IN-VITRO ANTIBACTERIAL ACTIVITIES AND PRELIMINARY PHYTOCHEMICAL SCREENING OF THE AQUEOUS AND ETHANOLIC EXTRACTS OF ZINGIBER OFFICINALE

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1 Ife Jurnal f Science vl. 15, n. 1 (2013) 93 IN-VITRO ANTIBACTERIAL ACTIVITIES AND PRELIMINARY PHYTOCHEMICAL SCREENING OF THE AQUEOUS AND ETHANOLIC EXTRACTS OF ZINGIBER OFFICINALE Bashir, S.F., * A.H. Kaw, J.A. Bala and Y.U. Dabai Department f Micrbilgy, Faculty f Science, Department f Medical Labratry Science, Faculty f Medicine, Bayer University, PMB 3011, Kan, Nigeria 3 Department f Public Health and Preventive Medicine, Faculty f Medicine, Usmanu Danfdiy University, PMB 2346, Skt, Nigeria (*Crrespndence authr: ahkaw@yah.cm; ) (Received: March 2013; Accepted: April 2013) 1 2 ABSTRACT Studies n the in-vitr antibacterial activities and phytchemical screening f the aqueus and ethanlic extracts f Zingiber fficinale (ginger) against sme clinical bacterial islates (Escherichia cli, Staphylcccus aureus and Pseudmnas aeruginsa) btained frm ear and urine samples were carried ut using standard methds. Phytchemical screening revealed the presence f flavnids, sterids, tannins and reducing sugars in bth extracts while alkalids and sapnins were nly present in the ethanlic and aqueus extracts respectively. In additin, a cream, sft and dark-brwn ethanlic extract as well as slid, sft and black aqueus extract were recvered. The biassay studies shwed that the ethanlic extract exhibited an activity against S. aureus with 8 mm zne f inhibitin at a cncentratin f 1000 µg/disc while n activity was recrded against E. cli and P. aeruginsa at the same disc ptency. On the ther hand, the aqueus extract was fund t be mre active against E. cli with 9 mm zne f inhibitin at a cmparatively higher cncentratin f 3000 µg/disc. The minimum inhibitry cncentratin ranged between 250 and 6000 µg/ml. Overall, the results f the study shwed that the ethanlic and aqueus extracts f Z. fficinale have antibacterial ptentials against the tested clinical bacterial islates, thus supprting the plant's ethnmedicinal uses. Keywrds: Zingiber Officinale, Extracts, Phytchemistry, Antibacterial Activity, MIC. INTRODUCTION Plant-derived prducts have been used fr medicinal purpses fr centuries. At present, it is estimated that abut 80.0% f the wrld ppulatin relies n btanical preparatins as medicines t meet their health care needs. Herbs and spices are generally cnsidered safe and prved t be effective against certain ailments. They are als extensively used particularly in many Asian, African and ther cuntries (Langner et al, 1998). Herbal medicine, smetimes referred t as 'Herbalism' r 'Btanical medicine', is the use f herbs fr therapeutic r medicinal value. A herb is a plant r plant part valued fr its medicinal, armatic r savry qualities (Kaw et al., 2011). Herbaceus plants prduce and cntain a variety f chemical substances that act upn the bdy fr the cure f infectins f micrbial rigin. Many drugs cmmnly used tday are f herbal rigin. Indeed, abut 25% f the prescriptin drugs dispensed in the United States cntain at least ne active ingredient derived frm plant material. Sme are made frm plant extracts; thers are synthesized t mimic a natural plant cmpund (Reagr et al., 2002). Amng the plants used as herbal medicine is ginger (Zingiber fficinale). Ginger is a perennial herb which grws frm undergrund rhizmes, and is ften mistakenly called the "rts". Btanically, it is the rhizme that prvides its slightly ht, citrus-like taste, and wnderful arma. Its btanical name is Zingiber fficinale, riginating frm the plant family called Zingiberaceae (Aliyu, 2006). Ginger is ne f the mst imprtant and widely grwn perennial rhizmatus herbs with ver 90 species. It is mstly grwn in Suth-Eastern Asia extending t Queensland and Japan. Ginger is mainly used as a spice and t sme extent fr medicinal purpses (Langner et al., 1998). Ancient civilizatins in India and China used fresh ginger t treat nausea, asthma, cughs, clic, heart palpitatins, lss f appetite and rheumatism, fever, swelling, dysentery, diarrhea and sre (Aliyu, 2006). It has been fund t stimulate circulatin and imprve

