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Complementary Therapies in Clinical Practice 18 (2012) 173e176

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Complementary Therapies in Clinical Practice
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Antimicrobial activity of Calendula officinalis petal extracts against fungi, as well as
Gram-negative and Gram-positive clinical pathogens
Efstratios Efstratiou a, Abdullah I. Hussain b, *, Poonam S. Nigam a, **, John E. Moore a, c,
Muhammad A. Ayub b, Juluri R. Rao d
Centre of Molecular Biosciences, Institute of Biomedical Sciences Research, University of Ulster, Coleraine BT52 1SA, UK
Department of Chemistry, Government College University Faisalabad, Pakistan
Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City, Hospital, Belfast BT9 7AD, UK
Agri-Food & Biosciences Institute (AFBI), Belfast BT9 5HX, UK

a b s t r a c t
Keywords: The aim of the present study was to assess the antimicrobial activity of methanol and ethanol extracts of
Calendula officinalis pot marigold (Calendula officinalis) petals against clinical pathogens. The antimicrobial potential of
C. officinalis extracts was evaluated against a panel of microorganisms isolated from patients at the
Disc diffusion assay
Resistant strain
Belfast City Hospital (BCH), including bacteria and fungi, using disc diffusion assay. Methanol extract of C.
Clinical isolates officinalis exhibited better antibacterial activity against most of the bacteria tested, than ethanol extract.
Both methanol and ethanol extracts showed excellent antifungal activity against tested strains of fungi,
while comparing with Fluconazole.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction It is known that carotenoids, which belong to the general group of
exogenous non-enzymatic bioactive components, are considered
Calendula officinalis L., a member of the Asteraceae family, is an provitamins because they can be converted to active vitamin A.7
annual plant with yellow to orange flowers, mostly seen in the Among the carotenoids, beta-carotene is the most important bioac-
Mediterranean region.1 Also known as pot marigold, it has been tive that can be provided through diet. It has been cited that beta-
cultivated as a food and medicinal plant since the Middle Ages. As carotene may work synergistically with vitamin E.8 Lycopene,
a medicine, it has found applications in the treatment of inflam- another carotenoid, has been found to have antioxidant, antimicrobial
mation and skin wounds.2 In the early Indian and Arabic cultures, as and antiproliferative properties. Research suggests that it can be very
well as in ancient Greece and Rome, C. officinalis was used as col- protective against prostate cancer.9
ourant for fabrics, foods and cosmetics.3 Its petals can be used to dye C. officinalis has been studied extensively for its beneficial effects
natural fabric such as wool, cotton, linen, hemp and silk.3,4 Among on humans. Literature has shown that a herbal tea made from C.
its extracts, lycopene and lutein give an orange to yellow colour, officinalis could improve the symptoms of colitis, duodenal ulcers
which makes extracts of C. officinalis a natural dye substance.5 and gastroduodenitis.9,10 Although considerable work has been
Nowadays, C. officinalis is approved for food use in U.S.A. and done on C. officinalis extracts, no report is available with in vitro
appears in the Food and Drug Administration’s list of GRAS antimicrobial studies of C. officinalis extracts against clinical isolates
(Generally Recognized as Safe) substances. It has a high economic and resistant strains. Therefore, the aim of the present study was to
value as a herbal medicine and is widely used in cosmetics, assess the antimicrobial activity of C. officinalis petal extracts
perfumes and pharmaceutical preparations as well as in food.1 against a range of clinical fungal and bacterial pathogens.
C. officinalis includes a high number of carotenoids such as fla-
voxanthin, lutein, rubixanthin, b-carotene, g-carotene, and lycopene.6 2. Experimental

2.1. Collection of plant materials and chemicals
* Corresponding author. Current address: Institute of Pharmaceutical Sciences,
University Sains Malaysia, Penang, Malaysia. Tel.: þ60 16 4080836.
The flowers of C. officinalis were obtained from botanical garden,
** Corresponding author. Faculty of Life & Health Sciences, University of Ulster
BT52 1SA, Northern Ireland, UK. Tel.: þ44 287 032 4053.
Government College University, Faisalabad, Pakistan. The petals
E-mail addresses: (A.I. Hussain), were separated and dried at room temperature. All culture media
(P.S. Nigam). were purchased from Oxoid Ltd. The other chemicals, solvents,

1744-3881/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.

