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MYCMED-586; No. of Pages 5

Journal de Mycologie Médicale (2016) xxx, xxx—xxx

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

The anti-candidal activity of Satureja

khuzistanica ethanol extract against clinical
isolates of C. albicans
L’activité anti-Candida de l’extrait éthanolique du Satureja
khuzistanica contre des isolats cliniques de C. albicans

M. Mahboubi *, N. Kazempour

Department of Microbiology, Medicinal Plants Research Center of Barij, PO 3795191916, Kashan, Iran

Received 21 January 2015; received in revised form 21 November 2015; accepted 22 November 2015

KEYWORDS Summary Candida albicans is the common cause of some infectious diseases such as vaginal
Satureja khuzistanica; candidiasis or candidemia. Due to the emergence of drug resistant isolates of C. albicans,
Ethanol extract; finding a new anti-Candida agent is a new strategy for current treatments. This study
Candida albicans; evaluated the anti-candidal activity of Satureja khuzistanica ethanol extract against clinical
Anti-candidal agent; isolates of C. albicans. S. khuzistanica ethanol extract from aerial parts of plant at full
Synergistic effect flowering stage was evaluated against 30 clinical isolates and two ATCC reference strains of
C. albicans by disc diffusion and micro-broth dilution assay. Also, in this study we evaluated
the synergistic effects of amphotericin B, clotrimazole and ketoconazole with S. khuzistanica
ethanol extract. The means of MIC and MFC of S. khuzistanica ethanol extract against clinical
isolates were 299.4 and 722.6 (mg/mL), respectively. S. khuzistanica ethanol extract in-
creased the anti-candidal effect of amphotericin B and ketoconazole, while it had no
synergistic effect on clotrimazole against clinical isolates of C. albicans. Therefore,
S. khuzistanica ethanol extract can be introduced as a new source of anti-candidal agent
against clinical isolates of C. albicans.
# 2015 Elsevier Masson SAS. All rights reserved.

Résumé Candida albicans est la cause fréquente de certaines maladies infectieuses telles que
MOTS CLÉS la candidose vaginale ou la candidémie. En raison de l’émergence de souches résistantes aux
Satureja khuzistanica ; médicaments, la recherche des nouveaux agents anti-Candida est une nouvelle stratégie pour les

* Corresponding author.
E-mail addresses: mahboubi1357@yahoo.com, mahboubi@barijessence.com (M. Mahboubi).

http://dx.doi.org/10.1016/j.mycmed.2015.11.003
1156-5233/# 2015 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
+ Models
MYCMED-586; No. of Pages 5

2 M. Mahboubi, N. Kazempour

Extraction à l’éthanol ;
Candida albicans ;
Agent anti-Candida ;
Effet synergique
traitements actuels. Cette étude a évalué l’activité anti-Candida de l’extrait alcoolique de
Satureja khuzistanica contre les isolats cliniques de C. albicans. L’extrait éthanolique de parties
aériennes de S. khuzistanica au stade de pleine floraison a été évalué contre 30 isolats cliniques
et deux souches de référence ATCC de C. albicans par diffusion du disque en gélose et par dosage
en micro-dilution. En outre, nous avons évalué les effets synergiques de l’amphotéricine B, du
clotrimazole et du kétoconazole avec l’extrait éthanolique de S. khuzistanica. Les moyennes de
MIC et MFC de l’extrait éthanolique de S. khuzistanica contre les isolats cliniques étaient de
299,4 et 722,6 (mg/mL), respectivement. L’extrait éthanolique de S. khuzistanica augmentait
l’effet anti-Candida de l’amphotéricine B et du kétoconazole, alors qu’il n’a eu aucun effet
synergique sur le clotrimazole contre des isolats cliniques de C. albicans. Par conséquent,
l’extrait éthanolique de S. khuzistanica peut être considéré comme une nouvelle source d’agent
anti-Candida contre les isolats cliniques de C. albicans.
# 2015 Elsevier Masson SAS. Tous droits réservés.