2 94 Bashir et al.: In-Vitr Antibacterial Activities and Preliminary Phytchemical... bld flw and has been used fr centuries t aid in digestin, inhibit vmiting and prevent mtin r sea sickness. In India, ginger is used t prevent heart attacks and relieve migraine headaches. It has been fund t reduce chlesterl levels and lwer bld pressure. Ginger has als been fund t have antimicrbial prperties as it cntains extremely high levels f phytchemicals (plant substances with a healing effect) (Cakir et al., 2004). In additin t its beneficial effects n the heart and its anti-cancer activities, ginger has been reprted fr its anti-inflammatry effects and has high cntents f anti-xidants prperties (Aliyu, 2006). Thus, with respect t histrical and cultural recrds f varius medicinal plants and their applicatins as therapeutics, the present study aimed at determining the in-vitr antibacterial effects f the aqueus and ethanlic extracts f Z. fficinale as well as the phytchemical screening f the extracts t determine their biactive cmpnents with a view t further substantiate its ethn-medicinal ptentials. MATERIALS AND METHODS Cllectin, Identificatin and Preparatin f the Plant Materials Fresh ginger plant rhizme was purchased frm a lcal market in the metrplitan Kan, nrthern Nigeria. Its btanical identity was first authenticated at the field using standard keys and descriptins as reprted by Sfwra (1993) and Aliyu (2006). A further cnfirmatin and authenticatin f the plant at the herbarium sectin f the Department f Plant Science, Bayer University Kan, Nigeria was carried ut, where vucher specimens were preserved and stred fr future reference. The rhizme was thrughly washed with sterile distilled water and then allwed t air-dry fr few days. It was then grund int pwder frm using pestle and mrtar, sieved thrugh 250µm mesh t btain fine pwder which was stred at rm temperature in sealed cntainers until required fr use. Extractin Prtcls The extractin prtcls described by Fatpe et al (1993) were adpted. Tw different slvents were used in the extractin prcess: 95% ethanl and water. Here, 100 grams f the fine grunded pwder f the rhizme was taken and perclated with a liter (1000 ml) f ethanl t give a dilutin rati f 1:10. This was allwed t stand fr a perid f tw weeks with regular shaking using magnetic shaker. The cntent was then filtered using Whatman N. 1 filter paper and the filtrate btained was transferred int a pre-weighed beaker and then evaprated t dryness in a water bath at 30 C until the slvent escaped cmpletely leaving behind the ethanlic extract. This was then weighed t find the percentage yield f the extract. Similarly, anther 100 grams f the pwder was perclated with a litre f sterile distilled water in Kilner jar. This was als placed in the magnetic shaker t ensure regular shaking fr a perid f ne week. The aqueus perclatin was allwed fr nly ne week as leaving it t perclate fr mre than this perid may lead t fungal grwth n the surface f the slutin. The aqueus slutin was filtered using Whatman N. 1 filter paper, pured int a pre-weighed beaker and evaprated t dryness in a water bath at 30 C until the slvent escaped cmpletely leaving behind the aqueus extract nly. The percentage yield f the extracts was als determined. Phytchemical Screening f the Extracts Test fr Alkalids This was carried ut qualitatively accrding t Cuili (1994). Using a pipette, 1.0 ml f the aqueus r ethanlic extract was placed in tw separate test tubes. Using a drpper, three drps f Dragendff's and Meyer's reagents were separately added. An range-red precipitate r turbidity with Dragendff's reagent r white precipitate with Meyer's reagent was indicative f the presence f alkalids. Test fr Sapnins This was carried ut accrding t the methd reprted by Brain and Turner (1975). T 0.5 g f the aqueus r ethanlic extract in a test tube, 5.0 ml f sterile distilled water was added and shaken vigrusly. A frth that persisted fr 15 minutes was indicative f the presence f sapnins. Test fr Flavnids This was carried ut in accrdance with the

3 methd as reprted by Sfwra (1993). A 4 mg weight f the aqueus r ethanlic extract and a piece f magnesium ribbn were added fllwed by cncentrated HCl drp-wise. A clr change frm crimsn t magenta indicated the presence f flavnids in the extract. Test fr Sterids This was carried ut accrding t Cuili (1994). A 2 grams weight f the aqueus extract was taken in a test tube and evaprated t dryness. The extract was then disslved in acetic anhydride fllwed by the additin f chlrfrm and then cncentrated sulphuric acid was added by the side f the test tube. Appearance f a brwn ring at the interface f the tw liquids and the appearance f vilet clur in the supernatant layer indicated the presence f sterids in the