# In Vitro Antimicrobial Activity Screening of Ethanol Extract of Lavandula stoechas and Investigation of Its Biochemical Composition

Authors: Kerem Canlı; Ali Yetgin; Atakan Benek; Mustafa Eray Bozyel; Ergin Murat Altuner
Journal: Advances in Pharmacological Sciences
 (2019)

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## Abstract

The aim of this study was to test antimicrobial activity of ethanol extract ofLavandula stoechas against 22 bacteria and 1 yeast. Also, biochemical composition of the extract was investigated. A wide range of Gram-positive, Gram-negative microorganisms, and multidrug resistant bacteria were selected to test the antimicrobial activity. As a result, the extract is observed to contain fenchone (bicyclo[2.2.1]heptan-2-one, 1,3,3-trimethyl-, (1R)-) and camphor (+)-2-bornanone) as major components and showed antimicrobial activity against all studied microorganisms except Escherichia coli ATCC 25922 and Klebsiella pneumoniae. The results of the study present that L. stoechas is active against MDR strains too.


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## Body

## 1. Introduction

The World Health Organization (WHO) has predicted increasing antimicrobial resistance as a major threat for the public health for the twenty-first century. In order to prevent spreading of antibiotic resistance infections, scientists have been conducting intensive researches to determine new antimicrobial agents. One way to prevent antibiotic resistance of microorganisms is by using new compounds that are not based on existing antimicrobial agents.TheLavandula genus is an important member of family Lamiaceae. It consists of 47 species of small evergreen shrubs having aromatic foliage and flowers [1]. Lavandula species are cultivated in France, Spain, and Italy. In Turkey, mainly two species, Lavandula stoechas and Lavandula angustifolia, and their subspecies and hybrid forms grow wildly or are cultivated [2]. The medicinal importance of the plant is well documented [3, 4], and the drugs prepared from this plant are registered in many Pharmacopeia [5]. L. stoechas L. is used in perfumery and cosmetics [6, 7]. Anticonvulsant, sedative, and antispasmodic activities were reported [8]. The essential oil (EO) of L. stoechas possesses weak antibacterial activity [9]. It is used in folk medicine as an antispasmodic, a sedative, and a diuretic and for rheumatic diseases [7]. The main purpose of this study was to investigate the antimicrobial activity of L. stoechas and reveal the major components of its ethanol extracts.


## 2. Materials and Methods

### 2.1. Endemic Plant Samples

Dried flowers ofL. stoechas L. were purchased from the local market in Canakkale, Turkey, and identified by Dr. Mustafa Eray Bozyel.


### 2.2. Disk Diffusion Test

Plant samples were dried after collection and ground into small pieces with a grinder. GroundL. stoechas samples were shaken in ethanol (Sigma-Aldrich) at 125 rpm for 2 days at room temperature. After that, all the mixture was filtrated through Whatman no. 1 filter paper into evaporation flasks. Filtrates were evaporated by a rotary evaporator (Buchi R3) at 45°C [10, 11]. Finally, the remnants were collected and weighed. 5.83, 23.4, and 35.1 mg samples were prepared. The activity of the extract was tested against 22 bacteria and 1 yeast, where most of the strains were standard; nostandard strains were isolated from food and the MDR strains were clinical isolates. Nonstandard strains were identified in Ankara University, Department of Biology and Duzce University, Department of medical. All bacterial strains were incubated at 37°C for 24 hours; however, Candida albicans was incubated at 27°C for 48 hours [11]. Each bacteria and yeast were inoculated into 0.9% sterile saline solution and adjusted to 0.5 McFarland standard, in order to standardize inocula to contain about 108 cfu·mL−1 for bacteria and 107 cfu·mL−1 for C. albicans [11]. The antimicrobial activity of ethanol extract of L. stoechas was tested by the disk diffusion test, as mentioned before [12]. Firstly, Mueller–Hinton agar (BD Difco, USA) was poured into 90 mm sterile Petri dish in order to reach a meant depth of 4.0 mm ± 0.5 mm. The extracts were loaded on 6 mm Oxoid Antimicrobial Susceptibility Test Disks. Disks were left to dry overnight at 30°C in sterile conditions in order to prevent any remaining of solvent, which may interfere with the results. After that, prepared microorganisms, which were inoculated into saline solution, were streaked on the surface of Petri dishes. These plates were left to dry for 5 minutes at room temperature in aseptic conditions [12]. Next, disks were tightly applied to the surface of plates. Finally, these plates were incubated, and inhibition zone diameters were recorded [12].Preculturing conditions for all microorganisms are as mentioned previously[13].


### 2.3. Broth Dilution Test

Broth dilution method for minimum inhibitory concentration (MIC) determination as described previously was employed [14]. Serial 2-fold dilutions were made to obtain a concentration range of 0.07–35.9 µg/mL. The MIC was defined as the lowest concentration of extract inhibiting any visible bacterial growth. All tests were conducted in triplicates.


### 2.4. GC-MS Analysis

For the identification of chemical components, each sample was analyzed by Agilent GC 6890N-Agilent MS 5973 equipped with HP5-MS capillary column (30m ∗ 0.25 mm; coating thickness 0.25μm). Analytical conditions were an injector temperature of 350°C; carrier gas helium at 1 mL/min; injection mode: split, split ratio 10 : 1; volume injected: 1 μL of sample in ethanol extract; oven temperature programmed from 40°C to 350°C at 4°C/min; pressure: 48.2 kPa; and split flow: 9.9 mL/min. The MS scan conditions were a transfer line temperature of 280°C, an interface temperature of 280°C, and an ion source temperature of 230°C. Identification of the components was conducted by matching the retention times against National Institute of Standards and Technology (NIST Mass Spectrometry DATA CENTER) data library, and crosscheck was applied with previously published data [15, 16] The chemical components found to be higher than 1% were accepted as the major components, and the list of these components and information regarding them are given in Table 1.Table 1
The major chemical components ofL. stoechas according to the GC-MS analysis.
No.
Retention time
Compound name
Formula
Molecular weight (g/mol)
Area (%)
1
14.529
Eucalyptol
C10H18O
154.249
6.22
2
16.940
Bicyclo[2.2.1]heptan-2-one, 1,3,3-trimethyl-, (1R)-
C10H16O
152.233
18.81
3
19.163
(+)-2-Bornanone
C10H16O
152.233
8.64
4
22.138
Fenchyl acetate
C12H20O2
196.286
1.10
5
24.593
Bornyl acetate
C12H20O2
196.286
3.40
6
26.015
Myrtenyl acetate
C12H18O2
194.270
5.18
7
30.067
Benzaldehyde, 2-hydroxy-4-methyl-
C8H8O2
136.148
1.64
8
31.687
Cubedol
C15H26O
222.366
1.10
9
32.350
Cubedol
C15H26O
222.366
1.08
10
32.854
Bicyclo[3.1.1]hept-3-en-2-one, 4,6,6-trimethyl-
C10H14O
150.218
1.40
11
33.844
Acetic acid, 4a-methyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-yl ester
1.42
12
34.749
Veridiflorol
C15H26O
222.366
4.50
13
35.121
Viridiflorol
C15H26O
222.366
2.99
14
37.869
Unknown
—
—
1.02
15
39.683
Andrographolide
C20H30O5
350.449
1.15
16
48.039
Palmitic acid
C16H32O2
256.424
2.48
17
52.456
Behenic alcohol
C22H46O
326.600
1.06
18
75.553
Tetrapentacontane
C54H110
759.451
1.48
19
77.908
Tetrapentacontane
C54H110
759.451
1.37
20
82.903
Tetrapentacontane
C54H110
759.451
4.84
21
86.068
Tetrapentacontane
C54H110
759.451
4.83
22
91.031
Tetrapentacontane
C54H110
759.451
2.53
23
91.398
Stigmast-5-en-3-ol, (3.beta.)-
C29H50O
414.707
3.25


