IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Antibacterial Activity of Calendula Officinalis Flowers In Vitro M. N. Hamad , H.J. Mohamme d* , M.A. Merdaw* Departme nt of Pharmacognosy and Medicinal Plants, College of Pharmacy, Unive rsity of Baghdad *Departme nt of Clinical Laboratory Sciences, College of Pharmacy, Unive rsity of Baghdad Received in : 14, January ,2010 Accepte d in : 27,February,2011 Abstract Flower samples of Calendula officinalis were collected from the gardens of the college of Pharmacy in Baghdad city to examine their antibacterial activity .The effect of ethanol crude extract of p etals and reproductive p arts of flowers in different concentrations were obtained and bioassayed in vitro for its bioactivity to inhibit the growt h of eight typ es of bacteria .The extracts of p etals p art were clearly sup erior for all bacteria esp ecially the bacteria Pseudomonas aeru ginosa (inhib ition zone was 25mm in the concentration 100mg/ml) from gram negative bacteria, and Staphy lococcus aureus (inhib ition zone was 14mm in the concentration 50mg/ml) from gr am p ositive bacteria, while the extracts of reproductive parts were less effective than p etals p art. These results confirm the antibacterial activity of Calendula officinalis flowers and supp ort the traditional use of the p lant in the therapy of bacterial infection. Key words: antibacterial activity , p etals p art, reproductive p arts Introduction The plant Calendula officinalis belon gs to the family : Asteracea [1]. Commonly known as English marigo ld or pot marigold, field marigold , garden marigold, gold bloom, holligo ld, maravilla and, marybud [2]. Its name comes from the Latin word, Calends , the first day of every month, because of its long flowering p eriod, because the flowers fol low the sun , it was linked to t he ast rological sign of summer [3]. Its bright orange p etals are v isible in gardens in every month of y ear; such is its hardy nature and widely distributed through Europe and the M editerranean. The orange p etals and the whole flower head ar e used medicinally [4,5]. M arigold flowers often used in food industry for their nutritive qualities as well as coloring of several culinary p roducts, is well known of c enturies in pop ular medicine because of their biolo gic activities. Their use for the p rep aration of cosmetic p roducts is well known also [6]. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 The major active constituents include sap onins , triterp endiol esters and flavonoids includin g hy p eroside and rutin, The orange flower contains a high content of caroteniodes includin g f lavo xanthin and auroxanthin [7,8,9]. Calendula has antibacterial and antifungal activity [10,11] , and it has been used for the treatment of burns, abrasions, skin inflammations, ulcers, wounds and eczema[12]. It has be en used internally for the treatment of gastiritis , bleedin g of duodenal ulcers and colitis [13] Researchers fro m Venezuela examined extracts of dried flowers from Calendula officinalis for its inhibitory effects on the human immunodeficien cy virus type1(HIV-1) [14] Calendula officinalis e xtracts show anti-cancer effects in vitro st udies on t umor cell lines, derive from Leukemias, melanomas, fibrosacomas, breast, p rost ate, cervix, lung and p ancreas [15]. Materials and Methods Plant material Flowers of Calendula officinalis were collected during Ap ril-M ay 2009 from the garden of the college of Pharmacy in Baghd ad city and was identified by Ali Abdul-hussein Al- M osawi (Prof. in college of Sciences/University of Baghdad). Extraction Extraction was carr ied out