extract. The same prcedure was fllwed fr the ethanlic extract. Test fr Tannins This was carried ut accrding t Cuili (1994). A 2 grams weight f the aqueus extract was diluted in sterile distilled water in a test tube. Then, 2-3 drps f 5% ferric chlride (FeCl 3) slutin were added. A green-black r blue-black cluratin was indicative f the presence f tannins in the extract. Same prcedure was adpted fr the ethanlic extract but using dimethylsulphxide (DMSO) as the diluent. Test fr Reducing Sugars This was carried ut accrding t the methd f Brain and Turner (1975). Here, ne gram each f the extracts was weighed and intrduced int separate test tubes. The ethanlic and aqueus extracts were diluted with 2.0 ml each f DMSO and sterile distilled water respectively. T the slutin thus btained were added Fehling's slutin and the mixture was then warmed. A brick-red precipitate at the bttm f the test tubes was indicative f the presence f reducing sugars in the extracts. Biassay Studies Bashir et al.: In-Vitr Antibacterial Activities and Preliminary Phytchemical... Cllectin, Identificatin and Authenticatin f Clinical Bacterial Islates The test rganisms (clinical bacterial islates) 95 included ne Gram-psitive bacterium (Staphylcccus aureus) and tw Gram-negative bacteria (Escherichia cli and Pseudmnas aeruginsa). They were cllected frm the Pathlgy Department f Murtala Mhammed Specialist Hspital (MMSH), Kan, Nigeria. Tw islates fr each species were cllected: ne islate frm urine sample and the ther frm ear swab. Identificatin and cnfirmatin f the islates was carried ut using the Gram's reactin, cultural, mrphlgical and bichemical tests (Chesebrugh, 2000). Preparatin f Extract-impregnated Paper Discs This was carried ut as described by Chesebrugh (2000). Here, discs f 6mm diameter were punched ut frm Whatman N.1 filter paper with the aid f paper puncher and placed int Biju bttles in batches f 100 discs. The discs were then sterilized by autclaving at 121 C fr 15 minutes and then allwed t cl. Preparatin f Extract Treatment Cncentratins Tw grams f ethanlic and aqueus extracts were each disslved in 2 ml f DMSO and water in separate Biju bttles t yield 1,000,000 µg/ml (1 g/ml) slutins f ethanlic and aqueus extracts respectively. These were labeled as ethanlic and aqueus stck slutins respectively. Frm the ethanlic extract stck slutin, 0.5 ml was measured and intrduced int a clean bttle cntaining 0.5 ml DMSO. This gave a cncentratin f 500,000 µg/ml. Then, 100 discs were added such that after even distributin (with the help f shaking at equilibrium) each disc absrbed 0.01 ml f the extract, which is equivalent t 5,000 µg/disc. Frm the same stck slutin, 0.4, 0.3, 0.2 and 0.1 ml was each pipetted int separate sterile bttles cntaining 0.6, 0.7, 0.8 and 0.9 ml f DMSO respectively. This was fllwed by the additin f 100 discs int each bttle such that each disc absrbed 0.01 ml f the slutin t arrive at cncentratins f 4000, 3000, 2000 and 1000 µg/disc respectively. These were stred in a refrigeratr at 4 C until required fr use. The same prcedure was repeated fr the

4 96 Bashir et al.: In-Vitr Antibacterial Activities and Preliminary Phytchemical... aqueus extract using sterile distilled water (Chesebrugh, 2000). Preparatin f Turbidity Standard and Standardizatin f Inculum The standard turbidity slutin (barium sulphate standard turbidity slutin) was prepared as described by Chesebrugh (2000). T 99 ml f sterile distilled water, 1 ml f cncentrated sulphuric acid (H SO ) was added t arrive at 1% 2 4 (v/v) slutin f the acid. A quantity (0.5 g) f barium chlride (BaCl.2H O: BDH) was 2 2 disslved in water until the final vlume f the slutin was 50 ml. Then, 0.6 ml f the barium chlride slutin was added t 99.4 ml f the 1% H SO slutin. This resulted in turbid slutin. A 2 4 small quantity f this turbid slutin was taken in a test tube, which served fr cmparisn during standardizatin f the incula. Fr standardizing the inculum, the test rganisms were subcultured nt nutrient agar (xid) plates and incubated at 37 C vernight after which perid an vernight clny was taken, transferred int a tube cntaining 2.0 ml f nrmal saline until the turbidity f the suspensin matched with the turbidity f the standard barium sulphate slutin. This was cnfirmed by cmparing the turbidity f the diluted inculum with that f the standard turbidity slutin against the backgrund f a printed white paper (where the inculum was mre turbid, a little mre nrmal