IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Antimicrobial Activities of Aqueous and Methanolic Extracts from Salvia officinalis and Salix acmophylla Used in the treatment of wound infection isolates M.R.Ali, A.S.Aboud Departme nt of biology, Collage of Science, Unive rsity of Al-Mustansiryah Abstract The aqueous and methanol extracts of Salvia officinals and Salix acmoplylla traditionally used for the treatment of infections disease were tested for their active against gram p ositive and gram negative bacteria isolated from wound infection culture using the broth dilution and disc diffusion melhod. Results of this st udy revealed the p rescence of p hy tochemical which were active against gram p ositive and negative bacteria. M ethanol extracts of both p lants showed the highest activity other the aqueous extract. The minimum inhibitory concentration (M IC) of the aqueous extracts on t he test organism was 25- 100 mg\ml,while that of t he methanol extract was ranged between 25 -50 mg\ml on the test organisms, the minimum bacterial concentration (M BC) ranging between 25-100 mg\ml for methanol extract , and 25-200 mg\ml for aqueous extracts. The highest activity at 100 and 121 o C was demonst rated by the methanol extracts of Salix acmophylla against Staphylococcus aureus and Klebsiella spp. While in methanol extracts of Salvia officinalis the 45 o C was the effective temp erature. In this st udy p lants extracts against gram negative bacteria showed activity in acidic p H only in contrast of gram p ositive bacteria which were constant in all p lants extract . Salvia officinalis contained essential elements at higher levels than Salix acmoplylla .Ca and Zn were p resent of high levels in Salix acmoplylla than other. The results of this st udy suggest the p ossibility of using the methanolic extracts of these plants in treating diseases caused by the test organisms, esp ecially when p repared at acidic p H. Introduction Plants p lay a vital role in maintaining human health and contribute towards the imp rovement of human life. They are imp ortant comp onents of medicines, cosmetics, dy es,and beverages etc. [1] Although hundreds of p lant sp ecies wer test ed for antimicrobial prop erties[2]. There are many cases of infection by drug resistant bacteria whereas few drugs are available effective for the treatment of such p atients. Thus, it is urgently necessary to discover or develop new drugs that are effective on such drug resistant bacteria. We have been try ing to discover novel comp ounds, such as antimicrobial comp ounds and inhibitors of drug resistance sy st ems in bacteria, [3] that are effective against multidrug-resistant bacteria. Though Salvia officinalis (sage) is known as one of the herbs t hat has antimicrobial activity , there are few p apers that have shown ther antibacterial activity , and have shown anti-fungal, anti-viral p rop erties that make it a useful weapon in combating many illnesses [4]. Salvia officinalis is cultivated in several countries mainly to obtain dried leaves to be used as raw material in medicine, p erfumery IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 and food industry [5]. Salvia comp rises one of the largest genera of flowering p lants in the world with 900 to 950 sp ecies occurring worldwide excep t in Aust ralia[6 and 7]. The European salvias, best known from Salvia officinalis L. (common sage), the sage of culinary and herbal uses, also offer several st riking ornamental sp ecies[8 and 9]. The dried root of Salvia (Dan-Shen in Chinese) is one of the most p op ular traditional herbal medicines in some Asian countries, and has been used extensively for the treatment of coronary artery diseases angina p ectoris, myocardial infarction , cerebrovascular diseases , various ty p es of