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Electronic Physician (ISSN: 2008-5842) http://www.ephysician.

ir
August 2016, Volume: 8, Issue: 8, Pages: 2752-2758, DOI: http://dx.doi.org/10.19082/2752

Effect of aqueous and ethanolic extracts of Lippia citriodora on candida albicans

Maryam Ghasempour1, Saeid Mahdavi Omran2, Ali Akbar Moghadamnia3, Faranak Shafiee4
1
DDS, MS, Faculty of Dentistry, Babol University of Medical Sciences, Babol, Iran
2
Ph.D., Cellular and Molecular Biology Research Center, Department of Medical Parasitology and Mycology,
Babol University of Medical Sciences, Babol, Iran
3
Ph.D., Department of Pharmacology and Toxicology, Faculty of Medicine, Babol University of Medical Sciences,
Babol, Iran
4
DDS, Postgraduate Student, Faculty of Dentistry, Babol University of Medical Sciences, Babol, Iran

Type of article: Original

Abstract
Introduction: Because of resistance and side effects to common antifungal drugs activity, the research on herbal
substances with antifungal activity is frequent. Lemon verbena (Lippia citriodora) is a member of Verbenaceae
family. The aim of this study was to determine the anti-candida activities of the ethanolic and aqueous extracts of
the lemon verbena leaves and compare them with nystatin and fluconazole.
Methods: In this 2015 study, 15 clinical isolates and standard strain of candida albicans PTCC 5027 were used,
and the inhibitory effects of the ethanolic and aqueous extracts, Nystatin and Fluconazole, were evaluated using
disk and well diffusion methods. Also, the minimal inhibitory concentration (MIC) was determined. Five
concentrations of aqueous and ethanolic extracts (156-2500 μg/ml), Nystatin (8-128 μg/ml) and Fluconazole (4-
64 μg/ml) were used in disk and well diffusion methods, and nine concentrations of aqueous and ethanolic
extracts (19-5000 μg/ml), Nystatin (0.5-128 μg/ml), and Fluconazole (0.25-64 μg/ml) were applied for MIC. Data
were analyzed using Tukey's post-hoc and one-way ANOVA tests. The significant level was considered p < 0.05
in the current study.
Results: In the well and disk diffusion techniques, limited growth inhibition halos were produced around some
clinical isolates at different concentrations of ethanolic extract; however, no growth inhibitory halo was observed
with any concentrations of the aqueous extract. The MIC values of ethanolic extract, aqueous extract, Nystatin
and Fluconazole for clinical isolated and standard strain were 833 ± 78.5and 625μg/ml; 4156 ± 67.4 and 2500
μg/ml; 10.13 ± 1.91 and 4 μg/ml; and 1.97 ± 0.25 and 1 μg/ml, respectively.
Conclusion: The results showed that the ethanolic extract was stronger than the aqueous extract of this plant,
which can be used as an alternative for drugs. It is recommended that the ethanolic extract of this plant be
investigated in vivo for better evaluation of its efficacy and properties.
Keywords: Anti-Candida albicans activity, Lippia citriodora, Ethanolic and Aqeoues extracts, Nystatin,
Fluconazole
1. Introduction
Oral candidiasis is one of the most common opportunistic infections with varying clinical manifestations that affect
the oral cavity. Based on previous studies, Candida albicans is the most common etiologic agent for this condition
(1, 2). Research has shown a significant correlation between Candida colonies in children and early carious lesions
during childhood (3, 4). Polyenes (nystatin) and azoles (fluconazole) are the most commonly-used local agents for
the treatment of candidiasis; however, due to the presence of resistant species of Candida, toxicity, and insufficient
bioavailability, only a small number of these agents are effective therapeutically (5). In recent decades, plant
derivatives have attracted a lot of attention (6). One of these plants with excellent therapeutic properties is Lippia
citriodora from the Verbena family. The plant is indigenous to North America; however, it has been planted in some
Corresponding author:
Dr. Faranak Shafiee, Faculty of Dentistry, Babol University of Medical Sciences, Babol, Iran. Tel: +98.9183733349,
Email: faranakshafiee@gmail.com
Received: November 21, 2015, Accepted: February 28, 2016, Published: August 2016
iThenticate screening: February 28, 2016, English editing: May 17, 2016, Quality control: August 02, 2016
© 2016 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-
NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
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European countries and in Iran in regions with moderate weather conditions (northern areas) under greenhouse
conditions due to its beneficial properties (7). The leaves of this plant have aromatic constituents, which are used to
prepare traditional tea. L. citriodora has been listed as one of the safe plants and use of its alcoholic and traditional
tea forms has been considered safe (8). Citral (geranial, neral), geraniol, lemonene, and cineol are the main
ingredients of the extract of L. citriodora (9). This plant is used in traditional medicine for the treatment of asthma,
spasms, colds, colic, diarrhea, malabsorption, and anxiety (10). Evaluation of the biological activity and ingredients
isolated from the extract and leaf extract of L. citriodora showed its anti-oxidative, anti-microbial, anti-
inflammatory, and anti-fungal activities and analgesic effects as a result of its phenolic acid, flavonoid, and
terpenoid compounds. The essential oils of L. citriodora decrease cellular lysis due to oxidative stresses, and they
are a good candidate for conditions leading to neuronal degradation (8, 10-13). The aim of the present study was to
determine the inhibitory effect of the aqueous and ethanolic extracts of L. citriodora leaves on Candida albicans
isolated from the dorsum of the tongue of patients and the standard strain in culture media.

