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GC-MS ANALYSIS AND ANTI-MICROBIAL ACTIVITY OF ESSENTIAL OIL OF MENTHA PIPERITA L. FROM KULLU-A NORTH INDIAN REGION OF HIGHER ALTITUDE HIMALAYAS

SHARMA VIVEK1*, SHARMA NISHA1, PATHANIA VIJAYLATA2, MALIK A. REYAZ1, SINGH BIKRAM2, GUPTA C. RAGHBIR1
  1. Department of Botany, Punjabi University Patiala-147002 (Punjab) India
  2. N.P.P. Division, I.H.B.T. (CSIR) Palampur-176061 (Himachal Pradesh) India
 
Corresponding Author : SHARMA VIVEK, Department of Botany, Punjabi University Patiala-147002 (Punjab) India, E-mail: vivek03sharma@rediffmail.com, Telephone: +91-98167-67189; +91-175-3046265
 
Received: 09 September 2010
Accepted: 24 November 2010
 
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Abstract

The essential oil analysis of Mentha piperita L. (Peppermint) has been done for the first time from study area of Northern Indian region of Kullu (1362m) district of Himachal Pradesh. The extraction yield for the essential oil of M. piperita L. was 0.42% for sample M-7. The oil was analyzed by GC-MS, the components of oil were identified by comparing their retention indices and mass spectra fragmentation patterns with those stored on the MS-computer library and also from the published literatures. The major constituents reported from essential oils of M. piperita were: L-Menthone (28.66%); Menthol (9.94%); Piperitone oxide (16.0%); Eucalyptol (7.03%); L-Menthone (3.13%); Isoneomenthol (2.93%); á-Phellandrene (3.21%); ä-3-Carene (3.27%); dl-Limonene (2.53%); á-Pinene (2.02%), etc. Furthermore, anti-microbial activity of oil was evaluated using agar well diffusion method. The anti-microbial test results showed that the oil had a potential anti-microbial activity against all seven Gram+ve and Gram-ve bacterial strains such as: Pseudomonas fluorescence, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Staphylococcus epidermis and Acenetobactor calcoaceticus. Essential oil showed maximum zone of inhibition and minimal inhibition concentration against Bacillus subtilis (MTCC-2451) and Pseudomonas fluorescence (MTCC-664) bacterial strains.



 

Key words

 
Mentha piperita L., Peppermint, Essential oil, GC-MS, Anti-microbial, Kullu (1362m), North India
 

Introduction

 
Mentha piperita L. (Peppermint) is a medicinally important plant that belongs to the family Lamiaceae (Kirethekar and Basu, 1985). This herb is frequently growing and cultivated in Europe, Asia, North America, Austraia and also in India for the production of peppermint oil. Peppermint is a non-native herbaceous plant, it is a perennial, which can reach up to 100 cm in height and has four-sided stem. The flowers are irregular in shape; they are pinkish or purplish (Clark and Menory, 1980).
 
Mentha piperita L. is a hybrid mint which originated probably due to accidental hybridization between M. aquatica and M. spicata. It is adapted to almost all areas and can be found in different altitudes. However, different hybridization experiments were carried out between M. piperita and other species of mints using techniques such as protoplast fusions (Krasnyanski et al., 1998; Sato et al., 1996).
 
The volatile oil extracted from the aerial parts of this herb is a source of commercial menthol. Peppermint leaves contain volatile oil that is composed of free menthol, monoterpene, menthofurane and traces of jasmine, which improve the oil’s quality remarkably (Dew and Evans, 1984). The essential oil of this species is most popular and widely used. It is employed for flavoring, pharmaceuticals, mouth washes, cough drops and confectionery. The oil has also antiseptic and local anesthetic properties. Peppermint oil or peppermint tea is often used to treat indigestion; it may also increase the flow of bile from the gall bladder (Mimica et al., 2003; Forster, 1996). Peppermint oils relaxing action acts as counterirritant and analgesic with the ability to reduce pain and improve blood flow to the affected area. Peppermint oil and menthol have moderate antibacterial effects against both Grampositive and Gram-negative bacteria (Diaz et al., 1988). Peppermint is also found to have anti-viral and fungicidal activity (Chaumont and Senet, 1978). Menthol is virucidal against influenza, herpes and other viruses. Aqueous extracts of peppermint leaves were anti-viral against influenza A, newcastle disease virus in egg and cell culture system was studied by Hirobe et al., (1994). Beside these, peppermint oil has been the subject of numerous other studies: Baser et al., (1999) studied the essential oils of Mentha species from Northern Turkey; Lawrence (1997) also reported the essential oils of peppermint; Gerherman et al., (2000) study the comparative analysis of some active principles of herb plants including M. piperita by GCMS. Aflatuni et al., (2000) studied the variation in the extract composition of mints of different origin cultivated in Finland. A comparative investigation on the essential oil composition of two Bulgarian cultivars of M. piperita L. was also carried out (Stojanova et al., 2000. Maffei (1999) studied the sustainable methods for a sustainable production of peppermint essential oil. Monoterpene composition of essential oil from M. piperita L. with regard to leaf composition using Solid- Phase Micro-extraction and GC-MS analysis was studied by Rohloff (1999). Variation of chemical composition of essential oil of M. piperita L. during the growing time was also investigated by Chalchat et al., (1997). Productivity and biochemical composition of M. piperita L. of different origins (Lithuanian, Polish and from Ukraine) was also studied by Dambrauskiene et al., (2008). Phenols and lactones in Italo-Mitcham peppermint oil M. piperita L. were studied by Naf and Velluz (1998).
 