hepatitis , chronic renal failure dy smenorrhea, and also t o imp rove microcirculation in human body [10 ]. An extract from Salvia officinalis (Sage) leaves showed antimicrobial activity against vancomycin-resistant enterococci (VRE). We isolated the effective comp ound and identified it as oleanolic acid, a triterp enoid.. These two comp ounds also showed antimicrobial activity against Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (M RSA)[11]. Willow and "bains," resp ectively, are common English and vernacular names for a number of sist er trees of the genera Salix. These are fast growing and y et medium-sized deciduous trees belonging to the p lant family Salicaceae. They are of enormous ecological and economic imp ortance [12]. As p er the Unani sy st em of medicine the leaves of willows give "cold dry " effect while the flowers disp lay "cold wet" effect. Sleeping on a bed of willow leaves is beneficial in treating heart p roblems and body p ain. A squash of fresh leaves is believed to control dy sentery, earache, worms, etc. Inhaling the aroma of fresh flowers of willow relieves headache and mental tension. A distillate extract of willow flowers is much more effective in relieving the above ailments.[13]. The goal of this invest igation was to discover p lant p roducts that inhibit micro-organisms, esp ecially that causes wound infection. Materials and Methods Col lection of plant samples The medicinal p lants used for the exp eriment were identified according to various literatures, and including other p ertinent taxonomic literature .Collected p lants were washed thoroughly and chopp ed into small p ieces shade dried and grinded into p owdered form. Clean and dry sep arating funnel was t aken. Test mi croorganisms Bacterial sp ecies Shigella dysenteriae ; Aeromonas hydrophila ; Escherichia coli ; Enterobacter spp ; Klebsiella spp ; Pseudomonas aeruginosa and Staphylococcus aureus were all obtained from the student laboratory in M ust ansiryah University . Culture medium and inocul um The st ock cultures of microorganisms used in this st udy were maintained on Plate Count Agar slants at +4°C. Cell susp ensions were p repared by inoculation of each bacteria into 10 ml of Nut rient broth. Incubation was p erformed at 37°C for 24 h. On t he next day M ueller-Hinton Agar (M HA) was p repared and cooled to 45°C. Bacterial susp ension was added into M HA to give a final concentration of 10 7 bacteria/ml and p lated out. Phytochemical screeni ng IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 The two p lant extracts were screened for p hy tochemical constituents by using st andard p rocedures of analysis [14 and 15]. Anti bacterial activity The p late-hole diffusion assay as described by [16] was used to determine the growt h inhibition of bacteria by the p lant extract. The isolated bacteria from wound infection were obtained. The tests were carried out by using a st ock concentration of 500mg/ml p repared by dissolving 1g of the methanol extract (M TE) and aquatic extract into 2ml of distilled water. Nut rient agar was p repared and 25ml each was p oured into st erile p etri dish. This was allowed to solidify and dry . Using a st erile cock-borer of 9mm diameter three equi-distant holes p er p late were made in the set agar and were inoculated with 0.5ml over night susp ension of the bacteria. Thereafter, the wells (holes) were filled with the extract solution at varying concentrations of 500mg/ml, 400mg/ml and 300mg/ml resp ectively. This was done in triplicate and the p lates were incubated at 37ºC for 18hours. T he antibacterial activities were observed and measured by using a transp arent meter rule and recorded if the zone of inhibition was ،10mm [17]. Minimum Inhi bitory Concentration (MIC) M IC is defined as the lowest concentration where no visible turbidity is observed in the test tube (bacteriostatic concentration). The Reuben et al.