saline was added until its turbidity matched with Macfarland standard f 3.30 x 10 cfu/ml). Susceptibility Testing f the Clinical Bacterial Islates This was carried ut using agar diffusin technique as described by Kirby-Bauer et al (1996). Here, nutrient agar (xid) plates were prepared and the surface f the agar was dried in a ht-air ven. Using sterile swab sticks, the nutrient agar plates were aseptically inculated with the test rganism. With the aid f sterile frceps and syringe, discs cntaining different cncentratins f the extract (5000, 4000, 3000, 2000 and 1000 µg/disc) were impregnated firmly n t the surface f inculated plates. Cntrl discs (30 µg/g chlramphenicl as a psitive cntrl while DMSO was used as a negative cntrl) were als incrprated nt the inculated plates. The discs were sufficiently spaced ut t prevent verlapping f the znes. The plates were then allwed fr the pre-diffusin time f 15 minutes after which they were incubated at 37±1 C fr 24 hurs. Diameters f znes f inhibitin were measured using millimeter rule and the results expressed in mm. Determinatin f the Minimum Inhibitry Cncentratin This was carried ut in accrdance with the methds described by Mrell et al. (2003) and Barnett (1992). Extract cncentratins f 2000, 1000, 500 and 250 µg/ml were prepared. A quantity (0.1 ml) f the suspensin f the test bacterium was inculated nt fresh nutrient agar (xid) plates at the different extract cncentratins. The plates were incubated at 37±1 C fr hurs. The lwest cncentratin f the extract that inhibited the grwth f the test bacterium was nted and recrded as the minimum inhibitry cncentratin (MIC). RESULTS Physical and Phytchemical Characteristics f the Extracts The results f the physical characteristics f the extracts shwed that yields f 9.2 and 8.6 grams f the aqueus and ethanlic extracts were btained respectively. The aqueus extractin was black, pungent, sft and slid while the ethanlic was dark-brwn, pungent, sft and creamy (Table 1). Table 1: Physical Characteristics f Ethanlic and Aqueus Extracts f Zingiber fficinale Extract Initial Final Clur Odur Texture Appearance weight (g) weight (g) Ethanlic Dark- Pungent Sft Creamy brwn Aqueus Black Pungent Oily Slid

5 Bashir et al.: In-Vitr Antibacterial Activities and Preliminary Phytchemical... Results f the phytchemical screening f the extracts shwed the presence f flavnids, sterids, tannins and reducing sugars in bth the aqueus and ethanlic extracts while alkalids and sapnins were nly present in the ethanlic and aqueus extracts respectively (Table 2). Table 2: Phytchemical Characteristics f Aqueus and Ethanlic Extracts f Z. Officinale Extract Alkalids Sapnins Flavnids Sterids Tannins Reducing sugar Ethanlic Aqueus KEY: + = present - = absent The results f the biassay studies shwed that the ethanlic extract was active nly at cncentratin f 1000 µg/disc and the activity was prnunced n S. aureus II while fr S. aureus I, the activity started at 4000 µg/disc. The ethanlic extract was fund t be active against P. aeruginsa II nly at 2000 µg/disc and at 3000 µg/disc against P. aeruginsa I while it was fund t be active at cncentratin f 4000 µg/disc against E. cli II and inactive at all at all cncentratins against E. cli I (Table 3). Table 3: Antibacterial Activities f the Ethanlic Extract f Zingiber Officinale Cncentratin f discs (µg/disc) Islates Psitive cntrl Negative cntrl Diameter f zne f inhibitin (mm) E. cli I E. cli II S. aureus I S. aureus II P. aeruginsa I P. aeruginsa II KEY: E. cli I, P. aeruginsa I and S. aureus 1 are islates btained frm urine samples. E. cli II, P. aeruginsa II and S. aureus II are islates btained frm ear swabs. The aqueus extract had its lwest activity at 3000 µg/disc, but it was mre prnunced against E. cli II with 9 mm zne f inhibitin while n E. cli I, there was n activity at all cncentratins. On S. aureus I and II, the extract was active at 4000 µg/disc with 8 mm zne f inhibitin each while n bth P. aeruginsa I and II, 3000 µg/disc was active with 8 mm zne f inhibitin each (Table 4). Table 4: Antibacterial Activities f the Aqueus Extract f Zingiber Officinale Cncentratin f discs (µg/disc) Islates Psitive cntrl Negative cntrl Diameter f zne f inhibitin (mm) E. cli I E. cli II S. aureus I S. aureus II P. aeruginsa I P. aeruginsa II KEY: E. cli I, P. aeruginsa I and S. aureus I are islates btained frm urine samples. E. cli II, P. aeruginsa II and S. aureus II are islates btained frm ear swabs.

