Abstract: Background and Objective: Mentha longifolia is an extremely flavorful and aromatic herb of the mint family native to many parts of Europe, Asia and Northern and Southern Africa also known as horse mint. Extracts of M. longifolia have been used for centuries as a food flavoring and a medicine. The present study was aimed to reinvestigate the essential oil of Mentha longifolia (L.) Huds. for its chemical composition and concomitantly anti-inflammatory, analgesic, antipyretic, antibacterial and fungicidal activity. Materials and Methods: The essential oil was extracted using hydrodistillation method and analyzed by GC-MS. Anti-inflammatory, anti-nociceptive and antipyretic activities of essential oil were experimentally determined using mice model. Antibacterial activity and antifungal activity were determined by using disc diffusion and agar dilution method, respectively. Results were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. Results: The major constituents identified in the essential oil of Chamoli collection were piperitenone oxide (60.44%), 1,8-Cineole (6.16%), piperitenone (3.46%), dl-limonene (2.72%) and piperitone (1.69%). The Joshimath collection showed the presence of piperitenone oxide (36.89%), cis-piperitone oxide (17.89%), camphor (3.11%), isophorone (2.52%), thymol (2.41%) and coumarin (1.41%) and the Badrinath collection contains cis-piperitone oxide (42.74%), piperitenone oxide (33.48%), 4-Hydroxypiperitone (3.26%), thymol (2.52%), 2-Isobutylcyclopent-3-en-1-one (1.57%) and piperitenone (1.02%). The other constituents were present in minor quantities (less than 1.0%) in all three collections. The essential oil of Mentha longifolia show insignificant anti-inflammatory and analgesic activity but shows significant antipyretic, antibacterial and antifungal activity. Conclusion: The study indicated the wide chemical diversity in the essential oil of Mentha longifolia (L.) Huds collected from different geographical regions. The variation in chemical constituents may also effect the biological activities of the samples collected from different locations.
INTRODUCTION
A number of Mentha species, indigenous to Himalayan region grow wild in India which include Mentha longifolia, Mentha piperata, Mentha spicata and Mentha arvensis1,2. Mentha longifolia has the widest natural geographical distribution of any Mentha species from Central Asia to Western Europe and in Sothern Africa with 22 subspecies. In India its distribution is limited to Western Himalayas from Kashmir to Garhwal and Ladahkh, coming down to the sub-Himalayan ranges in Jammu. Four varieties of Mentha longifolia (L.) Huds viz. M. longifolia (L.) Huds var. longifolia, M. longifolia (L.) Huds incana variety with slender interrupted spikes and Mentha longifolia (L.) Huds var. royleana with stout continous spikes have been reported from Uttarakhand2.
The plant Mentha longifolia (Labiatae) is generally known for its multiple pharmacological activities like, antipyretic, anti-inflammatory activities3,4, antioxidant5,6, hepatoprotective7, spasmodic, choleretic, CNS stimulative effects8, calcium channel blocking activity9, antimutagenicity10 as well as antimicrobial activities5,11-16. Several chemotype of Mentha longifolia are reported from different countries. The differences in their chemical composition may be because of both biotic (genetic, ontogeny, morphogenic) and abiotic (soil, climate, altitude and temperature) factors which may affect biosynthesis and growth.
Mentha longifolia have been reported to contain a range of components, including ceramides17, cerebrosides 17, glycosides18, flavonoids10. The main active component of Mentha longifolia was its essential oil, which was reported to govern its various properties. Among different chemotypes reported from different country. Piperitone oxide rich chemotype has been reported from Greece19, Itly20, Lithuania21, Tajakistan22 and Tutkey23.
Piperitone oxide and piperidinone oxide rich varieties has been reported from Bossnia24, Egypt25, Tajakistan22, Greece26, India27-30, Iran31, Jordan32, Pakistan33, Turkey23, Ujbekistan34, Lithuania21 and Morracco35. While carvone rich chemotype has been reported from Crete36, Crotia37, Greece26, India38, Iran39, Sudan40.
Linalool rich with piperitone oxide and/or has been reported from Turkey23. Carveol rich chemotype has been reported from Iran41, dihydrocarvone rich from Serbia42, Turkey23, Mentha furane rich chemotype from South Africa43, Menthone rich from South Africa44, puligone/menthone/ isomenthone rich chemotype from Tunesia5 and Israel45 and 1,8 cineole rich chemotype from Iran46.
Keeping in mind the above chemotypic diversity, in present communication M. longifolia from three different regions of Uttarakhand was taken to investigate its chemical diversity and various pharmacological activities like anti inflammatory, antinociceptive, antipyretic, antibacterial and antifungal activity of the essential oil.
