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Candida Albicans
Candida Albicans
ARTICULO ORIGINAL
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Abstract
Objective: To determine anti-Candida albicans activity, cytotoxicity and drug interaction of essential oils and extracts from plants collected in
Colombia.
Materials and methods: The antifungal activity was evaluated following the AFST-EUCAST protocol. With most active samples, the inhibition of the
formation of germ tubes and budding, the in vitro pharmacodynamics, using time-kill assays, and the interaction with itraconazole and amphotericin
B following the chequerboard technique were evaluated. The cytotoxicity assay for all samples was done using MTT.
Results: Strong activity in 17.57% of the samples was found. The lowest MIC values were obtained with Piper bredemeyeri Jacq and Lippia origanoi-
des Kunth (B) oils and Morinda royoc L extract. The three samples inhibited the formation of germ tubes and budding. P. bredemeyeri Jacq oil and M.
royoc L extract samples showed fungicidal activity at 2xMIC. A synergistic effect was obtained with the combination of P. bredemeyeri Jacq oil and
itraconazole, but not for the combination with amphotericin B. Active samples against C. albicans were not cytotoxic on Vero cells ATCC CCL-81,
excluding P. bredemeyeri Jacq oil.
Conclusions: The results of this study suggest that Colombian medicinal and aromatic plants represent an untapped source of compounds with
anti-C. albicans activity that could be a resource in the development of new therapeutic natural products.
Key words: Essential oils, extracts, Candida albicans, antifungal activity, cytotoxicity, synergism
Resumen
Objetivo. Determinar la actividad anti-Candida albicans, la citotoxicidad y la interaccin con antifngicos de aceites y extractos de plantas recolec-
tadas en Colombia.
Materiales y mtodos. La actividad antifngica fue evaluada siguiendo el protocolo Antifungal Susceptibility Testing Subcommittee of the European
Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST). Con las muestras ms activas se evalu la inhibicin de la formacin de tubo
germinal y la gemacin, la farmacodinamia mediante curvas de tiempo muerte y la interaccin con itraconazol y anfotericina B. Se determin la
citotoxicidad mediante la tcnica MTT.
Resultados. Se encontr actividad en 17,57 % de las muestras. La mayor actividad se obtuvo con los aceites de Piper bredemeyeri Jacq y Lippia origanoides
Kunth (B) y el extracto de Morinda royoc L. Las tres muestras inhibieron la formacin de tubo germinal y la gemacin. El aceite de P. bredemeyeri Jacq y el
extracto de M. royoc L mostraron actividad fungicida con dos veces la concentracin inhibitoria mnima. Se encontr un efecto sinrgico por la combinacin
del aceite de P. bredemeyeri Jacq e itraconazol, pero no con anfotericina B. Las muestras activas no fueron citotxicas, excepto el aceite de P. bredemeyeri Jacq.
Conclusin. Los resultados de este estudio sugieren que las plantas de Colombia son una fuente no explorada de compuestos con actividad anti-C.
albicans, tiles para el desarrollo de nuevos productos teraputicos.
Palabras clave: aceites esenciales, extractos, Candida albicans, actividad antifngica, citotoxicidad, sinergismo.
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tract showed fungicidal activity at 2 times the MIC In this study, oils and extracts from medicinal
after 8 hours of incubation (Figure 1b). At lower and aromatic plants were tested against two C.
concentrations (0.25, 0.5 and 1 times the MIC), we albicans strains and a clinical isolate. Currently,
GLGQRWQGVLJQLFDQWUHGXFWLRQLQ&)8PO (<3 - there is no agreement on the acceptable level
log10) compared with the starting inoculum. of activity for natural products when they are
compared to standard drugs. Aligiannis, et al.(16),
A synergistic effect was obtained for the combi- VXJJHVWHG D FODVVLFDWLRQ RI DQWLIXQJDO DFWLYLW\
nation of itraconazole and P. bredemeyeri Jacq in plant derivates (antifungal activity based on
(FICI range 0.09-0.13), but no interaction was MIC results) as follows: strong inhibitors, MIC up
detected for the combination of P. bredemeyeri to 0.5 mg/ml; moderate inhibitors, MIC between
Jacq and amphotericin B (FICI=1.06). 0.6 and 1.5 mg/ml; weak inhibitors, MIC above
1.6 mg/ml. According to these criteria, we found
According to the American National Cancer Ins-
strong anti-C. albicans activity in 17.57% of the
titute (USA) criteria samples with anti-C. albicans
evaluated samples (Table 1).
