https://doi.org/10.33263/LIANBS11.1417
Alexandru Mihai Grumezescu1*, Carmen Mariana Chifiriuc2, Ioana Marinaş2, Crina Saviuc2, Dan Mihaiescu1, Veronica Lazǎr2
1Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Polizu Street 1-7, Romania
2Faculty of Biology, University of Bucharest, Aleea Portocalelor 1-3, Romania
*Correspondence :grumezescu[at]yahoo.com
Abstract
Essential oils extracted by microwave assisted hydrodistillation technique from Ocinum basilicum and Mentha piperita were characterized by GC-MS. An adapted diffusion method was used in order to assess the potentiator effect of the essential oils on the susceptibility of Staphylococcus aureus clinical and reference strains to some of the currently used antibiotics, i.e. clindamycin, ciprofloxacin, tetracycline, gentamicin, penicillin and erythromycin.. The Ocimum basilicum and Menthapiperita essential oils, exhibited a strong, strain specific influence on the antibiotic susceptibility of the tested strains.
Keywords: Ocimum basilicum, Mentha piperita, Staphylococcus aureus, antimicrobial activity
1. Introduction
Aromatic plants are used in folk medicine as antimicrobial agents and their essential oils have been known to have antibacterial and antifungal proprieties [1]. Essential oils and their components are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers, and their exploitation for potential multi-purpose functional use [23]. Mentha piperita, commonly called peppermint, is a well-known herbal remedy used for a variety of symptoms and diseases (recognised for its carminative, stimulant, antispasmodic, antiseptic, antibacterial and antifungal activities) [4]. Traditionally, basil (O. basilicum) has been extensively used in food as a flavouring agent, and in perfumery and medical industries [5]. Recently the potential uses of O. basilicum essential oil, particularly as antimicrobial and antioxidant agents have also been investigated [6]. Bacteria can develop antibiotic resistance by altering the expression or function of their own genes or by acquiring new genes. The rate of resistance development by mutation depends upon the number of events required for resistant clones to reach clinically relevant levels. The larger a bacterial population is, the higher the probability that antibiotic pressure will aureus strains to different antibiotics. it is to be noted the fact that is the first report on the influence of select spontaneous or preexisting mutants with reduced susceptibility. The exchange and acquisition of new genetic material, by transduction, transformation or conjugation, contributes to the rapid horizontal dissemination of resistance determinants [7]. The emergence of multidrug resistant bacterial strains has become a major challenge in the treatment of infectious diseases and a top public health problem. The antibiotic-resistance of S. aureus strains is a serious concern besideits pathogenicity. Strains of S. aureus have been observed to show resistance against multiple antimicrobials. Various genetic determinants such as MecA (methicillin), TetK/M (tetracyclines), MsrA/B (macrolides), AacA-D (aminoglycosides), ErmA/B/C (macrolides, lincosamides and streptogramin B), and LinA (lincosamides) have been reported in human S. aureus isolates[8,9]. The emergence of methicillin resistant Staphylococcus aureus (MRSA) as a major nosocomial pathogen lead to the endemic spread of resistant strains in many hospitals [10]. In the present paper, we established the chemical composition of essential oils from Ocimum basilicum and Menthapiperitaby GC–MS method, and evaluated the influence of the essential oils on the susceptibility fo S. these essential oils on the antimicrobial effect of drugs on clinical strains.
2. Experiment details
2.1. Extraction and characterization of the essential oils.
Ocimum basilicum and Mentha piperitaplant materials were purchased from a local supplier and subjected to essential oil extraction. A Neo Clevenger type apparatus according to European Pharmacopoeia 6 was used performing two microwave assisted extractions from 225g plant material [11]. Subsequently, DMSO was added to form a stock solution (1:1 with essential oil v/v) which was kept in a cool place before use. Chemical composition was settled by GC-MS analysis. Gas chromatographic analysis was performed by using an Agilent 6890 Series GC System. Detection was carried out with a 5973 mass-selective single quadrupole detector (Agilent technologies). Operation control and data process were carried out by Agilent Technologies ChemStation software (Santa Clara, CA, USA). The mass spectrometer was calibrated before use with perfluorotributylamine (PFTBA) as a calibration standard. The working conditions were: H2-carrier gas, flow: 1,2 ml/min, temperature program 50/300oC with a ramp rate of 5oC/min; the temperature of the injector and of the detector was 250oC, and a DB5-MS (30m; 0.25 mm id; 0.25 µm) column.