Introduction Materials and methods

Vaginal yeast infections or vaginal candidiasis are common Plant materials

yeast infections in the women. About 75% of women have
one recurrence during their lives. Candida albicans is a
common type of yeast that causes candidiasis in the vagina
of infected women [8]. The symptoms of candidiasis are
abnormal vaginal discharge, itching and burning of the
vagina, pain with intercourse, painful urination and swell-
ing of the vulva [2]. Also, C. albicans can cause candidemia
or disseminated infections, which are the significant cause
of morbidity and mortality of patients [14]. Treatment of
Candidia infections are performed by miconazole, clotri-
mazole, tiaconazole, butaconazole, fluconazole, echino-
candines and amphotericin B and some other azole drugs
[18], but due to increasing resistance to azole drugs [17],
the scientists are interested in finding the new sources of
antifungal agents among the prescriptions in traditional
medicine [13,20]. Satureja khuzistanica Jamzad (Marzeh-
E-Khuzistani) is a member of Labiatae family. Although, it is
used in folk medicine for the treatment of different ail-
ments such as cramp, muscle pains, nausea, indigestion,
diarrhea and infectious diseases but the reputation of this
plant is for its pain relieving and antiseptic effects
[5,19,27]. Recent studies have been confirmed some bio-
logical activities such as anti-inflammatory effects [15,25],
antinociceptive and analgesic effects [22] according to
its traditional applications. The antiseptic effects of
S. khuzistanica were confirmed against bacteria [1,3] yeast
and filamentous fungi [6,23]. Although, there are some
investigations in the regards of its antiseptic activities
against Pseudomonas aeruginosa [1], Listeria monocytoge-
nes [3], Staphylococcus aureus [4] and yeast like Crypto-
coccus neoformans [6], Candida albicans [4] and
filamentous fungi such as Aspergillus flavus, A. niger, Peni-
cillium sp., Fusarium sp., Alternaria sp., Rhizopus sp. and
Mucor sp. [23], but there is no study that evaluate the anti-
candidal activity of S. khuzistanica ethanol extract against
clinical isolates of C. albicans. In this investigation, we
evaluated the anti-candidal activity of S. khuzistanica etha-
nol extract against 30 clinical isolates and two ATCC strains
of C. albicans. Also this study evaluated the synergistic
effects of some antifungal agents and this extract against
these clinical isolates.
S. khuzistanica aerial parts at the full flowering stage were
collected from Lorestan Province in June 2014 and a voucher
specimen was authenticated under the number 168-1. Dried
aerial parts of plant were grinded. The extraction was per-
formed with ethanol: water (70:30, v/v). The dried plant was
mixed with solvent at the ratio of 1:10 (w/v) for 24 h at
ambient temperature in percolator. The mixture was sepa-
rated by the tap of percolator and then was filtered through
filter paper. The residue was rinsed with the same solvent
and the extract was dried under vacuum. The extraction
yield was 47%. The dried extract was kept in refrigerator at
4 8C until the analysis.
Microbial strains
This study was conducted on 30 clinical isolates of C. albicans
that were isolated from the vaginal samples (n = 21), oral
wounds of patients with AIDS (n = 6) and genital infections of
diabetic patients (n = 3). C. albicans ATCC 10231 and ATCC
66506 also were used in this study. The strains were cultured
on Sabouraud dextrose agar medium with chloramphenicol
and were identified as C. albicans on the basis of some
biochemical test’s results such as germ tube (positive),
chlamydospore formation, color of colony, absorption of
carbohydrate and fermentation tests. The possible presence
of C. dubliniensis in the clinical isolate panel was considered
by removing the strains with dark green color colonies on
ChromAgar Candida Agar at 37 8C after 24 h and the absence
of growth at 42—45 8C.
Antifungal activity evaluation
By using the methods of CLSI (NCCLS) (disc diffusion and
micro-broth dilution assays), the sensitivity of C. albicans
clinical isolates to S. khuzistanica ethanol extract was
determined. The optical density of fungal suspension at
530 nm was 1.0 (1  106 CFU/mL). Candidal suspensions
were cultured on Sabouraud dextrose agar using a sterile
cotton swab. The stock solution for extract was prepared
in dimethyl sulfoxide (DMSO) (0.1024 g/mL) and then it

Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
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MYCMED-586; No. of Pages 5
The anti-candidal activity of Satureja khuzistanica ethanol extract
was filtered. Subsequently, filter discs (6 mm in diameter)
(Padtan Teb Co, Tehran, Iran) were saturated with 15 and
20 ml of diluted extract. Clotrimazole (10 mg/disc), fluco-
nazole (10 mg) and amphotericin B (10 U/disc) (Rosco,
Diagnostica A/S, Taastrupgaardsvej 30 DK-2630 Taastrup)
were used as controls. The plates were incubated at 37 8C
for 48 h. The inhibition zone diameters were measured by
dial caliper and were recorded in millimeter  Standard
Deviation (mm  SD) [10]. The minimum inhibitory
concentration (MIC) and minimum fungicidal concentra-
tion (MFC) values of ethanol extract and amphotericin
B were determined by micro-broth dilution assay. 100 ml
of two-fold serially diluted compounds (3.1—0.0025 mg/mL)
in MOPS-buffered RPMI 1640 were poured in 96-well micro-
titer plates. After shaking, 100 ml of diluted microbial
suspensions (105 CFU/mL) was added to each well and
incubated at 35  2 8C. MICs were defined as the lowest
concentration of extract that inhibited the fungal
growth after 48 h. MFC values were the first well
showing no growth on Sabouraud dextrose agar with 2%
dextrose [9].
Synergistic activity
After determining the MIC values for clinical isolates, the MIC
value for 90% of isolates (MIC90) were determined and the
sub-inhibitory concentration of extract (1/4 or 1/2 of MIC90)
was inserted to Sabouraud dextrose agar with 2% dextrose.
The control group was the Sabouraud dextrose agar with 2%
dextrose without extract. Plates were inoculated with the
microbial suspensions (1  106 CFU/mL) by sterile swabs and
the antibiotic discs (clotrimazole [10 mg/disc], fluconazole
[10 mg] and amphotericin B [10 U/disc]) were put on the
plates and were incubated at 35  2 8C for 48 h. Then, the
diameter of inhibition zone was measured by dial caliper and
reported [7].
Statistical analysis
The data were analyzed in Graph Pad prism and the diffe-
rences between the results and its significance were esti-
mated by Anova One-Way in GraphPad Prism. The significant
level was 0.05.
Results
The antifungal activity of S. khuzistanica ethanol
extract against clinical isolates of C. albicans
In disc diffusion method, the antifungal activity of
S. khuzistanica ethanol extract against clinical isolates
revealed that the mean of inhibition zone diameter (mm)
of extracts (1.5 mg/disc = 11.3  0.62 mm) was lower than
synthetic antifungal agents (20.85  1.36, 26.3  2.5,
24.1  2.6 mm were for amphotericin B, clotrimazole,
ketoconazole, respectively). The means of MIC and MFC
values were 299.4  54.5 and 722.6  53.44 (mg/mL) for
S. khuzistanica ethanol extract while these amounts were
0.53  0.19 and 0.98  0.36 (mg/mL) for amphotericin B,
respectively (Fig. 1).
Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
3
Figure 1 The antifungal activity of S. khuzistanica extract
against clinical isolates of C. albicans by disc diffusion assay.
L’activité antifongique de l’extrait de S. khuzistanica extraire
les isolats cliniques de C. albicans par dosage en diffusion par la
méthode des disques.
Synergistic or antagonistic effects of
S. khuzistanica ethanol extract with antifungal
drugs
Synergistic activity: S. khuzistanica ethanol extract
(75 mg/mL) with amphotericin B, clotrimazole and ketoco-
nazole against clinical isolates of C. albicans exhibited no
significant synergistic effects. At this concentration, the
inhibition zone diameters (mm) of amphotericin B, and
clotrimazole were increased but these increases were not
significant (additive effect). Therefore, we evaluated the
synergistic effects of amphotericin B, clotrimazole and keto-
conazole with 150 mg/mL of S. khuzistanica ethanol extract
(1/2 MIC90). The results of increasing in ethanol extract
showed that there was a synergistic effect (P < 0.05) bet-
ween S. khuzistanica ethanol extract with amphotericin B
and ketoconazole, while this extract had no effect on clo-
trimazole activity (Table 1; Fig. 2).
Discussion
Medicinal plants are new sources for finding the antimicro-
bial agents. The antifungal activity of S. khuzistanica metha-
nol extract was confirmed against one isolate of C. albicans
[4]. In this investigation, the methanol extract from wild and
cultivated plant was investigated. The yield of extraction by
methanol solvent was much lower (2.7—3.1%) than the etha-
nol extraction from our study (47%). The anti-candidal eva-
luation of S. khuzistanica methanol extract (concentration
2.7 mg/mL) against one isolate of C. albicans exhibited that
the inhibition zone diameters were 15 and 16 mm, while the
MIC values were 4 and 1 mg/mL for wild and cultivated
S. khuzistanica methanol extract. The amount of MIC value
from our study was close to the MIC value of wild type
S. khuzistanica methanol extract [4]. Therefore, the anti-
candidal activity of S. khuzistanica methanol extract against
one isolate of C. albicans was similar to the anti-candidal
activity of S. khuzistanica ethanol extract against clinical
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MYCMED-586; No. of Pages 5

