Antibacterial Activity of Trichosanthes cucumerina Linn. Extracts

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1 ISSN Aville Online t Interntionl Journl of Phrmeutil & Biologil Arhives 2011; 2(2): ORIGINAL RESEARCH ARTICLE Antiteril Ativity of Trihosnthes uumerin Linn. Extrts L.D.A.M. Arwwwl 1*, M.I. Threw 2, L.S.R. Armewel 1, N. Fernndo 3 nd L.D. Guruge 3 *1 Industril Tehnology Institute, Buddhlok Mwth, Colomo 07, Sri Lnk 2 Institute of Biohemistry, Moleulr Biology nd Biotehnology, University of Colomo, No. 90, Cumrtung Munids Mwth, Colomo 03, Sri Lnk 3 Deprtment of Miroiology, Fulty of Medil Sienes, University of Sri Jywrdenpur, Gngodwil, Nugegod, Sri Lnk Reeived 02 Fe 2011; Revised 28 Mr 2011; Aepted 08 Apr 2011 ABSTRACT Trihosnthes uumerin Linn (Fmily: Cuuritee) is limer grown in Asin ountries inluding Sri Lnk, Indi, Mly Penisul nd Philippine. The present study ws onduted to evlute whether eril prts of T. uumerin n exert ntiteril tivity. The ntiteril tivity of hot wter extrt (HWE) nd old ethnoli extrt (CEE) of T. uumerin eril prts ws evluted y () vile olony ount nd () dis diffusion tehniques ginst grm (+) nd grm ( ) teril strins suh s Stphyloous ureus, Streptoous pyogenes, Esherihi oli nd Pseudomons eroginos. Results of the present study revel tht T. uumerin hs omponents tht n signifintly inhiit the growth of S. ureus,, E. oli nd P. eroginos. Of the two extrts tested, CEE ws found to exert onsistently etter ntiteril tivity thn HWE. In onlusion, T. uumerin extrts exhiited ntiteril tivity ginst grm (+) ve teril strins suh s S. ureus, nd grm (-) ve teril strins suh s E. oli nd P. eroginos. Key Words: Trihosnthes uumerin, Stphyloous ureus, Streptoous pyogenes, Esherihi oli, Pseudomons eroginos INTRODUCTION Wound infetions re ommon in developing ountries due to poor hygieni onditions. Stphyloous ureus, Streptoous pyogenes, Esherihi oli, Pseudomons eroginos, Streptoous pneumonie nd Klesiell pneumonie re some importnt teril strins using wound infetions [1]. A wide rnge of ntiiotis (e.g. erythromyin, tetrylines, trimethoprim, sulfonmides, gentmiin, et) re eing used t present for treting wound infetions [2, 3].Bteril resistne to ntiiotis is mjor therpeuti prolem nd the rte t whih new ntiiotis re eing produed is slowing [4]. The inresing filure of hemotherpeutis nd ntiioti resistne exhiited y pthogeni miroil infetious gents, hs led to the sreening of severl mediinl plnts for their potentil ntiteril tivity. There re severl reports in the literture regrding the ntiteril tivity of plnt [5, 6, 7] extrts. Trihosnthes uumerin Linn. (Fmily: Cuuritee) is one of the mediinl plnts tht is often used in Sri Lnkn trditionl systems of mediine for the preprtion of formultions used [8,9] to tret vriety of disese onditions. It is widely distriuted in Asin ountries inluding Sri Lnk, Indi, Mly Peninsul nd Philippine [8]. Experimentlly, T. uumerin of Sri Lnkn origin hs een shown to hve ntidieti [10], gstroprotetive [11], nti-inflmmtory [12], ntioxidnt [13], lipid lowering [14], tivities nd [15] non toxiity in rodents.aording to pulitions on therpeuti uses of T. uumerin, it is useful for tretment of wounds inluding oils, sores, skin eruptions suh s ezem nd dermtitis [8]. The presene of teri within wound use infetions nd dely the heling [2]. However, up to dte, no investigtions hve een *Corresponding Author: L.D.A.M. Arwwwl, Emil: menukrwwwl@yhoo.om