[18] was employed. In this method, t he broth dilution technique was utilized where the p lant extract was p repared to the highest concentration of 500mg/ml (st ock concentration) in st erile distilled water and serially diluted (two-fold) to a working concentration ranging from 0.780 mg/ml to 200mg/ml using nutrient broth and later inoculated with 0.2ml susp ension of the test organisms. After 18 hours of incubation at 37ºC, the test tubes were observed for turbidity . The least concentration where no turbidity was observed was determined and noted as the minimum inhibitory concentration (M IC) value. Minimum Bacterial Concentration (MBC) The M BC is defined as the lowest concentration where no bacterial growt h is observed (bacteriocidal concentration). This was determined from the broth dilution resulting from the M IC tubes by sub culturing to antimicrobial free agar as described by Usman et al., [19]In this technique, the contents of the test tubes resulting from M IC was streaked using a sterile wire loop on agar p late free of bacteria and incubated at 37ºC for 18 hours. The lowest concentration of the extract which showed no bacterial growt h was not ed and recorded as the M BC. The effect of heat and pH on medicinal plant e xtract The samples of p lant extract (one vial of 100 ml) were p rovided to determine the effect of heat on it, test samples were heated 45 ºC, 70 ºC, 100 ºC and 121 ºC for 15 min. [20]. To determine the effect of p H, extracts were treated at p H ranges of 3 to 8 using 1 N HCl and 1 N NaOH solutions resp ectively in series of test tubes for 1 h and then tested for antibacterial activity [21]. De termination of e ssenti al elements Three grams of dried p lants were taken and mixed with 8ml of concentrated H2So4 (98%) and 2ml of HClo3 (60%) in conical flask for 24 hours which covered by watch class. Then this mixture was left for 6 hours at t he sand bath at 80C° , until the digestion material was converted to a white p owder. Then add 8ml of deionized water to this p owder and the trace elements were determined by flame atomic absorp tion sp ectrop hotometer[22]. IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Re sult and Discussion The result of the Phy tochemical screening for Salvia officinalis and Salix acmophylla showed the same results waich are p resented in Table 1. This reveals a moderate concentration of alkaloids,coumarines,cardiacglycosides,ratenges, p henols, flavonoids, sap onins, tannins,essensial oil and terpenes some of which chemical comp ounds have been associated to antibacterial activities and thus have curative prop erties against p athogens [23] excep t st eroids, no one of both p lant extracts contains it. Phy tochemical constituents such as tannins, flavonoids, alkaloids and several other aromatic comp ounds are secondary metabolites of p lants that serve as defense mechanisms against p redation by many microorganisms [24]. This may therefore exp lain the demonst ration of antimicrobial activity of Salvia officinalis and Salix acmophylla. Regression analysis of t he relationship between size of inhibition zone (mm) and plant crude extract concentration (Log value) showed that there was a significant correlation between concentrations of tested p lant extracts and the mean inhibition zone of p athogenic isolates. The invitro antibacterial activities are shown in Table 2. As is shown, a wide sp ectrum activity against some of bacterial st rains was st udied. Amongst the Gram-positive and Gram-negative bacteria,Gram p ositive bacteria S. aureus were inhibited by both p lant extracts. Indifference methanol extract of Salvia officinalis was more effective in compare is on with the