IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Inhibition of Bacterial Growth by Lawsonia inermis (henna) Leaf Extracts In Vitro M. A. Merdaw Departme nt of Clinical Laboratory Sciences ,College of Pharmacy, Unive rsity of Baghdad Abstract Leaf sa mples of Lawsonia inermis were collected fro m Basrah city , South of Iraq to examin e their antimicrobial activity . The effects of water and chlorofor m crude extracts of the leaves in different con centrations were obtain ed and bioassay ed in vitro for its bioactivity to inhibit t he growt h of si x typ es of bacteria . The extract of water was clearly sup erior for all bacteria esp ecially the bacteria S taphlylococcus aureus (inhib ition zone was 21mm in concentration 70mg/ml) fro m gram p ositive bacteria, and Klebsiella pneumoniae (inhib ition zone was 20mm in the same concentration) , and the growt h of all bacteria was inhibited to varying degrees by increasin g the concentration of the henna leaves and are commonly known to p ossess antimicrobial activity . These results confirm the antibacterial activity of henna leaves and sup p ort the traditional use of the p lant in therapy of bacterial infections . Introduction The p lant Lawsonia inermis (henna) belon gs to the family Lythraceae and is best known for its colouring matter contained in the leaves [1] , and it is a glabrous much br anched shrub or quite a small tree with grayish –brown bark [2] . The henna p lant is one such p lant known since with healing att ributes and is now the subject of intense scientific st udy [3,4] . The p lant constituents are made up of mannite , tannic acid , mucila ge and gallic acid , but the main constituent is 2-hy droxy nap hthoquinone (lawsone) , known to be the major bioactive constituent , dried p owdered leaves of henna contain about 0.5-1.5% lawsone . Henna is naturalized and cultivated in the trop ics of America , Egy pt , India and parts of t he midlle east [2,5]. In early Islamic culture henna usa ge is very evident in the book Prop hetic M edicine where the medicinal p ractices of Prop het M ohammed (PBUH) , as mentioned by his followers and others that were close to him in his household , were recorded [6]. The therap eutic efficacies of many indigenous p lants for several disorders have been described by p ractitioners of traditional herbal med icines [7] . B ein g sources of many lif e sustaining metabolites , the research is still on for p lants t o be used in healin g . This in part is due to the growing p roblem of worldwide antibacterial resistance . Isolation of microbial agents less suscep tible to regular antibiotics and recovery of resistant isolates during antibacterial therap y is increasin g throughout the world [8,9] . Indeed the sy nthesis and evalution of antimicrobial activity of the natural naphthoquinone p roducts and their derivatives has been invest igated [10,11] . Although naphthoquinones do appear to exhibit a wide sp ectrum of biological activities , the mechanism/s of action r emains somewhat unclear[12] . However lawsone was shown to elicit in vivo lower toxic effects in mussel tissues than tissues in higher or ganisms . This may be due to the lower detectable levels of xanthine oxidase in the invertebrate mussels [13,14] . Leaves of henna are useful to bring down the severity of many medical p roblems like dy sentery , diseases of the sp leen , lumbago , bronchitis and syp hilitic eye infection [5] . Based on these observations this study was undertaken to determine the p ossible antimicrobial activities of Lawsonia inermis against different bacterial sp ecies . IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 Materials and Methods Plant material : The henna p lant Lawsonia inermis leaves samples used in this study were collected from Basrah city ,South of Iraq . Fresh leaves wer e dried in shad e , then wer e ground to p owder . Preparation of extracts : Dried Lawsonia inermis leaves were p owdered mechan ically and mixed with st erile distilled water in a conical flask and left to soak overnight at room temp erature , the residue was then filterated . The filterate was then mixed with the chloroform in a sep arating funnel , the mixture was shaken until sep aration was observed in form of two lay ers ; the water and the chloroform extract . The different lay ers were run out into sep arate beakers and p laced in an oven to dry at 50c .Residues and the extracts were made into susp ensions using st erile distilled water at concentrations (30 , 50 , 70 ) mg/ml . Bacterial species : Three types of gram p ositive bacteria namely Staphylococcus aureus , Bacillus subtilis and Streptococcus pyogen es , and three types of gram negative bacteria namely Escherichia coli , Pseudomonas aeruginosa and K lebsiella pneumoniae were used . The bacteria was cultered on nutrient brot h (oxoid) at 37c for 24h . De termination of diameter of growth inhibition : The agar well diffusion method was emp loyed . Four 9mm wells were made in each p late of solidified nutrient agar . 