MATERIALS AND METHODS
Plant source: Fresh plant material was collected from three different locations (Badrinath, Chamoli and Joshimath) in mountainous region of Uttarakhand, India and was identified by plant taxonomist Prof. Y.P.S. Pangty. The specimens have been deposited in the Department of Chemistry, Pantnagar for future reference.
Isolation of essential oil: About 1000 g of the fresh collected aerial parts of the herbs were subjected to hydro distillation for 8 h using Clevenger-type apparatus according to the European Pharmacopoeia47. The oil was extracted in dichloromethane (Merk, AR-grade) and dried over anhydrous sodium sulfate. The yield of oil in different collections ranged from 0.1-0.2% w/v and the oil was stored at 4°C for further analysis.
GC/MS analysis: GC-MS data were recorded on gas chromatograph HP 6890 with mass selective detector MS 5973 (Agilent technologies, USA) fitted with a HP-5 msec fused silica column (30 m×0.25, 0.25 μm film thickness). The carrier gas was helium and initial column temperature was 50°C rising 250°C at a rate 5°C min–1 the MS detector acquisition parameters. Transfer line held at 260°C and detector was held at 280°C. Detection was performed in the full scan mode from m/z 41-450. Components were identified with the aid of an automatic system of processing data of GC-MS supplied by NIST mass spectra library. Further confirmation was done by referring to Kovats index data generated from a series of alkanes (C9-C28)48.
Experimental animals: Animals [Swiss albino mice (R)] were procured from Lab animal division, Central Drug Research Institute, Lucknow. The mice were divided into four groups of six mice each for the experiments. They were housed in standard cages at a constant temperature of 22±1°C, relative humidity 55±5% with 12 h light-dark cycle (08:00-20:00) for one week at least before the experiment. The experimental protocol was approved by the Committee on Animal Research (ethical committee) with Registration No. 330/CPCSEA. All tests were conducted under the guidelines of the ethical committee for the study. The essential oil collected from Badrinath was used for evaluation of pharmacological activities.
Anti-inflammatory activity
Carrageenan-induced mice paw edema: The anti-inflammatory activity of Mentha longifolia essential oil (MLEO) was determined by the carrageenan-induced edema test in the right hind paw of mice. Male ICR mice (six per each group) were fasted for 24 h before the experiment with free access to water. Fifty microliter of 1% carrageenan suspension (Sigma Co., USA) in saline was injected into the plantar side of right hind paw of the mice49. Percentage reduction of edema was measured immediately at 1st, 3rd and 24th h after the administration of the carrageenan, using a plethysmometer. Indomethacin (10 mg kg–1 b.wt.) was administered intraperitoneally 30 min before carrageenan injection. MLEO (50 and 100 mg kg–1 b.wt.) was orally administered 60 min before carrageenan injection. The control was given in an equal volume of saline.
Formaldehyde induced paw edema: The formaldehyde induced arthritis essential oil was determined by following the method used50. One percent solution of formaldehyde (0.10 mL) was injection in the right hind paw of the mice on day 1 of the experiment. Doses of M. longifolia essential oil (50 and 100 mg kg–1 b.wt.) were administered orally daily in the morning till the end of the study period i.e., for 10 days. Ibuprofen at a dose of 10 mg kg–1 b.wt., orally given used as the standard drug. Saline water was used as negative control. In the evening hours, plethysmometrically, paw volume of all the mice was measured for 10 days.
Analgesic activity
Acetic acid-induced writhing response: The writhing test in mice was carried out by following the reported method51. In the brief the writhes were induced by intraperitoneal injection of 1.0% acetic acid (v/v, 0.1 mL/10 g b.wt.). There are two different doses (50 and 100 mg kg–1 b.wt.) of MLEO administered orally to each groups of mice, 60 min before chemical stimulus. Ibuprofen as a positive control was administered 30 min prior to acetic acid injection. The number of muscular contractions was counted over a period of 30 min after acetic acid injection. The data represented the total numbers of writhes observed during 10 min.
Hot plate method: It is a simple and sensitive method for studying analgesic and hyper analgesic reaction in mice and was performed to measure analgesic response latencies52.
Antipyretic activity: To evaluate the antipyretic activity of essential oils, pyrexia was developed by using yeast by method described by Rao et al.53. Pyrexia was induced by subcutaneous injection of 10 mL kg–1 of 20% suspension of Brewer’s yeast (Sacchromyces cerevisiae). The mice were allowed to remain quiet in the cage for 18 h for a rise in body temperature. At 19th h, the rates were again restrained to record their rectal temperature. Immediately, 0.1 mL/10 g of the MLEO in the doses of 50 and 100 mg kg–1 b.wt. and paracetamol (33 mg kg–1 b.wt.) was administered orally. Control group received 0.2 mL normal saline. Temperature was recorded at hourly interval in all the mice upto 3 h.