4 M. Mahboubi, N. Kazempour

Table 1 The synergistic activity of S. khuzistanica ethanol had the acceptable anti-candidal activity against clinical
extract and antibiotics (mm). isolates of C. albicans.
L’activité synergique de S. khuzistanica extrait de l’éthanol Due to the pain and inflammation as a result of C. albicans
et des antibiotiques (mm). infections, the application of S. khuzistanica as a treatment
can help reducing the pain and inflammation in women’s
Means  SE Lower Upper
vagina. The antinociceptive, analgesic [11,22], anti-inflam-
AP-Extract 20.8  0.12 17.8 22.4 matory [15,25] and immune-stimulatory effects of
(75 mg/mL) S. khuzistanica were confirmed. S. khuzistanica inhibits
AP-Extract 22.2  0.18 18.7 28.5 the expression of iNos gene in macrophages [15] and the
(150 mg/mL) activation of spinal glial cell [11] and activation of the serum
AP-Control 21.1  0.19 9 25.1 lysosyme [16] and finally decreases the pain and inflamma-
Keto-Extract 27.1  0.35 21.5 33.7 tion and increases the immune system of patients. There-
(75 mg/mL) fore, C. albicans infected patients may benefit from
Keto-Extract 31.25  0.36 23.5 34.7 S. khuzistanica ethanol extract by its analgesic, anti-inflam-
(150 mg/mL) matory and anti-candidal effects.
Keto-Control 27.9  0.32 18.4 36.3 So, it can be an alternative source for treatment of
Clo-Extract 23.9  0.23 18.4 28.4 C. albicans related diseases. More pharmacological, toxico-
(75 mg/mL) logical and clinical studies are required to confirm the
Clo-Extract 25.2  0.32 20.6 28.8 efficacy of S. khuzistanica ethanol extract against
(150 mg/mL) C. albicans infections.
Clo-Control 24.7  0.3 18.5 31.4 The synergistic effect of plant extracts and antibiotics has
been the subject of many other studies [7,12,21]. The
AP: amphotericin B; Ket: ketoconazole; Clo: clotrimazole.
synergistic effects of itraconazole and fluconazole with
Echinophora platyloba ethanol extract were confirmed [7].
isolates of C. albicans from this study. Therefore, due to the Our results showed potent synergistic effects of
high yield of extraction for S. khuzistanica ethanol extract S. khuzistanica ethanol extract and amphotericin B and
rather than its methanol extract and the adverse effects of ketoconazole as treatment for C. albicans infections. If
methanol residue for application in human uses, it is more the application and safety of S. khuzistanica ethanol extract
economical to do further investigations on S. khuzistanica were confirmed in clinics, the advantage of these kinds of
ethanol extract as antifungal agent against C. albicans. The studies would be using of lower amount of harmful anti-
high sensitivity of C. albicans to other species of Satureja biotics or decreasing the time of treatment in patients that
sp is reported [24,26]. Therefore, S. khuzistanica like finally leads to decreasing the cost of treatment.
S. abyssinica spp. Abyssinica, S. paradoxa and S. hortensis
Conclusion

This study introduces S. khuzistanica ethanol extract as a

new antifungal candidate against clinical isolates of
C. albicans. Furthermore, S. khuzistanica ethanol extract
had synergistic effects with amphotericin B and ketocona-
zole.

