested 3 Microorganisms 11 MIC (mg/ml) 3 Fruits 3 Leaves 3 Roots 2 Control A E C A E C A E C M AB Gram-positive S . aureus 2.50 0.62 0.62 2.50 1.25 2.50 >5.00 0.62 0.31 0.07 NT B . subtilis 1.25 0.15 0.31 1.25 0.31 0.31 2.50 0.62 0.31 0.01 NT B . cereus 1.25 0.15 0.31 2.50 0.62 0.62 5.00 1.25 0.62 0.03 NT C . diphtheriae 0.62 0.15 0.31 1.25 0.62 1.25 2.50 1.25 0.62 0.62 NT Gram-negative P . vulgaris >5.00 0.15 1.25 2.50 0.31 1.25 >5.00 2.50 1.25 0.15 NT E . coli 1.25 0.62 0.62 2.50 0.62 1.25 >5.00 2.50 1.25 0.15 NT S . marcescens >5.00 2.50 2.50 >5.00 5.00 >5.00 >5.00 >5.00 >5.00 2.50 NT S . typhimurium 5.00 1.25 2.50 >5.00 1.25 5.00 >5.00 >5.00 2.50 2.50 NT K . pneumoniae 2.50 1.25 1.25 2.50 0.31 2.50 >5.00 2.50 1.25 1.25 NT P . aeruginosa >5.00 1.25 2.50 >5.00 1.25 2.50 5.00 2.50 1.25 2.50 NT Yeast C . albicans >5.00 0.62 2.50 >5.00 1.25 2.50 >5.00 5.00 2.50 NT 0.15 Aqueous extract from T . terrestris leaves revealed close results to the fruit extract, except it was active against P . vulgaris with an MIC value of 2.50 mg/ml and resisted by S . typhimurium , S . marcescens and P . aeruginosa . Ethanol and chloroform extracts from leaves also possessed promising results against the tested bacteria with less action compared with fruit ethanol and chloroform extracts. The best MIC values were calculated against B . subtilis , P . vulgaris and K . pneumoniae ( MIC =0.31 mg/ml) using the ethanol extract and B . subtilis ( MIC =0.31 mg/ml) using chloroform extract. S . marcescens resisted the chloroform extract and was inhibited by the highest ethanol extract concentration. The different extracts from T . terrestris roots exhibited from moderate to no activity against tested bacteria. The weakest extract from the roots was the aqueous extract, which was only active against B . subtilis , B . cereus , C . diphtheriae and P . aeruginosa using high concentrations ( MIC =5.00 and 2.50 mg/ml). Chloroform extract showed high antibacterial activity against some of the tested bacteria, with all the calculated MIC values greater than those of ethanol extract from the same plant part. The standard drug Maxipime showed a greater antibacterial activity than that of most used plant extracts, and the strongest action was seen against B . subtilis ( MIC =0.01 mg/ml). Regarding antifungal activity, all aqueous extracts from different parts of T . terrestris were resisted by C . albicans . The strongest antifungal activity was observed using the ethanol extract from T . terrestris fruits with an MIC value of 0.62 mg/ml. The standard drug Amphotericin B achieved the highest antifungal activity against C . albicans ( MIC =0.15 mg/ml). DISCUSSION 1 All the extracts were able to inhibit the growth of one or more of the tested microorganisms. The Gram-positive bacteria B . subtilis , B . cereus and C . diphtheriae were the most sensitive strains to the ethanol extract of T . terrestris fruits ( MIC =0.15 mg/ml), while the strongest activity was demonstrated against the Gram-negative bacterium P . vulgaris ( MIC =0.15 mg/ml) using the same extract. The activity of the plant against both Gram-positive and Gram-negative bacteria may be indicative to the presence of broad spectrum antibiotic compounds or simply general metabolic toxins in the plant, in addition to the plant (fruits, leaves and root) content of pharmacological active metabolites like furostanol and spirostanol saponins (Kostova and Dinchev, 2005 2005 ), flavonoid glycosides (Saleh et al., 1982 1982 ), phytosterols and some amides (Xu et al., 2000 2000 ). The plant also contains a mixture of B-carboline alkaloids: harmane, norharmane, tetrahydroharmane, harmine, harmaline, harmol, harmalol, ruin and dihydroruin (Bourke et al., 1990 1990 ). Saponins have been reported to possess a wide range of biological activities. The toxicity of saponins to insects (insecticidal activity), parasite worms (anthelmintic activity), molluscs (molluscicidal), and fish (piscidal activity), and their antifungal, antiviral, and antibacterial activities are well documented (Lacaille-Dubois and Wagner, 1996 1996 ; Milgate and Roberts, 1995 1995 ). The mode of action of