Iranian Journal of Medical Microbiology

Mentha piperita L. with the common name “Peppermint” is one of the most important medicinal plants. This plant belongs to the family Lamiaceae, order lamiales. This plant is a natural hybrid of spearmint (M. spicata L.) and water mint (M. aquatica L.) (1). It has the following general properties: antioxidant, antimicrobial, antiviral, anti-seizure, anti-tumor, anti-allergic, anti-cancer, anticoagulant, analgesic and anti-insect (3). Its effective compounds include 1% volatile oil, resin, flavonoids, phenols, carotene, betaine and tannin (4). This study aimed to investigate the antimicrobial and antioxidant properties of M. piperita L. and to determine its main constituents using gas chromatography mass spectroscopy (GC-MS).
 

The plant was collected from Marivan area (Kurdistan Province, Iran) during summer 2017 and approved by the Faculty of Botany at Kashan University. The essential oil was extracted by microwave method (Using the Microwave Reactor, Milestone MicroSYNTH Model) without solvent. The main constituents of the plant’s essential oil were identified by gas chromatography/mass spectrometry (GC/MS) Agilent HP-6890 gas chromatography and Hewlett-Packard 6890-5972 (9). In this study, the antioxidant activity was investigated by β-carotene bleaching method (BHT standard as positive control) (10). The antioxidant activity was calculated using the "percent inhibition" formula:
I% = (A_sample - A_control) / (A_control(0) - A_control) × 100
where I% is the inhibition percentage, A_sample is the absorption of the sample, A_control is the absorbance of the control and A_control(0) is the absorbance of the control after the desired time. To investigate the antimicrobial activity, a set of microorganisms including Gram-positive and negative bacteria, fungi and yeast were used. The bacterial strains were cultured in Nutrient agar overnight at 37^°C and fungi and yeast were cultured in Sabouraud Dextrose Agar overnight at 30^°C.
The antimicrobial activity was determined by agar diffusion method. The microbial suspensions adjusted to 0.5 McFarland were cultured in Mueller Hinton Agar. At the end, the sample solutions were added to the wells. After 24 h, the diameter of growth inhibition zones were measured.
To obtain the minimum inhibitory conce-ntration (MIC), microdilution broth method was used according to CLSI (Clinical & Laboratory Standards Institute) (11). The samples were first diluted with dimethyl sulfoxide (DMSO) in sterile tubes containing Brain Heart Infusion (BHI) broth. The sample solutions, 0.5 MacFarland suspension and BHI broth were added to each well. The sample-free well was used as negative control. After 24 h, the lowest inhibitory concentration was considered as MIC. Rifampin and Gentamicin antibiotics (for bacteria) and Nystatin (for fungi) were used as positive controls.

 

The results obtained from analysis of the essential oil compounds using GC-MS are presented in Table 1. Menthol, neomentyl acetate and menthofuran were the main constituents of M. piperita L. essential oil, respectively and are highlighted in Table 1. In other studies, mentol, carvone, menton, neomentol and menthol acetate have been reported by Fadaie et al., (13). Mimica-Dukić et al., identified menthol (14) as the main constituents of M. piperita L. essential oil.
In other researches (reference), menthol has been found as a major component of the essential oil which is in accordance with our findings.
The lowest growth inhibitory concentration and diameter of growth inhibition zone of rifampin, gentamicin and nystatin antibiotics against the tested microorganisms are shown in Table 2 and those regarding the essential oil are shown in Table 3. The results showed that the essential oil has good inhibitory effect against Gram-positive bacteria. İşcan et al. and Sivropoulou et al. reported that peppermint essential oil had a greater effect against gram-positive bacteria than gram-negative bacteria (21, 22), which is similar to our findings. Tsai et al. reported the lowest inhibitory concentration of 0.08±0.00 % (v/v) for C. albicans and S. aureus (1). Afridi et al. reported the growth inhibition zone of 17±0.61 mm for S. aureus (23).
 
 

Area %	Kovats index	Retention time (min)	Compound	Number
0.67	939	6.34	alpha-Pinene	1
0.38	975	7.32	Sabinene	2
0.97	979	7.43	beta-Pinene	3
5.84	1031	8.99	1,8-Cineole	4
0.59	1098	10.09	trans-Sabinene hydrate	5
3.49	1152	12.98	L-Menthone	6
19.72	1164	13.34	Menthofuran	7
41.21	1171	14.05	Menthol	8
1.23	1237	15.74	Pulegone	9
0.28	1252	16.28	Piperitone	10
0.99	1295	16.71	1R-Menthyl acetate	11
21.49	1273	17.56	1S-Neomenthyl acetate	12
0.79	1305	17.87	Isomenthyl acetate	13
0.71	1419	20.93	Isocaryophyllen	14
0.82	1419	21.35	trans-Caryophyllene	15
0.52	1592	26.65	Viridiflorol	16
0.31	2100	39.25	Heneicosane	17

Nystatin		Gentamicin		Rifampin		Microorganisms
MIC (μg/mL)	IZ (mm)	MIC (μg/mL)	IZ (mm)	MICb (μg/mL)	IZa (mm)	Gram-positive bacteria
*	*	500	35	250	40	S. epidermidis
*	*	500	21	15.6	13	B. subtilis
*	*	500	21	250	10	S. aureus
						Gram-negative bacteria
*	*	500	18	250	8	S. dysenteriae
*	*	250	22	250	7	K. pneumonia
*	*	500	8	-	-	P. aeruginosa
*	*	500	21	-	-	S. paratyphi-A serotype
						Fungi
31.25	27	*	*	*	*	A. niger
31.25	30	*	*	*	*	A. brasilienis
125	33	*	*	*	*	C. albicans

Essential oil			Microorganisms
MIC (μg/mL)	IZ (mm)	Gram-positive bacteria
31.25	13	S. epidermidis
31.25	10	B. subtilis
62.50	10	S. aureus
62.50	10	S. dysenteriae
31.25	11	K. pneumonia
-	-	P. aeruginosa
-	-	S. paratyphi-A serotype
-	-	A. niger
-	-	A. brasilienis
-	-	C. albicans

The results of the β-carotene bleaching assay showed that the inhibitory potency for inhibiting linoleic acid oxidation for BHT standard and the essential oil was 96% and 93%, respectively. Accordingly, the percentage of inhibitory potency was very high, and this result indicated high antioxidant activity of M. piperita L. The high antioxidant activity may be due to the presence of a major menthol in peppermint essential oil. Yadegarinia et al. (19) and Tabatabaei et al. (26) reported the inhibitory potency of the essential oil 50.17% and 41.3%, respectively. Comparing the results of the present and other studies in Iran, it is found that the essential oil of M. piperita L. extracted from Marivan county has a higher antioxidant activity compared to that from other locations investigated.
Different extents of antimicrobial and antioxidant properties as well as distinct essential oil constituents in different studies may be due to differences in geographical areas, climatic conditions, harvesting times, storage conditions and duration, method and duration of extraction, tested microbial strains and the environment of different crops, etc.

 

The results of this study showed that the essential oil of M. piperita L. cultivated in Marivan has good antioxidant and antimicrobial activity. The major component of the oil is menthol. The essential oil effects could be attributed to their monoterpene compounds. The essential oil of this plant is one of the natural sources of antioxidants which could be suggested to replace the synthetic source.

 

We are grateful to Natural Essences Research Institute of Kashan University for supporting this work.

 

The authors reported no conflict of interest.