2. Material and Methods


2.1. Study design and setting
In this in vitro study, we used the standard strain of Candida albicans (PTCC 5027) procured from the Industrial
Microorganism Collection Center of Iran and 15 C. albicans samples isolated from the dorsum of the tongue of 4-
12-year-old children who were referred to the Department of Pediatric Dentistry of Babol University of Medical
Sciences. The inclusion criteria consisted of no use of anti-microbial mouthwashes during the past month and no use
of anti-fungal agents, antihistamines and corticosteroids, and a caries index > 4. The samples were collected after
briefing the children’s parents and obtaining informed written consent forms. The samples were immediately
cultured on Sabouraud dextrose agar plates (Scharlau-Merck, Germany) in association with chloramphenicol. After
fungal growth, a part of the colony was transferred to the CHROMagar culture medium (CHROMagar Candida,
Paris, France). C. albicans species was confirmed by the color produced on the CHROMagar medium and formation
of a germ tube in serum and production of vesicles in the cornmeal agar median (Himedia Laboratories, Ltd,
Mumbai, India) plus 1% Tween 80. Then, a suspension of 48-hour yeast colonies was prepared in the Sabouraud
Dextrose Agar with Chloramphenicol medium with the use of physiologic serum. Then, a standard 0.5 McFarland
concentration was prepared using the well technique with the disk placemat method. Microdilutions of yeasts were
prepared at 5×102 - 2.5×103 in each mL with the use of RPMI medium (Sigma-Aldrich, Germany).

2.2. Preparation of aqueous and ethanolic leaf extract of L. citriodora


L. citrodora leaves were collected from the Research Garden of Babol University of Medical Sciences in May 2015,
rinsed, dried at room temperature for 27 hours, and milled. To prepare the aqueous extract, 30 g of the dry powder of
the leaves were mixed with 100 mL of water at 70-80 °C, which had already been boiled, and stored at room
temperature for 24 hours. The resultant extract was filtered through Whatman filter papers (Whatman No. 1) and
placed in an oven at 40-50 °C for drying. The dry weight of the material was determined. This extract was
considered the aqueous extract of L. citriodora. To prepare the ethanolic extract, 75 g of the dry material and 250
mL of 95% dehydrated ethanol were poured in a beaker, and, after 48 hours, the resultant solution was filtered
through No. 1 Whatman paper filters and dried in a vacuum machine. The dry ethanolic extract was weighed. This
extract was considered the dry ethanolic extract.

2.3. Evaluation of the antifungal activity with the use of disk diffusion technique
Sterile paper disks were immersed in the final concentrations of 156-2500 µg/mL of the aqueous (dissolved in
water) and ethanolic (dissolved in dimethyl sulfoxide, DMSO) extracts, 4-64 µg/mL of fluconazole (dissolved in
water) and 8-128 µg/mL of nystatin (dissolved in dimethyl sulfoxide, DMSO) and then dried. The suspension
prepared from C. albicans in sterile physiologic serum was spread on a plate containing Sc medium using a sterile
swab. Five disks containing different concentrations of the aqueous and ethanolic extracts of L. citriodora, nystatin
and fluconazole were placed separately in each plate. The disks were placed at a distance of 15 mm from the plate
margin and 24 mm from the center of the adjacent disk. A disk with no preparation and a disk with 0.2% DMSO
were considered as negative controls. Then, the diameter of the transparent halo around each disk was measured.
Halo diameters ≥2 mm indicated antifungal activity (14). All of the plates were prepared in duplicate and incubated
at 37 °C for 48 hours.

