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Antioxidant and antibacterial effects of the essential oil and various extract of
Dionysia revolute Boiss.
Running head: Chemical composition and biological activity of essential oil and various
extracts of Dionysia revolute Boiss.
Abbass Mohebi –Poorkani1, Iman Mahdavi2, Fariba Sharififar3*, Hamid R. Shaterian1, Nasrin
Kazempour1, Alireza Nowroozi1, Nasim Hassanabadi2,Neda mohamadi4
RESEARCH ARTICLE
Word count: 2096
1
Department of Biology, University of Sistan-Balouchestan, Sistan-Balouchestan, Iran
Faculty of Veterinary, Bahonar University, Kerman, Iran
3
Herbal and Traditional Medicines Research Center, Faculty of Pharmacy, Kerman University of Medical Sciences,
Kerman, Iran
4 Ph.D student, Pharmaceutical Research Center, Institute of Neuropharmacology, Kerman University of Medical
Sciences, Kerman, Iran
2
*
Corresoponding author:Tel: 00983413205020; Fax: 00983413205017; fsharififar@kmu.ac.ir;
sharififar@yahoo.com
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Abstract
Objective: Dyonisia revoluta Boiss. is used in Iranian traditional medicine in gastric disordres
and for wound healing. There are no studies for this plant in the literature. We aimed to study the
chemical composition of the essential oil of the plant and assess the antioxidant and antibacterial
effects of its essential oil and different extracts of this plant. Materials and methods: Essential oil
was provided by steam distillation and analyzed with gas/chromatography and mass
spectrometry. Essential oil, and various extracts were evaluated for antioxidant effect using
diphenylpicrylhydrazil (DPPH) assay. Disk diffusion was used for antibacterial studies. Results
and conclusion: GC/MS analysis indicated the presence of acetophenone (29.94%0),
acetylphenol (20.23%), and benzaldehyde (14.26%) as main compounds of essential oil.
Ethanolic extract showed the highest inhibition DPPH radical with IC50 = 98.8µg/ml in
comparison to BHT (IC50 = 47.19µg/ml). Ethanolic and ethylacetate extracts showed inhibitory
effect against the major of tested bacteria except E. aerogenes and C. freundii. Our results
indicated that the ethanolic extract of this plant would be a good candidate for further studies.
The essential oil of this plant also exhibited notable antioxidant which needs to be studied using
the other methods to have valid results.
Key words: Dionysia revoluta; Essential oil; Extract; DPPH; Disk diffusion
1. Introduction
The role of oxidant agents has been known in pathogenesis of many disease such as diabetes,
cancer, cardiovascular disease, (1). Essential oils are aromatic secondary metabolites of
medicinal plants which as well as flavonoids and phenolics show different activities such as
antimicrobial, antioxidant, anticonvulsant, anti inflammatory, diuretic...(2-6). The essential oils
as well as phenols and flavonoids can act as antioxidants and antibacterial and have an important
role in to prevention of different disorders which are associated with oxidative factors or
bacterial agents (4, 7). Considering the problems with synthetic antioxidant and also the side
effects and tolerance to common antibacterial drugs, there are many interests in finding new
therapeutic agents especially from natural sources.
The genus of Dionysia belongs to Primulaceae family and comprises more than 27
perennial specific species in Iran which restrict distributed in dry mountains. Some of these
species also have been found in Iraq, Anatolia, Pakistan, Tadzikestan and Afghanistan (8). The
plant of Dionysia revulata (Boiss.) subspecies revoluta is one of these endemic plants which has
widely distribution in Iran. This plant is used in gastric disturbance and disinfection of
wounds(9) . Previous studies have revealed the antibacterial effect of this plant against of
Staphylococcos aureus in comparison to Erythromycin and Cephalexin (9). There are no study
for chemical composition and antioxidant and antibacterial effects of this plant in the literature.
In continuing the study of antioxidant and antibacterial effects of the extracts and essential oil of
medicinal plants, we have studied this plant essential oil and different extracts for evaluation of
these biological activity.
