Anti-Inflammatory Activity of a Mixture of Peppermint and Thyme Volatile Oils

Received: 13-Dec-2021;Accepted Date: Dec 27, 2021; Published: 03-Jan-2022

Introduction

The use of natural remedies is still increasing, despite the rapid development of the chemical drug industry. According to World Health Organization (WHO) more than 80% of the world’s population relies on traditional medicine for their primary healthcare needs. The newest developed products need to be safe, have better quality and effectiveness.

Inflammation is known today by the presence of five external sings: tumor-swelling of the tissue, calor-elevated tissue temperature, rubor-blood color-like redness of vascularized tissue at the inflammation site, dolor-intensive sensation of a noxious stimulus, and functio laesa, impaired function of the organ affected [1].

The research of the anti-inflammatory effect of plant extracts can lead to the development of new useful treatments to reduce inflammation [2].

Thymus vulgaris (thyme) and Mentha piperita (peppermint, mint) are members of the Lamiaceae family.

Thymus vulgaris is a perennial, aromatic herb, native to southern Europe. It is used for a long time as a flavoring agent, culinary herb and herbal medicine. The principal constituents of volatile oil of Thymus vulgaris are thymol and carvacrol, which have, antimicrobial, antioxidative, expectorant, antitussive, antispasmodic, anti-inflamatory, and antibacterial properties [3-9].

Mentha piperita is a perennial aromatic herb, native of the Mediterranean regions, and a natural hybrid between spearmint (Mentha spicata L.) and water mint (Mentha aquatic L.). It is used for flavor, fragrance, and in traditional medicine and drugs industry. The main chemical compounds of volatile are menthol and menthone. As medicinal herb is known for chemopreventive potential, antiallergenic effects, antitumor and antimicrobial, antioxidant properties and for digestive complaints [10-14].

Because there are research results about the anti-inflammatory activity of thyme [15] and peppermint volatile oils [16], our goal in this study is to report the anti-inflammatory activity and the chemical composition, emphasizing the marker compounds (menthol, menthone, thymol, and carvacrol) of a mixture of the two volatile oils in a definite proportion.

Material and Methods

Volatile oils

Mentha piperita and Thymus vulgaris volatile oils (Mayam®) were purchased from ELEMANTAL SRL, ORADEA, ROMANIA. The mixture of volatile oils (MVO) was prepared by homogenization of Mentha piperita volatile oil: Thymus vulgaris volatile oil 40:60 (V/V).

HPTLC analysis of volatile oils

Qualitative analyses for volatile oils have been performed according to TLC Atlas-Plant Drug Analyses [17]. 0.5 mL MVO (T5) was dissolved in HPLC grade toluene, and diluted in the same solvent to 10 mL. Reference substances, were prepared as follows: thymol (T1)-5 mg in 10 ml of toluene, carvacrol (T2)-10 μl in 10 ml toluene, menthol (T3)-50 mg in 10 ml toluene and menthone mixture of isomers (T4) 50 mg in 10 ml toluene. Then 10 μL of the samples and 10 μL of reference substances were loaded as 10 mm bands on 20 × 10 cm TLC plates (Silica gel 60F254 CAMAG LINOMAT 5 instrument, Camag, Switzerland). The mobile phase (1) was methylene chloride and mobile phase (2) toluene: ethyl acetate (97:3 V/V); After development, plates were dried and derivatised with anisaldehyde solution R1 reagent at 100^°C for 10 minutes. The fingerprints were evaluated in fluorescence mode at 254 nm and in daylight (after derivatisation) with WinCats and VideoScan software.

Gas chromatography analysis

Gas chromatographic analysis was performed using an AGILENT- 6890N, equipped with a flame ionization detector (FID) detector and a HP-5 (5% phenyl/95% dimethylpolysiloxane) fused silica capillary column (30 m × 0.32 mm; film thickness 0.25 μm). Nitrogen was the carrier gas (2.0 mL min^-1). The injector temperature was kept at 220^°C and the oven temperature program was from 60^°C to 160^°C at a rate of 2^°C min^-1. Detector (FID) was operated at 250^ºC. 0.2 mL MVO diluted to 10 ml with h-heptane. 1 μL were injected in split mode (50:1).

Experimental animals

Males Wistar rats (150 g-190 g) provided by Biobase of Cantacuzino Institute, Bucharest (Romania) with health certificate were used in the in vivo experimental studies.

All experiments in the present study conformed to regulations issued by FELASA (Federation of European Laboratory Animal Science Associations) and ARSAL (Romanian Association for Laboratory Animal Science) and were approved by the local ethics committees of ICCF-Bucharest for animal experimentation.

Animals were housed under standard environmental conditions, at 22 ± 2^°C, relative humidity 50%-60%, under a 12 h dark light cycle with free access to water and standard pellet diet. The animals were checked at the reception and kept 7 days for acclimatization in the ICCF vivarium, observing the clinical status, behavior and food consumption. The animals received standardized food and water ad libitum. Animals were deprived of food 12 hours prior to the dosing.

Anti-inflammatory test

The animals were were divided into different groups (n=6)<one irritated and treated with MVO (group I), one control group irritated and treated with commercial product, Voltaren forte gel, 23,2 mg/g diclofenac diethylamine (GlaxoSmithKline Consumer Halthcare SRL) (group II) and one control group irritated, but untreated (group III). For administration, 2% dilutions of MVO was prepared in a DMSO: ethanol: distilled water 10:10:80 (v/v); Measurement of rat paw volumes with induced inflammation was carried out using the Plethysmometer 7140 Ugo Basile (Italy), equipped with dedicated CUB software for data acquisition and storage.