2.4. Evaluation of the antifungal activity using the well technique


The suspension prepared from C. albicans in sterile physiologic serum was inoculated on a plate containing
Sabouraud dextrose agar culture medium along with chloramphenicol with the use of a sterile swab. After two

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hours, cavities measuring 6 mm in diameter were prepared in it. Different concentrations of the aqueous extract
(dissolved in water), ethanolic extract (dissolved in DMSO), fluconazole (dissolved in water) and nystatin
(dissolved in DMSO) were added to the wells. All of the plates were prepared in duplicate and incubated at 37 °C
for 48 hours.

2.5. Microdilution to determine the minimum inhibitory concentration (MIC)


MIC was determined with the use of CLSI-M27-A3 (Clinical and Laboratory Standard Institute) standard technique
to determine the inhibitory and lethal effect of the medication (15). Nine series of different concentrations of the
extract were prepared in duplicate at the final concentrations of 19‒5000 µg/mL, 0.25‒64 µg/mL of fluconazole,
0.5-128 µg/mL nystatin in a total volume of 100 µL in sterile 96-well ELISA plates with flat bottoms, containing
100 µL of RPMI. Then, the fungal suspension at concentrations of 5×10 2 - 2.5×103 yeast cells/mL was added to the
plates at a volume of 100 µL. All of the plates were prepared in duplicate and incubated at 35 °C for 48 hours.
Positive and negative controls and DMSO solution also were considered.

2.6. Statistical analysis


The data were analyzed using Tukey's post hoc and One-Way ANOVA tests using SPSS 18 software, and they were
expressed as the mean ± the standard error of the mean (SEM). The data obtained from the ethanolic and aqueous
extracts of lemon verbena were compared with the findings of the control group fluconazole and nystatin and p <
0.05 was considered statistically significant.

3. Results
In the present in vitro study, the samples were collected from 22 volunteers as follows: 10 subjects in the 4-6 age
group and 12 in the 7-12 age group, with mean DMFT score of 5.5 and 8.6 respectively. Of the 22 samples cultured,
two were not C. albicans samples, and no fungal growth was detected in five samples. The presence of green
colonies in 15 CHROMagar culture media indicated the growth of C. albicans (Figure 1). In addition, all of these
microorganisms produced germ tubes and vesicles (chlamydoconidium) in the cornmeal agar along with 1% Tween
80 in 13 plates (Figure 2). In this study, the anti-fungal activities of different concentrations of aqueous and
ethanolic extracts of L. citiodora leaves against C. albicans were evaluated with the use of disk diffusion, well, and
microdilution techniques in comparison to nystatin and fluconazole.

Figure 1. CHROMagar culture media indicating the growth of C. albicans

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In the well and disk diffusion techniques, small growth inhibitory halos were observed around a number of
clinically-isolated stains; however, no inhibitory halos were observed around the standard strains. In addition, the
aqueous extract exhibited no inhibitory halos in the disk diffusion and well techniques. The diameters of growth
inhibitory halos with nystatin-containing disks (with the minimum concentration of 8 µg/mL around the isolated and
standard stains) were 0.53 ± 0.53 and 0; the maximum diameters of growth inhibitory halos at a concentration of 128
µg/mL around clinically-isolated and standard stains increased to 16.13 ± 0.44 and 17 in a dose-dependent manner.
In addition, the growth inhibitory halos in the same order in the well technique with the minimum nystatin
concentration were 0.535 ± 0.55 and 0, and, with the maximum concentration, they were 17.8 ± 0.59 and 17. The
growth inhibitory halo diameters for 4 µg/mL of fluconazole with the clinically isolated and standard strains in the
disk diffusion technique were 0.57 ± 0.55 and 0, and in the well technique they were 0 and 0. With the maximum
concentration (64 µg/mL), the diameters were 15.53 ± 1.28 and 17, and 17.33 ± 0.36 and 18, respectively. MICs are
presented in Table 1. The MIC results showed a higher inhibitory effect of ethanolic extract of L. citriodora than its
aqueous extract. In addition, the standard strain exhibited higher sensitivity than the clinically isolated C. albicans
strains at the concentrations evaluated. Although no growth inhibitory halos were detected in the disks and wells
containing the aqueous extract, in the MIC technique, the growth inhibitions for the clinical and standard strains
were 833 ± 78.5 and 2500 µg/mL, respectively. The MIC of the ethanolic extract was 833 ± 78.5 and 625 µg/mL for
the isolated and standard strains, respectively, indicating higher inhibitory potential for the ethanolic extract than the
aqueous extract (p = 0). In addition, the MIC of nystatin for clinical and standard strains was 10.13 ± 1.91 and 4,
respectively, with 1.97 ± 0.25 and 1 µg/mL, respectively for fluconazole. Although the MIC of the ethanolic extract
of L. citriodora was higher than those of nystatin and fluconazole (p = 0), this extract exhibited anti-fungal activity.

