Antimicrobial Activity of Grapefruit Seeds Extracts (In Vitro Study)
Total Page：16
File Type：pdf, Size：1020Kb
ISSN: 1812–1217 Antimicrobial Activity of Grapefruit Seeds Extracts (In vitro Study) Waidulla N Al–Âni Department of Dental Basic Sciences BSc, PhD (Asst. Prof.) College of Dentistry, University of Mosul Nahla O M Tawfik Department of Dental Basic Sciences MBChB, MSc, PhD (Lec.) College of Dentistry, University of Mosul Enas Y Shehab Department of Dental Basic Sciences BSc, MSc (Asst. Lec.) College of Dentistry, University of Mosul الخﻻصة اﻷهداف: هتدف الدراسة إىل حتديدد العاللةدة ااددللل لاثدمالةم لالصداملائ اادلكح يلالبذدر لكدييلر البميد دميل المواا طرااقوا اللمو : أُسداملد ااصداملائ اادلكح يلالبذددر ﻹاثينددل ر ذ لكددييلر البميدد ددميل يل ةدد ٪٠٢ ﻹيلزن/ حثددمذ يلذلددمل اام ددة هدددلجر ثددد ملددمالةم اابددررا الايةرليددة اليتكةددةل يلاااةاكددل يلالباكةصددة المكريددة يلااكة ددل الكة ددلز يل دد يو العاددررا العلريددة يلي أي ددل حتديددد صددارا الادددلج رميةددة اث اتددلر ددل اماع لاةدد ااملادد يلارر دد العاللةددة اادددللل لاثددمالةم يلالعرميددل دد هدددلج الباررتبصددةديب ةدد يب ﻹ۱٠ ٢٪ ذ يل ﻹ٠ ٢٪ ذ لال اددة سددةرمل النتووجق : أعرددا ااصدداملائ ااددلكح لكددييلر البميدد ددميل ﻹ٠٢٪ ذ يف تددير الدراسددة اةثددة اجيل ةددةل يلهدددلج الهدد لاثددمالةم اةددد الدراسددةن حةددا أعرددا يددلبأ ةللةددة ددب اليلددر دارددلر همايلحدد ددل ﻹ۱٢ اددمذ – ﻹ ۱۱ اددمذ لاةلر ددة دد الباررتبصددةديب ﻹ۱٣ ادم - ٠٢ ادمذ يل ﻹ۷ ادم - ٠٠ اددمذ لا ا ةد ﻹ۱٠ل ٢٪ذ يل ﻹ٠ل ٢٪ذ عادا الاددرا أ دل ااصداملائ البذدر اددم ياردح أيدة اللةددة ددللل اﻹسوتنتجاج:: ميااددمل ااصاملائ االكح لكييلر البمي ميل اللةة ملةدل ثد اجلمالةم ممل جيااه صاذدم بكةاح ملةد لﻹساملدا لرهم أيل اةم ABSTRACT Aims: The purpose of this study is to determine the antimicrobial activity of aqueous and alcoholic extract of grapefruit (Citrus Paradisi Rutaceae) seeds. Materials and Methods: Aqueous and alcoholic (ethanol) extracts of grapefruit seeds "GSE" in 20% (w/v) concentration were investigated for activity against Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumonia, Candida albicans and a mixed oral flora. The level of antimicrobial effects was established using in vitro disc diffusion method. Their antibacterial and antifungal activity was compared to the activity of Chlorhexidine (CHX) solution in two concentrations (0.12 and 0.2%) as a control. Results: The aqueous GSE (20%) solution used in this study gave positive results with lethal effect on the tested organisms with zones of inhibition ranging from 10mm-18mm in diameter, which is comparable to that of chlorhexidine (7mm - 22mm) and (13mm - 20mm) for 0.12% and 0.2% solutions respectively. Ethanolic GSE did not show any antimi- crobial activity. Conclusions: Aqueous GSE has a good antimicrobial effect, which makes it a good natural preparation for use as antiseptic or disinfectant. Key Words: Grapefruit Seeds. chlorhexidine and aqueous extract. Al–Âni WN, Tawfik NO, Shehab EY. Antimicrobial Activity of Grapefruit Seeds Extracts (In vitro Study) Al–Rafidain Dent J. 2011 11(2): 341-345. Received: 16/5/2010 Sent to Referees: 16/5/2010 Accepted for Publication: 28/6/2010 INTRODUCTION ed by the facts that they contain different The use of herbal medicine is widely classes of polyphenolic flavenoids that spread and growing (1). Plants have been have been shown to exert antibacterial, exploited for the treatment of many infec- antifungal and antioxidant activities (4, 5). tions and diseases because those plants Grapefruit seed extract (GSE) is a com- readily contain substances for defense mercial product derived from the seeds against attacks by insects, herbivores and and pulp of grapefruit (6, 7). The present microorganisms (2). The citrus fruits make study contributes to the identification of a group of plants of great medicinal im- the antibacterial and antifungal effects of portance (3). The therapeutic efficacy of the self – made aqueous and ethanolic ex- citrus fruits such as grape fruits is support- tracts of GSs and to compare those effects Al – Rafidain Dent J www.rafidaindentj.net 341 Vol. 11, No2, 2011 Al–Âni WN, Tawfik NO, Shehab EY with an important orally used antiseptics ed against one fungal isolates (Candida solution chlorhexidine gluconate (CHX) in albicans) by the disc diffusion method. 0.12% and 0.2% concentrations. Colonies were streaked on Muller- Hinton agar. Within 15 minutes after the plates were inoculated, a prepared discs MATERIALS AND METHODS were applied to the surface of the inoculat- 1. Preparation seeds for extraction: ed plates by sterile forceps, taking care The seeds of the grape fruits were air-dried that each disc is 15mm apart from edge of for two weeks to prevent loss of active the plate. Then plates were placed in an components. Then were ground into pow- incubator at 37C for 24 hours for bacteria der with electrical blender. Water and eth- (13) and for 48 hours for Candida albicans . anol alcohol were used for the extraction After incubation, the plates were ex- (8). amined, the zone of inhibition of growth 2. Preparation of aqueous extract: were noted and measured. The antimicro- Forty grams of dry seeds powder were bial activity of each extract was measured placed in 160ml of sterile distilled water from the diameters of zone of inhibition and left at room temperature for 24 hrs, the for each organism and this was compared mixture was filtered firstly by gauze and with that of CHX solution (0.12% and secondly by filter paper (Whatman No. 1). 0.2%). After filtration, it was placed into incuba- tor at 37C°. The liquid was evaporated, and the precipitated extract was left at the RESULTS base of the baker (9). The antimicrobial effects of self made 20% (w/v) GSE (aqueous and ethanolic) 3. Sterilization of aqueous extract: extracts and CHX solution (0.12% and Five ml of distilled water was added to 1 0.2%) are shown in Table (1). gram of plant extract powder to produce The ethanolic extract showed no zone 200mg/ml (20%w/v) solution, sterilization of inhibition. The aqueous extract (20%) was carried out by passing through a ster- was active against all gram negative bacte- ile filter membrane 0.22µg (10). ria (Figure, 1). GSE produced the largest 4. Preparation of alcoholic extract: zone of inhibition for normal flora (18mm) Twenty grams of dry seeds powder was (Table, 1), but exerted lower activity on added to 200ml of ethanol. The same pro- the growth of Staphylococcus aureus cedure as in aqueous extract was ap- (10mm). In comparison, CHX 0.2% (con- pliedbut the solvent for extraction was trol I) showed the largest zone of inhibi- 95% ethanol (9). tion against normal flora (20mm), while 5. Sterilization of alcoholic extract: exerted lower activity on the growth of S. Five ml of Dimethyl sulfoxide was added aureus, Proteus sp. and Klebsiella sp to 1gm of seed extract powder and steri- (18mm). The zone of inhibition to CHX lized by pasteurization. The same concen- 0.12% (control II) is 7, 11, 18 and 22mm tration of (20%w/v) is as aqueous extract for S. aureus, Proteus sp., Klebsiella sp, (10). and normal flora respectively. The aque- 6. Sensitivity test (disc diffusion method): ous extract (20%) was active against Can- Susceptibility testing was done on each dida albicans with a zone of inhibition of isolate (in triplicate) for each type of the 12mm in diameter (Table, 1). extract, CHX solution ( 0.12 and 0.2% ) Both CHX solutions (0.12% and were used as control using disc diffusion 0.2% ) showed approximately the same method(11). Discs were prepared by adding activity on the Candida albicans isolate 1ml of seed extract to 10 discs (12). tested (13mm and 12mm respectively) Four types of bacterial isolates were when compared with the aqueous GSE as used; Staphylococcus aureus, Proteus vul- shown in Figure (1), Table (1). garis, Klebsiella pneumonia and a mixed oral flora. The antifungal activity was test- 342 Al – Rafidain Dent J Vol. 11, No2, 2011 Antimicrobial effect of grapefruit seeds extracts 25 20 15 10 5 0 Inhibition zone) ( mm Ca. Al. St.Au. Pr.sp. Kl.sp. Nor. Fl. GSE (20%) CHX (0.2%) CHX (0.12%) Figure (1): Comparison between the effect of 20% aqueous GSE and CHX solutions( 0.2% & 0.12%) on different types of microorganisms. Table (1): Comparisons between the antimicrobial activity of the 20% aqueous GSE with CHX solutions Inhibition Zone ( mm ) Types of microorganism GSE (20%) CHX (0.2%) CHX (0.12%) Candida albicans 12 13 12 Staph. aureus 10 18 7 Proteus sp. 16 18 11 Klebsiella sp. 16 18 18 Normal flora 18 20 22 *Diameter of zone of inhibition in mm (Data represented as mean of three reading);**Disc diameter = 6mm (17). DISCUSSION regarding their biological properties In this study, solutions of 20% con- Techniques of solubility of these substanc- centrated GSE (aqueous and ethanolic) es are among other problems. Normaliza- were tested for antibacterial properties tion of methods and laboratory conditions, against a number of gram negative and in addition to the techniques used (extrac- dissolution and dispersion, culture لgram positive organisms. The ethanolic tion extract showed no activity against all test medium) for testing biological and phar- microorganisms. This is in agreement with macological activities of solutions at the Adedeji et al. where negative results were definitive stage of laboratory screening are also obtained. Kroum et al. found that desirable, in order to provide a common methanolic extract were more active than basis for the comparison of results ob- ethanolic extract, but many studies showed tained in various parts of the world in dif- that ethanolic GSE possess good antibacte- ferent organisms tested under similar la- rial activities against many types of mi- boratory conditions. croorganisms (6, 15 & 16). The method of ex- The aqueous GSE has consistently traction will influence the chemical com- good antimicrobial activity against all bio- position, and thus, can have repercussion type tested.