6 98 Bashir et al.: In-Vitr Antibacterial Activities and Preliminary Phytchemical... Results f the minimum inhibitry cncentratins (MICs) f the ethanlic extract n the test rganisms were 4000, 8000, 250, 6000 and 4000 µg/ml against E. cli II, S. aureus I, S. aureus II, P. aeruginsa I and P. aeruginsa II respectively. Fr the Table 5: MICs f the Aqueus and Ethanlic Extracts f Zingiber Officinale Islates Aqueus extract (µg/ml) Ethanlic extract (µg/ml) E. cli II S. aureus I S. aureus II P. aeruginsa I P. aeruginsa II aqueus extract, the MICs fr E. cli II, S. aureus I, S. aureus II, P. aeruginsa I and P. aeruginsa II were 3000, 4000, 3000, 6000 and 3000 µg/ml respectively (Table 5). KEY: E. cli I, P. aeruginsa I and S. aureus I are islates btained frm urine samples. E. cli II, P. aeruginsa II and S. aureus II are islates btained frm ear swab. DISCUSSION The activities f the aqueus and ethanlic extracts f Z. fficinale against the bacterial islates in this study culd be attributed t the presence f the biactive ingredients such as alkalids, tannins and sapnins in the extracts. These cmpunds have been reprted t have exhibited antibacterial ptentials (Fatpe et al., 1993; Cakir et al., 2004; Kaw et al., 2011). Accrding t the present study, preparing an extract with rganic slvent such as alchl has been shwn t have exhibited a better antibacterial activity. This is in accrdance with the results earlier reprted by Kwa et al (2012) wh reprted ethanl as the best slvent that disslves multivariable type f cmpunds. The effectiveness f the rganic slvent (ethanl) might be attributed t tw reasns: firstly, the nature f bilgically-active cmpnents; their activities culd be enhanced in the presence f rganic slvents. Secndly, the strength f the extractin capacity f the ethanl culd have allwed mre f the biactive cnstituents respnsible fr the antibacterial activities. On the ther hand, the ethanlic extract was fund t be mre active against the Gram-psitive islates (S. aureus) while the aqueus extract was fund t be mre active against the Gram-negative islates (P. aeruginsa and E. Cli). This variability culd be related t the ability f the different slvents t disslve sme f the chemical cnstituents in the extracts. Hwever, the resistance f E. cli I t bth extracts (Tables 3 and 4) culd be attributed t the presence f phytchemicals that have pr slubility in the gut as reprted in Table 2 r the antibitics taken by the patient(s) frm which it was islated; as such it culd have develped resistance t such antibitics and becme a resistant strain (Kubmarawa et al., 2008; Kumurya et al., 2010). It culd als be as a result f crss resistance that might have been develped by the bacterium. The findings f the present study have shwn that the islates btained frm the ear swabs exhibited a greater susceptibility t the extracts than thse btained frm the urine samples. This bservatin culd be attributed t the fact that the rganisms frm urine (E. cli I, S. aureus I and P. aeruginsa I) might have been subjected t varius drugs and antibitics than thse frm the ear swabs (E. cli II, S. aureus II and P. aeruginsa II). In additin, the kidney, which acts as an imprtant rgan fr excreting the urine, is als invlved in metablizing drugs and antibitics taken up by a patient. Thus, resistance culd be develped by such rganisms. On the ther hand, the rganisms frm the ear swabs culd be less subjected t antibitics unless in rare cases, hence might nt develp such a resistance. CONCLUSIONS AND RECOMMENDATIONS The results btained in this study justify the ppularity enjyed by this plant and it als

