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Hexane, acetone and methanol extracts obtained from Psidium guajava leaves were studied for their antifungal properties against Trichophyton rubrum, Trichophyton tonsurans, Sporotrix schenckii, Microsporum canis, Cryptococcus neoformans, Candida parapsilosis,and Candida albicans by using the agar disk diffusion technique. Compared to control, hexane extract showed the best antifungal activity, being active against all the tested dermatophytes. Methanol and acetone extracts also showed relevant activity. The phytochemical analysis of the hexane extracts revealed the presence of flavonoids, terpenoids and coumarins, whereas alkaloids, carbohydrates and saponins were not detected. Since the bioactive compounds in the hexane extract inhibit the growth of microorganisms, it could be considered for future development of new anti-skin disease agents.
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Journal of Medicinal Plants Research Vol. 6(41), pp. 5435-5438, 25 October, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR12.240
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Antifungal activity of Psidium guajava organic extracts
against dermatophytic fungi
Padrón-Márquez Beatriz1, Viveros-Valdez Ezequiel1, Oranday-Cárdenas Azucena1 and
Carranza-Rosales Pilar2*
1Departamento de Química Analítica, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León,
San Nicolás de los Garza, Nuevo León, México.
2División de Biología Celular, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social,
Monterrey, Nuevo León, México.
Accepted 22 March, 2012
Hexane, acetone and methanol extracts obtained from Psidium guajava leaves were studied for their
antifungal properties against Trichophyton rubrum, Trichophyton tonsurans, Sporotrix schenckii,
Microsporum canis, Cryptococcus neoformans, Candida parapsilosis, and Candida albicans by using
the agar disk diffusion technique. Compared to control, hexane extract showed the best antifungal
activity, being active against all the tested dermatophytes. Methanol and acetone extracts also showed
relevant activity. The phytochemical analysis of the hexane extracts revealed the presence of
flavonoids, terpenoids and coumarins, whereas alkaloids, carbohydrates and saponins were not
detected. Since the bioactive compounds in the hexane extract inhibit the growth of microorganisms, it
could be considered for future development of new anti-skin disease agents.
Key words: Antimycotic activity, dermatophytes, inhibition zone, crude extracts, Psidium guajava.
INTRODUCTION
Many skin diseases such as tinea and ringworm caused
by dermatophytes are prevalent in tropical and
subtropical regions. In general, these fungi live in the
dead, top layer of the dermis and in moist areas of the
body. They can penetrate into the cells and cause itching,
swelling, blistering and scaling. Dermatophytes are
important causes of acute or chronic deep-seated human
infections, especially recurrent mucosal, cutaneous, or
nail infections that can be severe in debilitated or
immunocompromised individuals (Debruyne and
Coquerel, 2001; Welsh et al., 2010). However, the toxicity
of currently available antifungal therapies, as well as the
increasing of drug-resistance among the etiologic agents
has driven the research towards the study of new
antimicrobial agents from natural products (Khan et al.,
2012; Ajose, 2007). Based on reports that plants have
*Corresponding author. E-mail: pilarcarranza@cibinmty.net. Tel:
52+(81) 8190 4036. Fax: 52+(81) 8190 4035.
developed mechanisms of defense to protect themselves
against biotic and abiotic threats, including infections by
pathogens like fungi, bacteria, and viruses, recent
interest has been focused to the search of plant-derived
fungicides and antimicrobials (Gurgel et al., 2005;
Wojtaszek, 1997).
