PHARMACEUTICAL AND BIOLOGICAL EVALUATIONS February 2016; vol. 3 (Issue 1): xxx-xxx. www.onlinepbe.com ISSN 2394-0859 Research Article Comparative study of phytochemical and synergistic antibacterial activity of Tribulus terrestris (L.) and Pandiaka heudelotii (Moq.) Hien on some clinical bacterial isolates S. Abubakar1,2*, B. O. Akanbi3, V. A. Etim1, O. Segun2, J. C. Ogbu3 1 Biotechnology and Genetic Engineering Laboratory, Sheda Science and Technology Complex, P.M.B. 186, Garki, Abuja, Nigeria 2 Department of Biological Sciences, University of Abuja, P.M.B 117, Nigeria 3 Department of Microbiology, University of Abuja, P.M.B. 117, Abuja, Nigeria ABSTRACT *For correspondence Dr. S. Abubakar, Biotechnology and Genetic Engineering Laboratory, Sheda Science and Technology Complex (SHESTCO), P.M.B. 186, Garki, Abuja, Nigeria. Email: salisuabubakar99 @yahoo.com Objective: This research work focus on synergistic effect of ethnomedicinal plants namely, Tribulus terrestris and Pandiaka heudelotii methanolic and aqueous leaves extracts on two species of clinical bacterial isolates namely, Staphylococcus aureus and Escherichia coli, using disc diffusion method. Methods: The leaves were collected, air dried, pulverized and extracted by maceration. The extracts were subjected to preliminary phytochemical screening using standard procedures. The susceptibility test of individual plant as well as the synergistic effect of combinations of the plants extract was carried out using disc diffusion method. Results: Collectively the plant extracts revealed the presence of alkaloids, flavonoids, steroids, saponins, glycosides, amino acids, reducing sugars and tannins. The in-vitro antimicrobial activity of the crude extracts was examined against both the plants leaves extract showed antibacterial activity against the tested microorganisms. However, some of the eluents collected from methanolic extract fractions of column chromatography were also showed activity on tested organisms. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the leaves extract were determined within the ranges of (125 to 250 mg/ml) and (250 to 500 mg/ml) respectively, the highest synergistic activity was attained against E. coli by combined both methanolic P. heudelotii and T. terrestris extract which showed a zone of (27.54±00). Conclusions: The result indicates antibacterial activity of the leaves extract of P. heudelotii in combination with that of T. terrestris against the clinical bacterial isolates tested and laid credence to the advantage of the combination of the two plants over the two other individual forms. Statistically using, Microsoft excel version 2010. Received: 19 January 2016 Accepted: 08 February 2016 Keywords: Antibacterial, Phytochemical screening, Synergistic effect, Tribulus terrestris, Pandiaka heudelotii ©Pharmaceutical and Biological Evaluations 1 Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. ‘maasun kadangaree’ (lizard’s spears). Traditionally in Burkina Faso P. heudelottii has being used as a tonic drink for parturient, as a spasmolytic, in the treatment of, blennorrhoea, annexite salpingitis, in the treatment of women genital apparatus inflammation, in the treatment of malaria 13. The plant is highly esteemed by traditional healers and used in treatment of asthma, bleeding in facilitating delivery, boils, bronchitis, cold, cough, colic, debility, dropsy, dog bite, dysentery, ear complications, headache, leucoderma, pneumonia, renal complications, scorpion bite, snake bite, and skin disease. Traditional healers claim that addition of this plant would enhance the efficacy of any drug of plant origin. Introduction Herbal medicine represents one of the most important fields of traditional medicine all over the world. To promote the proper use of herbal medicine and determine their potential as source of new drugs, it is essential to study medicinal plants which have folklore reputation in more intensified way. However, over 80% of the World’s population uses plant as their primary source of medication.1 T. terrestris is a flowering plant in the family Zygophyllaceae, blooms with small yellow flowers containing only 5 petals. It grows with multiple stems that spring out from one crown. It has pinnate leaves made of two rows of little leaflets which are arranged opposite each other along the stem. The fruit appears a week after blooming; its seeds are firm and stiff and have two sharp spines. The common name for T. terrestris is devil’s eyelashes, puncture vine or cat’s head.2 The aim of this present work