8 and 17.4. Preparation of plant extract 3.12 Briefly. B. 2. Efstratiou et al. For the positive (UUC collection) control. / Complementary Therapies in Clinical Practice 18 (2012) 173e176 standards used were. to a final concentra. Department of Bacteriology. CM0003. C. The greater the inhibition zone.Bacillus subtilis NCTC 10400 [JEM7]. Bacillus cereus NCTC 7464 [JEM8]. Aspergillus fumigatus 27.174 E.2. respectively. coli NCTC 25922 [JEEM17]. positive and Gram-negative bacteria. UK. BT9 7AD. The materials were stirred at 350 rpm. However. The results from the disc diffusion method against the fungal Whereas. Furthermore. officinalis petals extracts were dissolved Klebsiella aerogenes NCTC 9528 19  1b 13  1a 39  2c in their respective solvents to a final concentration of 10 mg/mL. As a positive E. Antimicrobial activity individually tested against a panel of pathogenic microorganisms including:. Antimicrobial activity was assessed by measuring the Fluconazole (Sigma.5 and Exophiala extract showed better antibacterial activity (28 and 18 mm. Oxoid). Plates were incubated at extracts. Antimicrobial tests were then carried out by NCCLS disc diffusion method. obtained by methanol filtered and solvent was then evaporated under vacuum using the and ethanol. coli [JEM4] 14  2a 14  1a 35  2b solvents used. Ciprofloxacin (Fluka. C. aureus [JEM18] 22  2a 19  2a 30  2b For the antifungal test. coli (Ampicillin-resistant) 13  1b 10  1a 44  1c (4500 mg/disc) and placed on the inoculated agar. Pseudomonas aer.11 Methanol and ethanol extracts of C. officinalis petals. cereus [JEM8] 15  2b 10  1a 36  3c Escherichia coli (UUC collection) 10  1a 9  1a 44  2b rately impregnated with 15 mL of the 300 mg/mL plant extracts E.05). Candida glabrata against most of the bacteria than the ethanol extract. Candida krusei ATCC 6258. Antimicrobial activity better extraction solvent for extraction of polyphenols. aeruginosa [JEM16] 12  1a 10  1a 40  2b agar (NA) and potato dextrose agar (PDA) medium. 100 mL of suspension con. For Bacillus cereus NCTC 7464 14  1a 16  2a 38  3b the antibacterial test. Sciences. Results and discussion Ten grams of dried and ground petals (80 meshes) were trans- ferred into a flask containing 150 mL of the solvents (methanol and 3. 1 and 2).). while the fungal strains were cultured for 24e48 h at 37  C using Table 1 potato dextrose agar (PDA. faecalis [JEM10] 13 15 Bacillus pumilis [JEM15] 14  1a 13  1a 41 1b control. The results from the disc Staphylococcus aureus MSSA 25923 [JEM18]. respectively.2. Methanol is usually considered as 2. coli (UUC zones (IZ) (Table 1).1. coli NCTC 12900 [JEM4]. Paper discs for the positive a Inhibition zones including the disc diameter of 6 mm. officinalis petals were 3. the greater the collection). E. 37  C for 18e24 h for bacterial strains and for 24e48 h for fungal Ethanol (Rathburn Chemicals Ltd. which is the official strain repository of Northern ethanol extracts of C. that the methanol extract showed more inhibition albicans ATCC 90028. Bel. Extract yield ethanol) used for the extraction. into a small vial and stored at 4  C for further analysis. rotary evaporator. officinalis petals exhibited excellent antifungal Ireland Public Health Laboratory. Coleraine.4 g/100 g of dry petals. b Values are mean  standard deviation (SD) of three replicates. Negative controls were prepared using the same E.05) difference in activity between employed to dissolve the plant