Medicinal plants are considered a rich source for
antimicrobial agents, with the advantage that most of the
natural products used in traditional medicine are readily
available in rural areas at relatively lower cost than
modern medicines (Mahesh and Satish, 2008; Mann et
al., 2008). Plants generally produce many secondary
metabolites which constitute an important source of
microbicides, pesticides and pharmaceutical drugs. In
relation to this, the common guava tree (Psidium guajava
Linn.), a member of the Myrtaceae family, has been
reviewed extensively by Gutiérrez et al. (2008) in order to
highlight the pharmacologic effects of the extracts
obtained from its fruits, leafs, bark or roots. The
investigators found that extracts from P. guajava have
antispasmodic and antimicrobial properties for the
5436 J. Med. Plants Res.
Table 1. Antifungal activity of P. guajava leave extracts
Microorganism
Zone of inhibition (mm)
Organic extract
Control*
MeOH
Acetone
Candida albicans
11 ± 2
17 ± 2
45 ± 5
Candida parapsilosis
17 ± 3
10 ± 1
25 ± 5
Cryptococcus neoformans
11 ± 1
18 ± 3
35 ± 3
Microsporum canis
-
-
30 ± 4
Microsporum gypseum,
-
-
43 ± 5
Trichophyton tonsurans
19 ± 3
-
44 ± 4
Trichophyton rubrum
10 ± 2
13 ± 1
50 ± 6
Sporotrix schenckii
-
11 ± 2
47 ± 4
*Ketoconozole was used as positive control.
treatment of diarrhea and dysentery. Additional
pharmacological properties attributed to extracts from P.
guajava include antioxidant, hepatoprotective, antialler-
gic, antigenotoxic, antiplasmodial, cytotoxic, cardioactive,
anticough, anti-inflammatory, antinociceptive, hypogly-
cemic and antidiabetic activities, thus, supporting its uses
in traditional medicine. Previous works have also showed
important antifungal activity of the tinctures (Dutta et al.,
2000). Because little is known about the antifungal
properties of organic extracts prepared from P. guajava
leafs, this study was performed to assess the efficacy of
the organic extracts against dermatophyte fungi.
MATERIALS AND METHODS
Sample collection and processing
P. guajava (Linn) (Myrtaceae) was collected in San Nicolás de los
Garza, Nuevo León, México, during May and June, 2000; it was
identified in the Department of Botany by Dr. Marcela González
Alvarez. A plant specimen was deposited in the ethnobotanical
collection of the FCB-UANL herbarium (voucher specimen number:
024884).
Leaves from the plant were dried at room temperature, and 30 g
of the dry-powdered material were sequentially extracted by
maceration with hexane, acetone and methanol (3 times, 24 h
each). The plant:solvent ratio was 1:5 (w/v). After filtration and
concentration under reduced pressure, the percentage (w/w yield)
of extracts from P. guajava was hexane (5.5), acetone (11.6) and
methanol (19.8).
Microbial suspension
Clinical isolates of Candida albicans, Candida parapsilosis,
Cryptococcus neoformans, Microsporum canis, Microsporum
gypseum, Trichophyton tonsurans, Trichophyton rubrum, and
Sporotrix schenckii were maintained at 4°C in Sauboraud Dextrose
Agar (SDA) culture medium. The dermatopyhtes were subcultured
on SDA slants and incubated at 35°C for 7 to 14 days, depending
on the microorganism. The mycelial growth was scraped aseptically
and suspended thoroughly in sterile distilled water. The suspension
was standardized spectrophotometrically to an absorbance [also
called optical density (OD)] of 0.600 at 450 nm. These adjusted
suspensions approximately corresponded to 0.5 to 2.5 103
cells/ml and were used as inoculum for antifungal susceptibility
testing (Chandrasekaran and Venkatesalu, 2004).
Antifungal assay
Antifungal activity tests were performed by using the disk diffusion
agar method (Bauer et al., 1966). Test plates were prepared with
20 ml of sterile SDA. The standardized fungal suspension was
applied on the solidified culture medium by using sterile cotton
swabs and allowed to dry for 5 min. A sterile paper disk (Whatman
AA disk, 6 mm) was impregnated with 10 μl of a stock solution (50
mg/ml) from each crude extract. The disks were aseptically
transferred on the inoculated agar plates and incubated for 48 h to
7 days, depending of the tested fungi. Antifungal activity was
determined by measuring clear zones of inhibition around the test
crude extract discs. The clear zones indicated the fungicidal effect
while fungi static effect referred to the unclear zone of inhibition.