extracts for the same p lant ,while in Salix acmophylla the cold aqueous extract was p ossessed antibacterial highly than methanol and hot aqueous extract.All Gram negative bacteria i.e. E. spp ,S. dysenteriae, A . hydrophila were found to be resistant to all of the extracts of Salvia officinalis , Excep tionally K .spp, E . coli and P. aeruginosa showed zone of inhibition . Where as all gram negative bacteria i.e. S. Dysenteriae, A . hydrophila, K . spp , E . coli and P . aeruginosa gave antibacterial activity as zone of inhibition around the extract of Salvia officinalis, but only E . spp was resist ant t o all of the extracts p reparation. The demonst ration of antibacterial activity against both gram p ositive and gram negative bacteria may be indicative for the p resence of broad sp ectrum antibiotic comp ounds [25].Several workers have reported that many p lants p ossess antimicrobial p rop erties including the p arts which include; flower, bark, st em, leaf, e.t.c. It has been shown that when solvents like ethanol, hexane and methanol are used to extract p lants, most of them are able to exhibit inhibitory effect on bot h gram p ositive and gram negative bacteria [26]. Out of the two solvents used for extraction, the methanol extracts showed the highest activity against the test organisms, followed by the aqueous extracts(hot & cold). Different solvents waer been reported to have the cap acity to extract different p hy toconstituents depending on their solubility or p olarity in the solvent [24]. M ethanol extracts in this st udy might have had higher solubility for more p hy toconstituents, consequently the highest antibacterial activity . The demonst ration of antimicrobial activity by water extracts p rovides the scientific basis for the use of these p lants in the traditional treatment of diseases, since most traditional medicine men use water as t heir solvent in which the decoctions are prepared. The minimum inhibitory concentration M IC and minimum bactericidal concentration M BC results are shown in Tables 3,4,5 and 6 resp ectively. These tables reveal that t he ranges of activity for both M IC and M BC are 0.780 to 200mg/ml. The highest M IC and M BC values is an indication that either the plant extracts are less effective on some bacteria or that t he organism has the IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 p otential of developing antibiotic resistance, while the low M IC and M BC values for other bacteria is an indication of the efficacy of the plant extracts. Result of the effect of temp erature on the p lant extracts showed that various temp erature ranges of 45, 70, 100 and 121 o C had various effects on the antimicrobial activity of the extracts (Figs 1, 2, 3, 4,5and 6), The highest activity (diameter of zone of inhibition 30 mm) at 100 and 121 o C was demonst rated by the methanol extracts of Salix acmophylla against S. aureus and K.spp , whether as A . hydrophila lose activity at the same temp erature. While in methanol extracts of Salvia officinalis the 45 o C was the effective temp erature (diameter of zone of inhibition 25 mm).,while A.. hydrophila had the constant activity in different used temp eratures .As can clearly be seen by these figures, the rest bacteria did not hav resp onse to these temp eratures in each methanol,and both of aqueous extracts (no z one of inhibition ) . The activity is slightly increased at acidic p H (3 to 5). While at alkaline p H the activity of the p lant extracts is reduced excep t for A.. hydrophila in each of p lant exracts (Figs. 7,8,9,10,11and 12). The antibacterial activity of the extracts is slightly increased at acidic pH. Increase in activity of p hy otoconstituents in the p resence of acidic medium has earlier been reported [27]. The local app lication of these p