0.5 ml of each concentration of the extracts was measured out and mixed with 0.3g of agar . The well was carefully filled with this mixture and left to solidify . Each of the plates was seeded with each of the d ifferent or ganisms and incubated in the incubator for 24h . The measurements were then taken again by using linear measurement method [2,15] . Results and Discussion The p lant leaves of Lawsonia inermis are commonly used in v arious dy e industries for commer cial p urp oses. M any st udies showed that these leaves containes n aphthoquinone (lawsone) in high er concentrations which was p roved to have analgesic, anti inflammatory and antipyretic effects in rat models. Inhibitory action of henna was shown against both gram p ositive and gram negative bacteria . Ant ibacterial activity is recorded when the zone of inhibition is greater than 6mm [2]. Results as shown in Table 1and 2 indicated that all of the two tested leaf extracts of henna p lant (Lawsonia inermis) at different concentrations supp ressed the growt h of the tested bacteria at varying degr ees . Extract of water was clearly sup erior in bioactivity as comp ared to that of chloroform . The maximum inhibition zone was found in 70mg\ml water extract concentration and it was 21mm in the bacterial isolate of Staphylococcus aureus , while the least inhibition zone in the same concentration was 14mm for the p athogen Pseudomonas aeruginosa . The activity increased with the increase of the extract concentration . Even in the lowest concentration of water extract i.e., 30mg\ml , all of the bacterial isolates showed substantial inhibition in their resp ective growt h . The minimu m inhib ition zone in 30mg\ml concentration was observed in Ps.aeruginosa with 9mm inhibition zone Table (2). The chloroform extract of the plant showed maximum activity against the Klebsiella pneumoniae where the zone of inhibition was 12mm and the weakest activity was observed against Streptococcus pyogenes and Pseudomonas aeruginosa (8mm) in the same concentration (30mg\ml) .This data is in close agr eement with p revious reports elsewhere using the same p lant [2,5] . It is not p ossible to make a direct correlation between the observed activity of the p lant extracts in vitro and the actual effects when used in vivo for the diseases observed by the indigenous p eople and traditional healers [16] . Therefore , it is imp ortant that the p lant should also be further investigated to evaluate the sign ificance of these extracts , clin ical role IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 and the medical sy st em of indigenous p eople . Additional deep research is ne cessary to isolate and characterize their active co mpounds for p harmacological test ing. Conclusions It is clear that Lawsonia inermis leaves as an extract may be useful as an antimicrobial agent against the above mentioned bacteria . Further studies need to be undertaken regarding toxicity , safety and absorp tion p attern of the active ingredients . Isolation of the active ingr edient from Lawsonia inermis will facilitate further studies . The present study identifies henna as source of biolo gical antimicrobial, since it showed a high activity against wide sp ectrum of bacteria to be killed without any side effects and/or bacterial resist ance as current sy nthetic antibiotics are doing and this sp ecifity app ears as additional p oint in the natural antibiotics research. Re ferences 1.Habbal, O.A.; AL- jabri, A.A.; EL-Ha g, A.H.; AL-M ahrooqi, Z.H. and AL-Hashmi, N.A. (2005) , In vitro antimicrobial activity of Lawsonia inermis Linn (henna). A p ilot st udy on the Omani henna . Saudi M ed J 26(1): 447-50 . 2.M uhammad, H.S. and M uhammad, S.(2005).The use of Lawsonia inermis Linn. (henna) in the management of burn wound infections. African J of Biotechnology .4(9) :934-37. 3.Singh, A. Sin gh, D.K.