### 2.5. Controls

Empty sterile disks and extraction solvent (ethanol) were used as negative controls. Ciprofloxacin and gentamicin used as pozitif controls (Table2).Table 2
Pozitif controls (inhibition zones in mm).
Ciprofloxacin
Gentamicin
B. subtilis DSMZ 1971
36
30
C. albicans DSMZ 1386
—
—
E. aerogenes ATCC 13048
30
23
E. durans
24
14
E. faecalis ATCC 29212
19
13
E. faecium
28
28
E. coli ATCC 25922
—
20
K. pneumoniae
30
22
L. innocula
18
13
L. monocytogenes ATCC 7644
20
28
P. aeruginosa DSMZ 50071
28
15
P. fluorescens P1
19
12
S. enteritidis ATCC 13075
36
24
S. infantis
24
24
S. Kentucky
34
13
S. typhimurium SL 1344
35
23
S. aureus ATCC 25923
22
24
S. epidermidis DSMZ 20044
34
25
—: no activity observed.


### 2.6. Statistics

The statistical analysis was executed using a parametric method, the one-way analysis of variance (ANOVA), with a significance level of 0.05. In order to put forward any correlation between concentration and antimicrobial activity, Pearson correlation coefficient was calculated. All statistical analysis were conducted by using R Studio, version 3.3.2 [17].


## 2.1. Endemic Plant Samples

Dried flowers ofL. stoechas L. were purchased from the local market in Canakkale, Turkey, and identified by Dr. Mustafa Eray Bozyel.


## 2.2. Disk Diffusion Test

Plant samples were dried after collection and ground into small pieces with a grinder. GroundL. stoechas samples were shaken in ethanol (Sigma-Aldrich) at 125 rpm for 2 days at room temperature. After that, all the mixture was filtrated through Whatman no. 1 filter paper into evaporation flasks. Filtrates were evaporated by a rotary evaporator (Buchi R3) at 45°C [10, 11]. Finally, the remnants were collected and weighed. 5.83, 23.4, and 35.1 mg samples were prepared. The activity of the extract was tested against 22 bacteria and 1 yeast, where most of the strains were standard; nostandard strains were isolated from food and the MDR strains were clinical isolates. Nonstandard strains were identified in Ankara University, Department of Biology and Duzce University, Department of medical. All bacterial strains were incubated at 37°C for 24 hours; however, Candida albicans was incubated at 27°C for 48 hours [11]. Each bacteria and yeast were inoculated into 0.9% sterile saline solution and adjusted to 0.5 McFarland standard, in order to standardize inocula to contain about 108 cfu·mL−1 for bacteria and 107 cfu·mL−1 for C. albicans [11]. The antimicrobial activity of ethanol extract of L. stoechas was tested by the disk diffusion test, as mentioned before [12]. Firstly, Mueller–Hinton agar (BD Difco, USA) was poured into 90 mm sterile Petri dish in order to reach a meant depth of 4.0 mm ± 0.5 mm. The extracts were loaded on 6 mm Oxoid Antimicrobial Susceptibility Test Disks. Disks were left to dry overnight at 30°C in sterile conditions in order to prevent any remaining of solvent, which may interfere with the results. After that, prepared microorganisms, which were inoculated into saline solution, were streaked on the surface of Petri dishes. These plates were left to dry for 5 minutes at room temperature in aseptic conditions [12]. Next, disks were tightly applied to the surface of plates. Finally, these plates were incubated, and inhibition zone diameters were recorded [12].Preculturing conditions for all microorganisms are as mentioned previously[13].


## 2.3. Broth Dilution Test

Broth dilution method for minimum inhibitory concentration (MIC) determination as described previously was employed [14]. Serial 2-fold dilutions were made to obtain a concentration range of 0.07–35.9 µg/mL. The MIC was defined as the lowest concentration of extract inhibiting any visible bacterial growth. All tests were conducted in triplicates.