lants involves the addition of high doses of p otash which is a st rong basic salt, and for the fact that the activity of the extracts reduced at alkaline p H in this st udy , it may exp lain why the plant concoction is taken for a longer p eriod of time before any curative effect is noticed. In this st udy , it was noticed that gram p ositive bacteria S. aureus gave constant result in all plant extracts. While p lant extracts against gram negative bacteria esp ecially E.coli and E.spp showed activity in acidic p H only, and this activity was st able in all p lant extracts app lication. As well as lactose fermented bacteria K.spp is inhibited in different p H ,but with low inhibition zone, similar to PS.aeruginosa . Ten elements, Ca, Co, Cu, M n, Fe, K, Na, P, Zn and Pb, were determined in Salvia officinalis and Salix acmophylla Table 7 . Salvia officinalis contained essential elements (M n, Fe, K,Na,P and Pb,) at higher levels than Salix acmophylla. Ca and Zn were p resent at high levels in Salix acmophylla than other. Therefore, it may not p roduce any health risks for human consumption, if other sources of t oxic metal contaminated food are not taken at t he same time. Re ferences 1-Khare, C.P. (2004). Ency clop edia of Indian M edicinal Plants, Rational West ern Therapy and other Traditional Usage, Botany (Sp ringer Verlag Berlin M eidelberg) . 406-407. 2-Nair, M .G. and Burke, B.A. (1990 ).Ant imicrobial metabolite and related comp ounds. Journal of Agri- cultural and Food Chemistry 38, 1093-1096. 3-Horiuchi, K.; Shiota, S.; Hatano, T. ; Yoshida, T. ; Kuroda, T. and Tsuchiya, T.(2007). Ant imicrobial Activity of Oleanolic Acid from Salvia officinalis and Related Compounds on Vancomycin-Resistant Enterococci (VRE). Biol. Pharm. Bull. 30(6) 1147—1149 . 4-Eidi,A. ; A. and, Baharc, M .(2006). Effects of Salvia officinalis L. (sage) leaves on memory retention and its interaction with the cholinergic sy st em in rats. Ap p lied and Basic Nut ritional Science.22(3): 321-326. 5- Santos,P.C.; Seabra, R.M .; Andrade, P.B.and Fernandes,F.M . (2002). Phenolic antioxidant comp ounds p roduced by in vitro shoot s of sage (Salvia officinalis L.). Plant Sci. 162: 981 – 987. 6- Clebsch , B . (1997). A book of salvias: Sages for every garden. Timber. Press,. Port land , OR . IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 7-Lasseigne ,F.T ; Stuart, L.W; Frank A .B. and Thomas, G.R.(2007). Day /Night Temp erature Affects Growt h and Phot osy nthesis of CultivatedSalvia Taxa. J. Amer. Soc. Hort . Sci. 132(4):492–500. 8-Reales, A. (2004). Numerical taxonomy st udy of Salvia sect. Salvia (Labiatae). Bot. J. Linn. Soc. 145:353–371. 9-Kintzios, S. E. (2000). Sage: The Genus Salvia.. CRC Press. p p . 27. 10-Jiang ,R.W.;Lau, K.M .; Hon ,P.M .; Thomas ,C.W.M ; Woo ,K.S.;and Fung ,K.P.(2005).Chemistry and Biological Activities of Caffeic Acid Derivatives from Salvia Miltiorrhiza. Crr.M ed.Chem.12:237-246. 11- Kumiko,H.;Sumiko,S.H.;Tsut omu,H.;Takashi,Y.;Teruo,K.;andTomofusa,T.A..(2005).Antimicroi al activity of oleanolic acid from Salvia officinalis andrelated comp ounds on vancomycin resistance entero coocci (VRE).Bio. and pharma.ball.30(6):1147-1149. 12- Argus, G. W.( 1997). Infragener ic classification of Salix (Sa licaceae) in the New World’, Sy st . Bot. M onogr. 52: 121. 13- Noatay,K.L.(2003).The bat wood from cold climes.Agriculture Tribune,Chandigarh ,India. 14- Trease,G.E. and Evans W.C. (2002).Pharmacology .15thg Edn.Saunders Publishers ,London.pp :42-393. 15- Harbome , J. B. (1993) . Phy tochemist ry .Academic Press, London , p p :89-131. 16-Ogundipe, O.O.; M oody , J.O.; Fakeye, T.O. and Ladip, O.B. (2000). Ant imicrobial activity of Mallotus oppositifolium extractives. Afr. J. M ed. M ed. Sci. Vol. 29: 3/4, p p 281-283. 