Essential oils are chemical products produced by odoriferous glands from a variety of plants. These essential oil have many health benefits i.e. antiseptic, anti-inflammatory and antimicrobial activities. So due to these medicinal properties present study was designed to analyze essential oil of Thymus vulgaris and Mentha arvensis for their chemical composition and biological activities.
Essential oil from these plants were extracted by hydrodistillation method, and analyzed by GC-MS. To test the microbial activity of these oil disk diffusion method and micro wells method were used. For free radical scavenging DPPH assay was used. However total phenolic content was measured by colorimetric method.
The GC-MS analysis of T. vulgaris oil showed the presence of 47 chemical compounds among which thymol, terpinene, p-cymene and carvacrol were major. However essential oil of M. arvensis showed the presence of 28 constituents, among which the Menthone, Menthol, Isomenthone, Eucalyptol, neo-Menthol, cis-Piperitone oxide, Linalool, Thymol, Limonene, and α-Phellandrene were major. Essential oil from both these plant tested for antimicrobial activity showed that the T. vulgaris oil was effective against seven bacterial strains and the essential oil of M. arvensis was effective against six bacterial strain. The antioxidant activity of both samples by DPPH assay which showed positive result.
As both species showed the presence of active components, positive microbial activities, and antioxidant activity so, research should be carried on for further biological activities of these oil for betterment of living beings.
Uçucu yağlar, antiseptik, antiinflamatuvar, antimikrobiyal gibi çeşitli etkileri olan, farklı bitkilerin koku salgı bezleri tarafından üretilen kimyasal ürünlerdir. Bu çalışma, esansiyel yağların bu tıbbi yararları gözönünde bulundurularak Thymus vulgaris ve Mentha arvensis’in uçucu yağlarının kimyasal bileşimlerinin ve biyolojik aktivitelerinin analizi amacıyla tasarlanmıştır.
Bitkilerden elde edilen uçucu yağlar hidrodistilasyon metodu ile ekstrakte edilmiş ve GC-MS ile analiz edilmiştir. Yağların mikrobiyal aktivitesini test etmek için disk difüzyon yöntemi ve mikro kuyucuk metodu kullanılmıştır. Serbest radikal temizleyici etki DPPH yöntemi ile, fenolik içerik ise kolorimetrik metod ile ölçülmüştür.
Her iki uçucu yağın GC-MS analizi sonucunda, T. vulgaris yağının yapısında aralarında timol, terpinen, p-simen ve karvakrolün bulunduğu 47 kimyasal bileşiğin varlığı; M. arvensis yağının içeriğinde ise menton, mentol, izomenton, ökaliptol, neo-mentol, cis-piperiton oksit, linalol, timol, limonen ve α-felandren bileşenlerini içeren 28 kimyasal bileşenin varlığı tespit edilmiştir. Antimikrobiyal aktiviteleri incelendiğinde, T. vulgaris yağının 7 bakteri suşuna karşı, M. Arvensis uçucu yağının ise 6 bakteri suşuna karşı etkili olduğu belirlenmiştir. Her iki uçucu yağın antioksidan aktivitesi, DPPH testi ile pozitif sonuç göstermiştir.
Her iki uçucu yağın aktif bileşenlerinin varlığı, pozitif mikrobiyal ve antioksidan aktiviteleri, bu yağların biyolojik aktiviteleri ile ilgili daha ileri çalışmalara ihtiyaç olduğunu göstermektedir.
Since ancient times, people used herbs and parts of plants as food and medicine and with the passage of time instead of plant parts, plant products are also utilized for phytotherapy like essential oils from aromatic plants, are used to treat different aliments due to wide array of biological activities i.e. antiseptic, anticancer, spasmolytic, hepatoprotective, antimicrobial, antioxidant, and pesticidal . These essential oil are mostly utilized in aromatherapy to treat different disorders. The essential oils are chemical products with low boiling point, greatly soluble in organic solvents, produced by odoriferous glands in a great variety of plants. These oils accumulate in all parts (leaves, barks, woods, flowers, fruit, rhizomes and seeds) of the plant. These oil contain different chemical compounds due to which they have many important pharmacological properties. Some of the chemical components (d-limonene, d-carvone or geranyl acetate) of these essential oils are used at industrial level as a scents, cosmetics, detergents, taste makers, cleansers and as solvents.
