STUDY OF CHEMICAL COMPOSITION OF ROSEMARY ESSENTIAL OIL FROM WESTERN MOROCCO AND EVALUATION OF ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY OF ITS EXTRACTS

STUDY OF CHEMICAL COMPOSITION OF ROSEMARY ESSENTIAL

OIL FROM WESTERN MOROCCO AND EVALUATION OF

ANTIOXIDANT AND ANTIBACTERIAL ACTIVITY OF ITS

EXTRACTS

Sara HAIDA1*, Fatima Zahra ESSADIK1, Abdelaziz KRIBII1, Amar HABSAOUI1, Khadija OUNINE2, Abdessamad BENMOUMEN3 and Abderahim KRIBII1

1

Laboratory of Separation Processes, Team of Environment and Applied Chemistry, Faculty

of Sciences, Ibn Tofail University, Kenitra – Morocco.

2

Laboratory of Biology and Health, Applied Microbiology Team, Faculty of Sciences, Ibn

Tofail University, Kenitra – Morocco.

3

Laboratory of plants Biology and microorganisms, Faculty of Science, Mohammed I

University, Oujda - Morocco.

ABSTRACT

This work is inscribed in the frame of valorization of medicinal and

aromatic plants from Morocco. The plant chosen for this study is

rosemary (Rosmarinus officinalis L.). it can be found in the west of

Morocco. The volatile fraction obtained by hydrodistillation (0.85%)

was analyzed by GC-MS process and its chemical composition was

determined. α-pinene, camphor, verbenone and camphene were the

main components of the essential oil. The phenolic compounds of

rosemary are obtained by maceration and decoction according to

suitable extraction protocols. The best extract yields are recorded by

the decoction. UV-visible spectrophotometry analysis showed that the

highest contents of total polyphenols and flavonoids are obtained in the

methanol extract by decoction. The antioxidant activity of different

extracts was evaluated by free radical scavenging method with DPPH• (2,2-diphényl-1-picrylhydrazyl), aqueous extract obtained by decoction seems to be the most

active (IC50 = 0.138 mg/mL). The rosemary extracts were tested for antibacterial activity by

sterile disc diffusion method, using three strains of different bacteria (Escherichia coli,

Volume 4, Issue 7, 307-323. Research Article ISSN 2277– 7105

Article Received on 05 May 2015,

Revised on 30 May 2015, Accepted on 24 June 2015

*Correspondence for Author

Sara HAIDA

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

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 0.25μm 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 1µL.

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:

2.5. Phytochemical tests

To realize the phytochemical tests, extracts of rosmary (MEM, MED, AEM and AED) are

diluted in suitable solvents.

2.5.1. Flavonoids

The presence or absence of the flavonoids in one extract can be detected by a simple and

rapid test with magnesium. This test involves putting 5 mL of each extract in tubes, add 1 mL

of concentrated HCl and 0.5 g of magnesium turnings. The presence of flavonoids is

indicated by the transfer of the color to red or pink.[8]

2.5.2. Saponins

The saponins are characterized by a foam index. Their detection is performed by adding, in a

test tube, a little water to 2 ml of extract, after stirring, the mixture is allowed to stand 20 min

and the content of saponin is measured by measuring the height of the foam formed.[8] • No foam: negative test (-)

• Foam height lower than 1 cm: weak positive test (+) • Foam height between 1 and 2 cm: positive (++) • Foam height over than 2 cm: very positive test (+++)

Petroleum ether Extraction

Water Extraction Methanol Extraction

(5 h) (24 h)

(Agitation)

(5 h) (24 h)

(Agitation)

(Filtration, centrifugation and evaporation)

(Filtration, centrifugation and evaporation)

Residue

Dry plant material (70 g)

EEM

MEM

EED

Residue

MED

AEM AED Extraction by maceration

Extraction by decoction Extraction by Soxhlet

2.5.3. Tannins: The presence of tannins is confirmed by adding, to 1 mL of extract, 1 mL water and 1 to 2 drops of dilute solution of ferric chloride. The color turns to blue in the

presence of gallic tannins and to greenish blue in the presence of catechin tannins.[9]

2.6. Quantitative estimation of total polyphenols

The protocol used in the laboratory is based on that described by Singleton and Rossi,[10] with some modifications. To 0.5 ml of each sample dilution or gallic acid (used as a standard), we

add 1 mL of Folin-Ciocalteu reagent diluted 10 times. After 2 min, 0.8 ml of sodium

carbonate solution (7.5%, w/v) is added and the whole is incubated for 30 min in darkness at

room temperature. A blanc is prepared in parallel under the same conditions with distilled

water instead of the extract solution, and the absorbance of each solution is determined at 765

nm. The same procedure is repeated to all standard Gallic acid solutions (0 – 0.25 mg/mL).