antibacterial effects of saponins seems to involve membranolytic properties, rather than simply altering the surface tension of the extracellular medium, thus being influenced by microbial population density (Killeen et al., 1998 1998 ). Flavonoids are phenolic structures containing one carbonyl group. Since they are known to be synthesized by plants in response to microbial infection (Dixon et al., 1983 1983 ), it should not be surprising that they have been found in vitro to be effective antimicrobial substances against a wide array of microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls (Cowan, 1999 1999 ). More lipophilic flavonoids may also disrupt microbial membranes (Tsuchiya et al., 1996 1996 ). Alkaloids isolated from plant are commonly found to have antimicrobial properties (Omulokoli et al., 1997 1997 ). Alkaloids may be useful against HIV infection (Sethi, 1979 1979 ) as well as intestinal infections associated with AIDS (McDevitt et al., 1996 1996 ). Berberine and harmane are important representatives of the alkaloid group. The mechanism of action of highly aromatic planar quaternary alkaloids such as berberine and harmane is attributed to their ability to intercalate with DNA (Phillipson and O′Neill, 1987 1987 ). The best MIC values against most bacteria were seen in the ethanol extract from the plant fruits followed by leaf extracts and finally root extracts which were resisted by most of the Gram-negative bacteria specially S . marcescens . Candida albicans remains the most common infection-causing fungus; about 45% of clinical fungal infections are caused by C . albicans (Gupta et al., 2004 2004 ). The present study shows that ethanol extract from T . terrestris fruits had potent anti- C . albicans activity with an MIC value of 0.62 mg/ml. The antifungal activity of T . terrestris may be attributed to various chemicals detectable in its extracts such as saponins (Zhang et al., 2006 2006 ). The action mechanisms of saponins may lie in damage to the membrane and leakage of cellular materials, ultimately leading to cell death (Mshvildadze et al., 2000 2000 ). All parts (fruits, stems plus leaves and roots) of Turkish and Iranian T . terrestris showed antibacterial activity against Enterococcus faecalis , S . aureus , E . coli and P . aeruginosa (Abbasoglu and Tosun, 1994 1994 ; Kianbakht and Jahaniani, 2003 2003 ) in contrast to aerial parts of Yemeni T . terrestris which had no detectable antibacterial activity against these bacteria (Awadh-Ali et al., 2001 2001 ), and only fruits and leaves of Indian T . terrestris were active exclusively against E . coli and S . aureus (Williamson, 2002 2002 ). It can be argued that antibacterial activity of Iraqi T . terrestris is similar to Turkish and Iranian T . terrestris . Different results concerning the antibacterial activity of T . terrestris might be due to different geographic sources of the plant used, different types of strains used, and different assay methods. CONCLUSION 1 Since Iraqi T . terrestris demonstrated activity against the most prevalent Gram-negative bacteria in urinary infections, namely E . coli , the use of the plant as a urinary anti-infective is validated, scientifically supported by the results obtained in this work. References U Abbasoglu , F Tosun . Antimicrobial activity of Tribulus terrestris L. growing in Turkey . Hacettepe Universitesi Eczacilik Fakultesi Dergisi , 1994 . 14 81 - 85 . . Antimicrobial activity of Tribulus terrestris L. growing in Turkey K Abeywickrama , GA Bean . Toxigenic Aspergillus flavus and aflatoxins in Sri Lankan medicinal plant material . Mycopa-thologia , 1991 . 113 187 - 190 . . Toxigenic Aspergillus flavus and aflatoxins in Sri Lankan medicinal plant material NA Awadh-Ali , WD Jülich , C Kusnick , U Lindequist . Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities . J Ethnopharmacol , 2001 . 74 ( 2 ): 173 - 179 . DOI: 10.1016/S03788741(00)00364-0 http://doi.org/10.1016/S03788741(00)00364-0 . Screening of Yemeni medicinal plants for antibacterial and cytotoxic activities CA Bourke , MJ Carrigan , RJ Dixon . Upper motor neuron effects in sheep of some betacarotine alkaloids identified in Zygophyllaceous plants . Aust Vet J , 1990 . 