therefore, is to evaluate the implication of the individual and synergistic antibacterial activity of two ethnomedicinal plant of Nigeria namely: - T. terrestris and P. heudelotii towards achieving greater potency at significantly well-defined concentration ratios. Based on our knowledge, little or nothing is known about the combined activity of the Nigerian T. terrestris and P. heudelotii or has not been documented. T. terrestris is used in folk medicine as tonic, aphrodisiac, analgesic, astringent, stomachic, anti-hypertensive, diuretic, lithontriptic and urinary anti-infective.3 The antimicrobial effects of T. terrestris from other countries have been reported. Turkish and Iranian spp. showed activity against all test bacteria oral pathogens4 while the Yemeni species showed no detectable antimicrobial activity against any of the reference bacteria.2 The T. terrestris extract is also used for urinary dysfunction, asthma and opthalmia and different,5,6 and has been shown to have antihypertensive and vasodilatory properties.7,8 It may also protect against oxidative stress and exhibits antitumor, cytotoxic, antifungal and antihelmentic properties.9,10 T. terrestris is also used in folk medicine as tonic, aphrodisiac, analgesic, astringent, stomachic, anti-hypertensive, diuretic, lithon-triptic, and urinary antiinfective.11 Materials and Methods This research work was carried out in the Sheda Science and Technology Complex/Microbiology laboratory of the University of Abuja Main Campus, Gwagwalada, Abuja, FCT, North Central Zone of Nigeria. The FCT is located at Longitude 9 ºN and Latitude 7 ºE with a land mass of 7, 315 sq km of which Gwagwalada occupies 1, 043 sq km. It is situated within the Savannah region with moderate climatic conditions. Collection/identification of plant materials Fresh leaves of P. heudelotii and T. terrestris were collected in the month of August, 2013. The plants were further identified and authenticated by a trained taxonomist Mr. O. Segun of biological sciences department, University of Abuja, Gwagwalada, Abuja, Nigeria. The authenticated specimens were deposited at the biological garden, University of Abuja, Nigeria. P. heudelotii is a long branched herb, 2-3 ft, high; tips of calyx and bracteole often reddish.12 Belongs to the Amaranthaceae family, occurring from Senegal to South Nigeria and across central Africa to Sudan. Its common name in Hausa is ©Pharmaceutical and Biological Evaluations 2 Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. and finally allowed to stand for two weeks with constant shaking at regular intervals at room temperature (30±5oC). Thereafter, the percolates were filtered using Whatman No. 1 filter paper and solvent (methanol) were evaporated at room temperature to obtain the methanolic extract of the leaves, which was subsequently stored in sterile bottles until required for use. The same procedures was used to obtained the aqueous extract. Sterilizations This was carried out by use of autoclave (Serado, Model YX-241D), which was done at a temperature of 1210C, pressure of 15psi for fifteen minutes. It was used to sterilize media, glass wares before use. Collection and confirmatory test of the isolates The test organisms (Staph. and E. coli) were clinical isolates obtained from the diagnostic laboratory of University of Abuja Teaching Hospital, Gwagwalada, Abuja, Nigeria.The identity and purity of the test organisms were confirmed as follows: Extracts sterilization and concentration The stock solutions of the P. heudelotii and T. terrestris extracts (both aqueous and methanolic) were prepared in screw capped bijoux bottles. 0.5 g of each of the extract powder was weighed on an analytical weighing balance and dissolved in 1 ml of methanol to arrive at 500 mg/ml concentration of stock solution. The stock solutions were filter-sterilized using a membrane filter (pore size 0.45 µm) (Sterlitech). Three varied extract concentrations 500 mg/ml, 250 mg/ml and 125 mg/ml, were prepared from the stock solutions using double dilution. The sterile extracts were stored aseptically under refrigeration temperature at 40C for further use.17 Staphylococcus aureus An inoculum of the test organism was cultured on mannitol salt agar. The growth produced yellowish colonies. This is due to their ability to use mannitol as food source leading to the production of acidic byproducts of fermentation that will lower the pH of the medium and caused the pH indicator phenol red to turn yellow. The colonies were further subjected to Gram staining and were gram