The reading of the absorption allows to determining the concentration of polyphenols in each

solution with reference to a calibration straight plotted from gallic acid solutions previously

prepared. The total phenolic content is expressed as milligrams of gallic acid equivalents per

gram of dry matter (mg GAE/g DM).

2.7. Quantitative estimation of flavonoids content

The estimation of the flavonoid content was determined by colorimetry according to the

method of Zhishen et al. [11] with some modifications. 1 mL of each sample dilution or of

the quercetin is mixed with 3 mL of distilled water. After stirring, a volume of 0.3mL of

sodium nitrite solution (5%, w/v) is added and the mixture was stirred well. 5 min later, 0.2

mL of aluminium chloride solution (10% w/v) is added, the whole is incubated in darkness at

room temperature for 30 min, and then 0.5 mL of sodium hydroxide is added to the mixture.

The absorbance of the solution is measured at 510 nm. In this case the calibration straight is

plotted from the quercetin solutions (0 - 0.5 mg/mL) previously prepared. The total

flavonoids content is expressed as milligrams of quercetin equivalents per gram of dry matter

(mg QE/g DM).

2.8. Evaluation of antioxidant activity

To study the antioxidant activity of different extracts, we opted for the reduction method of

standard. The solution of DPPH• is prepared by solubilization of 6 mg of DPPH• in 200 ml of ethanol.

The method involves mixing in a tube, 2 mL of the solution of DPPH freshly prepared with

100 mL of each solution. A negative control (or blanc) is prepared in parallel with ethanol.

The all is then incubated in the dark for 30 min and the absorbance is measured at 517 nm.

Three optical density measurements were determined for each solution.

The evaluation of the antioxidant activity is expressed as percentage inhibition of the DPPH

radical according to the following relationship.[13]

Where, %IP is inhibition percentage and Abs is the absorbance.

This formula allows to draw the straight line which represents the variation of inhibition

percentage as a function of the different concentrations of each sample (y = ax + b). From

this straight, it is possible to deduce the concentration which can reduces 50% of DPPH• for each sample studied and for ascorbic acid. This concentration called IC50 is generally

calculated according to the following equation:

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

Briefly, inoculum is prepared by inoculating each culture streaked on nutrient agar medium to

obtain isolated colonies. After incubation for 18 hours at 37°C, 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 µL

of the diluted extracts and are placed on the inoculated plates, these plates are incubated at

37°C for 24 hours. To compare the results, seven antibiotics (Norflaxacin 5 µg, Erythromycin

15 UI, Fusidic acid 10 µg, Tetracycline 30 UI, Sodium Cefotaxime 30 µg, Amoxicilline 25

µg and Gentamicine 10 µg) 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

RT:3,20 - 70,28

5 10 15 20 25 30 35 40 45 50 55 60 65 70

Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R el at ive A bu nd an ce 7,66 8,10 19,08 9,29 12,08 23,51 28,07 36,23 38,23 13,68 16,30 41,65 45,59 51,29 _55,41 33,45

7,08 _{62,31 69,04}

56,34

NL: 1,22E10 TIC F: MS pesticideso c_29_mai1 4_1

Figure 2: Chromatogram of rosemary oil essential.

