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

Calendula ofcinalis L., a member of the Asteraceae family, is an
annual plant with yellow to orange owers, mostly seen in the
Mediterranean region.1 Also known as pot marigold, it has been
cultivated as a food and medicinal plant since the Middle Ages. As
a medicine, it has found applications in the treatment of inam-
mation and skin wounds.2 In the early Indian and Arabic cultures, as
well as in ancient Greece and Rome, C. ofcinalis was used as col-
ourant for fabrics, foods and cosmetics.3 Its petals can be used to dye
natural fabric such as wool, cotton, linen, hemp and silk.3,4 Among
its extracts, lycopene and lutein give an orange to yellow colour,
which makes extracts of C. ofcinalis a natural dye substance.5
Nowadays, C. ofcinalis is approved for food use in U.S.A. and
appears in the Food and Drug Administrations list of GRAS
(Generally Recognized as Safe) substances. It has a high economic
value as a herbal medicine and is widely used in cosmetics,
perfumes and pharmaceutical preparations as well as in food.1
C. ofcinalis includes a high number of carotenoids such as a-
voxanthin, lutein, rubixanthin, b-carotene, g-carotene, and lycopene.6
exogenous non-enzymatic bioactive components, are considered
provitamins because they can be converted to active vitamin A.7
Among the carotenoids, beta-carotene is the most important bioac-
tive that can be provided through diet. It has been cited that beta-
carotene may work synergistically with vitamin E.8 Lycopene,
another carotenoid, has been found to have antioxidant, antimicrobial
and antiproliferative properties. Research suggests that it can be very
protective against prostate cancer.9
C. ofcinalis has been studied extensively for its benecial effects
on humans. Literature has shown that a herbal tea made from C.
ofcinalis could improve the symptoms of colitis, duodenal ulcers
and gastroduodenitis.9,10 Although considerable work has been
done on C. ofcinalis extracts, no report is available with in vitro
antimicrobial studies of C. ofcinalis extracts against clinical isolates
and resistant strains. Therefore, the aim of the present study was to
assess the antimicrobial activity of C. ofcinalis petal extracts
against a range of clinical fungal and bacterial pathogens.
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: ai.hussain@yahoo.com (A.I. Hussain), p.singh@ulster.ac.uk 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),
Ethanol (Rathburn Chemicals Ltd.), Ciprooxacin (Fluka, 17850),
Fluconazole (Sigma, F8929).
2.2. Preparation of plant extract
Ten grams of dried and ground petals (80 meshes) were trans-
ferred into a ask containing 150 mL of the solvents (methanol and
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
ltered and solvent was then evaporated under vacuum using the
rotary evaporator. The concentrated/dried extract was transferred
into a small vial and stored at 4  C for further analysis.
2.3. Antimicrobial activity
37  C for 18e24 h for bacterial strains and for 24e48 h for fungal
strains. Antimicrobial activity was assessed by measuring the
diameter of the growth inhibition zone (IZ) in millimeters (including
disc diameter of 6 mm) for the test organisms compared to controls.
3. Results and discussion
3.1. Extract yield
The extract yield from C. ofcinalis petals, obtained by methanol
and ethanol, were found to be 19.8 and 17.4 g/100 g of dry petals.
Methanol was observed to be a better solvent for extraction,
compared to ethanol. The effect of the extraction solvents were
statistically signicant (p  0.05). Methanol is usually considered as
better extraction solvent for extraction of polyphenols.11

Methanol and ethanol extracts of C. ofcinalis petals were
individually tested against a panel of pathogenic microorganisms
including:- Bacillus subtilis NCTC 10400 [JEM7], Pseudomonas aer-
uginosa NCTC 27853 [JEM16], Bacillus cereus NCTC 7464 [JEM8],
Escherichia coli (UUC collection), ampicillin-resistant E. coli (UUC
collection), E. coli NCTC 12900 [JEM4], E. coli NCTC 25922 [JEEM17],
Staphylococcus aureus MSSA 25923 [JEM18], Klebsiella aerogenes
NCTC 9528 [JEM2], Enterococcus faecalis NCTC 775 [JEM10], Bacillus
pumilis [JEM15], Klebsiella pneumoniae 700603 [JEM19]; and path-
ogenic fungi including: Candida albicans 0103 (UUC collection), C.
albicans ATCC 90028, Candida krusei ATCC 6258, Candida glabrata
ATCC 2001, Candida parapsilosis ATCC 22019, Aspergillus avus GC
6158, Aspergillus fumigatus 27.5, Aspergillus niger 27.5 and Exophiala
dermatitidis GC 7895. The pure bacterial and fungal strains were all
obtained from the Microbiology Laboratory, School of Biomedical
Sciences, University of Ulster, Coleraine, Northern Ireland, UK.
Whereas, isolated JEM strains were obtained from the MicroARK
culture collection, which is the ofcial strain repository of Northern
Ireland Public Health Laboratory, Department of Bacteriology, Bel-
fast City Hospital, Belfast, BT9 7AD, UK. Bacterial strains were
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
potato dextrose agar (PDA, CM0057, Oxoid).
3.2. Antimicrobial activity
Methanol and ethanol extracts of C. ofcinalis petals exhibited
varying antimicrobial activity, as shown by the growth inhibition
zones (IZ) (Table 1). The greater the inhibition zone, the greater the
antimicrobial activity (Figs. 1 and 2). The results from the disc
diffusion method indicated that the tested C. ofcinalis petals
extracts had comparable antibacterial effects against both Gram-
positive and Gram-negative bacteria. Furthermore, it can be seen
from Table 1, that the methanol extract showed more inhibition
against most of the bacteria than the ethanol extract. However,
against S. aureus MSSA 25923 and E. faecalis NCTC 775, the ethanol
extract showed better antibacterial activity (28 and 18 mm,
respectively) than the methanol extract (18 and 14 mm,
respectively).
The results from the disc diffusion method against the fungal
strains are shown in Table 2. It can be seen that both methanol and
ethanol extracts of C. ofcinalis petals exhibited excellent antifungal
activity. The results were comparable with the standard drug,
Fluconazole.
Table 1
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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