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HAIDA et al.
Research Article
Laboratory of Biology and Health, Applied Microbiology Team, Faculty of Sciences, Ibn
Tofail University, Kenitra Morocco.
3
Article Received on
05 May 2015,
Revised on 30 May 2015,
Accepted on 24 June 2015
*Correspondence for
Author
Sara HAIDA
Laboratory of Separation
Processes, Team of
Chemistry, Faculty of
University, Kenitra
Morocco.
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HAIDA et al.
Staphylococcus aureus and Pseudomonas aeruginosa). The results show that only apolar
extracts responded positively to two of three bacterial strains tested.
KEYWORDS: Rosmarinus officinalis L., GC-MS, phenolic compounds, flavonoids,
antioxidant activity, antibacterial activity.
1. INTRODUCTION
Plants have been used by Humans since antiquity to handle common infectious diseases.
Some of these traditional treatments are always included as part of the usual treatment of
various diseases.[1] These plants are an important reservoir of potential compounds, which
have the advantage to be a big diversity of chemical structures possessing a very wide range
of biological activities.[2]
Currently, the development of bacterial resistance in antibiotics and toxicity of synthetic
antioxidants led the researchers to tap into the world of plants particularly in medicinal and
aromatic plants, for their antioxidant and antibacterial properties; these plants are used to
protect the human body against oxidation reactions and cellular pathogens.[3]
The biological properties are mainly due to polyphenols, which are secondary metabolites
widely spread in plants and known for their beneficial health effects.[4]
Among the inventory of medicinal plants from Morocco, rosemary (Rosmarinus officinalis
L.) is a shrub of the Lamiaceae family, which is widely used in traditional medicine for its
biological properties.[5] The aim of our work is to study the chemical composition of the
essential oil of rosemary in western Morocco, to demonstrate the wealth of this plant into
polyphenols and evaluate the antioxidant and antibacterial activity of its extracts.
2. MATERIALS AND METHODS
2.1. Plant material
The harvesting of rosemary samples is conducted in full bloom in March in the Gharb region
(western Morocco). The aerial parts (leaves, flowers, twigs) are collected in the early
morning.
2.1. Extraction of essential oil
Essential oil of Rosmarinus officinalis L. was extracted by hydrodistillation using a
Clevenger apparatus type. A flask of 2 L is charged with 250 g of freshly harvested plant
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material and distilled water (about 2/3 of the flask) and a few grains of carborundum, the
whole is boiled for 5 hours. After extraction, the obtained condensate is constituted by the
essential oil, immiscible with water of yellow color, and the hydrosol of white color loaded
by soluble compounds (or partially soluble) in water which can be concentrated by
performing a salting out.
2.2. GC-MS analysis
The essential oil was analyzed by gas chromatography coupled to mass spectrometry. Perkin
Elmer Trademark was apparatus used in this analysis; it consisted of chromatograph Clarus
680 type coupled to a mass spectrometer Clarus SQ type 8C. The capillary column used is a
Rxi-5ms 30m long, 0.25mm diameter and 0.25m thick. The oven temperature was
programmed at 60 C for 5 minutes and gradually rose up to 300 C at 2 C/min for the
remaining 10 minutes. The carrier gas used was helium with a flow rate of 1 mL/min. The
sample was injected with a volume of 1L.
Mass spectra of different compounds identified are recognized and affirmed by using the
database of mass spectra of pure products (NIST: National Institute of Standards and
Technology).[6] Retention indices of the various compounds were compared by referring to
the literature.[7]
2.3. Extraction of phenolic compounds
The extraction protocols are described and detailed in figure 1. Two types of extraction were
employed to isolate polar products: the extraction by maceration (at room temperature) and
the extraction by decoction (reflux). In each method the dry plant material, finely crushed, is
extracted with methanol, the residue obtained after filtration and drying is then extracted with
distilled water.
The apolar compounds are obtained by Soxhlet extraction with petroleum ether. At the end of
these protocols are obtained:
- MEM, AEM: Methanol and aqueous extracts obtained by maceration.
- MED, AED: Methanol and aqueous extracts obtained by decoction.
- EEM, EED: Extracts obtained by Soxhlet extraction with petroleum ether.
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(5 h)
EEM
EED
Methanol
Extraction
Petroleum ether
Extraction
Residue
(24 h)
(Agitation)
MEM
MED
(5 h)
Water
Extraction
Residue
AEM
AED
Extraction by maceration
Extraction by decoction
Extraction by Soxhlet
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(1,1-diphenyl-2-picrylhydrazyl),
[12]
modifications.
described
by
Sanchez-Moreno
with
some
of the mothers solutions for various extracts and ascorbic acid (0 - 0.15 mg/mL) used as
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Where IC50 is concentration required to reduces 50% of DPPH, a is the slope of straight and
b is the intercept of the straight.