at room temp erature under normal cond itions. Dried flowers of Calendula officinalis were p owdered and subjected to extraction in a so xhlet app aratus at room temperature using ethanol. The collected extracts were concentrated by evap oration under room temp erature. The extracts were made in to susp ensions using dimethy l sulfoxide solvent(DM SO) at concentrations (50,75,100) mg/ml from the petals p art of flower and the same concentrations from the reproductive parts of flower [16]. Micro-organi sms use d Gram-positive bacteria: Staphy lococcus aureus, Bacillus subt ilis , Staphy lococcus epidermidis. Gram-negative bacteria: Pseudomonas aeru ginosa, Escherichia coli, Prot eus mirabilis, Klebsiella p neumoniae and Enterococcus p neumoniae. Preparation of inoculum Susp ension of organism was p repared as p er M cfarland nephalometer st andard[17].A 24hours old culture was used for the prep aration of bacterial susp ensions. The susp ension of organism was made in a st erile isotonic solution of sodium chloride(0.9%w/v) and the turbidity was adjusted such that it contained ap p roximately 1.5x10 8 cells/ml. Agar well diffusion method The medium was p rep ared by dissolving all the ingredients in distilled water and subjected to sterilization in an autoclave at 121˚c for 15 minutes. The Petri p lates were washed thoroughly and st erilized in hot air oven at 160˚c for (1.5) hour .30ml of st erile nutrient agar was seeded by organisms (about 2ml according to M cfarlands standard). Pores IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 were mad e on the medium usin g a st erile borer and 0.1ml of the extracts were added to resp ective p ore. The Petri p lates seeded with organisms containin g extracts were kep t in refrigerator at 4˚c for 1hour to facilitate the diffusion of the extracts into the media. Aft er diffusion the Petri p lates were incubated at 37˚c for 24 hours in an incubator and zone of inhibition was observed and measured using a scale. The results of the antibacterial activity of p etals and reproductive p arts of flower of Calendula officinalis are tabulated in Table(1)and Table(2). Results and Discussion The essential oil of the flowers inhibited the growt h in vitro of Gra m p ositive bacteria such as Bacillus subt ilis and Staphy lococcus aureus ,and Gram negative bacteria such as Escherich ia coli and Pseudomonas aeru ginosa ,these results are in agr eement with some p revious studies [7,18,19]. Antibacterial activity is recorded when the zone of inhibition is greater than 6mm[20]. Results as shown in (Table1) indicated the antibacterial activity of p etals of Calendula officinalis to all isolates in all concentrations except the concentration (100mg/ml) can not inhibit t he growt h of Staphy lococcus ep idermidis ,Bacillus subtilis and Proteus mirabilis. The maximu m inhibition zone was found in( 100mg/ml) concentration and it was 25 mm in the bacterial isolate Pseudomonas aeruginosa whi le the minimum inhib ition zone in the same concentration was 8 mm for the isolate Enterococcus p neumoniae. In (Table2) the results shows that the antibacterial activity of reproductive p arts of Calendula officinalis is less than the p etals and it has no effect in all concentrations against the isolate Enterococcus p neumonia ,and also indicated that the maximum inhib ition zone was observed in Pseudomonas aeruginosa in the concentration (100mg/ml) and it was 24 mm while the concentrations (50mg/ml,75mg/ml) has no effect against this bacterium , the minimu m inhibition zone was 7 mm in Staphy lococcus epidermidis in the concentration (50mg/ml) and there was no effect at the