Due to different biological activities, a strong interest is developed from last few years in essential oils collected from different odoriferous plants . Until now about 3000 essential oils are observed from different plants species, out of these 300 have commercial importance as medicine, nutriment and cosmetics. One of the famous aromatic plant families is Lamiaceae, this family is important due to production of their essential oil from odoriferous plants. This family consists of about 252 genera and more than 6700 species . Essential oils from the members of this family known to possess antiseptic, anti-inflammatory and antimicrobial activities . So due to vast medicinal properties present study was designed to analyze essential oil from two members (Thymus vulgaris and Mentha arvensis) of this family.
Whole plant of T. vulgaris L. (Figure 1) at flowering stage were gathered from Ganga Choti, a mountain lying in Bagh, Azad Kashmir, Pakistan and the fresh aerial parts of M. arvensis L. (Figure 2) were collected from Muzaffarabad, Azad Kashmir, Pakistan. Plant species were identified with the help of literature and authenticated by taxonomist. Samples of plants are shad dried, converted into fine powder and stored at room temperature for later use.
Extraction of essential oil was done by hydrodistillation method with Clevenger apparatus shown in Figure 3, following the protocol of European Pharmacopoeia , . Sixty gram of dried thyme powder with 2.5-L water was poured into the apparatus and extraction was carried out for 2 h until no more oil was left. Thyme oil was decanted after every fraction and collected in Eppendorf tubes. The oil was dried with anhydrous sodium sulfate. After that the oil was stored at refrigerator at 4°C.
For extraction of oil from M. arvensis the protocol of Souza et al.  was followed, 1 kg of fresh leaves were grind and put into the distillation apparatus. The remaining container was filled with 2.5 L distilled water the processes was carried out for 2 h to obtain maximum oil. Same process was repeated until no more oil was left. Mentha oil was decanted after every fraction and collected in Eppendorf tubes. The oil was dried on anhydrous sodium sulfate and stored at 4°C before GC-MS Analysis.
Both samples of essential oil were analyzed by GC-MS (Hewlett-Packard model 6890 and Agilent 7890A, USA) by following the protocol of Negahban and Saeedfar . Essential oil were observed by Hewlett-Packard model 6890 (Figure 4) working at ionization energy of 75 eV with a DB-5 capillary tube of 0.25 mm diameter, 0.25 μm film thickness and 30 m length. The temperature was set to increase with regular interval of time from 60 to 23°C at 5°C/min. Helium was used as a carrier gas at 1.5 mL/min. MS source temperature was retained at 20°C, sample injection volume was 3 μL, split ratio was 1:60, interface temperature was 230°C and mass scan was 30–655 atomic mass unit.
The analysis was carried on Agilent 7890A attached with FID (Flame Ionization Detector), narrow capillary column (0.25 mm diameter, 0.25 μm film thickness and 30 m length). The temperature was set to increase with regular interval of time from 60 to 230°C at 5°C/min. Nitrogen gas was used as Carrier gas at 2 mL/min. Quantitative data were obtained from FID area percentages.
For identification of compounds after the oil at same conditions n-alkanes series was injected to calculate the retention index (RI) of all volatile constituents. The constituents were identified by comparison of their RI with the saved library data such as New York mass spectral library, Wiley Library and by comparison of the fragmentation patterns of the mass spectra with those reported in the literature , .