activity were not cytotoxic on Vero cells excluding
P. bredemeyeri Jacq oil (IC50=15.2 3) (Table 1).
Previous studies have shown the antifungal ac-
tivity in extracts and oils of plants belonging to
Discussion
Piper, Morinda, and Lippia genus (17-19). Lpez, et
al.(19), determined a MIC value of 100.000 g/
Plants, marine organisms, and microbes usua-
ml against C. albicans of the methanolic extract
lly produce biologically active compounds as a
from the plant Piper lanceafolum, using an agar
defense against predators and competition with
neighbors. Thus, it seems logical that most of diffusion method. In contrast, in this study, the
the drugs derived from natural sources have an- microdilution standard method AFST-EUCAST
ticancer or anti-infective properties. Important was used, and lower MICs with P. bredemeyeri
antifungal agents such as polyenes, echinocan- Jacq were found (GM-MIC of 157.5, 176.8, and
dins/pneumocandins, aureobasidins and sorda- 222.7 g/ml, with the three evaluated yeasts)
rins had their origin in natural products (15). (Table 1).
a b
10 10
8 8
Log 10 CFU/mL
Log 10 CFU/mL
6 6
4 4
2 2
0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
Time (h) Time (h)
c d
10 10
8 8
Log 10 CFU/mL
Log 10 CFU/mL
6 6
4 4
2 2
0 0
0 4 8 12 16 20 24 28 0 4 8 12 16 20 24 28
Time (h) Time (h)
Figure 1. Time-kill plots against C. albicans ATCC 90028 of a) P. bredemeyeri Jacq oil, b)
M. royoc L y P. bredemeyeri -DFTH[WUDFWFDPSKRWHULFLQH%DQGGLWUDFRQD]ROH*URZWK
control; 0.25x MIC; c 0.5x MIC; d[0,&;0,& 4x MIC
Lippia origanoides 512271 Oil 396.9 500 500 52.3 11.5 0.8
Lippia origanoides 512075 Oil 250 250 315 74.6 16.9 0.75
Lippia origanoides 512075 Oil 500 500 500 60.4 11.2 0.83
Piper bredemeyeri Jacq. 516939 Oil 157.5 176.8 222.7 15.2 3 0.81
Turnera aff. diffusa Willd. ex
516293 Oil * 353.6 500 52.2 5.2 0.93
Schult.
Lippia origanoides Kunth (A) 517741 Oil 500 500 500 31.4 5.6 0.75
Lippia origanoides Kunth (B) 517741 Oil 157.5 157.5 198.4 31.4 5.6 0.75
Lippia origanoides Kunth (C) 517741 Oil 500 396.9 396.9 34.4 5.9 0.76
Piper hispidum Sw. 519969 Oil 250 280.6 250 51.7 9.3 0.8
Lippia origanoides 512087 Oil 500 500 500 104.4 5.9 0.98
This is one of the few studies focused on evalua- The concentration at which P. bredemeyeri Jacq
ting the anti-C. albicans activity of L. origanoides and L. origanoides Kunth (B) oils were able to
oils using a standard method for determination inhibit the germ tube formation of C. albicans
of MIC by broth dilution of fermentative yeast. JPO RU JPO DUH ZLWKLQ WKH
Oliveira, et al .(20), also found important activi- concentration ranges published for this activity
ty against C. albicans in an oil of L. origanoides with Melaleuca alternifolia oil (tea tree oil) (0.25
using an agar diffusion method; however, we and 12.5% (v/v, approximately 2.25 and 112.5
cannot compare our results with theirs, because g/ml, respectively) (24). Those results make our
they used a technique that determined inhibi- QGLQJV SURPLVLQJ LI ZH FRQVLGHU WKDW WHD WUHH
tion zone (mm) but not MIC (g/ml). oil is used as commercial product for treatments
of fungal infections as oropharingeal and vagi-
Candida albicans is a dimorphic fungus, able to nal infections by C. albicans (24-26).
JURZDV\HDVWRUODPHQWRXVIRUP7KH\HDVWWR
hyphal transition begins with the formation of Candida albicans has isotropic growth by budding
a germ tube and it is the initial stage of hyphal and several antifungal drugs and plant extracts can
formation (21). The oils from P. bredemeyeri Jacq inhibit budding of yeast cells (21, 27). In this study the
and L. origanoides Kunth (B) and the M. royoc L treatment of the strains and the clinical isolate with
extract inhibited the germ tube formation. It is P. bredemeyeri Jacq and L. origanoides Kunth (B)
possible that those oils and extracts are acting oils, and M. royoc extract, decreased blastoconidia
on an important process in the morpho-trans- budding at 250 g/ml compared with the negative
formation of C. albicans, as wall or membrane control (p<0.05). Previous studies have suggested
integrity or reorganization of the cytoskeleton (22, that the deleterious effect of the essential oil on the
23)
. This characteristic could make a new antifun- cell wall of the fungus could be the main reason for
gal more selective towards the infecting germ the decrease in the rate of yeast budding, because
than towards the host cells. the cell wall is necessary for cell division (27).