positive. The test organism was inoculated on a nutrient agar slant in a bijoux bottle and refrigerated at 4oC.14 Phytochemical screening of the extracts The aqueous and methanolic extracts of P. heudelotii and T. terrestris were analysed for the presence of alkaloids, flavonoids, tannins, steroids, saponins, glycosides, amino acids and reducing sugars using method adopted from.18-20 Escherichia coli The isolate obtained was plated on Eosin methylene blue agar (EMB) by streaking from 18-24 hours. Colonies with distinctive metallic green sheen were observed which indicate a positive result for E. coli. The colonies were further subjected to Gram staining and was gram negative. The test organism was inoculated on a nutrient agar slant in a bijoux bottle and refrigerated at 4oC.15 Antibacterial testing of the extracts Agar disc diffusion method modified by Khan and Saeed was employed.21 The agar-disc diffusion method was employed to determine the growth inhibition abilities of the test organisms by the extracts. Discs of about 6mm were made from Whatman’s No. 1 filter paper using paper puncher. The discs were transferred into bijou bottles and sterilized in the oven at 121oC for 15mins.The sterilized discs were aseptically placed into each of the labeled bijou bottles to obtained a disc potency of 500 mg/disc, 250 mg/disc, and 125 mg/disc respectively of each extracts. Mueller-Hinton agar was prepared according to manufacturer’s guideline and 25 ml Sample preparation and extractions The fresh leaves were allowed to dry under shade to constant weight and pulverized using clean wooden mortar and pestle. The contents were stored in air-tight containers until required for further analysis.16 50 g of the fine powder of the leaves of P. heudelotii and T. terrestris was weighed and suspended in a bottle after which it was percolated with 500 ml of 95% methanol ©Pharmaceutical and Biological Evaluations 3 Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. each was poured into sterile Petri dishes. The agar was allowed to solidify and dry. The agar was aseptically inoculated uniformly with the test organism using streak method, it was allowed undisturbed for 30 minutes. With the aid of sterile forceps, impregnated paper discs containing P. heudelotii and T. terrestris extracts at different concentrations were arranged radially and pressed firmly onto the inoculated agar surface to ensure even contact. Each disc was sufficiently spaced out and kept at least 15mm from the edge of the plate and 25 mm from disc to disc to prevent overlapping of zones and the plates were incubated at 37oC for 24 hours. With the aid of a meter rule, the zone diameters of inhibition of growths were measured and recorded to the nearest millimeter.17,22 The same procedure where applied for the synergy and for the different eluents collected from column chromatography. Synergistic activity The synergistic activity was achieved by combining the different extracts at 1:1,T. terrestris methanolic + P. heudelotii aqueous, P. heudelotii aqueous + T. terrestris aqueous, P. heudelotii methanolic + T. terrestris aqueous and T. terrestris methanolic + P. heudelotii methanolic, to obtained disc potency of 500mg/disc of each combinations. Thin layer chromatography The crude methanolic and aqueous extracts of P. heudelotii and T .terrestris were subjected to thin layer chromatographic analysis using different solvent systems. The spots were visualized using UV-lamp. Column chromatography The crude methanolic extract of P. heudelotii and T. terrestris as observed in TLC plate indicated some components at 70/30 ethylacetate/n-hexane, 50/50 ethylacetate/nhexane, completely soluble methanol; hence the need for proper separation with column chromatography. The glass column was washed, dried, and clamped vertically onto a retort stand; the column was packed using the dry pack method. A piece of cotton wool was inserted into a clean dry glass column followed by 20 g adsorbent silica gel of mesh size 120-150 mm was slowly poured into the column. The column was tapped gently to give a uniform packing. 