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MYCMED-816; No. of Pages 4

Journal de Mycologie Médicale xxx (2018) xxx–xxx

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Original article

Antifungal activity of Camellia sinensis crude extracts against

four species of Candida and Microsporum persicolor
S. Akroum
Department of Applied Microbiology and Food Sciences, Faculty of Natural and Life Sciences, University Mohammed Seddik Ben Yahia, Jijel, BP 98 Ouled Aissa,
18000 Jijel, Algeria

ARTICLEINFO ABSTRACT

Article history: Objective of the study. – Candidiasis and dermatophytoses are benign infections in humans and animals,
Received 4 February 2018 but they are very dreaded diseases in immunocompromised individuals. These infections become
Accepted 16 June 2018 resistant to different treatments which make them more dangerous. In this work, we tried to find a new
Available online xxx way for treating them. So we were interested in the antifungal activity of Camellia sinensis (tea); this
plant is known to have many health benefits.
Keywords: Materials and methods. – We tested the ability of the acetone and aqueous crude extracts of the plant to
Acetone crude extract inhibit in vitro the growth of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and
Aqueous crude extract
Microsporum persicolor. Then, the antifungal activity against these species was tested in vivo in mice.
Camellia sinensis
Results. – The results showed that the acetone crude extract had the most important in vitro activity against
Antifungal activity
Candida all the fungi. But in vivo it was only the most active against Candida albicans, Candida glabrata, Candida
Microsporum persicolor tropicalis and Microsporum persicolor. Candida krusei was more sensitive to the aqueous crude extract.
Conclusion. – These results indicated that tea could be considered to treat infections caused by the five
tested species.
§C 2018 Elsevier Masson SAS. All rights reserved.

1. Introduction
Candidiasis and dermatophytoses are the most common human
infections in the world [1]. They are generally treated with a wide
variety of antifungal drugs [2], but they became increasingly
resistant to different treatments, especially when they are caused
by Candida, Microsporum and Trichophyton species [3,4]. These
infections can be acute or chronic and in some cases they require a
combination of several drugs [4,5]. But the treatments can show
significant liver toxicity which limits their use [6]. Also, mycosis
are very dangerous in immunocompromised people. In fact, they
may take on dramatic proportions from patient to patient [7].
Camellia sinensis (tea) is one of the most popular beverages in
the world [8]. The traditional medicine recommends this plant for
several health benefits because it prevents and treats many human
diseases: cancers, infections, cardiovascular and neurological
diseases [9].
In this work, we tested the antifungal activity of tea against four
Candida species known to be pathogenic to humans and animals
(Candida albicans, Candida glabrata, Candida tropicalis and Candida
E-mail address: muorka00@yahoo.com
krusei) and also against Microsporum persicolor. The activity was
tested in vitro and in vivo for the acetone and the aqueous crude
extracts of the plant.
2. Materials and methods
Camellia sinensis was bought from herbalists and identified at
the Department of Botany, University of Jijel (Algeria).
The yeasts provided from the hospital of Jijel (Algeria) and the
university hospital center of Constantine (Algeria). They were
isolated from different patients.
2.1. Preparation of the crude extracts
The acetone crude extract was prepared by maceration of 10 g
of plant in 100 mL of acetone/water (70/30, V/V) and the aqueous
crude extract was obtained using 100 mL of sterile water. The
maceration was done in an electric shaker during 2 hours. Then,
the solvents were recovered by filtration using the Whatman No.1
paper and evaporated with the rotary evaporator. The obtained
crude extracts were diluted in sterile water to obtain suspensions
of 1 mg/mL [10,11].