2. Materials and methods
2.1. Plant materials
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The plant was gathered from the mountains of Sarikhani in Buanat in Fars Province at april,
2011. The plant was dried in shade and was confirmed by Dr. Mirtadzadini, Bahonar University.
A voucher specimen was depoisted in Herbarium Center of Bahonar University.
2.2. Isolation of essential oil
The amount of 200g of dried aerial parts of the plant was subjected to hydrodistillation for 3 h.
using cleavenger apparatus to yeild 0.75% essential oil.
2.3. Plant extraction
The ethanolic, aqueous and ethyl acetate extracts of the plant were provided by succhelet
methods for 4h. and concentarted under vacuume to drying. The extracts and essential oil were
stored at -20◦C until experiment.
2.4. Gas Chromatography (GC) of essential oil
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The analysis of the essential oil was performed on an gas chromatograph Agilent HP-6890
(Agilent Technologies, Palo Alto, CA, USA) with a HP-1MS 5% phenylmethylsiloxane capillary
column (30 m × 0.25 mm, 0.25 μm film thickness; Restek, Bellefonte, PA) equipped with an
Agilent HP-5973 mass selective detector in the electron impact mode (Ionization energy: 70 eV).
The temperature of injector and detector was set at 250 ◦C and 230 ◦C respectively. the
temperature of oven was set at 40◦C at first for 1 min, and raised to 250 ◦C at 3◦C/min. Hellium
was used as carrier (flow rate1ml/min. Diluted samples (1/100 in acetone, v / v ) of 1,0 l were
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injected manually in the splitless mode.
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2.5. Gas chromatography/mass spectrometry (GC/MS)
Essential oils analysis was done with the same GC conditions (oven temperature, column, flow
rate ) using an Agilent-HP 6890 gas chromatograph equipped with an Agilent 5973 mass
selective detector in the electron impact mode (70 ev). Injector and MS transfer line temperatures
were set at 250 ◦C and 230 ◦C, respectively.
2.6. Qualitative and quantitative analysis
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For identifying the composition of the oil, relative retention time and mass spectra of each
component was compared to standards. confirmation of compounds was performed by
comparison of their mass spectra and retention indices relative to n-alkanes of with data
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in the NISt and Wiley library (10).
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2.7.DPPH free radical scavenging assay
Antioxidant potential of the essential oil and extracts of the plant was evaluated using DPPH
scavenging assay. 50µl of different concentrations of each sample was added to 5 ml of
methanolic solution of 0.004% DPPHand incubated at room temperature for 30min., absorbance
was read at 517nm(11) . Butylated hydroxytoluene (BHT) was used as reference drug.The
percent of inhibition was calculated as follow:
I%= (Acon-Asam)/Asam × 100, where Acon and Asam are the absorbance of control and sample
respectively.
2.8. Antibacterial effect using disk diffusion method
Antibacterial effect of the essential oil and extracts was studied by disk diffusion method against
seven Gram-positive and gram-negative strains of Staphyloccocus aureus, Ataphyococcus
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epidermidis, Pseudomons aeroginosa, Proteus mirabilis, Entrobacter aerogenes, Acinetobacter
boumannii and Citrobacter freundii. The plant extracts were diluted in alcoholic solvents and
prepared in concentration, of which 20 μl was droped on disk of 6 mm in diameter and the
concentrations to give 100µg/disk and 10µl/disk of plant extracts and essential oil respectively.
Turbidities of tested bacterial strains were prepared to 0.5 McFarland and inocula were spread on
Mueller Hinton Agar (MHA) and incubated at 37 Cº for 18-24 hours (12). Inhibition zone were
measured using vernier caliper. The tests were undertaken triple and the results were reported as
mean ± SD.
3. Results and discussion
3.1. GC/MS analysis of essential oil
The essential oil obtained from D. revoluta was pale yellow in with distinct, sharp odor. Forthy
–eight compounds were identified, representing 94.51% of the total oil components (Table 1). As
shown, the major components of the oil were found to be acetophenone (29.94%0), acetylphenol
(20.23%), benzaldehyde (14.26%). Phenyl propanoids and benzene derivatives comprise the
highest percentage of the total oil (66.21%). Oxygenated sesquiterpenes constitute about 12.00%
of the oil. Our results showed differences in composition with respect to data in the literature,
such as those reported for the essential oil derived from the D. diapensifolia. It is for the first
time that the composition of the essential oil of D. revoluta has been reported.