## 2.4. GC-MS Analysis

For the identification of chemical components, each sample was analyzed by Agilent GC 6890N-Agilent MS 5973 equipped with HP5-MS capillary column (30m ∗ 0.25 mm; coating thickness 0.25μm). Analytical conditions were an injector temperature of 350°C; carrier gas helium at 1 mL/min; injection mode: split, split ratio 10 : 1; volume injected: 1 μL of sample in ethanol extract; oven temperature programmed from 40°C to 350°C at 4°C/min; pressure: 48.2 kPa; and split flow: 9.9 mL/min. The MS scan conditions were a transfer line temperature of 280°C, an interface temperature of 280°C, and an ion source temperature of 230°C. Identification of the components was conducted by matching the retention times against National Institute of Standards and Technology (NIST Mass Spectrometry DATA CENTER) data library, and crosscheck was applied with previously published data [15, 16] The chemical components found to be higher than 1% were accepted as the major components, and the list of these components and information regarding them are given in Table 1.Table 1
The major chemical components ofL. stoechas according to the GC-MS analysis.
No.
Retention time
Compound name
Formula
Molecular weight (g/mol)
Area (%)
1
14.529
Eucalyptol
C10H18O
154.249
6.22
2
16.940
Bicyclo[2.2.1]heptan-2-one, 1,3,3-trimethyl-, (1R)-
C10H16O
152.233
18.81
3
19.163
(+)-2-Bornanone
C10H16O
152.233
8.64
4
22.138
Fenchyl acetate
C12H20O2
196.286
1.10
5
24.593
Bornyl acetate
C12H20O2
196.286
3.40
6
26.015
Myrtenyl acetate
C12H18O2
194.270
5.18
7
30.067
Benzaldehyde, 2-hydroxy-4-methyl-
C8H8O2
136.148
1.64
8
31.687
Cubedol
C15H26O
222.366
1.10
9
32.350
Cubedol
C15H26O
222.366
1.08
10
32.854
Bicyclo[3.1.1]hept-3-en-2-one, 4,6,6-trimethyl-
C10H14O
150.218
1.40
11
33.844
Acetic acid, 4a-methyl-1,2,3,4,4a,5,6,7-octahydronaphthalen-2-yl ester
1.42
12
34.749
Veridiflorol
C15H26O
222.366
4.50
13
35.121
Viridiflorol
C15H26O
222.366
2.99
14
37.869
Unknown
—
—
1.02
15
39.683
Andrographolide
C20H30O5
350.449
1.15
16
48.039
Palmitic acid
C16H32O2
256.424
2.48
17
52.456
Behenic alcohol
C22H46O
326.600
1.06
18
75.553
Tetrapentacontane
C54H110
759.451
1.48
19
77.908
Tetrapentacontane
C54H110
759.451
1.37
20
82.903
Tetrapentacontane
C54H110
759.451
4.84
21
86.068
Tetrapentacontane
C54H110
759.451
4.83
22
91.031
Tetrapentacontane
C54H110
759.451
2.53
23
91.398
Stigmast-5-en-3-ol, (3.beta.)-
C29H50O
414.707
3.25


## 2.5. Controls

Empty sterile disks and extraction solvent (ethanol) were used as negative controls. Ciprofloxacin and gentamicin used as pozitif controls (Table2).Table 2
Pozitif controls (inhibition zones in mm).
Ciprofloxacin
Gentamicin
B. subtilis DSMZ 1971
36
30
C. albicans DSMZ 1386
—
—
E. aerogenes ATCC 13048
30
23
E. durans
24
14
E. faecalis ATCC 29212
19
13
E. faecium
28
28
E. coli ATCC 25922
—
20
K. pneumoniae
30
22
L. innocula
18
13
L. monocytogenes ATCC 7644
20
28
P. aeruginosa DSMZ 50071
28
15
P. fluorescens P1
19
12
S. enteritidis ATCC 13075
36
24
S. infantis
24
24
S. Kentucky
34
13
S. typhimurium SL 1344
35
23
S. aureus ATCC 25923
22
24
S. epidermidis DSMZ 20044
34
25
—: no activity observed.


