Complementary Therapies in Clinical Practice 18 (2012) 173e176
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Complementary Therapies in Clinical Practice
journal homepage: www.elsevier.com/locate/ctcp
Antimicrobial activity of Calendula ofcinalis 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
a
Centre of Molecular Biosciences, Institute of Biomedical Sciences Research, University of Ulster, Coleraine BT52 1SA, UK
b
Department of Chemistry, Government College University Faisalabad, Pakistan
c
Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City, Hospital, Belfast BT9 7AD, UK
d
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 ofcinalis pot marigold (Calendula ofcinalis) petals against clinical pathogens. The antimicrobial potential of
Antimicrobial
C. ofcinalis 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 ofcinalis 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.
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 ofcinalis 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 owers, 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 inam- 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. ofcinalis was used as col- protective against prostate cancer.9
ourant for fabrics, foods and cosmetics.3 Its petals can be used to dye C. ofcinalis has been studied extensively for its benecial 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, ofcinalis could improve the symptoms of colitis, duodenal ulcers
which makes extracts of C. ofcinalis a natural dye substance.5 and gastroduodenitis.9,10 Although considerable work has been
Nowadays, C. ofcinalis is approved for food use in U.S.A. and done on C. ofcinalis extracts, no report is available with in vitro
appears in the Food and Drug Administrations list of GRAS antimicrobial studies of C. ofcinalis 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. ofcinalis petal extracts
perfumes and pharmaceutical preparations as well as in food.1 against a range of clinical fungal and bacterial pathogens.
C. ofcinalis includes a high number of carotenoids such as a-
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.: 60 16 4080836.
The owers of C. ofcinalis were obtained from botanical garden,
** Corresponding author. Faculty of Life & Health Sciences, University of Ulster
BT52 1SA, Northern Ireland, UK. Tel.: 44 287 032 4053.
Government College University, Faisalabad, Pakistan. The petals
E-mail addresses:
[email protected] (A.I. Hussain),
[email protected] 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 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ctcp.2012.02.003
174 E. Efstratiou et al. / Complementary Therapies in Clinical Practice 18 (2012) 173e176
standards used were; Methanol (Fisher Scientic, M/4056/17), 37 C for 18e24 h for bacterial strains and for 24e48 h for fungal
Ethanol (Rathburn Chemicals Ltd.), Ciprooxacin (Fluka, 17850), strains. Antimicrobial activity was assessed by measuring the
Fluconazole (Sigma, F8929). diameter of the growth inhibition zone (IZ) in millimeters (including
disc diameter of 6 mm) for the test organisms compared to controls.
2.2. Preparation of plant extract
3. Results and discussion
Ten grams of dried and ground petals (80 meshes) were trans-
ferred into a ask containing 150 mL of the solvents (methanol and 3.1. Extract yield
ethanol) used for the extraction. The materials were stirred at
350 rpm, 35 C for 24 h using an orbital shaker.11 The sample was The extract yield from C. ofcinalis petals, obtained by methanol
ltered and solvent was then evaporated under vacuum using the and ethanol, were found to be 19.8 and 17.4 g/100 g of dry petals.
rotary evaporator. The concentrated/dried extract was transferred Methanol was observed to be a better solvent for extraction,
into a small vial and stored at 4 C for further analysis. compared to ethanol. The effect of the extraction solvents were
statistically signicant (p 0.05). Methanol is usually considered as
2.3. Antimicrobial activity better extraction solvent for extraction of polyphenols.11
Methanol and ethanol extracts of C. ofcinalis petals were 3.2. Antimicrobial activity
individually tested against a panel of pathogenic microorganisms
including:- Bacillus subtilis NCTC 10400 [JEM7], Pseudomonas aer- Methanol and ethanol extracts of C. ofcinalis petals exhibited
uginosa NCTC 27853 [JEM16], Bacillus cereus NCTC 7464 [JEM8], varying antimicrobial activity, as shown by the growth inhibition
Escherichia coli (UUC collection), ampicillin-resistant E. coli (UUC zones (IZ) (Table 1). The greater the inhibition zone, the greater the
collection), E. coli NCTC 12900 [JEM4], E. coli NCTC 25922 [JEEM17], antimicrobial activity (Figs. 1 and 2). The results from the disc
Staphylococcus aureus MSSA 25923 [JEM18], Klebsiella aerogenes diffusion method indicated that the tested C. ofcinalis petals
NCTC 9528 [JEM2], Enterococcus faecalis NCTC 775 [JEM10], Bacillus extracts had comparable antibacterial effects against both Gram-
pumilis [JEM15], Klebsiella pneumoniae 700603 [JEM19]; and path- positive and Gram-negative bacteria. Furthermore, it can be seen
ogenic fungi including: Candida albicans 0103 (UUC collection), C. from Table 1, that the methanol extract showed more inhibition
albicans ATCC 90028, Candida krusei ATCC 6258, Candida glabrata against most of the bacteria than the ethanol extract. However,
ATCC 2001, Candida parapsilosis ATCC 22019, Aspergillus avus GC against S. aureus MSSA 25923 and E. faecalis NCTC 775, the ethanol
6158, Aspergillus fumigatus 27.5, Aspergillus niger 27.5 and Exophiala extract showed better antibacterial activity (28 and 18 mm,
dermatitidis GC 7895. The pure bacterial and fungal strains were all respectively) than the methanol extract (18 and 14 mm,
obtained from the Microbiology Laboratory, School of Biomedical respectively).