https://doi.org/10.1016/j.mycmed.2018.06.003
1156-5233/§ C 2018 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Akroum S. Antifungal activity of Camellia sinensis crude extracts against four species of Candida and
Microsporum persicolor. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.003
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2.2. Preparation of the fungal suspensions
Four yeasts were tested: Candida albicans, Candida glabrata,
Candida tropicalis and Candida krusei. Each one was cultured in
Sabouraud’s dextrose broth and incubated at 30 8C for 24 hours.
Then, cells were filtered and rinsed with a normal phosphate-
buffered saline suspension (PBS). The fungal suspensions were
prepared and diluted with PBS to obtain 107 CFU/mL [3]. For the in
vivo tests against the yeasts, the inoculum size sufficient to cause
the death of untreated mice was 1.5 × 104 CFU/mL; it was
determined in a preliminary experiment.
Microsporum persicolor was cultured in Sabouraud agar and
incubated at 28 8C for 14 days. After this, a conidial suspension was
prepared by submerging the mycelium surface with PBS and
filtering conidia. The concentration applied was 107 conidia/mL [12].
2.3. In vitro antifungal activity
The crude extracts were diluted in distilled water to obtain
different concentrations from 0.005 to 0.200 mg/mL. The fungal
suspensions were inoculated on Petri dishes containing Sabouraud
agar and different doses of the crude extracts. An incubation time
of 24 hours at 30 8C was applied for yeasts. M. persicolor required
48 hours at 28 8C. The antifungal activity was expressed with
growth or no growth and the minimum inhibitory concentration
(MIC) was determined for each crude extract [11].
2.4. In vivo antifungal activity against yeasts
C57BL6 mice were immunosuppressed with 2 mg of cyclo-
phosphamide given intraperitoneally during 4 days before
infection, then on the third day post infection. The animals were
kept in clean cages and served autoclaved food and water in order
to maintain a sterile environment throughout the experiment
[13]. The infections were induced intravenously and after 24 hours,
the crude extracts were administered intravenously during 5 days
at a concentration of 20 mg/mouse/day. We used groups of animals
containing five mice each. The mortality was monitored daily
[14]. As controls we used two groups of animals: the first was
immunosuppressed, infected and not administered with the
extracts. And the second one was not immunosuppressed, but
infected and administered with 100 mg of the tested extract. The
first group gave 100% of mortality and the second 0% of mortality.
2.5. In vivo antifungal activity against M. persicolor
C57BL6 mice were immunosuppressed by a subcutaneous
injection of 500 mg of estradiol valerate 4 days before the infection.
For each mouse, we shaved a little surface of skin and sprayed on it
10 mL of the fungal suspension. When the mycosis appeared, the
crude extracts were orally administered during 5 days at a
concentration of 20 mg/mouse/day diluted into 250 mL of distilled
sterile water. As with the previous activity, the animals were kept
Fig. 1. Determination of the CMI for aqueous crude extract against Candida albicans. A. Medium with 0.005 mg/mL of the extract. B. Medium with 0.010 mg/mL. C. Medium
with 0.020 mg/mL. The MIC was determined at 0.020 mg/mL.
Please cite this article in press as: Akroum S. Antifungal activity of Camellia sinensis crude extracts against four species of Candida and
Microsporum persicolor. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.003
in clean cages and served autoclaved food and water. Each group
contained five animals. The skin lesions were scored on a scale
from 0 (no visible lesions) to 3 (significant crusting and erythema)
[12]. As controls we used two groups of animals: the first was
immunosuppressed, infected and not administered with the
extracts. And the second one was infected and administered with
100 mg of the tested crude extract, but it was not immunosup-
pressed. The first group showed significant crusting and erythema
and in the second group, the infection did not appear.
The experiments were conducted according to the Algerian
Association of Experimental Animal Sciences. Permission to use
the animals was approved by the Department of Applied
Microbiology and Food Sciences, Faculty of Natural and Life
Sciences, University Mohammed Seddik Ben Yahia.
3. Statistical analysis
The mean values of the in vitro and in vivo antifungal activities
were calculated using Microsoft Office Excel 2007. The in vitro
antifungal activity experiments were repeated twice and the
results were expressed as MICs mean value T standard error of the
mean (SEM). For the in vivo antifungal activity, we used groups of
animals which contained five mice each. The results were obtained by
calculating the percentage of mortality.
4. Results
4.1. In vitro antifungal activity
The lowest concentrations of the crude extracts that gave no
growth were registered to all the species (Fig. 1). The results
showed that the acetone crude extract was active against all the
fungi. Candida albicans was the most sensitive species towards this
extract, followed by Candida glabrata and M. persicolor. The
aqueous crude extract had a good activity only against Candida
albicans; all the other species required greater MICs (Table 1).
4.2. In vivo antifungal activity against yeasts
The in vivo activity against yeasts showed that the acetone
crude extract was more active against Candida albicans, Candida
glabrata and Candida tropicalis (Figs. 2–4). Candida albicans
required the lowest dose to give 100% of viability, so it was the
most sensitive species. But, the infection caused by Candida krusei
was resistant even the administration of 100 mg of the acetone
crude extract (Table 2).
On the other hand, the aqueous crude extract was only active
against Candida albicans and Candida krusei: it gave 100% of
viability with 60 mg and 100 mg respectively (Fig. 5). These
experiments were repeated twice and gave exactly the same
results.
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Table 1
MICs of the in vitro antifungal activity (expressed in mg/mL).