17-Kudi, A.C.; Umoh, J.U.; Eduvic, L.O.and Getu, J. (1999). Screening of some Nigerian M edicinal p lants for Antibacterial Activity . J. Ethanopharm. 67:225-228 18-Reuben,K.D; Abdulrahman,F.I.; Akan,J.C.; Usman, H.; Sodipo, O.A. and Egwu, G.O. (2008). Phy tochemical Screening and In Vitro Ant imicrobial Investigation of the M ethanolic Extract of Croton Zambesicus M uell ARG. Stem Bark. European Journal of Scientific Research,23(1):134- 140. 19-Usman, H.; Abdulrahman, F.I. and Ladan ,A.H. (2007). Phy tochemical and Ant imicrobial Evaluation of Tribulus terrestris L. (Zy gop hy laceae). Growing in Nigeria. Res. J. Bio. Sci. M edwell Journals, 2007 2(3): 244-247. 20-Franz,C.M .;Toit,D.M .;Holy,H.A.;Schillinger,U. and Holzapfel, W.H.(1997). Production of nisinlike bacteriocins by lactococcus lactis st rains isolated from vegetables. J. Basic. M icrobiol. 37:187-196. 21-Doughari, J. H., Pukuma, M . S. and De, N.(2007). Ant ibacterial effects of Balanites aegyptiaca L. Drel and Moringa oleifera Lam. on Salmonella typhi .African Journal of Biotechnology Vol. 6 (19), p p . 221 22-Beyenbach, K.W.(1990). Transp ort of magnesium across biological membranes.M agnes Trace Elem.9 :233 –254 23-Nweze, E.T.; Okafor, J.I.and Njoku, O. (2004). Ant imicrobial Activities of M ethanolic extract of Trumeguineesis (Schumm and Thorn) and M orinda lucinda Benth used in Nigerian Herb. M edicinal Practice. J. Bio. Res. Biotechnol. 2 (1): 34-46. 24-Doughari,J.H. (2006). Ant imicrobial Activity of T a m a r in d u s in d ica Linn .Trop ical Journal of Pharmaceutical Research; 5 (2): 597-603. 25-Srinivasan, D.; Perumalsamy , L.P.and Nathan, S. S. T(2001).Ant imicrobial activity of certain Indian medicinal p lants used in folkloric medicine. J Ethnop harm; 94: 217-222 26-Bushra, N.R. and. Ganga,T. D.( 2003). Ant ibacterial activity of selected Seaweeds from Kovalam sout h West coast of India. Asian. J. of M icrobiol. Biotech. Env. Sc. 5(3): 319-322 27-M olan, P.C.(1992). The antibacterial activity of honey . The nature of antibacterial activity . Bee World. 73: 59 – 76 IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Table (1) Phytochemical screening of Methanol , Hot water and Col d water extract of Sal via officinalis and extract of Salix acmophylla So : Salvia officinalis. Sa : Salix acmophy lla. Number Constituents Methan ol extract Hot water extract Col d water extract S o S a S o S a S o S a 1 Alkal oids i.Dragendorff 's test ii.M eyer's test + + + + + + 2 Phenols + + + + + + 3 Cardiac glycosi des Killer-killanis test + + + + + + 4 Flavonoi ds i.Shinoda' s t est ii.FeCl3 test + + + + + + 5 S aponins Frot hing test + + + + + + 6 Terpenes Salkowski test + + + + + + 7 S teroids Libarman- Burchard's test - - - - - - 8 Tani ns i.FeCl3 test ii.Lead acetate test + + + + + + 9 Ratenge s + + 10 Coumarines + + 11 Essensial oil + + IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Table 2: Ant ibacterial Activity of Plant Extracts against Test Organisms Extract/concentration Mg/ml Zone of inhibition (mm) Co. P.a. E.sp p K.sp p A.h. S.d. E.c. S.a. M ethanol Extract of Salvia officinalis 500 400 300 25 22 20 - - - 10 9 9 - - - - - - 30 25 22 30 24 20 Hot aqueous Extract of Salvia officinalis 500 400 300 20 20 18 - - - 9 - - - - - - - - 28 25 21 12 11 9 Cold aqueous Extract of Salvia officinalis 500 400 300 20 20 17 - - - 10 9 - - - - - - - 20 15 13 20 15 11 M ethanol Extract Salix cmophylla 500 400 300 25 20 18 - - - 15 10 10 22 22 20 15 15 10 14 13 10 27 18 16 Hot aqueous Salix acmophylla 500 400 300 25 25 21 - - - 11 10 10 21 19 18 12 10 9 14 14 10 20 18 16 Cold aqueous Salix acmophylla 500 400 300 25 25 20 - - - 18 15 12 20 20 18 15 15 13 13 13 12 30 29 26 Control(water) - - - - - - - - Control(M ethanol) - - - - - - - - IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Tab le 3:Minimum Inhibitory Concentration (MIC) values for Bacterial Isolates Against Salvia officinalis extracts Bacter ia Extract concentration (mg/ml) 0.780 1.560 3.125 6.25 12.5 25 50 100 200 M H C M H C M H C M H C M H C M H C M H C M H C M H C P.a. - - - - - - - - - - - - - - - I I I + + + + + + + + + E.