2 IJPBA, Mr - Apr, 2011, Vol. 2, Issue, 2 Arwwwl et l. / Trihosnthes uumerin nd ntiteril tivity rried out to sientifilly evlute the ntiteril potentil of T. uumerin. Therefore, the im of the present investigtion ws to evlute the ntiteril tivity of T. uumerin ginst some seleted teril strins tht re known to use wound infetions. MATERIALS AND METHODS Plnt mteril T. uumerin plnts were olleted from Western provine of Sri Lnk etween the periods of August Septemer. The plnt ws identified nd uthentited y the urtor of Ntionl Herrium, Royl Botnil Grdens, Perdeniy, Sri Lnk. A vouher speimen (TS 01) ws deposited in the Industril Tehnology Institute, Colomo 7, Sri Lnk. Preprtion of hot wter extrt (HWE) T. uumerin eril prts were ut into smll piees nd ir dried. Plnt mteril (60 g) ws oiled in 1.9 L of distilled wter (DW) nd the finl volume ws redued to 240 ml y gentle oiling over 4 h nd freeze dried (yield 12.5% dry weight sis). A stok solution ws prepred t onentrtion of 1000 µg/ml from the HWE y dissolving 10 mg of the extrt in 10 ml of DW nd sterilized y utolving t 110 o C for 10 min. nd stored t - 20 o C until use. Preprtion of old Ethnoli extrt (CEE) T. uumerin eril prts were ir dried in the shde, ut into smll piees nd merted with ethnol (500 ml) nd kept for 48 h t room temperture (28-30 o C). The extrt ws filtered nd evported to dryness under redued pressure t 50 o C (yield 7.5% w/w dry weight sis). A stok solution ws prepred t onentrtion of 1000 µg/ml from the CEE y dissolving 10 mg of the extrt in 10 ml of 1% Tween 80 solution nd sterilized y utolving t 110 o C for 10 min. nd stored t - 20 o C until use. Seletion of Bteril strins Antiteril tivity of T. uumerin extrts ws evluted ginst the following teril strins otined from the Ntionl Colletion of Type Cultures (NCTC): grm positive teril strins, Stphyloous ureus (NCTC 25923), Streptoous pyogenes (NCTC 20258) nd grm positive teril strins, Esherihi oli (NCTC 25922), Pseudomons eroginos (NCTC 20620). Preprtion of growth medium for Bteril strins CM 55 gr (40 g), gr powder (5 g) nd Mueller Hinton gr (13 g) were dded to eker ontining 1L of distilled wter. The mixture ws then heted while stirring until ll the solid prtiles were dissolved ompletely. The finl ph of the medium ws djusted to 7.4 ± 0.2 nd utolved t 121 o C for 15 min. under pressure of 15 psi. When the temperture of the medium ws redued to 40 ± 5 o C, humn lood (70 ml) ws dded fter seprting the serum, mixed well, nd poured into petri dishes (15 ml/petri dish) nd llowed to set t room temperture. These lood gr pltes were stored t 4 o C until use. Evlution of Antiteril Ativity of the HWE nd the CEE Antiteril tivity ws evluted y use of () vile olony ount tehnique nd () dis diffusion tehnique Vile Colony Count Tehnique This ssy ws rried out ording to the method desried y Weersekr nd o workers [16]. One hundred miro liters (ontining 5 x 10 8 olony forming units) of eh teril (Stphyloous ureus, Streptoous pyogenes, Esherihi oli nd Pseudomons eroginos) suspension ws dded to vils ontining 900 μl from the solutions of the HWE (500 µg/ml) nd the CEE (500 µg/ml) respetively. The ontent of eh vil ws mixed well nd kept in n inutor t 37 o C for 60 min. For the ontrol tues, the sme teril suspension (5 x 10 8 olony forming units in 100 μl) nd 900 μl of sterile norml sline ws dded, mixed well nd kept in n inutor t 37 o C for 60 min. Susequently, 100 μl from eh tue ws