## 2.6. Statistics

The statistical analysis was executed using a parametric method, the one-way analysis of variance (ANOVA), with a significance level of 0.05. In order to put forward any correlation between concentration and antimicrobial activity, Pearson correlation coefficient was calculated. All statistical analysis were conducted by using R Studio, version 3.3.2 [17].


## 3. Results and Discussion

The diameters of inhibition zones, which were measured in millimeters, are given in Table3 as the mean values of three parallels with standard errors. No activities were observed for the negative controls. Furthermore, statistical analysis proved that there are no significant differences between the activities of three parallels of each extract volumes (p>0.05). On the other hand, a weak positive correlation is observed between the activities of extracts and the volumes tested, with a Pearson correlation coefficient of 0.3239.Table 3
Disk diffusion test result forL. stoechas (inhibition zones in mm).
5.83 mg loaded disk
23.4 mg loaded disk
35.1 mg loaded disk
Bacillus subtilis DSMZ 1971
11.00 ± 0.00
13.00 ± 0.00
14.00 ± 0.58
Candida albicans DSMZ 1386
7.00 ± 0.00
9.00 ± 0.00
11.00 ± 0.00
Enterobacter aerogenes ATCC 13048
—
—
8.00 ± 0.00
Escherichia coli
—
—
8.00 ± 0.00
Escherichia coli ATCC 25922
—
—
—
Enterococcus durans
—
10.00 ± 0.00
11.00 ± 0.00
Enterecoccus faecalis ATCC 29212
7.00 ± 0.00
9.00 ± 0.00
11.00 ± 0.00
Enterecoccus faecium
11.00 ± 0.58
16.00 ± 0.58
15.00 ± 1.15
Klebsiella pneumoniae
—
—
—
Listeria innocua
—
8.00 ± 0.00
10.00 ± 0.00
Listeria monocytogenes ATCC 7644
9.00 ± 0.00
14.00 ± 1.15
13.00 ± 0.58
Pseudomonas aeruginosa DSMZ 50071
—
8.00 ± 0.00
10.00 ± 0.00
Pseudomonas fluorescens P1
7.00 ± 0.00
9.00 ± 0.00
13.00 ± 0.00
Staphylococcus aureus
10.00 ± 0.00
10.00 ± 0.00
14.00 ± 0.58
Staphylococcus aureus ATCC 25923
12.00 ± 0.58
13.00 ± 0.00
18.00 ± 0.00
Salmonella enteritidis ATCC 13076
7.00 ± 0.00
9.00 ± 0.00
10.00 ± 0.00
Staphylococcus epidermidis DSMZ 20044
15.00 ± 0.00
19.00 ± 0.58
20.00 ± 0.00
Salmonella infantis
—
—
10.00 ± 0.00
Salmonella kentucky
—
7.00 ± 0.00
10.00 ± 0.00
Salmonella typhimurium SL1344
8.00 ± 0.58
—
10.00 ± 1.15
Acinetobacter baumannii MDR
—
10.00 ± 0.00
11.00 ± 0.00
Proteus vulgaris MDR
10.00 ± 1.15
14.00 ± 1.15
11.00 ± 1.15
Streptococcus pneumoniae MDR
10.00 ± 0.00
14.00 ± 1.15
13.00 ± 0.58
—: no inhibition.In addition, the results of broth dilution test (MIC values) are given in Table4.Table 4
MIC values forL. stoechas (MIC values in µg/mL).
MIC
Bacillus subtilis DSMZ 1971
35.9
Candida albicans DSMZ 1386
35.9
Enterobacter aerogenes ATCC 13048
—
Escherichia coli
—
Escherichia coli ATCC 25922
—
Enterococcus durans
35.9
Enterecoccus faecalis ATCC 29212
35.9
Enterecoccus faecium
17.95
Klebsiella pneumoniae
—
Listeria innocua
35.9
Listeria monocytogenes ATCC 7644
35.9
Pseudomonas aeruginosa DSMZ 50071
35.9
Pseudomonas fluorescens P1
35.9
Staphylococcus aureus
17.95
Staphylococcus aureus ATCC 25923
17.95
Salmonella enteritidis ATCC 13076
35.9
Staphylococcus epidermidis DSMZ 20044
8.98
Salmonella infantis
—
Salmonella kentucky
35.9
Salmonella typhimurium SL1344
35.9
Acinetobacter baumannii MDR
35.9
Proteus vulgaris MDR
35.9
Streptococcus pneumoniae MDR
35.9
—: no inhibition.According to Table3, L. stoechas has antimicrobial activity against all studied microorganisms except Escherichia coli ATCC 25922 and Klebsiella pneumoniae. Three of them have high susceptibility (15–25 mm); seven of them have moderate susceptibility (14–10 mm); and eleven of them have low susceptibility (9–7 mm). L. stoechas shows antimicrobial activity against all tested MDR bacteria. These results are important since antimicrobial activity of this species were determined against large range of Gram-negative and Gram-positive bacteria.According to GC-MS results, fenchone(bicyclo[2.2.1]heptan-2-one, 1,3,3-trimethyl (1R)-), and camphor (+)-2-bornanone) are mainly found in the composition of L. stoechas ethanol extract. Similar results were obtained when compared with previous researches [18–20] (Figure 1).Figure 1