Ketoconazole ( 250 μg) disks were used as a standard reference or
positive controls, and the solvent or empty disks were used as
negative controls. All assays were performed in triplicate.
Phytochemical screening
The phytochemical constituents of the plant were determined in
accordance with the methods described by Harborne (1984). The
colour intensity of extracts and/or the appearance of solids in them
during the identification reactions allow a semi-quantitative
evaluation of the presence of secondary metabolites.
RESULTS
Organic extracts from P. guajava leaves were
investigated for their antifungal effect against clinically
important dermatophytes fungi. Compared to control, the
best activity found in our investigation was observed with
the hexane extract, which inhibited all the tested
dermatophytes (Table 1). However, methanol and
acetone extracts showed relevant activity against 70% of
the strains. C. neoformans was the most sensitive fungi,
and the acetone extract was the most active (18 ± 3). M.
gypseum and M. canis were inhibited by the non polar
Beatriz et al. 5437
Table 2. Phytochemical screening of P. guajava leaf extracts.
Organic extract
Alk
Flav
Coum
Sap
Phenolics
Sesq
CHO
Terp
Hexane
-
+
+
-
+
+
-
++
Acetone
-
+
-
+
+
-
+
+
Methanol
-
++
+
+
++
+
+
++
Alk, Alkaloid; Flav, flavonoids; Coum, coumarins; Sap, saponins; Sesq, sesquiterpene lactones; CHO, carbohydrates; Terp, terpenoids;
(++), abundant; (+), present; (), absent.
extract, whereas C. parapsilosis was the most resistant,
with inhibition zones of 25 ± 5 mm showed by the positive
control (Ketoconozole) and 17 ± 3 mm by the methanol
extract.
The secondary metabolites that were identified in the
non-polar extract were flavonoids, terpenoids and
coumarins; only the methanol and acetone extracts
showed carbohydrates and saponins; no alkaloids were
detected in any extracts (Table 2).
DISCUSSION
Previous studies have shown the fungicidal effect of
organic extracts derived from plants, and also has been
shown that the activity of secondary metabolites may
vary depending on the type of solvent used. In
accordance with the last, antifungal activity has been
reported in polar compounds such as glycosilated
flavonoids, and saponins isolated from polar extracts
(Kim et al., 2010; Lanzotti et al., 2012), and, in non-polar
compounds, like terpenoids (Wang et al, 2011; Singh et
al., 2011).
In the present study, methanol and acetone extracts
showed comparable activity against the fungal strains;
similar results were obtained by Nair and Chanda (2007),
with inhibition zone diameters of 7.5 to 18 mm against
Candida spp and C. neoformans (9 ± 1.15), the best
antifungal activity was showed by the hexane extract.
With regard to our results, diverse authors have found
that antifungal activity relies on the organic solvents
used. For example, Machado et al. (2009) demonstrated
antimicrobial activity in the methanol extracts, while Tay
et al. (2004) and Cardoso et al. (2010) reported activity
with acetone and hexane extracts, respectively. The
observed activity for the hexane extract is acceptable,
considering that a crude extract was used, and the active
compound could be diluted. It is possible that isolating
the active compound or compounds will provide better
fungicide activity. The above results suggest that P.
guajava could be an important source of non-polar com-
pounds with antimicrobial activity. Regarding to the last,
reports about antibacterial and antifungic compounds
isolated from leaves of P. guajava showed that in the
polar extract (alcoholic), flavonoids such as quercetin and
its glycosides derivates are responsible of the strong
antibacterial activity, including against C. albicans (Arima
and Danno, 2002; Metwally et al., 2010). In the non-polar
extract (toluene), the terpenoids betulinic acid and lupeol
were isolated; these compounds showed antifungal
activity against Colletotrichum camelliae, Fussarium
equisitae, Alternaria alternate, Curvularia eragrostidies
and Colletrichum gleosproides (Ghosh et al., 2010). Both
triterpenoids have been previously reported possessing
antifungical activity against S. schenckii, M. canis, C.