inoulted on to lood gr pltes nd inuted for 24 h t 37 o C. At the end of 24 h inution, the olony ount in eh plte ws estimted nd expressed s olony forming units per milliliter (fu/ml). Plnt extrts tht ompletely inhiited the growth of speifi teril strin (i.e. no olony growth) were tested further s desried elow. 1 in 2 dilution series (250, 125, 62.5, 31.25, 15.62, 7.81 µg/ml) of the HWE nd CEE extrts were prepred from the stok solution (500 µg/ml) of eh extrt. Then, 900 μl of eh dilution ws introdued to teril suspension (5 x 10 8 olony forming units in 100 μl) nd sujeted to the sme proedure s desried ove. As the positive ontrol, gentmiin ws used t onentrtions of 250, 125, 62.5, 31.25, 15.62, 7.81 µg/ml. All experiments were performed in triplite. Dis Diffusion Tehnique Dis diffusion method [17] ws employed for sreening ntiteril tivities of the HWE nd 809

3 Arwwwl et l. / Trihosnthes uumerin nd ntiteril tivity IJPBA, Mr - Apr, 2011, Vol. 2, Issue, 2 the CEE. Briefly, suspension of eh teril strin (100 µl of 1 x 10 5 CFU/ml) ws spred on lood gr pltes prepred s desried previously. Sterile diss (6 mm in dimeter) impregnted either with 100 µl of the stok solutions (1000 µg/ml) whih equls to 100 µg/dis of the HWE, CEE or gentmiin (s the positive ontrol) were pled on the inoulted lood gr pltes. The test pltes were inuted t 37 o C for 24 h nd visully exmined for visile growth of the teril strins. After 24 h, the dimeters of the inhiition zones were mesured in millimeters. To detet whether the extrt ould medite dose dependnt hnge, sterile diss (6 mm in dimeter) ontining different doses of the HWE, CEE or gentmiin (100 µg/dis 12.5 µg/dis) were pled on the surfe of the lood gr plte nd sujeted to the sme experimentl proedure s mentioned ove. All the experiments were performed in triplite. Sttistil Anlysis Dt re given s mens ± S.E.M. Sttistil omprisons were mde using one wy ANOVA followed y Dunns Multiple rnge test. A P vlue 0.05 ws onsidered s signifint. RESULTS Effets of the HWE nd the CEE on vile olony ount As evident from (Tle 1) oth HWE nd CEE demonstrted signifint (P 0.05) ntiteril effets ginst S. ureus,, E. oli nd P. eroginos. On ompring the two extrts ginst the tested teril strins, the CEE ws found to e more effetive s n ntiteril gent thn the HWE. Thus, onentrtion of µg/ml of HWE nd µg/ml of CEE required to ompletely inhiit the growth of ove tested orgnisms. Further, S. ureus nd S. pyogenes pper to e more suseptile thn E. oli nd P. eroginos to the ntiteril tions of the HWE nd CEE. However, the positive ontrol, gentmiin ws found to e more potent ntiteril gent thn oth CEE nd HWE. Tle 1. Antiteril tivity of the hot wter extrt (HWE) nd the old ethnoli extrt (CEE) of Trihosnthes uumerin y vile olony ount ssy Conentrtions (µg/ml) of Trihosnthes uumerin extrts tht ompletely inhiited growth of Bteril strin Type of extrt E. oli P. eroginos S. ureus Hot wter Extrt(HWE) ± ± 0 Cold Ethnoli Extrt (CEE) ± ± 0 Gentmyin 7.03 ± ± ± ± ± ± ± ± 0 Vlues re expressed s men ± S.E.M., n = 3, Signifintly (P 0.05) different from nd, Signifintly (P 0.05) different from nd, Signifintly (P 0.05) different from nd Effets of the HWE nd the CEE on Dis Diffusion As shown in (Tle 2) oth HWE nd CEE showed signifint (P 0.05) ntiteril effets ginst S. ureus,, E. oli nd P. eroginos in the dis diffusion ssy. When ompring the two extrts ginst the tested teril strins, the CEE demonstrted