Disclosure of interest

The authors declare that they have no competing interest.

Acknowledgements

This study is supported by Medicinal Plants, Research Center

of Barij, Kashan, Iran (Grant No.: 9323).

References

[1] Abbasi A, Bahador A, Esmaeili D, Mahbubi A, Amiri M, Amiri M.

Figure 2 The synergistic evaluation of S. khuzistanica ethanol
extract (74.9 and 149 mg/mL) and antibiotics.
L’évaluation synergique de l’extrait éthanolique de
S. khuzistanica (74,9 et 149 mg/mL) et des antibiotiques.
The study of inhibitory effects of Satureja khuzistanica against
MDR isolates of Pseudomonas aeruginosa. Int J Curr Microbiol
App Sci 2014;3:614—8.
[2] Achkar JM, Fries BC. Candida infections of the genitourinary
tract. Clin Microbiol Rev 2010;23:253—73.
[3] Akbari-Shahabi S, Assmar M, Massih A, Ghaemi N, Issazadeh K,
Shokri-Fashtali S. Evaluation of antibacterial activity of Satu-
reja khuzistanica essential oil against standard and isolated

Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
+ Models
MYCMED-586; No. of Pages 5
The anti-candidal activity of Satureja khuzistanica ethanol extract
strains of Listeria monocytogenes. Zahedan J Res Med Sci
2014;16:38—41.
[4] Amanlou M, Fazeli MR, Arvin A, Amin HG, Farsam H. Antimicro-
bial activity of crude methanolic extract of Satureja khuzista-
nica. Fitoterapia 2004;75:768—70.
[5] Amin GR, Ministry of Health Publications. Popular Medicinal
Plants of Iran, Vol. 1. Tehran; 1991.
[6] Amirrajab N, Zarrin M, Sadeghi-Nejad B. In vitro antifungal
activity of Satureja khuzistanica Jamzad against Cryptococcus
neoformans. Pakistan J Med Sci 2010;26:880—2.
[7] Avijgan M, Mahboubi M, Moheb Nasab M, Ahmadi Nia E, Yousefi
H. Synergistic activity between Echinophora platyloba DC etha-
nolic extract and azole drugs against clinical isolates of Candida
albicans from women suffering chronic recurrent vaginitis. J
Mycol Med 2014;24:112—6.
[8] Biggs WS, Williams RM. Common gynecologic infections. Prim
Care 2009;36:33—51.
[9] CLSI. Reference method for broth dilution antifungal suscepti-
bility testing of filamentous fungi; approved standard CLSI
document M38-A2. Wayne: Clinical and Laboratory Standards
Institute; 2008.
[10] CLSI. Method for antifungal disk diffusion susceptibility test-
ing of filamentous fungi; proposed guideline. CLSI document
M51-P. Wayne: Clinical and Laboratory Standards Institute;
2009 .
[11] Esmaeili-Mahani S, Ebrahimi B, Abbasnejad M, Rasoulian B,
Sheibani V. Satureja khuzestanica prevents the development
of morphine analgesic tolerance through suppression of spinal
glial cell activation in rats. J Nat Med 2015;69:165—70. http://
dx.doi.org/10.1007/s11418-013-0796-6.
[12] Garcia-Gomes AS, Curvelo JA, Soares RM, Ferreira-Pereira A.
Curcumin acts synergistically with fluconazole to sensitize a
clinical isolate of Candida albicans showing a MDR phenotype.
Med Mycol 2012;50:26—32.
[13] Goun E, Cunningham G, Chu D, Nguyen C, Miles D. Antibacterial
and antifungal activity of Indonesian ethnomedical plants.
Fitoterapia 2003;74:592—6.