albicans and C. neoformans (Shai et al., 2008). Taking
the lasts reports into account, and the similitude of our
results, it can be possible that the same compounds
could be responsible for antidermatophyte activity of the
P. guajava leaves.
Conclusion
The results of this study indicate that the leaves of P.
guajava contain bioactive compounds, like flavonoids and
terpenoids which inhibit the growth of dermatophytic fungi
thereby providing an additional alternative source of
antifungal compounds.
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... Most studies on the antifungal activity of P. guajava have been carried out with preparations of the leaf from the plant. Thus, extracts from this part of the plant obtained with various organic solvents or hot water elicited broad and meaningful activity against species in the genera Candida, Saccharomyces, Cryptococcus, Trichosporon, Aspergillus, Sporothrix, and Microsporum [198][199][200][201][202][203]. The antifungal activities in the leaf preparations potentiated that of fluconazole [203,204] and have been attributed to phenolic compounds including tannins, coumarins, and flavonoids such as quercetin, and/or terpenoids [198,201,203,361]. ...
... Thus, extracts from this part of the plant obtained with various organic solvents or hot water elicited broad and meaningful activity against species in the genera Candida, Saccharomyces, Cryptococcus, Trichosporon, Aspergillus, Sporothrix, and Microsporum [198][199][200][201][202][203]. The antifungal activities in the leaf preparations potentiated that of fluconazole [203,204] and have been attributed to phenolic compounds including tannins, coumarins, and flavonoids such as quercetin, and/or terpenoids [198,201,203,361]. The former supposition is supported by the activity of tannic and flavonoid fractions from the leaf against several species of Candida [204] and the inhibitory effects of plant phenolic compounds on the biosynthesis of ergosterol for assembling the fungal plasma membrane [365]. ...
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Abstract: Essential oils (EOs) were extracted from the leaves of two Psidium guajava (P. guajava) cultivars (white and pink fruit forms) from the local fields of Faisalabad, Punjab Pakistan. Essential oils were analyzed by GC/MS and the antioxidant, antimicrobial and anti-haem biocrystallisation activities were assessed. GC/MS profiles revealed 40 and 57 compounds with total percentage composition 92.55 % and 86.89 % in P. guajava white and pink cultivars, respectively. The major compounds (>5 %) found in EO of P. guajava white were caryophyllene (9.08 %), dihydrocarveol acetate (7.04 %), nerolidol (6.69 %) and caryophyllene oxide (6.18 %) whereas α-phellandrene (11.66 %), eucalyptol (10.01 %), α-terpineol (7.78 %) and spathulenol (5.71 %) in P. guajava pink. Essential oils showed moderate antioxidant potential. The P. guajava pink cultivar had comparatively high antioxidant potential than P. guajava white while both had comparable antimicrobial potential. Essential oils from both of the cultivars of P. guajava exhibited poor anti-haem biocrystallization activity. Results revealed that the EO of P. guajava pink cultivar has high phenolic content as compared to P. guajava white and thus the greater antioxidant potential. Whereas the antimicrobial potential of both was almost equal. The results also revealed that both EOs have poor antimalarial activity in comparison of standard drug. Both qualitative and quantitative variations were observed in chemical composition of both EOs.
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Psidium guajava L. leaves were subjected to extraction, fractionation and isolation of the flavonoidal compounds. Five flavonoidal compounds were isolated which are quercetin, quercetin-3-O-α-L-arabinofuranoside, quercetin-3-O-β-D-arabinopyranoside, quercetin-3-O-β-D-glucoside and quercetin-3-O-β-D-galactoside. Quercetin-3-O-β-D-arabinopyranoside was isolated for the first time from the leaves. Fractions together with the isolates were tested for their antimicrobial activity. The antimicrobial studies showed good activities for the extracts and the isolated compounds.
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