3.2. Antioxidant activity
The results of antioxidant effect show that ethanolic extract and essential oil of D. rivoluta can
potentially reduce the stable DPPH radical to yellow color. The highest potential of DPPH
radical scavenging was due to the ethanolic extract with IC50 value of 98.8 µg/ml in comparison
to BHT (IC50= 47.19). The essential oil, aqueous and ethyl acetate extract exhibited inhibition
with IC50 values of 0.77µl/ml, 126.0 and 264.8µg/ml respectively in a dose dependent manner.
The highest percentage of DPPH inhibition percentage of ethanolic extract was 81.6% (200
μg/ml).The lower antioxidant activity of fractions like essential oil or ethyl acetae extract might
be due to the absence and/or lower amount of the donor groups such as phenolics or terpenes in
these fractions. Monoterpenes with methylene group exhibit potent antioxidant effect which
attributed to the presence of methylene groups in monoterpene hydrocarbons (13).
3.3. Results of antibacterial effect
The result of antibacterial activity of the essential oil and different extracts of D. rivoluta have
been shown in Table 2. The ethanol extract showed antibacterial activity against several of
Gram-positive and Gram-negative strains except E.aerogenes and C.freundii. Amongst the
tested strains, A. boumannii and and S. aureus showed most sensitivity to ethanolic extrac (
inhibition zone diameter 11 and 12 respectively). The ethyl acetae extract exhibited weaker
effect than that provided by ethanolic extract against S. aureus, A. baumannii, P. earoginosa and
P. mirabilis. This fraction and the essential oil have more prominent inhibitory effect against C.
freundii. and A. boumannii The other strains showed resistance to the essential oil. All the other
strains were resistant to essential oil except E. aerogenes and C.freundii bacteria. No
antibacterial effect was shown by aqueous extract.
4. Conclusion
It is for the first report for the antioxidant and antibacterial effects of the genus Dionysia. And it
is the second study for the chemical composition of this genus. Our results indicated that the
ethanolic extract of this plant would be a good candidate for further studies. The essential oil of
this plant also exhibited notable antioxidant which needs to be studied using the other methods to
have valid results.
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5. Acknowledgement
Authors are so grateful from Dr. Mirtadjaldini for his help for the identification of the plant.
References
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Losso JN, Shahidi F, Bagchi D. Anti-angiogenic functional and medicinal foods
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Bakkali F, Averbeck S, Averbeck D, Idaomar M. Biological effects of essential oils- A review. Food
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Sharififar F, Mirtajaldini M, Azampour M, Zamani E. Essential oil and methanolic extract of
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Sharififar F, Yassa N, Mozaffarian V. Bioactivity of major components from the seeds of Bunium
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Sharififar F, Yassa N, Shafiee A, . Antioxidant activity of Otostegia persica ( Labiateae) and its
constituents. Iranian Journal of Pharmaceutical Research. 2003:235-9.
8.
Mozaffarian V. A dictionary of Iranian plant names. Tehran: Farhang Mo’asern PubL; 1998.
9.
Saadatmand S, Alaediny A, Salehy M. Antibacterial effects of dionysia revoluta essence on
staphylococcos aureus and determination of biochemical component by GC-MASS method. The
Quarterly Journal of Biological Sciences Spring 2009; 1(2):57-63. 2009;1:57-63.
10.
Adams R. Identification of essential oil components by gas chromatography/mass spectroscopy.
illinoise: Allured Publishing Co.; 2001.
11.
Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000;14:3238.
12.
Bradshaw LJ. Laboratory Microbiology. New York: Saunders College Publishing; 1992.
13.