2.8. Preliminary antibacterial activity
The aim of this part is to assess the antibacterial activity of the extracts of rosemary. To
highlight this activity, we used diffusion method using sterile disks (or aromatogram). This
method relies on the diffusion of substances to be studied within a Petri dish, in a solid
nutrient medium.Three bacterial strains were used:1) - Staphylococcus aureus (Gram
positive) isolated in the laboratory. 2) - Pseudomonas aeruginosa MC1 (Gram-negative),
isolated from the water table M'nasra Kenitra (Morocco) by direct seeding on cetrimide agar
medium.[14] 3) - Escherichia coli (Gram negative), isolated from the discharges of abattoirs
and domestic waste water of Kenitra (Morocco).[15]
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Briefly, inoculum is prepared by inoculating each culture streaked on nutrient agar medium to
obtain isolated colonies. After incubation for 18 hours at 37C, 4 to 5 colonies are isolated
with a loop and transferred into a tube of broth Muller Hinton. Then, 1 mL from 108 CFU/mL
bacterial suspensions is spread on the Muller Hinton Agar plates. After 15 min, the excess is
removed until the agar dries. Filter paper discs (6 mm in diameter) are impregnated with 5 L
of the diluted extracts and are placed on the inoculated plates, these plates are incubated at
37C for 24 hours. To compare the results, seven antibiotics (Norflaxacin 5 g, Erythromycin
15 UI, Fusidic acid 10 g, Tetracycline 30 UI, Sodium Cefotaxime 30 g, Amoxicilline 25
g and Gentamicine 10 g) are tested on three bacterial strains. The diameters of the
inhibition zones are measured in millimeters.
3. RESULTS AND DISCUSSION
3.1. Quantitative results extractions
The obtained essential oil yield is of the order of 0.55%. This value, although small, is within
the AFNOR standards (0.5-2%).[16] The variation in quantity of volatile compounds which
may be driven by steam are influenced by various factors such as the flowering period, the
soil, the climate.[17] and the extraction method. [18] The yield of hydrosol (0.30%) is less than
that of essential oil. It is constituted of soluble compounds or partially soluble in water
isolated by salting out. There is thus obtained 0.85% of volatile products.
The results of extraction by maceration show that extractable polar extracts with methanol
and water (MEM, AEM) represent 72% of the products extracted against only 28% of apolar
compounds extracted with petroleum ether (EEM).
Compared with the results obtained by maceration, and from the same starting amount, the
polar compounds obtained by decoction (MED, AED) represent 74% of the products extracts
against 26% of apolar compounds (EED).
3.2. Analysis of essential oil by GC-MS coupling
Figure 2 and 3 show the chromatograms of the essential oil and the corresponding hydrosol.
The chemical composition of the rosemary essential oil and hydrosol is shown in Table 1.
Through analysis by GC-MS coupling, we identified in the essential oil 28 compounds
representing 96.24% in total.
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NL:
1,22E10
TIC F: MS
pesticideso
c_29_mai1
4_1
95
90
85
80
75
70
65
8,10
60
55
50
45
19,08
40
9,29 12,08
23,51
35
30
28,07
36,23
25
38,23
13,68
20
16,30
15
41,65
10
5
45,59
51,29
33,45
7,08
55,41
56,34
62,31
69,04
0
5
10
15
20
25
30
35
40
Time (min)
45
50
55
60
65
70
100
NL:
1,99E9
TIC F: MS
pesticideso
c_29_mai1
4_4
95
90
85
80
75
70
65
60
55
50
45
7,43
40
18,88
35
30
25
36,14
20
7,58
15
38,17
20,30
10
8,04
28,00
12,15
16,41
13,81
5
4,88
24,15
28,28 33,54
41,65
45,58
49,80 50,12
55,80
0
5
10
15
20
25
30
35
Time (min)
40
45
50
55
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RI**
936
950
978
1013
1015
1025
1051
1082
1086
1110
1123
1137
1150
1164
1176
1183
1235
1270
1318
1376
1421
1455
1479
1484
1496
1503
1520
1578
The differents classes of chemical compounds, identified in rosemary essential oil, are
classified according to their proportions in Table 2. These compounds generally have a
monoterpene skeleton, which may be connected to an alcohol, ketone, or ester.
Table 2: Chemical classes of compounds identified in the essential oil
Chemical classes
Compounds
-pinene, camphene, -pinene,
-terpinene, Monoterpenes
terpinene, p-cymene, limonene, terpinolene.
Chrysanthenone, camphre, pinocarvone, verbenone and
Monoterpene ketones
piperitenone.