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - K.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - A.h. - - - - - - - - - - - - - - - - - - - - - - - - - - - S.d. - - - - - - - - - - - - - - - - - - - - - - - - - - - E.c. - - - - - - - - - - - - - - - - - - - I I + + I + + + S.a - - - - - - - - - - - - - - - I - - + - - + I - + + - Key: - = R esistance (growth of bacteria) + = Concentrations show no turbidity (inhibition of bacterial growth) I = least concentration showing no turbidity (MIC) M=Methanol extract H= Hot aqueous extract C= C old aqueous extract Tab le 4: Minimum Bacterial Concentration (MBC) Values for Bacterial Isolates Against Salvia officinalis extracts Bacter ia Extract concentration (mg/ml) 0.780 1.560 3.125 6.25 12.5 25 50 100 200 M H C M H C M H C M H C M H C M H C M H C M H C M H C P.a. - - - - - - - - - - - - - - - B B B + + + + + + + + + E.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - K.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - A.h. - - - - - - - - - - - - - - - - - - - - - - - - - - - S.d. - - - - - - - - - - - - - - - - - - - - - - - - - - - E.c. - - - - - - - - - - - - - - - - - - - - - B B - + + B S.a - - - - - - - - - - - - - - - B - - + - - + B - + + - - = Resis tance (gro wth of bact eria) + = Con centratio ns sh ow no tu rbid it y (in hib it io n of bact erial gro wth ) B= = Min imum Bacteri cidal (MBC) M=Meth anol extract , H= Hot aq ueous extract C= Col d aqu eous extract S.d = S. dyse ntriae; S.a = S. aureus; A.h.= A . hydrophila; E.c.= E. coli ; E.spp. = Enterobac ter spp: K.spp = Klebsiella spp: P.a.=P . aeruginosa S. d = S. dysentriae; S.a = S. aureus; A.h.= A . hydrophila; E.c.= E. coli ; E.spp. = Enterobacter spp: K.spp = Klebsiella spp: P. a.=P . aeruginosa IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Tab le 5: Minimum Inhibitory Concentration (MIC) values for Bacterial Isolates Against Salix acmophylla extracts Bacter ia Extract concentration (mg/ml) 0.780 1.560 3.125 6.25 12.5 25 50 100 200 M H C M H C M H C M H C M H C M H C M H C M H C M H C P.a. - - - - - - - - - - - - - - - I I I + + + + + + + + + E.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - K.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - A.h. - - - - - - - - - - - - - - - - - - I - - + I I + + + S.d. - - - - - - - - - - - - - - - - - - - - - - - - - - - E.c. - - - - - - - - - - - - - - - - - - - I I + + I + + + S.a - - - - - - - - - - - - - - - - - I - - + I I + + + + Key: - = R esistance (growth of bacteria) + = Concentrations show no turbidity (inhibition of bacterial growth) I = least concentration showing no turbidity (MIC) M=Methanol extract H= Hot aqueous extract C= Cold aqueous extract Tabl e 6: Minim um B acterial Co ncentration (MB C) Val ues f or B acterial Is ol ates Ag ai nst Sal ix acmop hyll a extracts Bacter ia Extract concentration (mg/ml) 0.780 1.560 3.125 6.25 12.5 25 50 100 200 M H C M H C M H C M H C M H C M H C M H C M H C M H C P.a. - - - - - - - - - - - - - - - B - - + B B + + + + + + E.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - K.sp p - - - - - - - - - - - - - - - - - - - - - - - - - - - A.h. - - - - - - - - - - - - - - - - - - B - - + B B + + + S.d. - - - - - - - - - - - - - - - - - - - - - - - - - - - E.c. - - - - - - - - - - - - - - - - - - - B B + + - + + B S.a - - - - - - - - - - - - - - - - - B B B + + + + + + + - = Resistance (growt h of bact eria) + = Concentrations show no turbidity (inhibition of bacterial growth) B= = Minimum Bactericidal (MBC) M=Methanol extract H= Hot aqueous extract C= C old aqueous extract S.d = S. dyse ntriae; S.a = S. aureus; A.h.