5 g of the crude extract was pre-absorbed with silica gel which was allowed to dry and then loaded into the glass column. Silica gel was again added on top of the sample layer before addition of solvent. The elution started with 100% nhexane, n-hexane / ethyl acetate 90:10, ethyl acetate / n-hexane 80:20, ethyl acetate (100%), methanol (100%). Methanol washed out the extract completely due to the high polarity of the sample26. Minimum inhibitory concentration (MIC) MIC is defined as the lowest concentration where no visible turbidity is observed in the test tube (bacteriostatic concentration). The broth dilution technique was utilized where the plant extract was prepared to the highest concentration of 500 mg/ml (stock concentration) in sterile distilled water and serially diluted to 250 mg/ml, 125 mg/ml, 62.5 mg/ml and 31.25 mg/ml.0.2 ml suspension of the test organisms were transferred into 20 ml Mueller Hinton broth and 0.2 ml of each extracts concentrations were added and the test tubes were incubated for 24 hours at 37 ºC, the test tubes were observed for turbidity with the aid of a spectrophotometer. The least absorbance value was determined and noted as the MIC value.23 Minimum bactericidal concentration (MBC) The MBC is the lowest concentration of antibacterial substance required to produce a sterile culture.24 This was determined from the broth dilution resulting from the MIC tubes by streaking the contents of the tubes and incubated at 37ºC for 18 hours. The lowest concentration of the extract which showed no bacterial growth was noted and recorded as the MBC.25 ©Pharmaceutical and Biological Evaluations Statistical analysis Statistical analysis was carried out using Microsoft excel version 2010. An ANOVA (Analysis of Variance) was conducted to find the 4 Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. significant differences of the extracts at (P <0.05). Table 1 showed the physical characteristics of the extracts. These includes, the solvent of extraction used, initial weight of the extracts, final weight of the extracts, percentage yield of the extracts, colour and the texture of the extracts observed. Results and Discussion Physical characteristics and percentage yield of P. heudelotii and T. terrestris extracts. Table 1: Physical characteristics of the extracts. Plants Solvents T. terrestris P. heudelotii T. terrestris P. heudelotii Methanol Methanol Aqueous Aqueous I.W- Initial weight I.W (G) F.W (G) 25.0 25.0 25.0 25.0 2.7 2.7 4.8 4.2 %Yield 9.3 8.0 10.0 12.2 Colour Texture Dark green Dark green Light brown Dark brown gluey gluey Powdery gluey FW- Final weight Table 2: Phytochemical constituents of T. terrestris and P. heudelotii extracts. Phytochemical Alkaloids Flavonoids Steroids Amino acids Saponins Glycosides Tannins Reducing sugars T. terrestris Methanol + + + + + + P. heudelotii Methanol + + + + + + Aqueous + + + + + Aqueous + + + + + - + = Presents; - = Absents Table 3: Average zone of inhibition in (mm) of T. terrestris and P. heudelotii extracts. Test organism Extract Staphylococcus aureus PA PM TA TM PA PM TA TM Escherichia coli Extract concentrations in (mg/disc) 500mg/disc 250mg/disc 125mg/disc 10.13±01 14.51±00 14.04±04 19.23±00 14.14±00 15.53±00 14.60±01 19.88±00 8.42±00 11.23±05 12.83±01 11.41±00 10.79±00 12.85±01 10.58±03 17.94±00 NA= No Activity ©Pharmaceutical and Biological Evaluations 5 NA 9.27±03 8.27±00 NA 7.18±04 9.72±00 NA 9.72±00 Positive control (streptomycin) 10µg/disc 22.12±00 22.12±00 22.12±00 22.12±01 21.23±00 21.23±00 21.23±00 21.23±00 Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. Table 2 showed the results of qualitative phytochemical screening of both the methanolic and aqueous extracts of T. terrestris and P. heudelotii extracts, each solvents divulged the present of various phytochemical constituents while Alkaloids and steroids were present in both the solvents used for the extractions. zones of inhibition in millimeter using disk diffusion method (mean±SD) of the triplicate. Table 6: Activity of the different fractions of methanolic T. terrestris on S. aureus and E. coli. Table 4: Synergistic average zone of inhibition in (mm) of T. terrestris and P. heudelotii extracts. Test organis ms E. coli S. aureus Negativ e control (DMSO ) Concentration (500 mg/disc) TM/PA PA/TA PM/TM PM/TA 19.53± 00 20.55± 00 NA 19.65± 01 18.00± 01 NA 27.54± 00 25.50± 03 NA 7:3 hex : ethyl 8:2 ethyl : hex 9:1 ethyl : hex 100% methanol + _ + _ + + + + Table 1 showed the total percentage (%) yield of the two solvent used for the extraction namely; methanol and aqueous, both the aqueous and methanolic extract yield (10.0 g and 9.3 g) and (12.2 g and 8.0 g) for T. terrestris and P. heudelotii respectively, from the original weight of 25 g. The qualitative phytochemical analysis of the T. terrestris and P. heudelotii leaf extracts in Table 2. The result showed that the aqueous and methanolic leaf extracts of T. terrestris contains alkaloids, steroids, saponins and reducing sugars with the addition of