[14] Hajjeh RA, Sofair AN, Harrison LH, Lyon GM, Arthington-Skaggs
BA, Mirza SA, et al. Incidence of bloodstream infections due to
Candida species and in vitro susceptibilities of isolates collect-
ed from 1998 to 2000 in a population-based active surveillance
program. J Clin Microbiol 2004;42:1519—27.
[15] Jalalvand M, Shahsavari R, Sheikhian A, Mosayebi G. Satureja
khozestanica essential oil (SKEO) inhibits iNOS gene expression
Please cite this article in press as: Mahboubi M, Kazempour N. The anti-candidal activity of Satureja khuzistanica ethanol extract against
clinical isolates of C. albicans. Journal De Mycologie Médicale (2016), http://dx.doi.org/10.1016/j.mycmed.2015.11.003
5
in Lipopolysaccharide stimulated J774A.1 macrophage cell line.
Life Sci J 2013;10:230—5.
[16] Khosravi A, Yavari V, Alishahi M, Moosavi SM, Ghorbanpoor M,
Darvish Bastami K, et al. Effects of Oliviera decumbens and
Satureja khuzestanica hemological extract on some immuno-
logical and haematological parameters of Cyprinus carpio.
Camp Clin Pathol 2012;22:339—42.
[17] Mishra NN, Prasad T, Sharma N, Payasi A, Prasad R, Gupta DK,
et al. Pathogenicity and drug resistance in Candida albicans and
other yeast species. A review. Acta Microbiol Immunol Hung
2007;54:201—35.
[18] Mora-Duarte J, Betts R, Rotstein C, Colombo AL, Thompson-
Moya L, Smietana J, et al. Comparison of caspofungin and
amphotericin B for invasive Candidiasis. N Engl J Med 2002;
347:2020—9.
[19] Nafisy AT. A review of traditional medicine in Iran. Isfahan:
Isfahan University Publications; 1989.
[20] Navarro Garc½´a VM, Gonzalez A, Fuentes M, Aviles M, Rios MY,
Zepeda G, et al. Antifungal activities of nine traditional Mexi-
can medicinal plants. J Ethnopharmacol 2003;87:85—8.
[21] Olajuyigbe OO, Afolayan AJ. Synergistic interactions of metha-
nolic extract of Acacia mearnsii De Wild. with antibiotics
against bacteria of clinical relevance. Int J Mol Sci 2012;13:
8915—32.
[22] Saberi A, Sepehrib G, Esmaeili-Mahani S, Rasouliani V, Esmaeil-
pour K, Hajralizadeh Z, et al. Satureja khuzestanica extract
elicits antinociceptive activity in several model of pain in rats. J
Appl Sci 2013;13:729—35.
[23] Sadeghi-Nejad B, Shiravi F, Ghanbari S, Alinejadi M, Zarrin M.
Antifungal activity of Satureja khuzistanica (Jamzad) leaves
extracts. Jundishapur J Microbiol 2010;3:36—40.
[24] Sahin F, Karaman I, Gulluce M, Ogutcu H, Sengul M, Adiguzel A,
et al. Evaluation of antimicrobial activities of Satureja horten-
sis L. J Ethnopharmacol 2003;87:61—5.
[25] Tavafi M, Ahmadvand H, Tamjidipoor A, Delfan B, Khalatbari
AR. Satureja khozestanica essential oil ameliorates progression
of diabetic nephropathy in uninephrectomized diabetic rats.
Tissue Cell 2011;43:45—51.
[26] Tolossa K, Asres K, El-Fiky FK, Singab ANB, Bucar F. Composition
of the essential oils of Satureja abyssinica spp. abyssinica and
Satureja paradoxa: their antimicrobial and radical scavenging
activities. J Essent Oil Res 2007;19:295—300.
[27] Zargari A. Medicinal plants, Vol. 4. Tehran, Iran: Tehran Uni-
versity Press; 1990.