The time-kill studies are useful for the evaluation and C. parapsilosis has been demonstrated. Un-
of the pharmacodynamics characteristics of new like P. bredemeyeri Jacq oil only showed activity
antimicrobial agents (28). The oil of P. bredemeyeri against C. krusei (unpublished data).
Jacq and the extract of M. royoc L showed a fun-
gicidal activity on C. albicans at 2 x MIC after 4 Potential synergy of essential oils with antibiotics
and 8 hours, respectively. It is clinically more im- has been previously considered with the aims of
SRUWDQWWRQGIXQJLFLGDOFRPSRXQGVWKDQIXQ- increasing the rate of fungal killing, shortening
gistatic, particularly in HIV patients, because the the duration of therapy, avoiding the emergence
prophylactic use of fungistatic drugs has been of drug resistance, expanding the spectrum of
associated with an increased frequency of innate activity, and decreasing drug-related toxicity by
or acquired drug resistance in clinical isolates (29). allowing lower doses of antifungal agents to be
administered (33). In this study, a synergistic effect
Chemical analyses of most active essential oils was obtained when itraconazole and the oil from
against C. albicans have been carried out in our P. bredemeyeri Jacq were combined (FICI range,
laboratory. The main compounds of P. bredeme- 0.09-0.13). No interaction was detected with the
yeri Jacq oil were terpenes alpha-pinene (20.3%) combination of the oil of P. bredemeyeri Jacq and
and beta-pinene (32.3%) (unpublished data). amphotericin B (FICI=1.06).
Other studies have demonstrated that those ter-
The criteria of cytotoxic activity for the crude ex-
penes act on cellular integrity, inhibition of the
tracts as established by the American National
respiration and ion transport processes, and in-
Cancer Institute (USA), is an IC50 of less than 30
crease membrane permeability in C. albicans (30,
g/ml (34). According to these criteria, we consi-
31)
. Hence, it is possible that the antifungal acti-
der that oils with anti-C. albicans activity and M.
vity of P. bredemeyeri Jacq oil could be explained
royoc L extract were not cytotoxic on Vero cells
by a higher concentration of those monoterpe-
(IC50JPOH[FOXGLQJWKHRLORIP. bredeme-
ne hydrocarbons. On the other hand, chemical
yeri Jacq (IC50=15.2 3 g/ml) (Table 1). Gonz-
analyses of oil from L. origanoides Kunth (B) have
lez, et al..(35), orally administered an extract of M.
also been carried out in our laboratory. The main
royocLQUDWVDQGWKH\GLGQRWQGWR[LFHIIHFWV
components were the oxygenated monoterpenes
Both our results of in vitro citotoxicity, as well as
thymol (43.8%) and carvacrol (17.3%). The antimi-
the toxicity in vivo demonstrated by Gonzlez, et
crobial activities of those terpenes have also been
al..(35), suggest that M. royoc L extract may be a
demonstrated, and their mechanism of action has candidate for developing an antifungal of natu-
been associated with membrane permeability (20). ral origin against C. albicans.
7R WKH EHVW RI RXU NQRZOHGJH WKLV LV WKH UVW The discovery of a novel natural product for
time that antifungal activity of M. royoc L extract therapeutic use is a slow process. For example,
is described. Morinda genus contains substan- Taxol, an anticancer agent developed from the
tial amounts of anthraquinones, especially in the plant Taxus brevifolia, was discovered after a
roots. The antifungal activity of those quinones random screening of 35,000 plant samples that
has been demonstrated (32), so it is possible that took more than 25 years (36, 37).
anti-C. albicans activity of M. royoc L extract may
be produced by the presence of such molecules. In conclusion, the results of this study suggest that
Colombian medicinal and aromatic plants repre-
In our laboratory, the antifungal activity of L. sent an untapped source of compounds with anti-
origanoides Kunth (B) and M. royoc L extract C. albicans activity that could be a resource in the
against Aspergillus fumigatus,$DYXV&NUXVHL development of therapeutically natural products.