The in vivo method detection of the anti-inflammatory effect has been modified for topical administration of test samples [18]. The anti-inflammatory treatment was applied daily for 3 days before inducing the experimental edema, as well as 2 hours before, as follows: 0.7 ml of the test sample were administered by epicutaneous route (topical) in a diluted form 2%, by gentle massage in order to facilitate the skin absorption, into the right hind paw of the animals in group I.

The reference products were administered as likewise into the right hind paw of the animals in groups group II.

Control group III received pretreatment only with vehicle solution (10% (v/v) DMSO, 10% (v/v) ethanol, 80% (v/v) AD), in similar quantity.

Anesthetized rats in all groups received by a subcutaneous injection 0.05 ml sterile saline containing 1% (w/v) carrageenan into the plantar subaponevrosis of the right hind paw.

At 1, 2, 3 and 4 hours following the carrageenan injection, paws edema was evaluated.

In order to make the readings, the inflamed paw was visually marked at the level of the lateral malleolus, then inserted into the immersion fluid of the device measuring cell up to the marked level, with 4 plethysmometric measurements for each animal and for each moment established according to the protocol with data storage through the plethysmometer software.

Statistics

Data were reported as mean ± standard deviation (DS). Differences between groups were analyzed by one way ANOVA and Tukey’s post-hoc test of Microsoft Excel

Results

HPTLC profiles of the MVO are shown in Figures 1 and 2 and compared to reference substances thymol and carvacrol are shown in Figure 3 and to menthol and menthone mixture of isomers in Figure 4. HPTLC analysis was made following the chromatographic conditions from European Pharmacopoeia 9.0 [19] Monographs for Peppermint oil and Thyme oil.The MVO/T5 shows thymol/T1 (Rf~0.6) and his isomer carvacrol/T2 (Rf~0.56) as two pink zones in system 1 and is also is characterized by the prominent blue zone of menthol/T3 (Rf~0.27) and of menthone mixture of isomers/T4 (Rf between 0.04-0.58).The MVO chromatographic profile, analyzed by GC is presented in Table 1. The GC of the MVO resulted in the identification of 11 compounds. Chemical compounds found in MVO were terpenes. The major compounds were thymol, menthol, and p-cymene. The chromatographic profile of MVO shows the presence of all 4 marker compounds of the Thymus vulgar is volatile oil (thymol and carvacrol) and Mentha piperita volatile oil (menthol and menthone)

Figure 1: Chromatograms of the MVO in HPTLC analysis (SYSTEM 1).

Figure 2: Chromatograms of the MVO in HPTLC fingerprints in SYSTEM 2.

Figure 3: Comparison between MVO and thymol and carvacrol fingerprints in SYSTEM 1.

Figure 4: Comparison between MVO and menthol and menthone mixture of isomers in SYSTEM 2

Table 1: List of the compounds present in the MVO analyzed by GC to show the retention time and the area percentage of each compound.

Anti-inflammatory test

The average values of the volumes measured for each animal/ moment of measurement were calculated, then the average values per group/moment of measurement and the standard deviation.

Table 2 presents the average values for the measured volumes and standard deviations (SD) for the initial moment (before injection) and the next measurements

Table 2: Effect MVO and Voltaren upon carrageenan-induced paw edema. Average paw volumes and SD (ml)

Discussion

Until now there are research studies made only for individual volatile oils of the two species. For Thymus vulgaris the results for anti-inflammatory properties of diet administration of volatile oil in different doses (1250, 2500, 5000 ppm), on paw edema induced by carrageenan in topical administration, shows a descrease of paw swelling depending on the time and dose administrated [20]. The anti-inflammatory activity of Mentha piperita volatile oil in croton oil induced mouse ear edema model, in comparison with indomethacin (300 μg per ear) was significantly higher at all the administrated doses (200, 400 and 800 μg) (16).

The results of the anti-inflammatory activity of 2% MVO are presented in the Figure 5, showing the value of average paw volume of treated and control group animals at 1, 2, 3 and 4 hours after the injection of carrageenan. Maximum anti-inflammatory activity was observed in group 2, treated with 2% Voltaren (23,2 mg/g diclofenac diethylamine). Group 1 treated with 2% MVO showed a slightly weaker anti-inflammatory effect compared with group treated with Voltaren (23,2 mg/g diclofenac diethylamine), but differed significantly from the control, at all measurement times.

Figure 5: Evolution of carrageenan-induced acute edema (differences in average volumes per group)

Carrageenan injection produced evident paw edema in all groups since 1 hour and the evolution of the edema in presented in Figure 5.

Table 2 shows the paw volume of the rats at regular intervals of 1 hour. Carrageenan injection produced evident paw edema in all groups since 1 hour and the evolution of the edema.

Conclusion

This work showed the chromatographic profile anti-inflammatory activity of a mixture of Mentha piperita and Thymus vulgaris volatile oils. The data reported suggests the therapeutic potential of the mixture as a source for the development of novel anti-inflammatory agents. Therefore, combination of volatile oils should attract the interest of researchers and pharmaceutical companies for further investigations.

Acknowledgments

This work was supported by the ANCSI program POCA1- A1.2.3-G-2015, Project title<New technologies and natural derived products for human health use>, Ctr. No. 60/05.09.2016, ID P_40_406, SMIS 105542, Ctr. D, No 36/07.11.207

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