2.2. Microbial strains.
Four S. aureus strains of clinical origin (two wound secretions and two blood cultures), as well as the S. aureus ATCC 25923 reference strain were tested. Isolates were identified by using an automatic Vitek II system [12].
2.3. The antibiotic potentiator effect of the essential oils.
An adapted diffusion method was used in order to assess the potentiator effect of the essential oils and of ther analytical standards on the antibiotic susceptibility of the tested S. aureus strains to some of the currently used antibiotics, chosen according to CLSI reccomandations, i.e. clindamycin, ciprofloxacin, tetracycline, gentamicin, penicillin and erythromycin. Standardized antibiotic discs have been placed on the Mueller Hinton agar medium distributed in Petri dishes, previously seeded with a bacterial inoculum with a density adjusted by the aid of the 0.5 McFarland standard. Stock solutions of the R. officinalis essential oil and eucalyptol were spoted on the antibiotic discs. The plates were incubated 24h at 37°C, and the differences between inhibition zones diameters were quantified and compared with the growth inhibition zones obtained for the respective antibiotics.
3. Results and Discussions
There are many commercial basil varieties having different chemical properties. Basil has been clasiffied according to different geographical origins. They are the European chemotype, from Italy, France, Bulgaria, Egypt, and South Africa, having linalool and methyl chavicol as main components; Tropical chemotype, from India, Pakistan and Guatemala, being rich in methyl cinnamate; Reunion chemotype, from Thailand, Madagascar and Vietnam, being characterized by high concentrations of methyl chavicol [13, 14]. There is also a eugenol-rich chemotypefrom North Africa and Russia. The chemistry of peppermint oil is very complex and highly variable. The relative concentrations vary depending on climate, cultivar, and geographic location [15, 16, 17]. As a result of GC–MS analyses, the essential oil composition is listed in Table 1 for Mentha piperita and Table 2 for Ocimum basilicum. Mentha piperita essential oilproved to be rich in β-pinene, limonene, menthone, isomenthol, menthol while Ocimum basilicum in estragole, O-methyleugenol, cis-geraniol and eucalyptol.
Table 1. The main compounds and their percentage in Mentha piperitaessential oil.
| Peak | R.T. | % | compounds |
| 1 | 4.882 | 3.57 | α-Pinene |
| 2 | 5.24 | 0.09 | Camphene |
| 3 | 6.046 | 4.277 | β-Pinene |
| 4 | 6.684 | 0.211 | Ethylamylcarbinol |
| 5 | 6.841 | 0.04 | α -Phellandrene |
| 6 | 7.21 | 0.161 | α -Terpinen |
| 7 | 7.613 | 5.823 | Limonene |
| 8 | 8.486 | 0.061 | τ -Terpinen |
| 9 | 9.36 | 0.14 | m-Cymene |
| 10 | 9.83 | 0.322 | Geraniol |
| 11 | 11.162 | 1.456 | Isopulegol |
| 12 | 11.476 | 21.009 | Menthone |
| 13 | 11.733 | 12.437 | Isomenthol |
| 14 | 12.248 | 34.825 | Menthol |
| 15 | 12.573 | 3.365 | α -Terpineol |
| 16 | 12.785 | 0.108 | Carveol |
| 17 | 13.827 | 0.896 | Pulegone |
| 19 | 15.427 | 3.461 | Menthol, acetate |
| 20 | 17.722 | 0.535 | β -Bourbonene |
| 21 | 17.946 | 0.225 | β -Copaene |
| 22 | 18.618 | 1.579 | α -Caryophyllene |
| 23 | 19.245 | 0.08 | τ-Muurolene |
| 24 | 21.285 | 0.228 | δ -Cadinene |
Table 2. The main compounds and their percentage in Ocimum basilicum essential oil.