greter ntiteril tivity thn the HWE. As in the olony ount method, S. ureus nd pper to e more suseptile thn E. oli nd P. eroginos for the ntiteril tions of the HWE nd CEE. The positive ontrol, gentmiin ws found to e more potent ntiteril gent thn oth CEE nd HWE. Tle 2. Antiteril tivity of the hot wter extrt (HWE) nd the old ethnoli extrt (CEE) of Trihosnthes uumerin y dis diffusion ssy Dimeter of inhiition zones (in mm) Bteril strins Cold ethnoli extrt (CEE) Hot wter extrt (HWE) Gentmiin 50 µg/dis 50 µg/dis 50 µg/dis S. ureus 16.8 ± ± ± ± ± ±

4 Arwwwl et l. / Trihosnthes uumerin nd ntiteril tivity E. oli 26.3 ± ± 1.1 P. eroginos 24.5 ± ± ± ± µg/dis 25 µg/dis 25 µg/dis S. ureus 10.4 ± ± ± ± 0.3 E. oli 17.8 ± ± 0.6 P. eroginos 22.5 ± ± ± ± ± ± µg/dis 12.5 µg/dis 12.5 µg/dis S. ureus 4.8 ± ± ± ± 0.5 E. oli 12.5 ± ± 1.1 P. eroginos 17.5 ± ± ± ± ± ± µg/dis 6.25 µg/dis 6.25 µg/dis IJPBA, Mr - Apr, 2011, Vol. 2, Issue, 2 S. ureus 0.0 ± ± ± ± ± ± 1.5 E. oli 0.0 ± ± ± 1.1 P. eroginos 0.0 ± ± ± 1.4 Vlues re expressed s men ± S.E.M., n = 3, Signifintly (P 0.05) different from, Signifintly (P 0.05) different from DISCUSSION The tivity of plnt extrts ginst teri hs een studied for yers, ut more ggressively during the lst three dedes [18]. The inresing filure of hemotherpeutis nd ntiioti resistne exhiited y pthogeni miroil infetions gents hs led to the sreening of severl mediinl plnts for their potentil ntimiroil tivity [19].The present study revels tht T. uumerin hs omponents tht n exert signifint ntiteril tivity ginst S. ureus,, E. oli nd P. eroginos tht re known well s wound pthogens [2]. Furthermore, the CEE n exert onsistently etter ntiteril tivity thn the HWE. This differene in tivity of the HWE nd CEE my e relted to the polrity of ntiteril ompounds present in T. uumerin. Some other mediinl plnts hve lso een reported to hve strong ntiteril effets in orgni extrts thn queous extrts. Exmples inlude Cthrnthus roseus [20] nd Aporos lindleyn [21]. A zone of inhiition 9-15 mm is n indition [22] of strong ntimiroil tivity. E. oli nd P. eroginos were found to e most suseptile to T. uumerin with n inhiition zone greter thn 9 mm t very low onentrtion (12.5 µg/dis) of oth extrts. Phytohemil onstituents suh s tnnins, sponins, flvonoids, lkloids nd severl other romti ompounds re seondry metolites of plnts tht serve s defense mehnisms ginst predtion y mny miroorgnisms, insets nd other herivores [23]. Phytohemil investigtions revel tht T. uumerin extrts lso ontin tnnins, sponins, flvonoids nd lkloids s mjor [15] hemil onstituents. Therefore, these phytohemils my ontriute to its ntiteril tivity. Both E. oli nd P. eroginos were found to e more suseptile thn S. ureus nd to the tion of the T. uumerin extrts. This is possily due to the differenes in hemil omposition nd struture of ell wll of the miroorgnisms. In onlusion, T. uumerin extrts exhiited ntiteril tivity ginst oth grm (+) ve teril strins suh s S. ureus, nd grm (-) ve teril strins suh s E. oli nd P. eroginos mediting the presene of rod spetrum of ntiteril ompounds in the plnt. ACKNOWLEDGEMENT This work ws supported y the Ntionl Siene Foundtion (grnt numer NSF/SCH/2005/13). REFERENCES 1. Mertz P, Ovington L. Wound heling miroiology. Dermtol Clin 1993; 11: Lullmnn H, Mohr K, Ziegler A, Bieger D. Color Atls of Phrmology. 2 nd edn. Pulition of Thieme Stuttgrt. USA

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