(2001), M olluscicidal activity of Lawsonia inermis and its binary and tertiary combinations with other p lant derived mollusc icides .Indian J E xp Biol 39(3): 263-8 4.Az aizeh, H.S.; Fulder, K. Said, O.(2003),Ethno-Botanical knowledge of local Arab p ractitioners in the M iddle Easter region. Fitot erap ia ,74 : 98-108 5.Bhuvaneswari, K.; Gana Poongothai, S. ; Kuruvilla, A. and App ala Raju, B.(2002), Inhibitory concentrations of Lawsonia in ermis dry p owder for urinary p athogens. Indian J of Pharmacolo gy 34: 260-63 6.AL-Arnaoutt, S. AL-Arnaoutt, A.K.(1987), In Al-Jozieh IK Prop hetic M edicine Beirut: AL- Risala publishin g. 7.Natarajan, V.; Venugop al, P.V. M enon, T.(2003), Effect of Azadarichta indica (n eem) on the growt h p attern of dermatop hy tes. Indian J M ed. M icrobiol.21: 98-101. . 8.Bonjar, S.G.H.(2004), Screening for Ant ibacterial p rop erties of some Iranian p lants against two strains of Escherichia coli. Asian J.Plant Sci.3(3) : 310-14. 9.Bhavani, S.M . Ballow, C.H.(2000), New agents for gr am-p ositive bacteria. Current op inion in M icrobiol 3: 528-34. 10.Oliveira,C.G.T .; M irranda, F.F.; Ferreira, V.F.; Freitas, C.C.; R abello, R.F.and Carballido, J.M . and Correa, L.C.D.(2001), Sy nthesis and antimicrobial evalution of 3-hy drazino- naphthoquinone as analogs of l apachol.J Brazillian Chem Soc 12: 339-45. 11.Riffel, A.; M edina, L.F.; Stefani, V. ; Santos, R.C.; Bizani, D.and Brandelli, A.(2002), In vitro antimicrobial activity of a new series of 1,4-nap hthoquinones .Braz J M ed Biol Res 35:811-18. 12.Heinrich, M .; Barnes, J.; Gibbons, S. and Williamson, E.M .(2004), In: Fundamentals of p harmacognosy and p hytotherapy : Important natural p roducts and p hytomedicines in Pharmacy and M edicine . London:Elsevier Health Sci. 13.Lall ,N.; Das Sarma, M .; Hazra, B.and M ey er, J.M . (2003), Ant imy cobacterial activity of diospy rin derivatives and st ructural analogue of diospy rin against Mycobcterium tuberculosis in vitro. J Antimicrobial Chemother 51: 435-8. 14.Osman, A.M .; Rott eveel, S.; Den Best en, O.J.and Van Noort , P.C.(2004), In vivo exp osure of Dreissena p olymorp ha mussles t o the quinines mendione and lawsone .J App l Toxico l 24(2):135-41. 15.Abdulmoneim, M .A.(2007), Evalution of Lawsonia inermis Linn.(Sud anese Henna)leaf Extracts as an Ant imicrobial A gent. Res J of Biolo gical Sciences 2(4); 419-23 . IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 2009 16.Goun, E.G.; Cunningham, D.C.; Nguy en, C. and M iles, D.(2003), Antibacterial and antifungal activity of Indonesian ethnomedical p lants. Fitoterap ia, 74:592-96. Table (1): In vitro antibacterial activity of Lawsonia inermis leaf extracts on gram positive bacteria Zone of inhibition mm,diameter Concentration mg/ml Extract Organisms 11 17 21 30 50 70 water 9 16 19 30 50 70 chloroform Staphyloco ccus aureus 11 14 17 30 50 70 water 8 12 15 30 50 70 chloroform Streptococcus pyogenes 11 15 18 30 50 70 water 10 12 17 30 50 70 chloroform Bacillus subtilis IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.22 (4) 200 Table(2):In vitro antibacterial activity of Lawsonia inerm is leaf e xtracts on gram negative bacteria Zone of inhibition mm, diameter Concentration mg/ml Extract Organisms 13 17 19 30 50 70 water 11 17 18 30 50 70 chloroform Escherichia coli 9 11 14 30 50 70 water 8 11 14 30 50 70 chloroform Pseudomonas aeruginosa 13 18 20 30 50 70 water 12 17 20 30 50 70 chloroform Klebsiella pneumoniae 2009) 4 (22 المجلد مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة Lawsonia( مستخلصات ورق نبات الحنة باستعمالبكتیريتثبیط النمو ال inermis (خارج الحي میسون عبد الزهرة مرداو جامعةبغداد، كلیةالصیدلة، فرع العلوم المختبریةالسریریة الخالصة ر . من مدینة البصرة لفحص نشاطها الضد مایكروبي Lawsonia inermis جمعت اوراق نبات الحنة تم االختبا نمو فيالحیوي خارج الحي لمعرفة تأثیر مستخلصات الماء والكلوروفورم لالوراق بتراكیز مختلفة ومالحظة تأثیرها المثبط ة المستخلص المائي كان اوضح تأثیرا من مستخلص الكلوروفورم. ستة انواع بكتیریة على كل انواع البكتیریا المدروس من البكتیریا الموجبة ) مل/ ملغم70ملم عند التركیز21منطقة التثبیط ( Staphylococcus aureus بكتیریا السیما من البكتیریا السالبة لصبغة ) نفسهمم عند التركیز20منطقة التثبیط ( Klebsiella pneumoniaeلصبغة كرام وبكتیریا ة ی ونمو كل االنواع البكتیریة ثبط بدرجات مختلفة تختلف بأختالف تركیز ورق الحنة مما یعني امتالك نبات الحنة الكفاكرام .الضد مایكروبیة . تعمال التجاري للنبات في عالج االصابات البكتیریة ھذه النتائج تثبت النشاط الضد بكتیري لورق الحنة وتدعم االس