Toxicity: The acute toxicity test in mice and rats was carried out. Female mice (R) (20-30 g) were divided into test and control groups comprising of 6 animals in each group. The test was performed by using increasing oral doses of MLEO (500, 800 and 1000 mg kg–1 b.wt.), in 10 mL kg–1 volume to different test groups. Control group received saline solution (10 mL kg–1). The experimental mice were allowed for food, were all kept under regular observation for 48 h, for any mortality or behavioral changes.
Antibacterial activity: The essential oils were screened against pathogenic bacteria’s including one gram-negative (Salmonella enterica enterica) and 1 gram-positive (Staphylococcus aureus). These were procured from Department of Pharmacology, College of Veterinary and Animal sciences, G.B. Pant University of Agriculture and Technology, Pantnagar, U.S.Nagar, U.K., India. Essential oil was dissolved in 10% Tween 20 which was also used as control. Antibacterial activity was determined by using the disc diffusion method54.
Fungicidal bioassay: The tested phytopathogenic fungi including Rhizoctonia solani, Sclerotium rolfsii and Fusarium oxysporum were procured from Department of Pathology, College of Agriculture, G.B. Pant University of Agriculture and Technology, Pantnagar, U.S.Nagar, U.K., India. The antifungal activity was measured by agar dilution method and expressed as percentage inhibition against the mycelia growth diameter55.
Statistical analysis: Data were expressed as Mean±SE. Results were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test and p<0.05 was considered to be statically significant.
RESULTS AND DISCUSSION
The results of GC-MS analysis of Mentha longifolia collected from different locations viz: Chamoli, Joshimath and Badrinath of Garhwal region (Uttarakhand) in the month of October during flowering stage are recorded in Table 1. The GC-MS analysis of the essential oil collected from Chamoli, revealed the presence of 20 compounds contributing to 82.11% of the oil. The major constituents identified in the oil were piperitenone oxide, 1,8-Cineol, piperitenone, dl-limonene and piperitone. The other constituents were present in minor quantities (less than 1.00%) which include α-pinene, β-pinene, 3-octanol, p-cymene, β-phellandrene (trans) decahydronapthalene, p-cresol, linalool oxide, α-terpinolene, p-cymene-8-ol, α-copaene, caryophyllene oxide, m-ethyl cumene and (t) muurolol.
In the essential oil of M. longifolia collected from Joshimath. sample, 13 compounds were identified which contributed to (76.75%) of the total oil. The major identified compounds were piperitenone oxide, cis-piperitone oxide, camphor, isophorone, thymol and coumarin. The other minor components present were piperitenone, resorcinol, sesamol, α-thujone, thymol and spathulenol.
In the essential oil of M. longifolia collected from Badrinath, 34 components were identified which contributed to 99.67% of the total oil. cis-piperitone oxide, piperitenone oxide, 4-Hydroxypiperitone, thymol, 2-Isobutylcyclopent-3-en-1-one and piperitenone, were identified as major components. The other minor components (less than 1.0%) were 3-Octanol, p-cymene, dl-limonene, trans (β) ocimene, linalool, 3-octanyl acetate, isophorone, borneol, 4-Terpineol, p-cymene-8-ol, α-terpineol, piperitone, β-bourbonene, β-elemene, β-caryophyllene (E) β-farnesene, dehydroaromadendrene, germacrene-D, δ-cadinene, Spathulenol, caryophyllene oxide, widdrol, muurolol, α-cadinol, trans-piperitenone oxide, resorcinol, cis-iso piperitenone and Z-cinerolone.