Figure 2. Chlamydoconidium in the cornmeal agar

Table 1. Means and standard deviation of minimal inhibitory concentration (MIC) of Aqueous and Ethanolic
extracts of Lippia citriodora, Nystatin and Fluconazole against Candida albicans
Extracts Range (μg/ml) MIC (μg/ml)
Candida albicans (Clinical isolate, No. 15) Candida albicans (PTCC 5027)
Aqueous 19 -5000 4156 ± 67.4 2500
Ethanolic 19 -5000 833 ± 78.5 625
Nystatin 0.5 -128 10.13 ± 1.91 4
Fluconazole 0.25 - 64 1.97 ± 0.25 1

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4. Discussion
The present study evaluated the anti-fungal effects of aqueous and ethanolic extracts of L. citriodora leaves on C.
albicans. The results showed that 15 colonies of these fungal species were green in color in the CHROMagar culture
medium, indicating the presence of C. albicans. In addition, all of these colonies produced germ tubes, and 13
strains produced vesicles (Chlamydoconidium) in the cornmeal agar medium. A previous study showed the anti-
oxidative activity of leaf extract of L. citriodora. In addition, the anti-microbial and anti-fungal properties of L.
citriodora leaves have been attributed to aromatic and polyphenolic compounds in the leaves (16-18). In the present
study, the effects of aqueous and ethanolic extracts of L. citriodora leaves were evaluated using the disk diffusion,
well diffusion, and MIC techniques. In the well and disk diffusion techniques, limited growth inhibition halos were
produced around some clinical isolates at different concentrations of the ethanolic extract; however, no growth
inhibitory halos were observed with any concentrations of the aqueous extract. Absence of growth inhibitory halos
around disks containing the extract might be attributed to the fact that, in the present study, a lower concentration of
the extract was used compared to previous studies. In addition, since the effective material in the well and disk
diffusion technique gradually diffuses into the environment and affects the margins of the growing colonies, the
fungal species in question had the opportunity to make use of its defensive mechanisms to counteract the material’s
toxicity through a proper technique; however, in the micro-dilution technique, mixing of the extract with the culture
medium resulted in the presence of the effective material in all the areas of the environment. Therefore, it can be
expected that the threshold of response of each fungal species to the anti-fungal agent will be different (19). The
MICs of ethanolic extract, in the present study, on clinically isolated and standard strains were 833 ± 78.5 and 625
µg/mL, respectively, which were much lower than those in a 2005 study by Oskay, which showed that the MIC of
methanolic extract of L. citriodora leaves on C. albicans was 6 mg/mL (20). In the present study, inhibition
occurred at a lower concentration than that in the study by Oskay, which might be attributed to higher levels of
polyphenols and aromatic compounds in the extract (13, 17). In the present study, the MICs of the aqueous extracts
with clinically isolated and standard strains were 4156 ± 67.4 and 2500 µg/mL respectively, indicating a lower
inhibitory potential of the aqueous extract than the ethanolic extract. In a 2013 study by Koohsari, the antibacterial
effects of aqueous and ethanolic extracts of L. citriodora were evaluated at concentrations of 125-1000 mg/mL on
nine pathogenic species with the use of disk diffusion and well techniques. The results showed that the ethanolic
extract exhibited an antibacterial effect; however, the aqueous extract had no inhibitory effect on the pathogenic
species. The results showed that E. faecalis, S. epidermidis, and S. aureus were the most sensitive bacteria among
gram-positive bacteria and that Y. enterocolitica was the most resistant gram-negative bacterial species (14). Most
plant extracts have anti-microbial properties and consist of saturated organic or aromatic compounds. In most cases,
ethanolic or methanolic solvents are used for their initial extraction and, typically in the majority of studies, the use
of water is avoided for extraction of plant derivatives (21). Since the presence of phenolic components in plant