GC-MS chromatography ofL. stoechas.The antimicrobial effect of the plant is known from previous investigations, but there is no broad-spectrum study like this [18, 20]. It has also been reported for the first time that the plant’s ethanol extract is effective against multidrug-resistant microorganisms, which is one of the most important health hazards in the world [21].Acinetobacter species, particularlyAcinetobacter baumannii, have become significant pathogens especially in the nosocomial setting. A. baumannii has progressively been implicated in serious nosocomial infections, including bloodstream infection (BSI), nosocomial and ventilator-related pneumonia, and meningitis. These infections are particularly common in critically ill patients, with mortalities as high as 40–64% for pneumonia and 17–46% for BSI [22]. The extensive use of broad-spectrum antibiotic agents within hospitals has led to the rapid emergence of multidrug-resistant (MDR) A. baumannii strains. Only a few antimicrobial agents are active against MDR A. baumannii infections [22]. In our study, we observed 11 mm of inhibition zone against A. baumannii (MDR) strain. Moreover, we have found 14 mm of inhibition zones against Proteus vulgaris (MDR) and Streptococcus pneumoniae (MDR) strains. Our results present that L. stoechas is active against MDR strains too.Previous studies tested the antimicrobial activity ofL. stoechas and found MIC values between 0 and 6500 µg/mL [23]. When the result of this study were compared with the results of the previous study, it may be observed that the MIC values obtained in our study were much lower, which is better. The reason of this difference should be related with the strains used in two studies. Although B. subtilis, S. aureus, S. typhimurium, E. coli, and C. albicans were used in both studies, the strains were different. Only L. monocytogenes ATCC 7644 were common in these two studies but the MIC values were different. This difference could be related with the composition of extracts which were directly affected by the environment; those plant samples were collected.Another previous study was tested the antimicrobial activity of two subspecies ofL. stoechas. The only common microorganism used in our study and this previous study was C. albicans. They observed a MIC value > 100 µg/mL for both subspecies, which are quite higher than our results [24]. But this difference is logical since the plant samples, C. albicans strain, and collection area were different.Ez Zoubi et al. [25] tested the antimicrobial activity of the essential oil of L. stoechas against E. coli, K. pneumoniae, Proteus mirabilis, and P. aeruginosa. As a result, they observed MIC values between 2.5 and 10 µg/mL. Although the strains of these microorganisms were not defined, the difference in the MIC values between this study and our study was mainly due to using different types of extracts, namely, essential oil and ethanol extracts.


## 4. Conclusion

Our study clearly presents thatL. stoechas should have a possible medicinal uses, especially against MDR bacteria. However, further researches are needed in order to analyse the active substances and their activity mechanisms in details.



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*Source: 3201458-2019-01-10.xml*