Comparison of the results recorded in Table 1 revealed a significant difference in the chemical make up of the essential oils of Mentha longifolia collected from different locations. Particularly, cis-piperitone oxide (42.47%) was the major component in Badrinath collection and higher in amount collected from Joshimath(25.47%) and absent in Chamoli56. This compound was also present (7.04%) in Sirmaur, Kashmir oil28, 23.20% from Harsil, India 29 and was absent in Mana30 and Iran collection46. Piperitenone oxide (60.44%) present in essential oil from Chamoli was close to the earlier reported 58.90%56 but greater than in other samples of Badrinath (33.48%), Joshimath (36.89%), Harsil (18.60%)29 and Sirmaur (54.23%)28. This was found to be present (21.20%) in Mana sample30,45 and (18.90%) in Israel sample47. The species growing in lower elevations was different than M. longifolia (L.) Huds. Subsp. himalaiensis growing in higher elevations. Carvone earlier reported (9.80%) and (0.09%)28,56, it was found to be absent in other semples30,45. Similarly 4-Hydroxy piperitone oxide (1.50%) reported earlier56 was not present in any of the collections. Camphor (3.11%), coumarin (1.41%), nonen-2-one (2.37%) sesamol (0.52%) and α-thujone (0.35%) present in the samples from Joshimath, were absents in other samples reported earlier. Sample from Joshimath contained Isophorone (2.52%) greater than that present in the sample from Badrinath (0.74%) but was absent in the oil from Chamoli. This compound was not present in M. longifolia analysed earlier. Furthermore, there was great variation in the minor components of the essential oil. The difference in the chemical makeup of the oils from different locations may be due to difference in ecological conditions altitudes and temperature of the collection sites. Many M. longifolia sp. growing in mediterranean region are piperitenone oxiderich chemotypes20,57,58 and piperitone oxide type. Samples contained cis-piperitone oxide but not trans-piperitone oxide in all the collections whereas sample from Sirmaur contained (24.06%) and Mana (48.70%) contains trans-piperitone oxide but not cis isomer. However, could not find any collection which is Carvone type growing in higher elevations. Though Carvone type are reported from Iran39and Sudan40 and pulegone type from Jordan25 and Tunisia5. The results are significant from chemical diversity point of view.
Anti-inflammatory activity
Carrageenan-induced mice paw edema: The anti-inflammatory effects of the essential oil on carrageenan-induced edema in the mice right hind paw are presented in Table 2. There was a gradual increase in edema paw volume of mice in the control and sample group. However, in the standard drug treated group a significant reduction in edema was observed at 24th h (37.19%). The essential oil could not produce significant reduction in paw volume compared to control. The inhibitory effect of the oil recorded with a dose level of 50 and 100 mg kg–1 in 24 h were 3.57 and 7.22%, respectively in 24 h.
| Table 1: | Variation in the essential oil compostion (% in oil) of Mentha longifolia from different regions |
| t: Trace (less than 1%) | |
| Table 2: | Acute anti-inflammatory activity of essential oils of, Mentha longifolia (Mean±SE, n = 6) |
One-way ANOVA followed by Dunnett’s multiple comparison test, aSignificant (p<0.05) as compared to control, bSignificant (p<0.05) as compared to drug, MLEO: Mentha longifolia essential oil | |
Sub-acute anti-inflammatory activity: No significant inhibition was generated at doses of 50 and 100 mg kg–1 MLEO as compared to the control (Table 3) in sub-acute anti-inflammatory activity, where arthritis was induced by formaldehyde injection on day zero and the samples were administered orally daily for 10 days. During the investigation the essential oil was found to be insignificant comparatively standard drug, ibuprofen.
| Table 3: | Effect of essential oil of Mentha longifolia on formalin induced sub acute inflammation (Mean ± SE, n = 6) |
One-way ANOVA followed by Dunnett’s multiple comparison test, aSignificant (p<0.05) as compared to control, bSignificant (p<0.05) as compared to drug, MLEO: Mentha longifolia essential oil |
|
| Table 4: | Anti-nociceptive activity of essential oils of Mentha longifolia (Hot Plate Method) (Mean±SE, n = 6) |
One-way ANOVA followed by Dunnett’s multiple comparison test, aSignificant (p<0.05) as compared to control, bSignificant (p<0.05) as compared to drug, MLEO: Mentha longifolia essential oil |
|
| Table 5: | Anti-nociceptive activity of essential oils of Mentha longifolia (Writhing effect) (Mean±SE, n = 6) |
One-way ANOVA followed by Dunnett’s multiple comparison test, aSignificant (p<0.05) as compared to control, bSignificant (p<0.05) as compared to drug, MLEO: Mentha longifolia essential oil | |
Analgesic activity
Hot plate test: The hot plate test was useful for the evaluation of centrally acting analgesics which are known to elevate the pain threshold of mice towards heat59. The low reaction time shown by the mice treated with the plant oil suggests that it is not a centrally acting analgesic and thus is ineffective (Table 4).
Acetic acid-induced abdominal writhing test: Data recorded in Table 5 on the acetic acid-induced writhing responses in mice are indicative of no analgesic activity of essential oil. Ibuprofen significantly reduced writhing responses induced by acetic acid when compared with the control group. There was no significant effect of MLEO at 50 and 100 mg kg–1 in decreasing writhing responses in mice and showed inhibition of only 1.15 and 4.41% compared to control (Table 5).