| Peak | R.T. | % | compounds |
| 1 | 5.867 | 2.077 | Eucalyptol |
| 2 | 8.839 | 6.254 | cis-Geraniol |
| 3 | 10.613 | 67.291 | Estragole |
| 4 | 14.968 | 1.439 | Eugenol |
| 5 | 15.618 | 1.898 | β -Isosafrole |
| 6 | 16.256 | 7.36 | O-Methyleugenol |
| 7 | 16.983 | 1.96 | α -Farnesene |
| 8 | 17.375 | 0.385 | Guaiene |
| 9 | 18.615 | 1.06 | Caryophyllene |
| 10 | 21.282 | 0.591 | δ-Cadinene |
| 11 | 21.876 | 2.43 | cis- α -Bisabolene |
All the isolates were susceptible to clindamycin, ciprofloxacin and tetracycline, except for the S. aureus 5W, a tetracycline resistant strain. The four tested strain proved to be resistant to penicillin, erytromycin and oxacillin, irrespective to their isolation source (table 3). The results presented in Table 3 are demonstrating that the tested essential oils are representing antibiotic potentators, with a notable effect on the oxacillin susceptibilityin the presence of O. basilicum stock solution supplementation.
Table 3. The growth inhibition zone diameters obtained for the tested antibiotics in the presence of Menta piperita and Ocinum basilicum Staphylococcus aureus strains.
Staphylococcus aureus 2
| MP | OB | Control | |
| Clindamycin | 36 | 36 | 32 |
| Ciprofloxacin | 23 | 32 | 28 |
| Tetracycline | 17 | 23 | 22 |
| Gentamicin | 21 | 25 | 19 |
| Penicillin | 11 | 12 | 0 |
| Erythromycin | 23 | 28 | 22 |
Staphylococcus aureus 3
| MP | OB | Control | |
| Clindamycin | 36 | 40 | 38 |
| Ciprofloxacin | 27 | 27 | 25 |
| Tetracycline | 22 | 22 | 20 |
| Gentamicin | 22 | 22 | 19 |
| Penicillin | 12 | 0 | 0 |
| Erythromycin | 11 | 12 | 10 |
Staphylococcus aureus 4
| MP | OB | control | |
| Clindamycin | 40 | 36 | 34 |
| Ciprofloxacin | 22 | 26 | 24 |
| Tetracycline | 26 | 20 | 20 |
| Gentamicin | 23 | 23 | 20 |
| Penicillin | 18 | 16 | 15 |
| Erythromycin | 21 | 12 | 11 |
Staphylococcus aureus 5
| Antibiotics | MP | OB | Control |
| Clindamycin | 28 | 26 | 25 |
| Ciprofloxacin | 25 | 24 | 23 |
| Tetracycline | 28 | 8 | 0 |
| Gentamicin | 19 | 19 | 18 |
| Penicillin | 8 | 0 | 0 |
| Erythromycin | 0 | 0 | 0 |
MP= M. piperita, OB =O. basilicum essential oil;
However, the modulation of the inhibition zone diameter varied with the tested strain and antibiotic, demonstrating the necessity of the study extension on a statistically significant number of strains belonging to different species, with different antimicrobial susceptibility profiles, in order to confirm the potentiating effect of these oils on the antimicrobial activity of the existent drugs and indepth research for the identitification of the molecular mechanisms of this synergic action.
4. Conclusions
The results of the present study are demonstrating that the Ocimum basilicum and Mentha piperita essential oils could potentiate the antimicrobial activity of the existent antibiotics, representing a possible solution to the perpetual need for effective antibiotics and to the lack of new structures with potent microbicidal activity.
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