Monoterpenols
Linalol, borneol, terpinen-4-ol, -terpineol et geraniol
-caryophyllene, -humulene, -bisabolene,
Sesquiterpenes
-ylangene, Germacrene D, -curcumene,
-muurolene, -cadinene and caryophyllene oxide.
Monoterpene esters
Bornyl Acetate.
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%
53,35
20.60
10.63
8.30
3.36
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HAIDA et al.
Flavonoids
+ (Red)
+ (Red)
-
Tannins
+ (Blue)
+ (Blue)
+ (Green)
+ (Green)
Saponosids
+++
+++
From these results, we can conclude that flavonoids and gallic tannins are present in the
methanol extracts (MEM, MED) and catechin tannins are present in aqueous extracts (AEM
AED), contrariwise saponins exist mainly in MEM and AED extracts.
3.4. Phenolic compounds content
Due to the regression equation (y = 6.631x + 0.036, R2 = 0.997) of the calibration straight
(Figure 4) which is drawn from a series of increasing concentration solutions of gallic acid,
we calculated the content of total polyphenols in each extract. This value is expressed in
milligrams of gallic acid equivalents per gram of dry matter (mg GAE/g DM).
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The value of the flavonoids content is expressed in milligrams of quercetin equivalents per
gram of dry matter (mg QE/g DM).
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Flavonoids content
(mg QE/g DM)
2.186 0.179
2.643 0.116
-
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Equation
501.1x + 1.627
132.4x + 1.181
112.5x 1.949
354.5x + 1.146
26.91x + 0.888
0.443x + 0.971
R2 values
0.991
0.988
0.994
0.994
0.991
0.998
IC50 (mg/mL)
0.097
0.364
0.462
0.138
1.825
111
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HAIDA et al.
It has been shown that antioxidant molecules such as ascorbic acid, flavonoids and tannins
reduce and discolored DPPH due to their ability to give hydrogen.[20] Therefore, the
polyphenols in the extracts of rosemary are probably responsible for the antioxidant activity
of these extracts. However, it seems that the high antioxidant activity of the aqueous extract
AED is probably due to the significant presence of saponins in this extract, which have an
antioxidant effect.[21]
Further, some research has shown that the antioxidant activity of rosemary extracts in
particular is produced by phenolic diterpenes which are essentially rosmarinic acid, carnosol,
rosmanol, and carnosic acid.[22]
3.5. Antibacterial activity of the extracts of Rosemary
The preliminary test results of the antibacterial activity of different extracts of rosemary and
antibiotics are shown in Tables 6 and 7.
Table 6: Results of testing antibacterial activity of different extracts of rosemary
Inhibition diameter (mm)
Extracts
Disc load
E. coli
S. aureus
P. aeruginosa
5L
Hydrosol
9
15.5
5L
Essential oil
9
9
5L
MED
5L
MEM
5L
AED
5L
AEM
5L
EED
15.5
5L
EEM
20
(E. coli : Escherichia coli, S. aureus : Staphylococcus aureus, P.aeruginosa : Pseudomonas
aeruginosa), (- : no inhibition zone).
Table 7: Antibacterial activity of antibiotics studied.
Antibiotiques
Disc
load
Norfloxacin
5g
Erythromycin
15UI
Fusidic acid
10g
Tetracyclin
30UI
Sodium
30g
cefotaxime
Amoxycillin
25g
Gentamicin
10g
24
(- : no inhibition zone).
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Acceptance limits
sensitive
resistant
Critical
concentrations
(g/mL)
19
15
11
23
<19
<15
<11
<23
5
4
20
2
15
<15
10
16
18
15
<18
<15
8
6
S.
aureus
26.5
32
-
P.
aeruginosa
-
8
8
8
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HAIDA et al.
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Also, assay results showed that the highest concentrations of total polyphenols (3.094 0.069
mg GAE/g DM) and flavonoids (2.643 0.116mg QE/g DM) are recognized in the decoction
of methanol extract. Therefore, the decoction seems to be the best technique for the extraction
of total polyphenols and flavonoids.
The DPPH test results showed that the aqueous extract obtained by decoction (AED) is most
active with IC50 = 0.138 mg/mL, this activity is not negligible compared to that of ascorbic
acid (IC50 = 0.097 mg/mL) which is a good antioxidant.
The results of the antibacterial testing of the extracts compared to those of commercial
antibiotics used in parallel, have shown that only apolar extracts, obtained either by
hydrodistillation or by extraction with petroleum ether, responded positively on two of the
three strains used. The essential oil and hydrosol demonstrated antibacterial activity, against
Escherichia coli, of the order of 37.5% compared to Gentamicin. The EEM extracts, EED,
essential oil and hydrosol proved active against Staphylococcus aureus, but the highest
antibacterial power is obtained with the EEM extract, with 62.5% inhibition compared to the
Fusidic acid.
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