Candida albicans Candida glabrata Candida tropicalis Candida krusei M. persicolor

Acetone crude extract 0.005 T 0.000 0.010 T 0.005 0.045 T 0.005 0.050 T 0.000 0.020 T 0.005
Aqueous crude extract 0.020 T 0.005 0.120 T 0.015 0.080 T 0.010 0.090 T 0.005 0.180 T 0.010
Griseofulvin 0.020 T 0.005 0.035 T 0.005 0.040 T 0.005 0.040 T 0.000 0.060 T 0.000

4.3. In vivo activity against M. persicolor

The mycosis caused by M. persicolor was treated with the

administration of 80 mg of the acetone crude extract, so after four
days of treatment. But the aqueous crude extract was less active
against the species: skin lesions were also serious after the fifth
days of treatment (Fig. 6). For this mycosis the acetone crude
extract was the most suitable treatment (Fig. 7). The experiments
on M. persicolor were repeated twice and gave exactly the same
results.
Fig. 2. Percentage of mortality caused by Candida albicans after 5 days of treatment.

5. Discussion

Fig. 3. Percentage of mortality caused by Candida glabrata after 5 days of treatment.
Fig. 4. Percentage of mortality caused by Candida tropicalis after 5 days of treatment.
Table 2
Doses of extracts which gave 100% of viability.
Acetone crude extract
Aqueous crude extract
Fig. 5. Percentage of mortality caused by Candida krusei after 5 days of treatment.
The in vitro activity showed that the acetone and aqueous crude
extracts were active against the five species. But the acetone crude
extract gave the best MICs. Cheruiyot et al., 2015 have already
reported that the aqueous extract of Camellia sinensis was active
against several pathogenic fungi, especially against Candida
albicans, accorded with our findings. They also specified that the
hot aqueous extract was more active than the cold one. But they
reported that Candida glabrata was very sensitive to the aqueous
extract [11]. In our study, we observed that this species was more
inhibited by the acetone crude extract and the in vivo activity
confirmed this information. Our results could be considered as
interesting because Candida glabrata became increasingly resistant
to common antifungal treatment [15].
The in vivo activity also showed that the acetone crude extract
was the most active against Candida albicans, Candida glabrata,
Candida tropicalis and M. persicolor. A previous study has
established that the aqueous and the ethanol extracts were the
most efficacious for treating Candida infections and dermatophy-
toses [16]. This was in contradiction with our results because the
aqueous crude extract was only active against Candida albicans,
Candida glabrata and Candida krusei, and it required greater MICs.
The in vivo activity of this extract against M. persicolor was also
very low. To be more exact, the aqueous crude extract was more
active than the acetone only for the in vivo activity against Candida
krusei.
Candida albicans was the most sensitive species to our extracts.
Some studies have already reported that this species was very
Candida Candida Candida Candida
sensitive to tea extracts and resistant to the standard antifungal
albicans glabrata tropicalis krusei drugs [4,17]. By analogy, other research studies have demonstrated
that Candida albicans became more sensitive when tea extract was
40 mg 80 mg 100 mg –
60 mg – – 100 mg
combined with fluconazole, amphotericin B or copper sulphate,
also that the acetone extract of the plant was rich in catechins,
phytosterols, tannins and flavonoids which could be at the origin of
the antifungal activity of the plant [5,18,19].
The results indicated that Candida krusei was the most resistant
yeast, especially when it was tested in vivo. Cheruiyot et al.,
2015 showed that this species was sensitive to tea aqueous extract
once tested in vitro, but no work was found for the in vivo activity
[11]. These results were important because Candida krusei was
considered to be one of the most resistant Candida to the antifungal
drugs [4].
We also observed that M. persicolor mycosis was easily treated
with the acetone crude extract. The mycosis was entirely healed
with a low dose of this crude extract. So it could be suggested for

Please cite this article in press as: Akroum S. Antifungal activity of Camellia sinensis crude extracts against four species of Candida and
Microsporum persicolor. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.003
 G Model
MYCMED-816; No. of Pages 4
4 S. Akroum / Journal de Mycologie Me´dicale xxx (2018) xxx–xxx
Fig. 6. Results before and after treatment. A. Lesions and yellowing of hairs before treatment. B. Treatment with 100 mg of aqueous extract. C. Treatment with 80 mg of acetone
extract.
Figure 7. Skin lesions after five days of treatment. 0: no visible lesions; 1: rare and
superficial lesions; 2: the early crusting and erythema; 3: significant crusting and
erythema.
treating infection caused by this dermatophyte. No other data was
found about the antifungal activity of tea against M. persicolor.
6. Conclusion
The acetone crude extract of Camellia sinensis was highly active
in vitro against all the species. But once tested on mice, it was only
the most active against Candida albicans, Candida glabrata, Candida
tropicalis and M. pesicolor. The aqueous crude extract had the best
in vivo activity against Candida krusei.
Candida albicans was the most sensitive species when tested in
vitro and in vivo and this for the two extracts. M. persicolor mycosis
was healed with a low dose of the acetone crude extract, but it was
more resistant to the aqueous one.
Disclosure of interest
The authors declare that they have no competing interest.
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Microsporum persicolor. Journal De Mycologie Médicale (2018), https://doi.org/10.1016/j.mycmed.2018.06.003
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