67 248 - 251 . . Upper motor neuron effects in sheep of some betacarotine alkaloids identified in Zygophyllaceous plants LV Buwa , J van Staden . Antibacterial and antifungal activity of traditional medicinal plants used against venereal diseases . J Ethnopharmacol , 2006 . 103 ( 1 ): 139 - 142 . DOI: 10.1016/j.jep.2005.09.020 http://doi.org/10.1016/j.jep.2005.09.020 . Antibacterial and antifungal activity of traditional medicinal plants used against venereal diseases SC Chhabra , JF Shao , EN Mshiu . Antifungal activity among traditionally used herbs in Tanzania . The Dar Medicinal Journal , 1982 . 9 68 - 73 . . Antifungal activity among traditionally used herbs in Tanzania M Cowan . Plant products as antimicrobial agents . Clin Microbiol Rev , 1999 . 12 ( 4 ): 564 - 582 . . Plant products as antimicrobial agents R Dixon , P Dey , C Lamb . Phytoalexins: Enzymology and molecular biology . Adv Enzymol , 1983 . 55 1 - 69 . . Phytoalexins: Enzymology and molecular biology JN Eloff . A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria . Planta Medica , 1998 . 64 ( 8 ): 711 - 713 . DOI: 10.1055/s-2006-957563 http://doi.org/10.1055/s-2006-957563 . A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria N Gupta , A Haque , A Lattif , R Narayan , G Mukhopadhyay , R Prasad . Epidemiology and molecular typing of Candida isolates from burn patients . Mycopathologia , 2004 . 158 ( 4 ): 397 - 405 . DOI: 10.1007/s11046004-1820-x http://doi.org/10.1007/s11046004-1820-x . Epidemiology and molecular typing of Candida isolates from burn patients O Kandil , NM Radwan , AB Hassan , AM Amer , HA El-Banna . Extracts and fractions of Thymus capitatus exhibit antimicrobial activities . J Ethnopharmacol , 1994 . 44 ( 1 ): 19 - 24 . DOI: 10.1016/03788741(94)90094-9 http://doi.org/10.1016/03788741(94)90094-9 . Extracts and fractions of Thymus capitatus exhibit antimicrobial activities S Kianbakht , F Jahaniani . Evaluation of antibacterial activity of Tribulus terrestris L. growing in Iran . Iranian J Pharmacol Ther , 2003 . 2 22 - 24 . . Evaluation of antibacterial activity of Tribulus terrestris L. growing in Iran G Killeen , C Madigan , C Connolly , G Walsh , C Clark , M Hynes , B Timmins , P James , D Headon , R Power . Antimicrobial saponins of Yucca schidigera and the implications of their in vitro properties for their in vivo impact . J Agric Food Chem , 1998 . 46 ( 8 ): 3178 - 3186 . DOI: 10.1021/jf970928j http://doi.org/10.1021/jf970928j . Antimicrobial saponins of Yucca schidigera and the implications of their in vitro properties for their in vivo impact EW Koneman . Color Atlas and Textbook of Diagnostic Microbiology , 1995 . 2nd Ed : Roma A. Delfino , 550 - 605 . . Color Atlas and Textbook of Diagnostic Microbiology I Kostova , D Dinchev . Saponins in Tribulus terrestris —chemistry and bioactivity . Phytochem Rev , 2005 . 4 ( 2-3 ): 111 - 137 . DOI: 10.1007/s11101-005-2833-x http://doi.org/10.1007/s11101-005-2833-x . Saponins in Tribulus terrestris—chemistry and bioactivity M Lacaille-Dubois , H Wagner . A review of the biological and pharmacological activities of saponins . Phytomedicine , 1996 . 2 ( 4 ): 363 - 386 . . A review of the biological and pharmacological activities of saponins A le Grand , PA Wondergem , R Verpoorte , JL Pousset . Anti-infectious phytotherapies of the tree-savannah of Senegal (West-Africa) II. Antimicrobial activity of 33 species . J Ethnopharmacol , 1988 . 22 ( 1 ): 25 - 31 . DOI: 10.1016/0378-8741(88)90227-9 http://doi.org/10.1016/0378-8741(88)90227-9 . Anti-infectious phytotherapies of the tree-savannah of Senegal (West-Africa) II. Antimicrobial activity of 33 species SH Majeed , MJ Mahmood . Herbs and Medicinal Plants in Iraq between Traditional Medicine and Scientific Research , 1988 . 1st Ed : Baghdad Dar Al-Thaowra for Publishing , 40 (in Arabic) . Herbs and Medicinal Plants in Iraq between Traditional Medicine and Scientific Research J McDevitt , D Schneider , S Katiyar , 等 . Berberine: A Candidate for the Treatment of Diarrhea in AIDS Patients . Program and Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy , 1996 . : Washington, DC American Society for Microbiology , Abstr. 