extracts. . E.3. aerogenes [JEM2] 14  1a 12  1a 40  3b Paper discs were impregnated with 30 mL of the 10 mg/mL extracts Enterococcus faecalis NCTC 775 14  1a 18  2b 42  3c  1a  1a 36  2b (300 mg/disc) and placed onto the inoculated agar. obtained from the Microbiology Laboratory. cultured for 24 h at 37  C on nutrient agar (NA. Methanol and ethanol extracts of C. compared to ethanol. It can be seen that both methanol and culture collection. were found to be 19. Difference letter Negative controls were prepared again using the same solvents in superscript represent significant (p < 0. Methanol (Fisher Scientific. officinalis petals NCTC 9528 [JEM2]. activity. Disc diffusion method Bacteria Antibacterial activity of Calendula officinalis extracts in term of inhibition zone (IZ) in mma Methanol and ethanol extracts of C. CM0057. 35  C for 24 h using an orbital shaker. coli [JEM1] 21  2b 14  1a 36  2c (15 mL) of ciprofloxacin (4500 mg/disc) dissolved in both types of E. Bacillus extracts had comparable antibacterial effects against both Gram- pumilis [JEM15]. diameter of the growth inhibition zone (IZ) in millimeters (including disc diameter of 6 mm) for the test organisms compared to controls. ampicillin-resistant E. The results were comparable with the standard drug. Antibacterial activity of different Calendula officinalis extracts against clinical bacterial pathogens. coli [JEM17] 22  1b 18  1a 35  3c Staphylococcus aureus MSSA 25923 18  2a 28  2b 42  1c solvents employed to dissolve the plant extracts. M/4056/17). Klebsiella pneumoniae 700603 [JEM19].11 The sample was The extract yield from C. isolated JEM strains were obtained from the MicroARK strains are shown in Table 2. officinalis petals exhibited uginosa NCTC 27853 [JEM16]. and path. 17850).5. strains. dermatitidis GC 7895. The concentrated/dried extract was transferred Methanol was observed to be a better solvent for extraction. University of Ulster. S. fluconazole was prepared by dissolving it in both solvents Klebsiella pneumoniae [JEM19] 16  1a 14  1a 45  3b used to a final concentration of 1 mg/mL. Bacillus subtilis NCTC 10400 22  1b 18  2a 47  3c B. subtilis [JEM7] 14  1b 10  1a 39  3c taining approximately 108 colony-forming units (CFU)/mL of Pseudomonas aeruginosa NCTC 27853 19  2b 13  1a 42  2c bacterial cells and 104 cells/mL of fungi were spread on to nutrient P. The pure bacterial and fungal strains were all respectively) than the methanol extract (18 and 14 mm. Belfast. Methanol Ethanol Ciprofloxacin extract extract tion of 300 mg/mL. officinalis petals were dis- solved in methanol and ethanol. Klebsiella aerogenes diffusion method indicated that the tested C. aureus MSSA 25923 and E. ATCC 2001. Northern Ireland. Aspergillus flavus GC against S. control were impregnated with 30 mL of fluconazole (30 mg/disc). varying antimicrobial activity. paper discs (6 mm in diameter) were sepa. K. antimicrobial activity (Figs. 2. paper discs were impregnated with the same amount E. School of Biomedical respectively). Oxoid). fast City Hospital. as shown by the growth inhibition Escherichia coli (UUC collection). the ethanol 6158. Enterococcus faecalis NCTC 775 [JEM10]. Bacterial strains were Fluconazole. UK. it can be seen ogenic fungi including: Candida albicans 0103 (UUC collection). Aspergillus niger 27. Candida parapsilosis ATCC 22019. faecalis NCTC 775. The effect of the extraction solvents were statistically significant (p  0. from Table 1. F8929).