RT:3,73 - 59,72

5 10 15 20 25 30 35 40 45 50 55

Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 R el at ive A bu nd an ce 23,40 7,43 18,88 36,14 7,58 38,17 20,30 8,04 28,00 12,15 16,41

41,65 _{45,58 49,80}

24,15

13,81 33,54 50,12

4,88 28,28 55,80

Table 1: Chemical composition of rosemary essential oil

Compounds RT (min) % RI**

α-pinene* 7.66 30.78 936

Camphene* 8.10 7.25 950

β-pinene* 9.29 3.67 978

α-terpinene 11.25 0.38 1013

p-cymene 11.72 0.87 1015

Limonene 12.08 6.49 1025

γ-terpinene 13.68 2.31 1051

Terpinolene 15.37 1.60 1082

Linalol* 16.30 3.43 1086

Chrysanthenone 17.63 1.23 1110

Camphor* 19.08 9.54 1123

Pinocarvone 19.93 0.27 1137

Borneol* 20.41 3.33 1150

Terpinen-4-ol* 21.09 1.61 1164

α-terpineol 22.23 1.73 1176

Verbenone* 23.51 9.38 1183

Geraniol 26.26 0.53 1235

Bornyl acetate * 28.07 3.36 1270

Piperitenone 31.30 0.18 1318

α-ylangene 33.45 0.18 1376

β-Caryophyllene* 36.23 3.13 1421

α-humulene* 38.23 2.00 1455

Germacrene D 39.58 0.22 1479

α-curcumene 40.02 0.11 1484

α-muurolene 40.60 0.11 1496

β-bisabolene 41.65 1.10 1503

δ-cadinene 42.39 0.63 1520

Caryophyllene oxide 45.59 0.82 1578

* Compound also identified in hydrosol, ** Retention index.[7]

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 %

Monoterpenes α-pinene, camphene, β-pinene, α-terpinene,

γ-terpinene, p-cymene, limonene, terpinolene. 53,35

Monoterpene ketones Chrysanthenone, camphre, pinocarvone, verbenone and

piperitenone. 20.60

Monoterpenols Linalol, borneol, terpinen-4-ol, α-terpineol et geraniol 10.63

Sesquiterpenes

β-caryophyllene, α-humulene, β-bisabolene, α-ylangene, Germacrene D, α-curcumene,

α-muurolene, δ-cadinene and caryophyllene oxide.

8.30

3.3. Results of phytochemical tests

The phytochemical tests, carried out on polar extracts of rosemary, gave the results presented

in Table 3.

Table 3: Results of phetochemical tests (+): presence (-): absence

Extract Flavonoids Tannins Saponosids

MEM + (Red) + (Blue) +++

MED + (Red) + (Blue) -

AEM - + (Green) -

AED - + (Green) +++

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).

Figure 4: The calibration straight of gallic acid

The quantification of flavonoids is obtained through the calibration straight, which is the

regression equation (y = 1.584x-0.007, R2 = 0.999) formed by a primary standard "quercetin" at different concentrations (Figure 5). Only the methanol extracts (MEM and MED), having

The value of the flavonoids content is expressed in milligrams of quercetin equivalents per

gram of dry matter (mg QE/g DM).

Figure 5: The calibration straight of quercetin.

The total polyphenols and the flavonoids content of each extract are presented in Table 4.

These results show that methanol extracts provide the most important polyphenol levels in

both extractions (over 70% against less than 30% for aqueous extracts). In total, more

polyphenols obtained by decoction (58% of total polyphenols obtained by the two methods of

extraction), against only 42% obtained by maceration (about 16% more). What justifies, in

part, the higher yield obtained by this extraction method.

The results of the quantitative determination of flavonoids extract show that MED extract is

richer in flavonoids compared to the MEM extract. This shows that the extraction by

decoction can extract higher amounts of flavonoids with respect to the extraction by

maceration at room temperature.

Therefore, the decoction seems to be the best method of extracting polyphenols. However, we

must remain cautious with respect to the effect of temperature on the structural variation of

compounds, only the analysis of the chemical composition of these extracts and their

comparison can answer this question.

Table 4: The total polyphenols and the flavonoids content in the extracts of rosemary.

Extract Total polyphenols content

(mg GAE/g DM)

Flavonoids content (mg QE/g DM)

MEM 2.359 ± 0.086 2.186 ± 0.179

MED 3.094 ± 0.069 2.643 ± 0.116

AEM 0.943 ± 0.082 -

3.5. The antioxidant capacity of the extracts of rosemary

Evaluation of the antioxidant activity of rosemary extracts is performed by the DPPH• radical reduction test using the ascorbic acid as standard. The measurements of the absorbance, of

the various solutions prepared, are obtained using a UV-Visible spectrophotometer by

following the reduction of this radical which is accompanied by the change of the purple

color (DPPH•) to a yellow color (DPPH-H) measured at 517nm.