175 . Berberine: A Candidate for the Treatment of Diarrhea in AIDS Patients J Milgate , D Roberts . The nutritional and biological significance of saponins . Nutr Res , 1995 . 15 ( 8 ): 1223 - 1249 . DOI: 10.1016/0271-5317(95)00081-S http://doi.org/10.1016/0271-5317(95)00081-S . The nutritional and biological significance of saponins RA Mothana , U Lindequist . Antimicrobial activity of some medicinal plants of the island Soqotra . J Ethnopharmacol , 2005 . 96 ( 1-2 ): 177 - 181 . DOI: 10.1016/j.jep.2004.09.006 http://doi.org/10.1016/j.jep.2004.09.006 . Antimicrobial activity of some medicinal plants of the island Soqotra V Mshvildadze , A Favel , F Delmas , R Elias , R Faure , Q Decanosidze , E Kemertelidze , G Balansard . Antifungal and antiprotozoal activities of saponins from Hedera colchica . Pharmazie , 2000 . 55 ( 4 ): 325 - 326 . . Antifungal and antiprotozoal activities of saponins from Hedera colchica NCCLS (National Committee for Clinical Laboratory Standards) Performance Standards for Anti-Microbial Susceptibility Testing: Eleventh Informational Supplement , 2001 . : Wayne, PA, USA National Committee for Clinical Laboratory Standard , NCCLS, Document M100-S11 . Performance Standards for Anti-Microbial Susceptibility Testing: Eleventh Informational Supplement E Omulokoli , B Khan , S Chhabra . Antiplasmodial activity of four Kenyan medicinal plants . J Ethnopharmacol , 1997 . 56 ( 2 ): 133 - 137 . DOI: 10.1016/S03788741(97)01521-3 http://doi.org/10.1016/S03788741(97)01521-3 . Antiplasmodial activity of four Kenyan medicinal plants J Phillipson , M O′Neill . New leads to the treatment of protozoal infections based on natural product molecules . Acta Pharm Nord , 1987 . 1 131 - 144 . . New leads to the treatment of protozoal infections based on natural product molecules SM Saad Aldein . Medicinal Herbs , 1986 . 1st Ed : Baghdad Dar Al-Shoun Al-Thaqafia Al-Aama for Publishing , 70 (in Arabic) . Medicinal Herbs NA Saleh , AA Ahmed , MF Abdalla . Flavonoid glycosides of Tribulus pentandrus and T . terrestris Phytochemistry , 1982 . 21 ( 8 ): 1995 - 2000 . DOI: 10.1016/00319422(82)83030-6 http://doi.org/10.1016/00319422(82)83030-6 . Flavonoid glycosides of Tribulus pentandrus and T M Sethi . Inhibition of reverse transcriptase activity by benzophenanthridine alkaloids . J Nat Prod , 1979 . 42 ( 2 ): 187 - 196 . DOI: 10.1021/np50002a010 http://doi.org/10.1021/np50002a010 . Inhibition of reverse transcriptase activity by benzophenanthridine alkaloids H Tsuchiya , M Sato , T Miyazaki , S Fujiwara , S Tanigaki , M Ohyama , T Tanaka , M Iinuma . Comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant Staphylococcus aureus . J Ethnopharmacol , 1996 . 50 ( 1 ): 27 - 34 . DOI: 10.1016/0378-8741(96)85514-0 http://doi.org/10.1016/0378-8741(96)85514-0 . Comparative study on the antibacterial activity of phytochemical flavanones against methicillin-resistant Staphylococcus aureus EM Williamson . Major Herbs of Ayurveda , 2002 . : China Churchill-Livingstone , 312 . Major Herbs of Ayurveda YX Xu , HS Chen , HQ Liang , ZB Gu , WY Lui , WN Leung , TJ Li . Three new saponins from Tribulus terrestris . Planta Med , 2000 . 66 ( 6 ): 545 - 550 . DOI: 10.1055/s-2000-8609 http://doi.org/10.1055/s-2000-8609 . Three new saponins from Tribulus terrestris JD Zhang , Z Xu , YB Cao , HS Chen , L Yan , MM An , PH Gao , Y Wang , XM Jia , YX Jiang . Antifungal activities and action mechanisms of compounds from Tribulus terrestris L. . J Ethnopharmacol , 2006 . 103 ( 1 ): 76 - 84 . DOI: 10.1016/j.jep.2005.07.006 http://doi.org/10.1016/j.jep.2005.07.006 . Antifungal activities and action mechanisms of compounds from Tribulus terrestris L. The authors are grateful to Dr. Abdulazeez Shekho for the identification of plant species. We also thank all the staff of Microbiology Laboratory in Department of Biology, College of Education, University of Mosul, Iraq for making this study possible. 10.1631/jzus.B0720251 2007-11-11 2007-12-26 2008-01-16 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 09