Tepe B, Daferera D, Sokmen A, Sokmen M, Polissiou M. Antimicrobial and antioxidant activities
of the essential oil and various extracts of Salvia tomentosa Miller (Lamiaceae)
Food Chemistry. 2005;90:333-40.
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Table 1. The results of GC/MS analysis of the essential oil of D. revoluta
No
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12
13
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15
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28
29
30
31
32
33
34
35
36
37
38
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42
Retention
(Rt)
7.592
7.837
9.397
10.35
11.44
11.68
11.84
12.21
13.34
13.58
15.50
15.83
16.06
16.47
16.79
16.94
17.34
19.83
20.18
20.63
20.94
21.39
24.05
26.03
28.42
30.17
33.22
34.96
35.27
38.22
38.87
39.60
39.82
39.98
40.12
43.18
43.69
44.62
45.16
46.31
47.23
time Name
Percentage
Styrene
Heptanal
3,5-Xylenol
Benzaldehyde
6-Methyl-5-hepten-2-one
2,4-Heptadienal
2,3-Dehydro-1,8-cineole
3-Ethyl-1,4-hexadiene
Ο-Cymene
Salicylaldehyde
Acetophenone
Cis-Linalool oxide
1-Octanol
τ-Linalool oxide
6-Methyl-3,5-heptadiene-2-one
Nonanal
Linalool
2-Acetylphenol
Borneol
4-Terpineol
Methyl salicylate
β-Farencyl alcohol
2-Decenal
2,4-Decadienal
Eugenol
Geosmin
1,3-cyclopentadiene, 5,5-dimethyl-2-propyl
Dihydro-β-agarofuran
2H-benzocycloheptene-2-one,3,4,
4a,5,6,7,8,9octahydro-4a-methyl
Rosifoliol
Saelin 4,7(11)-diene
γ-Eudesmol
7-Epi-α-selinene
α-Eudesmol
β-Eudesmol
1H-cycloprop[e]azulene,1a,2,3,4,4a,5,6,7b
Fonenol
2-Propen-1-one,1,3-diphenyl
Paradisiol
3-phenylpropiophenone
Hexahydrofarensylalcohol
6
0.12
0.11
0.13
14.20
0.14
0.05
0.12
0.09
0.09
0.57
29.94
0.1
0.13
0.16
0.06
0.09
1.45
20.23
0.2
0.12
0.14
2.29
0.08
0.18
2.9
0.11
0.51
0.12
0.28
3.69
0.36
3.99
0.71
1.01
0.79
0.68
0.93
0.46
1.71
0.76
0.79
43
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45
46
47
47.66
49.49
53.35
54.93
55.78
Globulol
β-Phenyl-2-hydroxypropiophenone
Dibenzoylmethane
ο-Hydroxychalcone
Phytol
Total
Phenyl propanoids
Hydrocarbon monoterpenes
Oxygenated monoterpenes
Hydrocarbon sesquiterpenes
Oxygenated sesquiterpenes
0.23
0.54
0.65
2.11
0.39
94.51
66.21
.59
7.5
1.4
12
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Table 2. The results of Antimicrobial effect of the essential oil and different extracts of D.
revoluta by disk diffusion method (diameter of zone of inhibition in mm, mean value of three
independent experiments)
sample
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Microorganism
S.
S.
aureu epidermid
s
is
NI
NI
Essential
oil
Ethanol
11±1. 10± 0.6
extract
1
Ethylaceta 7±
NI
te extract
0.4
Aqueous
NI
NI
extract
NI: no inhibition
E.
aerogen
es
11± 0.7
C.
freund
ii
7± 0.3
P.
aeroginosa
P. mirabilis
NI
NI
NI
NI
NI
10± 0.9
9± 0.9
12± 0.6
7± 0.4
11±
0.6
8± 0.6
8± 0.3
8± 0.5
8± 0.4
NI
NI
NI
NI
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8
A. bauma
nnii
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Figure 1- The results of antioxidant evaluation of various extracts of D. revolute Boiss. using
DPPH scavenging assay in comparison to BHT
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Figure 2- The results of antioxidant evaluation of different concentration of essential oil of D.
revoluta using DPPH scavenging assay
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