concentration (100mg/ml) against this bacterium .From (Table 1) and (Table 2) its clear that the effective antibacterial activity of Calendula officinalis flower concentrated in their p etals more than the reproductive p arts and this confirm by p revious study [8]. It is not p ossible to make a direct correlation between the observed activity of the plant extracts in vitro and the actual effects when used in vivo for the diseases observed by the indigenous p eople and traditional healers [19] . Therefore , it is imp ortant that the p lant should also b e further investigated to evaluate the significance of these extracts , clinical role 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 Its clear that Calendula officinalis flowers as an extract may be useful as an antibacterial agent against the above mentioned bacteria . Amon g the organisms tested Pseudomonas aero ginosa was more suscep tible to the ethanolic extract of C alendula officinalis flower . Furt her pharmalogical and clinical st udies are required to understand the mechanism and the actual efficacy of this herb extract in treatin g various inf ections. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Re ferences 1. M uley ,B.P. ; Khadabadi, S. S. and Banarase,N.B. (2009),Phy tochemical Const ituents and Pharmalogical Activities of Calendula officinalis linn (Asteracea).Trop ical J. of Pharmaceutical Rese arch,October;8(5):455-456 2. Henrich, M .; Bames, J.;Gibbons, S. and. Williamsom, E.(2004),Fundamentals of Pharmacognosy and Phytotherapy .Churchill Livingst one,270. 3. Bezakova, L.; M asterova , I.; Paulikova ,I. and Psenak ,M .(1996), Inhibitory activity of isorhammntin glycosides from Calendula officinalis L.on the activity of lip oxy genase.Phar mazie, 51:126-7. 4. Pommier, P.;Gom ez, f.;Suny ach, M .;Came, C. and M ontbarbon, X.(2004),Phase111 randomized of C aleneula officinalis compared with troamine for the p reventation of acute dermatitis during irradiation for breast cancer .J.Cl in.Oncol.;22(8) :144-53. 5. SVKM , S.(2008), Ant imicrob ial activity of the leaf extracts of Calendula officinalis (LINN.).J. of herbal M edicine and Toxicology 2(2):65-66. 6. M arian, A.; Natalia, A.;and Carmen, C. (2004),Calendula officinalis flowers ,source of extracts with antioxidant activity .J. Chemist ry 13(2):169-176. 7. Braun, L. and Cohen, M . (2005),Herbs and natural supp lemants: an evidence –b ased guide Sy dney,Elsevier,98-100. 8. Roop ashree, T.S;Raman Dan g;Shobha R ani R.H and Narendra.(2008), Ant ibacterial activity of antip soriatic herbs: Cassia tora,M omordica char antia and Calendula officinalis. International J. of App lied Research in Natural Products.1(3), 20-28,Sep/Oct. 9. Neukirch,H.D.; Ambrosio,M .;Dalla Via, J.;and Guerriero, A.,(2004), Simultaneous quantitative determination of eight triterp enoid monoest ers from flowers of 10 Varieties of Phy tochem Anal.,15(1)30-35. 10. Tonks ,A.J.;Dudley ,E.;Port er, N.G.;Parton ,J.; Brazier, J.;Simth ,E.L and.;Tonks ,A.;(2007);A5.8-kDa comp onent of manuka honey stimulates immune cells via TLR4;J Leukoc Bio l.;82(5):1147-55. 11. Rossiter, K.;Reid, P.D.; Lwaleed, B.A.; Coop er, A.J.; Voegeli, D.; Coop er, R. and Getliff i K. (2006),Honey and angiogenesis;1 st International conference on the med icinal uses of Honey ;Kota Bharu;M alaysia. 12. Schulz, V.; Hanse l ,R.; Blumenthal, M . and Tyler, V. (2004) ,Rational Phy totherapy areferen ce guide for physicians p harmacists,Berlin,Sp ringer;344. 13. Bone, K. (2003), A clinical Guide to blend ing liqu id herbs. St Louis M issoun ,Churchill livinigst one,120-123. 