= A . hydrophila; E.c.= E. coli ; E.spp. = Enterobac ter spp: K.spp = Klebsiella spp: P.a.=P . aeruginosa S.d = S. dyse ntriae; S.a = S. aureus; A.h.= A . hydrophila; E.c.= E. coli ; E.spp. = Enterobac ter spp: K.spp = Klebsiella spp: P.a.=P . aeruginosa IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Tabl e 7: Esse n ti al e l e m e n ts co n ce n tra ti on of Salvia officinalis an d Salix acmophylla Elements Concentration Salvia officinalis Salix acmophylla Pb pp m 0.6 0.4 Na pp m 594 594 K % 1.2 0.44 Ca % 0.92 1.2 Fe pp m 700 200 Zn pp m 63.6 90.6 P % 0.35 0.13 M n pp m 5.7 3.1 Co pp m 2.5 0.5 Cu pp m 6.5 3.9 Fig 1. Effects of te m peratu re on antimicrobial acti vity of Me thanol extract Salix acmophylla Fi g 2. Effect of tem pe ratu re on an timicrobial acti vi ty of Hot aque ous extract Salix acmophylla Fi g 3. Effect of tem pe ratu re on antimicrobial acti vi ty of C ol d aque ous extract Salix acmophylla Fig 4. Effects of tem pe ratu re on an timicrobial acti vity of Meth an ol e xtra ct Salvia officinalis IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (3) 2010 Fig 5. Effe ct of tem pe ratu re on an timicrobial acti vity of Hot aqueous extract Salvia officinalis Fi g 6. Effect of tem pe ratu re on antimicrobial acti vi ty of C ol d aque ous extract Salvia officinalis Fig 7. Effects of pH on anti microbial activity of Methanol extract Sa Salix acmophylla Fig 8. Effect of pH on anti microbial activity of Hot aque ous extract Salix acmophylla IHJPAS IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (3) 2010 Fig 9. Effect of pH on anti microbial activity of Cold aque ous extract Salix acmophylla Fig 10. Effects of pH on anti microbial activity of Methanol extract Salvia officinalis Fig 11. Effect of pH on anti microbial activity of Hot aque ous extract Sal via officinalis Fig 12. Effect of pH on anti microbial activity of Col d aque ous extract Sal via offi cinal IHJPAS 2010) 3( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد الفعالیة المضادة للبكتریا للمستخلص المائي والكحولي لنبات المیرمیة والصفصاف .في عالج اخماج الجروح عملھ المست منعم رضوان علي ، انمار سعدي عبود المستنصریةالجامعة ، كلیة العلوم ، قسم علوم الحیاة -:الخالصة ــة ي عـالج االمـراض المعدی ي ھـذه الدراسـة اسـتعمال المسـتخلص المـائي والكحـولي لنبـات المیرمیـة والصفصـاف ـف م ـف ـت وذلك باختبار فعالیتھا ضد البكتریا الموجبة والسالبة لصبغة كرام المعزولة من اخماج الجروح وذلك باسـتعمال طریقـة االنتشـار كمـا . الموجبـة والسـالبة لصـبغة كـرامالتي تكون فعالة ضد البكتریا ئج الدراسة وجود مركبات كیمیائیة اظھرت نتا. اص باالقر ىلو بط االدـن غ التركیـز المـث د بـل للمسـتخلص حظت فعالیة عالیة للمستخلص الكحـولي لكـال النبـاتین مقارنـة بالمسـتخلص المـائي وـق اء المجھریـة المخ ن ،مـل/ ملغـم 100 – 25رة تبـالمائي تجاه االحـی راوح المسـتخلص الكحـولي ـم مـل / ملغـم 50 – 25بینمـا ـت اظھـر . مـل للمسـتخلص المـائي/ ملغـم 25 – 200مل للمسـتخلص الكحـولي و / ملغم 100- 25وقد بلغ التركز البكتیري االدنى ى فعالیـة عنـد درجـات الحـرارة ل المستخلص الكحـولي بینمـا ، K .sppو S. aureusم تجـاه بكتریـا 121، 100لصفصـاف اعـل ذه الدراسـة ان مسـتخلص النبـاتین اظھـر فعالیـة . م 45كان المستخلص الكحولي للمیرمیة فعال عند درجة حرارة ي ـھ كما لوحظ ـف ى الن ة كرام في الدالة الحامضـیة وعـل ان البكتریـا الموجبـة لصـبغة كـرام كانـت ثابتـضد البكتریا السالبة لصبغ یض ـف ع ةـق ي جمـی ـف ي بكما لوحظ احتواء نبات المیرمیة على العناصـر االساسـیة وبتراكیـز عالیـة مقارنـة . المستخلصات النباتیة نبـات الصفصـاف ـف ذه نتـائج وقد بینـت. نبات المیرمیةبحین لوحظ وجود عنصري الزنك والكالسیوم بمستویات عالیة في نبات الصفصاف مقارنة ـھ المكان است ك السـیماالمستخلص الكحـولي لكـال النبـاتین فـي معالجـة االمـراض المتسـببة بوسـاطة الجـراثیم عمالالدراسة انھ با تـل .المحضرة في اس ھیدروجین حامضي . . 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