7 ratinalizes the use f the plant fr the treatment f cutaneus and gastr-intestinal infectins. The range f bacteria inhibited suggests that the plant culd be used in the treatment f respiratry and urinary tract as well as wund infectins. The high vulnerability f P. aeruginsa t the extracts tested in this study is wrthy f nte as this bacterium is resistant t mst f the available disinfectants and antibitics (Neu, 1983; Timasz, 1994). It is therefre recmmended that further research needs t be carried ut using ther slvents t ascertain the antibacterial ptentials f the extracts. In additin, there is need t islate, purify and characterize these phytchemical cnstituents respnsible fr the bserved biactivities with a view t supplementing cnventinal drug develpment and/r prbably serve as lead cmpunds fr varius pharmaceuticals especially in develping cuntries like Nigeria. Finally, it wuld be wrthwhile t establish the txiclgical prperties f ginger by determining its minimum lethal dse (LC ). 50 REFERENCES Bashir et al.: In-Vitr Antibacterial Activities and Preliminary Phytchemical... Aliyu, B.S Cmmn ethn-medicinal plants f the semiarid regins f West Africa their descriptins and phytchemicals Triumph publishing cmpany Limited, Gidan Sa'adu Zungur, Kan, Nigeria. Barnett, M.E Micrbilgy Labratry Exercises. Wm. C. Brwn publishers, United States f America. Brain, K.R. and Turner, T.D Practical Evaluatin f Phyt-pharmaceuticals. Wright Scientechica,Bristl, United Kingdm. pp Cakir, A., Krdali, S., Zengin, H., Izumi, S. and Hirata, T Cmpsitin and antifungal activity f essential il islated frm Hypericum husspiflium and Hypericum heterrphyllum. Flavur and Fragrance Jurnal 19: Chesebrugh, M Medical Labratry Manual fr Trpical Cuntries Vlume II. Secnd editin, University Press, Cambridge, Great Britain, 377pp. Cuilel, I Methdlgy fr the Analysis f Vegetables and Drugs. Chemical Industry Divisin, NNIDO Rmania. pp Fatpe, M.O., Ibrahim, H. and Takeda, Y Screening f higher plants reputed as pesticides using the brine shrimp lethality assay. Internatinal Jurnal f Pharmacgnsy 31: Kaw, A.H., Suleiman, Z.A. and Yusha'u, M Studies n the antibacterial activities and chemical cnstituents f Khaya senegalensis and Ximenia americana leaf extracts. African Jurnal f Micrbilgy Research 5(26): Kirby, W.M., Bauer, A.W., Sherris, J.C. and Tutccch, M.C Antibitic susceptibility testing by a standardized single disc methd. American Jurnal f Clinical Pathlgy 45: Kubmarawa, D., Khan, M.E., Punah, A.M. and Hassan, M Phytchemical screening and antimicrbial efficacy f extracts frm Khaya senegalensis against human pathgenic bacteria. African Jurnal f Bitechnlgy 7(24): Kumurya, A.S., Kaw, A.H. and Uba, A Prevalence and in-vitr susceptibility studies f bacteria islated frm hspital patients presenting with titis media in Kan, Nigeria. Bilgical and Envirnmental Sciences Jurnal fr the Trpics 7(1): Kwa, A.M., Kaw, A.H. and Indabawa, I.I. (2012): Phytchemical screening and antibacterial activities f the aqueus and ethanlic extracts f the stem-bark and leaves f Bauhinia rufescens. Internatinal Jurnal f Bimedical and Health Sciences 8(1): Langner, E., Greifenberg, S. and Gruenwald, J Ginger: Histry and use. Advanced Therapy 15(1): Mrell, J.A., Mizer, H.E. and Granat, P.A Labratry Manual and Wrkbk in Micrbilgy: th Applicatin t Patient Care. 7 editin, McGraw- Hill cmpanies, New Yrk, USA. Neu, H.C The rle f Pseudmnas aeruginsa in infectins. Jurnal f Antimicrbials and Chemtherapy 11:1-13. Reagr, L., Gusman, J., McCy, L., Carin, E. and Heggers, J.P The effectiveness f related grape fruit-seed extract as an antibacterial agent I: An in-vitr agar assay. Jurnal f Alternative and Cmplementary Medicine 8: Sfwra, A Medicinal Plants and Traditinal Medicine in Africa. Spectrum bks l i m i t e d, Ibadan, Nigeria. pp Timasz, A Multiple-antibitic-resistant pathgenic bacteria: A reprt n the Rckefeller University Wrkshp. New England Jurnal f Medicine 330: