ISSN: 1812–1217

Antimicrobial Activity of 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

الخالصة األهداف: هتدف الدراسة إىل حتديدد العاللةدة ااددللل لاثدمالةم لالصداملائ اادلكح يلالبذدر لكدييلر البميد دميل المواا طرااقوا اللمو : أُسداملد ااصداملائ اادلكح يلالبذددر إلاثينددل ر ذ لكددييلر البميدد ددميل يل ةدد ٪ ٠٢ إليلزن/ 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 إل۱٠ ٢٪ ذ يل إل٠ ٢٪ ذ لال اددة سددةرمل النتووجق : أعرددا ااصدداملائ ااددلكح لكددييلر البميدد ددميل إل٠٢٪ ذ يف تددير الدراسددة اةثددة اجيل ةددةل يلهدددلج الهدد لاثددمالةم اةددد الدراسددةن حةددا أعرددا يددلبأ ةللةددة ددب اليلددر دارددلر همايلحدد ددل إل۱٢ اددمذ – إل ۱۱ اددمذ لاةلر ددة دد الباررتبصددةديب إل۱٣ ادم - ٠٢ ادمذ يل إل۷ ادم - ٠٠ اددمذ لا ا ةد إل۱٠ل ٢٪ذ يل إل٠ل ٢٪ذ عادا الاددرا أ دل ااصداملائ البذدر اددم ياردح أيدة اللةددة ددللل اإلسوتنتجاج:: ميااددمل ااصاملائ االكح لكييلر البمي ميل اللةة ملةدل ثد اجلمالةم ممل جيااه صاذدم بكةاح ملةد لإلساملدا لرهم أيل اةم

ABSTRACT Aims: The purpose of this study is to determine the antimicrobial activity of aqueous and alcoholic extract of grapefruit ( 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 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

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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-

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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. This is comparable to that of

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Al–Âni WN, Tawfik NO, Shehab EY proven topical antibacterial solutions chitin polymers which could be the cause (CHX). These results are also in agree- of death in those fungal strains (20). ment with several scientific studies which Despite the fact that CHX were found showed that GSE possess good antibacte- to be superior to the tested aqueous ex- rial activities against many types of mi- tract, the latter showed slight, but constant croorganisms (5, 6 & 15). Chemical research activity against all the tested bacteria and revealed the presence of , yeast. ascorbic acid, tocopherols, , li- CONCLUSIONS monoids, sterols and minerals in grapefruit The use of 20% concentration of GSE seeds and pulp extracts (4, 5 & 16). This bene- aqueous solution has effective anti bacteri- ficial antibacterial effect of GSE has been al activities against Staphylococcus aure- attributed to the antioxidant activity of us, Proteus vulgaris, Klebsiella pneumo- citrus flavonoids, such as naringenin and nia, Candida albicans and normal mouth hesperidine (4). The presences of those flora. The antibacterial activity of some of compounds were confirmed by thin layer that extracts was of such a level that it chromatography (5). Heggers et al. with aid would probably be therapeutically useful, of scanning transmission electron micros- because its activity was comparable with copy revealed GSE's antibacterial activity. one of most widely used antiseptic solu- It was evident that GSE disrupts the bacte- tion. rial membrane and liberate the cytoplas- mic contents with in 15 minutes after con- REFERENCES tact even at more diluted concentration (7). 1. Barnes J, Anderson LA, Pillipson JD. Fungi or yeast constitute a small pro- Herbal medicines. A guide for healthcare portion of the usual oral microbiota with professionals. 2nd edition, Pharmaceutical Candida species being the most common Press, London. 2002. P. 3. of the fungi present in healthy (30 – 45%) 2. Majorie M. Plants Products as antimicrobi- individual. Aqueous CHX solution has a al agents. Clini Micro Rev. 1999; 12: 564 – wide spectrum of antimicrobial activity at 582. low concentrations and is especially effec- 3. Hammer KA, Carson CF, Riley TV. tive against Candida albicans (11) and this Antimicrobial activity of essential oils and is in agreement of our result were both other plant extracts. J Applied Microb. CHX preparations had more or less same 1999; 86: 985 – 990. antifungal activity and this was compara- 4. Dembinski A, Warzecha Z, Konturek SJ, ble with that of our preparations. Ceranowicz P, Dembinski M, Pawlik WW, Krajewska et al. found that 33% Kusnierz – Cabala B, Naskalski JW. Ex- (m/V) GSE concentration exert a potent tract of grapefruit seed reduces acute pan- antifungal activity against the yeast like creatitis induced by ischemia / reperfusion fungi strains and had low activity against in rat; possible implantation of tissue anti- germatophytes and moulds (18), while oxidants. J Physiol Pharmacol. 2004; 55(4): Cvetnić and Vladimir – Kneźević re- 811 – 821. ported that GSE exhibit antifungal activity 5. Cvetnić Z, Vladimir – Kneźević S. Anti- against all tested strains of Candida albi- microbial activity of grapefruit seed and cans in concentrations from 8.25% to pulp ethanolic extract. Acta Pharm. 2004; 16.50% (m/V) (inhibition zones ranging 54: 243 – 250. from 9mm to 11mm) (5) and this is in 6. Reagor L, Gusman J, McCoy L, Carino E, agreement of our result. Heggers JP. The effectiveness of processed The inhibitory action of these natural grapefruit seed extract as antibacterial products on mould could involve cyto- agent: 1. An in vitro agar assay. J Altern plasmic membrane granulation, rupture Complement Med. 2002; 8: 325 – 332. and inactivation and / or inhibition of in- 7. Heggers JP, Cottingham J, Gusman J, tercellular and extracellular . The- Reagor L, McCoy L, Carino E, Cox R, se biological events could take place sepa- Zhao J. The effectiveness of processed rately or concomitantly culminating with grapefruit seed extract as an antibacterial mycelium germination inhibition (19). agent: II. Mechanism of action and in vitro Brull and Coote reported that plant lytic toxicity. J Altern Complement Med. 2002; enzymes act on fungal cell wall causing 8: 333 – 340. breakage of β-1,3 glycan, β-1,6 glycan and 8. Adedeji GB, Fagade OE, Oyelade AA. prevalence of Pseudomonas aeruginosa in

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clinical samples and its sensitivity to citrus 14. Kroum S A, Satta D, Lalaoui K. Antimi- extract. African J Bio Res. 2007; 10:183- crobial, antioxidant, cytotoxic activities 187. and screening of some Al- 9. Al-Joboory A, Al-Rawi M. Natural phar- gerian plants. Europ J Scien Res. 2009; macology. 1st ed. Baghdad: dar Al-Huriah; 31:289-295. 1994. 15. Von Woedtke Th, Schlüter B, Pflegel P, 10. AL- Nouman AY. Effect some plants Lindequist U, Jülich WD. Aspect of the extracts on the growth and metabolism of antimicrobial efficacy of grapefruit seed gram positive and gram negative bacteria. extract and its relation to preservative sub- Ph thesis. College of Science, Mosul Uni- stances contained. Pharmazie. 1999; 54: versity. 1998. 452 – 456. 11. Siqueira JF Jr, Rocas IN, Paiva SS, 16. Giuseppe G, Davide B, Claudia G, Ugo L, Guimaraes-Pinto T, Magalhaes KM, Lima Corrado C. Composition of Cit- KC. Bacteriologic investigation of the ef- rus . Molecules. 2007;12: 1641-1673. fects of sodium hypochlorite and chlorhex- 17. Lahlou M. Methods to Study the Phyto- idine during the endodontic treatment of chemistry and Bioactivity of Essential Oils. teeth with apical periodontitis. Oral Sur, Phytother Res. 2004; 18: 435–448. Oral Med, Oral Pathol, Oral Radiol and 18. Krajewska KE, Lukaszuk C, Niczyporuk Endo. 2007; 104: 122–130. W. Antifungal activity of 33% grapefruit 12. Pre Scott LM, Harley JP, Klein DA. water glycerol solution. J Eur Acad Derma- Micro biology. 3rd ed. Wm C Brown tol Venerol. 2003; 17: 486 – 487. Cowan communication, Inc., Iowa, USA; 1996. NM. Plant products as antimicrobial 13. Vandepitte J, Enghack K, Piot P, Heuch C. agents. Clin Micro Rev. 1999; 12:564-582. Basic Laboratory procedures in clinical 19. Brull S, Coote P. Preservative agents in bacteriology ( WHO ) - Genuva. 1991; foods: mode of action and microbial re- pp21-38; 82-95. sistance mechanisms. Inter J Food Mi- cro.1999; 50:1-17.

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