Antipyretic activity: The results of the antipyretic activity of the essential oil are presented in Table 6. Administration of the yeast to the rats produced significant increase in rectal temperature 18 h after yeast injection. The essential oil produced a moderate antipyretic activity. Paracetamol and MLEO showed significant antipyretic activity throughout the test period of 3 h. The essential oil showed maximum inhibition (37.61°C±0.01) at 100 mg kg–1 in 3 h and didn’t showed instant effect as the standard drug paracetamol (Table 6).
Acute toxicity: The sample was administered intraperitoneally and orally at doses of 150, 300, 450 and 600 mg kg–1 of body weight. The animals were observed during the first two hours for toxic signs and then mortality was recorded for each group at 24, 48 and 72 h after dose administration. Essential oil did not cause any behavioral changes and no death was observed. Thus it was considered to be a practically non-toxic substance.
Antibacterial activity: It has been reported that the essential oil components act on outer membrane permeability in gram-negative bacteria60. As recorded in Table 7, essential oil of Mentha longifolia (MLEO) showed moderate antibacterial activity against the tested pathogenic bacterial strains. The activity of essential oil showed a zone of inhibition of 18.70+0.6 mm against S. aureus and 17.13+0.3 mm against S. enterica enterica.
Antifungal activity: The results of antifungal activity assays against the three phytopathogenic fungi showed that the essential oil has inhibitory effect on the growth of fungi. At 250 ppm essential oil inhibited the growth of Fusarium oxysporum above 92% followed by Sclerotuim rolfsii (70.66%). The essential oil was found to be least effective against R. solani (Table 8).
Although leaf extract of Mentha longifolia was reported to show anti-inflammatory, analgesic and antipyretic activity3,4 but the essential oil of Mentha longifolia show insignificant anti-inflammatory and analgesic activity but shows significant antipyretic activity.
| Table 6: | Effect of essential oils of Mentha longifolia on yeast induced pyrexia in mice (Mean±SE, n = 6) |
One-way ANOVA followed by Dunnett’s multiple comparison test, aSignificant (p<0.05) as compared to control, bSignificant (p<0.05) as compared to drug, MLEO: Mentha longifolia essential oil |
|
| Table 7: | Antibacterial activities of the essential oils of Mentha longifolia |
| Values are means of three replications±SE, Means with the same letter are not significantly different at p<0.05, MLEO: Mentha longifolia essential oil | |
| Table 8: | Antifungal activities of essential oils of Mentha longifolia |
Values are means of three replications±SE, one-way analysis of variance (*Multiple comparisons versus control group (Dunnett's Method): p<0.050, MLEO: Mentha longifolia essential oil | |
It can be concluded that the activity of Mentha longifolia leaf extract was reported earlier are not due to essential oil constituents.
Since the essential oil consists of complex mixture of numerous components. The major or trace components may be responsible for the antimicrobial activities. Piperitone, cis-piperitone oxide, thymol, piperitenone oxide present in the oils investigated are also reported to be highly effective against various phytopathogens and bacterial strains61-63.
CONCLUSION AND FUTURE RECOMMENDATIONS
The study indicate the wide chemical diversity in the essential oils of Mentha longifolia (L.) Huds. collected from different geographical regions both qualitatively and quantitatively. It is reported that various biological activities in essential oils are associated with the synergetic effects of minor compounds. Based on these facts the diversified chemical composition of M. longifolia can be used for various biological activities, however, no report on pharmacological activity of M. longifolia essential oil exist, although work on leaf extracts is reported.
These results recommended that further work is required to check the genetic variability among the different populations using DNA and protein profiling.
SIGNIFICANCE STATEMENTS
The present study reveals the significant chemotypic diversity in the chemical composition of essential oils of Mentha longifolia (L.) Huds. with the population distributed in Mediterranean region and in Western Himalayan regions reported so far. The chemical variation may lead to variation in pharmacological activities. The present study will help to take up further studies on pharmacological activities with other chemovariants of Mentha longifolia (L.) Huds. to correlate the activities since this plant has many ethno-botanical uses. The study is of significant academic importance to study the biodiversity of medicinal and aromatic plants.
ACKNOWLEDGMENTS
Financial assistance from University Grants Commission, New Delhi (F. No. 41-234/2012(SR)) is acknowledged. Dr. D.S. Rawat plant Taxonomist, Department of Biological Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar, U.S. Nagar, Uttarakhand, India is thankfully acknowledged for identification of plant.