flavonoids and amino acid in methanolic extract while glycosides was only present in aqueous extract. The methanolic and aqueous leaf extract of P. heudelotii contains alkaloids, steroids, amino acids, and reducing sugars in addition flavonoids and tannins were present in methanolic extract while glycoside was present only on aqueous extract. E. coli. _ _ _ + + hex: n-hexane; ethyl: ethylacetate Table 3 and 4 showed the susceptibility test of individual extract of P. heudelotii and T. terrestris as well as the combinations (synergy) of the two plants extract on the two clinical isolates used S. aureus and E. coli at various concentrations. The extract showed broad spectrum action on both gram negative and gram positive bacteria, based on different concentrations of the extracts employed with ©Pharmaceutical and Biological Evaluations E. coli _ Table 5 and 6 showed the susceptibility of the tested clinical isolates on different fractions of methanolic extracts for both P. heudelotii and T. terrestris. Table 5: Activity of the different fractions of methanolic P. heudelotii on S. aureus and E. coli. S. aureus _ + _ + _ S. aureus _ hex: n-hexane; ethyl: ethylacetate 18.21± 03 20.54± 00 NA PA: P. heudelotii (aqueous). PM: P. heudelotii (methanol). TA: T. terrestris (aqueous). TM: T. terrestris (methanol); DMSO- Dimethylsulfoxide ; NA- No Activity Fractions 100% hex 7:3 hex : ethyl 8:2 ethyl : hex 9:1 ethyl : hex 100% methanol Fractions 100% hex The two extracts showed the presence of most of the secondary metabolites such as: - Tannins, steroids, saponins, alkaloids, amino acid, flavonoids and reducing sugar. Alkaloid contributes to a plant species fitness of survival.27 They often have pharmacological effects and are used as medication and recreational drugs.28 Similarly, tannins are well 6 Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. known for their antioxidant and antimicrobial properties as well as skin regeneration, antiinflammatory and diuretic properties.29,30 Flavonoids are widely recognized for exerting antioxidant, antimicrobial, anti-carcinogenic and antitumor properties.29,30 Many pharmacological activities such as antibiotic, antifungal, antiviral, hepato protective, and anti-inflammatory and ant-ulcer activities have been reported for saponins31 and steroids have been reported to exert analgesic properties.32 extract on both the test isolates were unable to determine within the prepared concentrations. The antimicrobial activity demonstrated by the combination of the two extracts is probably due to synergizing effect of the phytochemical result obtained. The bioactivity was prominent against E. coli at 500 mg/disc by PM/TM combination with 27.54±00 mm zone of inhibition. This clearly exhibits the advantage of the combination of the two plants over the two other individual forms coupled with enhanced synergistic activity. The antimicrobial susceptibility pattern of the crude extracts showed in table 3 the results shows that the methanolic and aqueous extracts of both T. terrestris and P. heudelotii shows activity on both the test organisms, which signified the broad action of the extracts. The methanolic extracts of both the samples shows high zone of inhibition ranges between (19.23±0, 14.51±00) on S. aureus and (19.88±00, 15.53±00) at 500 mg/disc each respectively. The aqueous extracts also illustrate zone within the range of (10.13±01 to14.04±04) on both the test isolates at 500 mg/disc. In similar study that involves the combination of two different extracts A. comosus and Allium sativum against S. typhi, the activity of each extract was significantly different from their combination and this shows an interaction between the two plants extracts.32 Soleimanpour4 evaluated the antimicrobial activities of an ethanol extract of T. terrestris alone and in combination with Capsella bursa-pastoris and Glycyrrhiza glabra against six pathogens namely Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus, Enterococcus faecalis S. aureus, and E. coli and the results shown that mixed extracts were more effective against all bacteria than any of the cases alone that indicates the synergistic effect between these three extracts. The MIC and MBC on table 7 indicates that both the methanolic and aqueous extract the MIC where determined within the ranges of 125 mg/ml to 250 mg/ml while the MBC where determined within the ranges of 250 mg/ml to 500 mg/ml. The MBC of P. Heudelotii aqueous Table 7: Minimum inhibitory/Bactericidal Concentration of P. heudelotii and T. terrestris extracts on S. aureus and E. coli. Test organism Extracts 500 mg/ml 250 mg/ml 125 mg/ml 62.5 mg/ml Staphylococcus aureus PA PM TA TM PA PM TA TM MIC MBC MIC MIC MIC MBC + MIC + MIC MIC + + MIC ++ ++ ++ ++ ++ + ++ + Escherichia coli ND MBC MBC ND MBC MBC - ND= MBC Not determined at prepared Concentrations, += Mild turbidity, ++=Strong turbidity ©Pharmaceutical and Biological Evaluations 7 31.25 mg/ml ++ ++ ++ ++ ++ ++ ++ ++ Abubakar S. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (1): xxx-xxx. part of Nara desert, Pakistan. Pak J Bot. 2010;42(2):839-51. 7. Phillips, S.J., R.P. Anderson and R.E. Schapire. Maximum entropy modeling of species geographic distributions. Ecol. Model., 190: 2006, 231-259. 8. Sharifi, A.M., R. Darabi and N. Akbarloo. Study of antihypertensive mechanism of Tribulus terrestris in 2K1C hypertensive rats: role of tissue ACE activity. Life Sci.,73 (23):2003, 2963-71. 9. Perveen, A., R. Abid and R. Fatima. Stomatal types of some dicots within flora of Karachi, Pakistan. Pak. J. Bot., 39(4):2007, 1017-1023. 10. Hu K, Yao X. Protodioscin (NSC-698 796): its spectrum of cytotoxicity against sixty human cancer cell lines in an anticancer drug screen panel. Planta Med, 2002;68:297-301. 11. Kianbakht S, Jahaniani F. Evaluation of Antibacterial Activity of Tribulus terrestris L., growing in Iran. Iranian J Pharmacol Therapeutics. 2003;2:22-4. 12. http://www.prota4u.org/protav8.asp?p=Pand iaka+heudelotii 13. Nacoulma OG. Plantes médicinales pratiques medicinales du Burkina Faso: cas du plateau central. Tome II.Thèse de doctorat d’état ès sciences naturelles, Université de Ouagadougou. 1996. 14. Clauditz A, Resch A, Wieland KP, Peschel A, Götz, F. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infection and immunity. 2006;74(8):4950–3. 15. Oyeleke SB, Manga BS. Essentials of Laboratory Practical in Microbiology. 1st edition. Tobest publisher. 2008; 94. 16. Fatope AO, Ibrahim H, Takeda Y. Screening of higher plants reputed as pesticides using brine shrimp lethality bioassay. International Journal of Pharmacognosy. 1993;31:250-6. 17. Okoli S, Iroegbu CU. Invitro antibacterial activities. African Journal of Biotechnology. 2004;4(9):946-52. 18. Harborne JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis, 2nd edn. Chapman and Hall, New York. 1984. Conclusions Based on the result of this research, there is clear evidence that the two plants can be a good source of bioactive substances that could possess broad spectrum activity. Therefore, there is an increasing need for researchers to investigate the synergistic capacity of plants. The results of the present research seem to be promising and may enhance the natural product uses, showing the potentiality of P. heudelotii and T. terrestris in the treatment of various infectious diseases caused by bacteria. It also indicates the need for understanding of synergism mechanism is fundamental to development of pharmacological agents to treat diseases causes by various bacteria using medicinal plants. Funding: No funding sources Conflict of interest: None declared References 1. Parekh J, Chanda S. In vitro antibacterial 2. 3. 4. 5. 6. activity of the crude methanol extract of woodfordi Fructose kurz. Flower (lythraceae). Brazilian Journal of Microbiology. 2007;38:204-7. Hashim S, Bakht T, Khan BM, Jan J. Medicinal properties, phyto-chemistry and pharmacology of tribulus terrestris L. (Zygophyllaceae). Pak J Bot. 2014;46(1):399-404. Ody P. The Complete Guide Medicinal Herbal. London: Dorling Kindersley; 2000, 223. 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Saunders; 2002: 481. 21. Khan FZ, Saeed A. Antimicrobial potentials of the constituents of sysmbrium irio: Hamdadmedicus. 2000;3(1):22-5. 22. Betoni JEC, Mantouani RP, Barbosa LN, Stasi LCD, Fernanda. A Synergism between plant extract and antimicrobial drugs used on S. aureus diseases. Memórias do Instituto Oswaldo Cruz, Rio De Janerio. 2006;101:387-90. 23. Usman H, Abdulrahman FI, Ladan AH. Phytochemical and Antimicrobial Evaluation of Tribulus terrestris L. (zygophylaceae). Growingin Nigeria. Research on Journal of Biological Science, MedwellJournals. 2007;2(3):244–7. 24. Cheesbrough M. Medicinal laboratory manual for tropical countries 2. That ford Press Limited ELBS Cambrige. 2000, 196205. 25. Vollekova, AD, Kostalova, Sochorova R. Isoquinoline Alkaloids from Mahonia aquifolium stem bark as active against Malassezia Sp. Folia Microbiology. 2001;46:107–11. 26. Salisu BK, Usman LA, Sani A, Muhammad NO, Akolade JO. Chemical composition and ©Pharmaceutical and Biological Evaluations 9