14. De Tommasi N,et al,(1990) , Structure and in vitro antiviral activity of sesquiterpene glycosides from Calendula officinalis .J.Nat Prod. Jul-Aug;53(4):830-5. 15. Jimenez-M edina E,et al,(2006), Anew extract of the p lant Calendula officinalis p roduces adual in v itro effect: cytotoxic anti-tumor activity and lymphocyte activation .BM C Cancer. M ay 5(6):119. 16. Shahid i Bonjar,G.H. (2004), Screening for antibacterial Prop erties of some Iranian Plants Agianst Tow Strians of Escherichia coli.Asian J. of Plant Sciences 3(3):310-314. 17. Ellen, J.B. and Sy dney, M .F. (1990). Baily and Scott diagnost ic microbiolo gy .8 th ed.,USA,M issouri.p .543.Evan WC.1996.Trease and Evans Pharmaco gnosy .14 th ed. London,En gland :W.B. Sounders comp any limited.p545-46. 18. Janssen ,A.M . et al .,(1986), Screening for antimicrobial activity of some essential oils by the agar overlay technique .Pharmaceutisch Weekblad,8:289-292. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 19. M uhammad ,H.S. and M uhammad, S. (2005),The use of Lawsonia iner mis L inn.(henna)in the management of burn wound infections. African J. of biotechnology .4(9),943-37. 20. Goun, E.G.; Cunningh am, D.C.;N guy en Cand M iles, D.,(2003),Antibacterial and antifungal activity of Indonesian ethanomedical plants.74;592-96. Table (1) : In vitro antibacterial activity of petals part of Calendula officinalis flower Zone of inhibition(mm, diameter) Concentration of the extract Bacteria 12 50mg/ml Bacillus subtilis 13 75mg/ml - 100mg/ml 14 50mg/ml Staphy lococcus aureus 12 75mg/ml 6 100mg/ml 10 50mg/ml Staphy lococcus epidermidis 12 75mg/ml - 100mg/ml 15 50mg/ml Escherich ia coli 14 75mg/ml 12 100mg/ml 13 50mg/ml Klebseilla p neumoniae 14 75mg/ml 20 100mg/ml 15 50mg/ml Pseudomonas aeru ginosa 17 75mg/ml 25 100mg/ml 11 50mg/ml Prot eus mirabilis 12 75mg/ml - 100mg/ml 18 50mg/ml Enterococcus p neumoniae 12 75mg/ml 8 100mg/ml IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (3) 2011 Table (2) : In vitro antibacterial activity of reproductive pa rts of Cal endul a officinalis flower Zone of inhibition(mm ,diameter) Concentration of the extract Bacteria 6 50mg/ml Bacillus subtilis 8 75mg/ml - 100mg/ml 10 50mg/ml Staphy lococcus aureus 8 75mg/ml - 100mg/ml 7 50mg/ml Staphy lococcus epidermidis 8 75mg/ml - 100mg/ml 10 50mg/ml Escherich ia coli 12 75mg/ml 5 100mg/ml 13 50mg/ml Klebseilla p neumoniae 12 75mg/ml 11 100mg/ml - 50mg/ml Pseudomonas aeru ginosa - 75mg/ml 24 100mg/ml 8 50mg/ml Prot eus mirabilis 10 75mg/ml - 100mg/ml - 50mg/ml Enterococcus p neumoniae - 75mg/ml - 100mg/ml 2011) 3( 24لوم الصرفة والتطبیقیة المجلدمجلة ابن الهیثم للع Calendula oثیر الضد بكتیري لنبات االقحوان أالت fficinalis الجسم خارج الحي *میسون عبد الزهرة مرداو ،*هدى جاسم محمد ،مها نوري حمد جامعة بغداد،كلیة الصیدلة،فرع العقاقیر والنباتات الطبیة جامعة بغداد،كلیة الصیدلة،فرع العلوم المختبریة السریریة* 2010 كانون الثاني 14:استلم البحث في 2011 شباط 27:بل البحث فيق الخالصة من حدیقة كلیة الصیدلة في مدینة بغداد لفحص نشاطها Calendula officinalisجمعت ازهار نبات االقحوان الحي لمعرفة تأثیر مستخلصات الكحول للبتالت واالجزاء التكاثریة للنبات الجسم ختبار الحیوي خارج تم اال. الضد بكتیري . انواع بكتیریة 8بتراكیز مختلفة لتثبیط نمو Pseudomonasبكتریا والسیمامستخلصات البتالت كان لها تاثیر أعلى وأوضح من تلك لالجزاء التكاثریة auroginosa من البكتریا السالبة لصبغة كرام وبكتریا ) یلترمل/ملغم 100ملمتر عند التركیز 25(طقة التثبیط كانت من Staphy lococcus aureus من البكتریا الموجبة لصبغة )یلترمل/ملغم 50ملمتر عند التركیز 14(منطقة التثبیط كانت ستعمال التجاري للنبات في ألوتدعم اCalendula officinalis زهار ألهذه النتائج تؤید النشاط الضد بكتیري . كرام .عالج االصابات البكتیریة .االجزاء التكاثریة، البتالت، النشاط الضدبكتیري: الكلمات المفتاحیة