Fig. Key: The central spot represents the positive control (ciprofloxacin). 3: Candida parapsilosis ATCC 22019. 1: Escherichia Coli JEM1. Typical agar plates showing the growth inhibition zones by Calendula officinalis methanol extract against bacterial strains. 3: E. Coli JEM17. Efstratiou et al. 4: Candida krusei ATCC 6258. 2: Bacillus cereus JEM8. 4: Staphylococcus aureus JEM18. . the dark spot stands for the C. 2: Candida glabrata ATCC 2001. 1. / Complementary Therapies in Clinical Practice 18 (2012) 173e176 175 Fig. E. officinalis extract and the white one for the negative control (methanol only). the dark spot stands for the C. 1: Candida albicans 0102. 2. Typical agar plates showing the growth inhibition zones by Calendula officinalis methanol extract agains fungal strains. officinalis extract and the white one for the negative control (methanol only). Key: The central spot represents the positive control (fluconazole).

Angelova I. Fraga SR. Antifungal activity a Inhibition zones including the disc diameter of 6 mm. pathogenic microorganisms. Khan ZA. Arshad MU. Wayne PA. Aspergillus flavus GC 6158 8  1a 7  1a 7  1a Aspergillus fumigatus 27. 11. Antibacterial potential of pot marigold. 4. Jacob RA. et al. activity of honey against community-associated methicillin-resistant Staph- crobial principles of C. Fluconazole. 1997. Vutreshni Bolesti reports in the literature have shown varying degree of anti. 12. Mathur R. officinalis extracts in term of inhibition zone (IZ) in mma Conflict of interest Methanol Ethanol Fluconazole None declared. Conclusion Approved Standard M2eA6. Isaac D. 16. Noor S. of the essential oil from Calendula officinalis L. Cortez DAG.. Further 2011. Bele C. the results of present investigation. Int J Cosmet Sci 2002. J Soc Integrat Oncol 2008.24(5):287e302. Antimicrobial activity of the leaf extracts of Calendula offi- cinalis (Linn.1007/s12161-011-9325-y. Dweck AC. Chakraborthy GS. NCCLS. HPLC analysis of carotenoids in four Bacillus subtulis. Sosa S.60:516e20. Escherichia coli. Staphylococcus aureus. Chakurski I. officinalis petals possessed good antimi.. Pham-Huy C.16 doi:10. Acta Biologica Szegediensis pneumoniae. albicans and Candida parapsilosis. Matev M. officinalis extracts. Comp Therap Clinic Prac 2008. Saar S. In another report.39:61e3. Food Anal Methods 2011. activity against fungal strains. Lycopene in the prevention of prostate cancer. In contrast to 2003.3(3):51e4. J Microb Antimicrob comparable with the standard antibiotic. extract extract Candida albicans 0103 10  1b 9  1b 7  0a Acknowledgements (UUC collection) b a b C. et al. / Complementary Therapies in Clinical Practice 18 (2012) 173e176 Table 2 investigated plant extracts may be used for the preservation of Antifungal activity of different Calendula officinalis extracts. officinalis leaf against 6.20:44e7. Goyal M. Tubaro A. Hussain AI. Dahan K. Stefanov G. The results of the present study indicate that the methanol and 14. Pseudomonas aeruginosa. Fennal M. Goyal and 8. Bohra A. as well as coagulase-negative duodenal ulcers and gastroduodenitis with a herbal combination of Symphitum Staphylococcus sp. albicans ATCC 90028 9  1 7  0 10  1 Candida krusei ATCC 6258 9  1a 10  1a 14  2b We would like to extend our special gratitude to Anisha Candida glabrata ATCC 2001 12  1a 14  2a 12  1a Majumdar and Ian Anderson who helped during the experimental Candida parapsilosis ATCC 22019 10  1a 10  2a 14  1b procedures and explained some techniques required. officinalis extracts 9. crobial potential.56(1):49e56. Free radicals. the topical antiinflamatory activity of Calendula officinalis flowers.1. Chatha SAS. Rathore HA. 10. Planta Med 1994. Some other officinalis and Calendula officinalis with and without antacids. Colourage 2009.15 extraction techniques and solvent systems on the extraction of antioxidant Varying degrees of antimicrobial activity might be due to the components from peanut (Arachis hypogaea L. Kearns A. ethanol and water extracts of C. C. Loughrey A. Becker H. . 6th ed.15(5):755e66. (Asteraceae) growing in Brazil. Pintea He H.13 cunninghamiana extracts. Antimicrobial effects of Calendula officinalis against human ethanol extracts of the C. against Escherichia coli. http://health. 5. The integrated antioxidant System.176 E. in superscript represent significant (p < 0. Nut Res 1995.47:37e40. 13. Rezende CM. 3.. Effect of baceterial and antifungal activities of C. J Herbal Med Tox 2008.4(2):89e96. antioxidants in disease and health.). who reported good antimicrobial activity of petroleum ether. Gazim ZC. Andrei S.6:29e36.5 9  1a 9  1a 11  1a Exophiala dermatitidis GC 7895 10  1a 10  1a 11  1a 1. Maeda Y.) hulls. Bissa S.14:77e82. Della LR. Both extracts showed antifungal activity that is 15. Performance standards for antimicrobial disc susceptibility test. Koichev A. Mathur14 observed good inhibition effect of C. Chakraborthy13 reported poor 7. variation of sources of microorganisms. Rao JR. The role of triperpenoids in extracts. National Committee for Clinical Laboratory Standards. Efstratiou et al. Int J Biomed Sci 2008. Difference letter Braz J Microby 2008. 1981. flowers.05) difference in activity between 2. chloroform. Dyeing of Nylon 6 fibres with Calendula officinalis & Casuarina Our results are in agreement with the finding of Chakraborthy. Klebsiella varieties of Calendula officinalis L. Candida albicans and Aspergillus niger. Mohamed RM. 4. Socaciu C. The yloccus aureus (CA-MRSA). b Values are mean  standard deviation (SD) of three raplicates. 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