The values obtained allowed us to plot the curves of the assay (absorbance versus

concentrations) for ascorbic acid and Rosemary extracts. These curves, having an exponential

pace, are represented by two linear parts: a descending line representing the reduction of

DPPH• radical and the horizontal one that indicates that reducing the DPPH• is total.

Figure 6 shows the reduction DPPH• curve depending on the concentration of ascorbic acid and Table 5 represents the IC50 values for each extract of rosemary.

These results show that ascorbic acid has a very powerful anti-radical activity with IC50

equal to 0.097 mg/mL, this value is close to that obtained by Bentabet N. and al.[19] which is of the order of 0.08 mg/mL. Among the five extracts, essential oil has the lowest antioxidant

power.

Figure 6: DPPH• reduction curve depending on the concentration of ascorbic acid.

Table 5: Values of IC50 of the different rosemary extracts.

Extract Equation R2 values IC50 (mg/mL)

Ascorbic acid 501.1x + 1.627 0.991 0.097

MED 132.4x + 1.181 0.988 0.364

MEM 112.5x – 1.949 0.994 0.462

AED 354.5x + 1.146 0.994 0.138

AEM 26.91x + 0.888 0.991 1.825

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

Extracts Disc load Inhibition diameter (mm)

E. coli S. aureus P. aeruginosa

Hydrosol 5μL 9 15.5 -

Essential oil 5μL 9 9 -

MED 5μL - - -

MEM 5μL - - -

AED 5μL - - -

AEM 5μL - - -

EED 5μL - 15.5 -

EEM 5μL - 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

Inhibition diameter (mm) Acceptance limits Critical concentrations

(μg/mL) E. coli S.

aureus

P.

aeruginosa sensitive resistant

Norfloxacin 5μg - 26.5 - ≥19 <19 5

Erythromycin 15UI - - - ≥15 <15 4

Fusidic acid 10μg - 32 - ≥11 <11 20

Tetracyclin 30UI - - - ≥23 <23 2

Sodium

cefotaxime 30μg - 8 8 ≥15 <15 10

Amoxycillin 25μg - 8 - ≥18 <18 8

Gentamicin 10μg 24 8 16 ≥15 <15 6

From these results we find that:

The essential oil and hydrosol of Rosmarinus officinalis have the same effect on Escherichia

coli with a 9 mm zone of inhibition, representing 37.5% inhibition compared to Gentamicin

which is the only antibiotic active against this strain. This strain showed a resistance against

methanol (MED, MEM), aqueous (AED, AEM) and petroleum ether (EED, EEM) extracts.

In comparison with Fusidic acid, the most active against Staphylococcus aureus strain is the

extract of petroleum ether, isolated at the end of extraction by maceration, which recorded the

most power inhibitor (62.5%) against this strain. Further, the extract of decoction of

petroleum ether and hydrosol showed significant antibacterial activity corresponding to

48.4%. However, the essential oil of Rosemary revealed only moderate activity with a 9 mm

zone of inhibition corresponding to 28.1% relative to Fusidic acid.

It turned out that no zone of inhibition around the discs was observed against Pseudomonas

aeruginosa. Thus, this bacterium has a very high resistance against potential antibacterial

action of Rosemary extracts.

From these results it seems obvious that only the apolar extracts, obtained either by

hydrodistillation or by petroleum ether extraction, which answered positively to tests

conducted with two of the three selected bacterial strains. As these volatils compounds are

easy to analyze by GC-MS, it would be interesting to know the responsible compound of this

antibacterial effect.

It has been demonstrated that the resistance of gram negative bacteria extracts and

particularly the essential oils is related in part to the complexity of the cell wall of these

organisms which contains an outer membrane, in contrast to the structure of the

Gram-positive bacteria.[23]

CONCLUSION

The analysis of the essential oil by coupling GC-MS, allowed us to determine its chemical

composition. We identified 28 compounds whose major products are α-pinene, camphene, camphor, β-pinene and verbenone.

Phenolic compounds of Rosemary are obtained by maceration and decoction, the best

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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