As a part of our investigation on aromatic medicinal plants, the aim of this work is to provide more information on the composition of essential oil obtained from M. piperita L. from a naturally grown species, collected from Kullu (1362m) of Himachal Pradesh region of Northern India. Thus, it is the first record of analysis of essential oil along with antimicrobial activity of M. piperita L. from the study area.
 

Experimental

 

Plant material

 
Fresh leaves of Mentha piperita L. were collected from Kullu (1362m) of Himachal Pradesh from Northern India, during the month of June, 2008 (Table 1). Specimens were authenticated by the Botanical Survey of India (BSI, Northern Circle), Dehradoon, Department of Biodiversity, I.H.B.T. (CSIR) Palampur and deposited in the herbarium of Department of Botany, Punjabi University, Patiala (Punjab) India.
 

Oil distillation

 
Five hundred grams fresh sample of leaves from study area were separated and ground, then immersed in water in a round bottom flask and hydrodistilled for 4h in a full glass Clevenger-type apparatus as recommended by British Pharmacopoeia giving yellowish oils. The essential oil was dried over anhydrous sodium sulphate (Merck) until the last traces of water were removed and then stored in a dark glass bottle at 4 ºC prior to GC-MS analysis (Adams, 1991).
 

Gas chromatography-Mass-spectrometry

 
GC-MS (70ev) data were measured on GC-MS (QP 2010 series Shimadzu, Tokyo, Japan) equipped with AOC 20i autosampler and BP-20 capillary column (SGC International Ringwood, Australia) of 30m length, 0.25mm i.d. and 0.25µm film thickness. Temperature was programmed from 70-220 ºC at a rate of 4 ºC/min, held isothermally at 70 ºC and 220 ºC for 4 and 5 min, respectively. Mass spectrometer source temperature, 200 ºC; interface temperature, 220 ºC; injector temperature, 220 ºC. Sample injection volume 2µL (diluted 5µL oil in 2mL dichloromethane, HPLC grade); split ratio, 1:50 and mass scan, 50-600 amu. Helium was used as a carrier gas with 1.1mL/min flow rate.
 

Identification of components

 
The retention index was calculated for all volatile constituents using a homologous series of n-alkanes. The components of oil were identified by matching their mass-spectra with those stored in the computer library such as Wiley (McLafferty, 1989), New York mass spectral (MS) library (Jennings and Shibamoto, 1980; Adams, 1989), National Institute of Standards and Technology (NIST) (Stein, 1990) and their retention indices (RI) either with authentic compounds or with published data in the literature based on retention indices of components on same phases of polar columns such as: BP-20, CW-20M, HP-20M and Supelcowax-10.
 

Microbial strains for anti-microbial activity

 
The microorganism strains used in the agar disc diffusion method were supplied by the Institute of Microbial Technology, Chandigarh, India. Grampositive bacteria: Bacillus subtilis (MTCC-2451), Staphylococcus aureus (MTCC-740), Staphylococcus epidermis (MTCC-435), Gram-negative bacteria: Escherichia coli (MTCC-443), Salmonella typhimurium (MTCC-1251), Pseudomonas fluorescence (MTCC-664) and Acenetobactor calcoaceticus (MTCC-127).
 

Anti-microbial screening

 
In vitro anti-bacterial activity of the M. piperita essential oil was studied against seven bacterial strains by the agar well diffusion method as described by Perez et al., (1990) with certain modifications. Nutrient agar (Hi Media, India) was used as the bacteriological medium. The anti-bacterial activity of essential oils was taken at different concentrations (10, 20, 30, 40, 50, 60 and 70µL/well). The nutrient agar was melted and cooled to 48-50oC and a standardized inoculum of 1 × 106 CFU/mL, (0.5 McFarland) was then added aseptically to the molten agar and poured into sterile petri dishes to give a solid plate. Wells were prepared in the seeded agar plates. The test compound was introduced in the well (8.5 mm). The plates were incubated overnight at 37°C. The anti-microbial spectrum of the oils was determined for the bacterial species in terms of zone sizes around each well. The diameters of zone of inhibition produced by the agent were compared with those produced by the commercial control antibiotics, 20µL each of amoxicillin and ciprofloxacin (5mg/mL of autoclaved distilled water). These are commonly used anti-biotics to treat infections caused by several Gram-positive and Gramnegative bacteria. For each bacterial strain positive controls were maintained. The experiment was performed three times to minimize the error and the mean values were presented.
 