Sciences, University of Ulster, Coleraine, Northern Ireland, UK. The results from the disc diffusion method against the fungal
Whereas, isolated JEM strains were obtained from the MicroARK strains are shown in Table 2. It can be seen that both methanol and
culture collection, which is the ofcial strain repository of Northern ethanol extracts of C. ofcinalis petals exhibited excellent antifungal
Ireland Public Health Laboratory, Department of Bacteriology, Bel- activity. The results were comparable with the standard drug,
fast City Hospital, Belfast, BT9 7AD, UK. Bacterial strains were Fluconazole.
cultured for 24 h at 37 C on nutrient agar (NA, CM0003, Oxoid),
while the fungal strains were cultured for 24e48 h at 37 C using Table 1
potato dextrose agar (PDA, CM0057, Oxoid). Antibacterial activity of different Calendula ofcinalis extracts against clinical
bacterial pathogens.
2.4. Disc diffusion method Bacteria Antibacterial activity of Calendula
ofcinalis extracts in term of inhibition
zone (IZ) in mma
Methanol and ethanol extracts of C. ofcinalis petals were dis-
solved in methanol and ethanol, respectively, to a nal concentra- Methanol Ethanol Ciprooxacin
extract extract
tion of 300 mg/mL. Antimicrobial tests were then carried out by
NCCLS disc diffusion method.12 Briey, 100 mL of suspension con- 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. aeruginosa [JEM16] 12 1a 10 1a 40 2b
agar (NA) and potato dextrose agar (PDA) medium, respectively. For Bacillus cereus NCTC 7464 14 1a 16 2a 38 3b
the antibacterial test, paper discs (6 mm in diameter) were sepa- B. 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. coli (Ampicillin-resistant) 13 1b 10 1a 44 1c
(4500 mg/disc) and placed on the inoculated agar. For the positive (UUC collection)
control, paper discs were impregnated with the same amount E. coli [JEM1] 21 2b 14 1a 36 2c
(15 mL) of ciprooxacin (4500 mg/disc) dissolved in both types of E. coli [JEM4] 14 2a 14 1a 35 2b
solvents used. Negative controls were prepared using the same E. 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. S. aureus [JEM18] 22 2a 19 2a 30 2b
For the antifungal test, C. ofcinalis petals extracts were dissolved Klebsiella aerogenes NCTC 9528 19 1b 13 1a 39 2c
in their respective solvents to a nal concentration of 10 mg/mL. K. 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. As a positive E. faecalis [JEM10] 13 15
Bacillus pumilis [JEM15] 14 1a 13 1a 41 1b
control, uconazole was prepared by dissolving it in both solvents Klebsiella pneumoniae [JEM19] 16 1a 14 1a 45 3b
used to a nal concentration of 1 mg/mL. Paper discs for the positive a
Inhibition zones including the disc diameter of 6 mm.
control were impregnated with 30 mL of uconazole (30 mg/disc). b
Values are mean standard deviation (SD) of three replicates. Difference letter
Negative controls were prepared again using the same solvents in superscript represent signicant (p < 0.05) difference in activity between
employed to dissolve the plant extracts. Plates were incubated at extracts.
E. Efstratiou et al. / Complementary Therapies in Clinical Practice 18 (2012) 173e176 175
Fig. 1. Typical agar plates showing the growth inhibition zones by Calendula ofcinalis methanol extract against bacterial strains. Key: The central spot represents the positive
control (ciprooxacin), the dark spot stands for the C. ofcinalis extract and the white one for the negative control (methanol only). 1: Escherichia Coli JEM1; 2: Bacillus cereus JEM8;
3: E. Coli JEM17; 4: Staphylococcus aureus JEM18.
Fig. 2. Typical agar plates showing the growth inhibition zones by Calendula ofcinalis methanol extract agains fungal strains. Key: The central spot represents the positive control
(uconazole), the dark spot stands for the C. ofcinalis extract and the white one for the negative control (methanol only). 1: Candida albicans 0102; 2: Candida glabrata ATCC 2001;
3: Candida parapsilosis ATCC 22019; 4: Candida krusei ATCC 6258.
176 E. Efstratiou 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 ofcinalis extracts. processed foods as well as pharmaceutical and natural therapies for
Fungi Antifungal activity of Calendula the treatment of infectious diseases in humans.
ofcinalis extracts in term of inhibition
zone (IZ) in mma Conict of interest
Methanol Ethanol Fluconazole None declared.
extract extract
Candida albicans 0103 10 1b 9 1b 7 0a Acknowledgements
(UUC collection)
b a b
C. 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.
Aspergillus avus GC 6158 8 1a 7 1a 7 1a
Aspergillus fumigatus 27.5 10 1a 9 1a 12 2a
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