extracts is one of the main reasons for their anti-microbial effects, it is possible that the presence of very small
amounts of phenolic compounds in the aqueous extract was the reason for the low anti-microbial effect of this
extract (17). Lozada, in a 2012 study, evaluated the effect of the essential oils of L. citriodora and L. origanoid on
growth inhibition of Moniliophthora roreri, and the results showed complete inhibition of fungal growth at
concentrations of 600‒1000 µg/mL (22). Observations in relation to the essential oils of L. citriodora showed its
high anti-bacterial activity against H. pylori (23). A 2010 study by Ramzi showed that L. citriodora extract has an
inhibitory effect on gram-positive bacteria, but it had no effect on Candida maltosa (24). In 2012, Ansari conducted
a study with 1/2, 1/4, 1/8, and 1/16 concentrations of 100µl of L. citriodora extract using disk diffusion technique to
determine MIC against S. aureus strain resistant to methicillin. The results showed that the inhibitory effect
increased with an increase in the concentration of the extract up to 55 µg/mL. In addition, the MIC was determined
at 15 µg/mL, and the results showed a high anti-microbial activity against methicillin-resistant S. aureus despite
very low toxicity against cells (25). The anti-bacterial activity of the extract might be attributed to the presence of
alkaloids, flavonoids, and tannin in its chemical structure (23). Tatsadjieu evaluated the antifungal activity of Lippia
rugosa leaves and showed that geraniol, neral, and geranial in the extract were the agents responsible for the anti-
fungal activity against Aspergillus flavus (26). A study in 2009 by Royaro showed that the extract of L. citriodora
leaves exhibited the highest anti-fungal activity at inhibitory concentrations of 99.21 and 65.5 µg/mL against C.
krusei and Aspergillus fumigatus, respectively (22). A study in 2012 by Lozada showed that the essential oils of L.
citriodora and L. origanoid resulted in 90% inhibition of fungal growth at a concentration of 200 µg/mL; however,
100% inhibition was achieved at concentrations in the range of 800‒1000 µg/mL (27). A study in 2007 by Oliviera
on the antibacterial activity of L. origanoid with the use of drop diffusion technique showed its inhibitory effects on
gram-positive microorganisms, including S. aureus and C. albicans (28). A study in 1994 by Villon and Chaumont
showed the antifungal activity of Lippia multiflora and Lippia chevalieri extracts against Aspergillus flavus. They
reported that trepenoids, especially citral and geraniol that are present in extract of L. citriodora, were responsible

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for the anti-fungal activity (29). Other anti-fungal agents are beta-caryophyllene and beta-caryophyllene oxide that
belong to cisquiterpens and are found in the plant extracts of Lippia rehmanii and Lippia citriodora (30). Despite the
complications reported in previous studies in relation to other antifungal agents, no side effects have been reported
after the use of leaf extract of L. citriodora; on the contrary, its positive protective effects on the general health have
been shown (7, 10, 11, 16, 30, 31).

5. Conclusions
The results of the present study showed that the aqueous extract, and particularly the ethanolic extract, of the leaves
of L. citriodora has activity against C. albicans, which might be attributed to the higher content of aromatic agents,
including essences and polyphenols, in the ethanolic extract compared to the aqueous extract. Use of this extract can
be suggested to volunteers, after evaluating them, as an agent against C. albicans.

Acknowledgments:
This study was based on post-graduate research conducted by Faranak Shafiee. The authors appreciated the financial
support of the Research Center of Babol University of Medical Sciences (grant number 9339423). We also thank
Mrs. Maryamosadat Shafiee for providing some practical works.

Conflict of Interest:
There is no conflict of interest to be declared.

Authors' contributions:
All authors contributed to this project and article equally. All authors read and approved the final manuscript.

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