Minimal inhibition concentration

 
The essential oil that exhibited considerable activity was diluted with nutrient broth (1:1) in a series of seven sets of three test tubes for different microorganisms (Aboaba et al., 2006). An aliquot of 1mL of the bacterial suspension (1x106) was inoculated into each tube. The control tubes were inoculated with same quantity of sterile distilled water and 75% ethanol. All tubes were incubated at 37°C for 24hrs. The lowest concentration that did not permit any visible growth when compared with the control was considered as the minimum inhibitory concentration. The contents of all tubes that showed no visible growth were cultured on nutrient agar, incubated at 37°C for 24hrs. The minimum bactericidal concentration was considered as the lowest concentration that could not produce a single bacterial colony.
 

Results and Discussion

 
The extraction yield for the essential oils of M. piperita L. from Kullu (1362m) was 0.42% (M-7) for the sample. The essential oil analysis led to the identification of 28 constituents representing 83.06% of the compositions of oil. The GC-MS chromatograph showing different peaks of the essential oil constituents (Figure 1).
 
In the essential oil sample, some major constituents reported were as follows: L-Menthone (28.66%); Menthol (9.94%); Piperitone oxide (16.0%); Eucalyptol (7.03%); L-Menthone (3.13%); Isoneomenthol (2.93%); á-Phellandrene (3.21%); ä-3-Carene (3.27%); dl-Limonene (2.53%); á-Pinene (2.02%) and some unidentified compounds with high percentage such as: 5-Methyl-2-(1-methylethylidene)-cyclohexanone (5.98%) was also reported along with some minor constituents (Table 2).
 
Previous investigations on M. piperita oil composition are consistent with our results in which menthone and menthol was found to be the major compounds (Lawrence 1997; Gerherman et al., 2000; Aflatuni et al., 2000; Stojanova et al., 2000; Maffei, 1999; Rohloff, 1999; Chalchat et al., 1997; Spencer et al., 1997).
 
Anti-microbial activity showed that, the inhibition zones were found increased considerably when the concentration rate increased. Therefore it can be said that quantity of the oil was important for inhibition effect. Among all Gram-positive bacterial growths, the maximum zone of inhibition was recorded against Bacillus subtilis (MTCC-2451) i.e. 61.9mm, followed by 37.6mm in Staphylococcus aureus (MTCC-740) and 32.8mm in Staphylococcus epidermis (MTCC-435) at 70µL/well (Figures 2, 3).
 
On the other hand four different Gram-negative bacterial strains were tested and among these microorganisms, Pseudomonas fluorescence (MTCC- 664) showed maximum zone of inhibition i.e. 54.7mm, followed by Acenetobactor calcoaceticus (MTCC-127) i.e. 37.1mm. The minimum zone of inhibition was recorded against the Salmonella typhimurium (MTCC- 1251) strain i.e. 29.9mm (Table 3).
 
The minimal inhibition concentration (MIC) was 2µL recorded in Gram-negative strain Pseudomonas fluorescence (MTCC-664) (Figure 4) followed by a Gram-positive strains Bacillus subtilis (MTCC-2451) 2.5µL (Table 4).
 
From these, it is concluded that the essential oil showed maximum zone of inhibition and minimal inhibition concentration against Bacillus subtilis (MTCC-2451) and Pseudomonas fluorescence (MTCC-664) bacterial strains, which indicate that M. piperita L. essential oil has capacity to inhibit the growth of both Grampositive and Gram-negative bacterial strains when used in a higher amount. Further, on the basis of previous studies on Mentha genus and present results of M. piperita L., which proved that, it is a medicinal and aromatic plant that acts as an important anti-microbial agent against many gram-positive and gram-negative bacterial strains and also has higher percentage of some most important chemical constituents.
 
According to Fleming (1998) reported that, due to many potent compounds such as menthol, menthone, limonene, etc., in M. piperita shows significant antimicrobial activity. These compounds have higher medicinal value especially in the treatment of dyspepsia, epigastric bloating, impaired digestion. Deans and Baratta (1998) also investigated that the compounds from M. piperita possess anti-microbial activity and suggesting that the M. piperita leaf extracts should contains the effective active constituents responsible for eliminating the bacterial pathogens.
 
Therefore, finally, it can be concluded that the active chemical compounds present in M. piperita L. should find place in treatment of various bacterial infections. The results from the present investigation are very encouraging and indicate that herb should be studied more extensively to explore its potential in the treatment of infectious diseases as well.
 

Acknowledgment

 
The authors are grateful to the Director, IHBT (CSIR), Palampur (H.P) India and Department of Botany, Punjabi University, Patiala (Punjab) India for providing necessary facilities and support.
 

Conflict of Interest

 
NIL
 

Source of Support

 
NONE
 

Tables at a glance

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Table 1 Table 2 Table 3 Table 4
 

Figures at a glance

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Figure 1 Figure 2 Figure 3 Figure 4
 

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