Essential oil of T. vulgaris was tested on six different strains of bacteria (Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhimurium) by using disk diffusion method. For the assay microorganism suspension (106 cells/mL) was poured on the solid media plate (Muller-Hinton agar). Filter paper (Whatman No. 1, pore size 11 μm) were impregnated with different concentrations (5, 10, 15 and 20 μL) of essential oil and put on the prepared agar. The plates were then inoculated for 26 h at 38°C. Ciprofloxacin (25 μg/disk) and cephalexin (15 μg/disk) were used as positive controls. The diameter of the zone of inhibition after incubation was calculated in millimeters and quantitative data was generated from these results.
The antibacterial activities of M. arvensis was tested with the help of broth dilution assay. The assay was performed in 0.2% sterile peptone water. The same bacterial strains were used for T. vulgaris antioxidant activity were suspended in that water with the turbidity visually corresponding to 0.6 Mc Farland. Different dilution (0.09–50.1%) of the oil were made. Fifty microliter of bacterial suspension was inoculated into micro wells with the same amount of essential oil dilution and incubated at 38°C for 26 h. After incubation minimum inhibitory concentration (MIC) was determined. Experiment was performed in triplicates.
Antifungal activities was carried out on Candida albicans by using Sabouraud agar and Malt Extract-Agar. For toxicity measurement poisoned food technique was used. For the assay inoculum was taken on 5 mm diameter disc and put at the mid of each petri plate containing PDA media. 0.5 mL of different concentration (0, 0.7, 1.30, 2.7, 5.5, 10.6, 100 μL mL−1) of both samples were then incubated at 30°C for 6–7 days with these culture. Fluconazole (15 μg/disk) was used as positive control. For quantitative data, percentage inhibition of mycelia was measured one by one.
To check percentage of phenolic contents, colorimetric method was used by following the protocol of . Gallic acid solution was used as a standard.
The essential oil of T. vulgaris was checked for anti-oxidant property on 2,2-diphenyl-1-picrylhydrazyl (DPPH) by following the protocol of , . For that, 5 mL of DPPH solution were taken with four different concentrations of extract. After incubation for 20 min the absorbance of sample was measured at 517 nm with spectrophotometer. Methanol was taken as blank and quercetin as reference compound.
Percentage inhibition was measured by following formula
Oil obtained from T. vulgaris is known as yellow oil. That yellow oil was subject for its chemical constituents result of the study showed the presence of 47 recognized chemical constituents shown in Table 1. The compounds with higher percentage were terpinolene (3.15%), p-cymene (8.53%), γ-terpinene (9.48%), carvacrol (3.35%) and thymol (60.55%). These result were agree with that of Negahban and Saeedfar , these researcher also showed the higher percentage of thymol (49%), β-cymene (19.99%), carvacrol (7.63%) and trans-caryophyllene (6.79%) in T. vulgaris. Miladi et al. also showed the higher percentage of thymol (52.19%) followed by content of carvacrol (6.72%) . In different studies the percentage of these chemical components are slightly vary from each other one of the research relate these variation to the different climatic conditions of the world which vary in different geographical region of the world. Quantity may also vary due to health and variety of the plant, age of the plant at the time of collection, method of drying and to the method of extraction of the oil from samples . The effect of different geographical zone on percentage composition was also confirmed by the research of Satyal et al.  who worked on thyme collected from different regions the result of their study showed that the T. vulgaris collected from France showed higher percentage of linalool (76.2%) and linalyl acetate (14.3%) where the thyme obtained from Serbia showed the higher presence of geraniol (59.8%) geranyl acetate (16.7%). In literature study it is also observed that the amount of carvacrol is also higher in other species such as Thymus pallescens de Noé an endemic plant of Algeria reported by Sadjia et al. . In our study the percentage of other components was observed less than 2%. A high correlation was observed between γ-terpinene and p-cymene with thymol and carvacrol contents in essential oil of T. vulgaris shown in Table 2.
|No.||Compounds||RIa||% RAb (means±SD)|
|26.||Thymol methyl ether||1237||0.65±0.15|
|27.||Carvacrol methyl ether||1245||0.40±0.13|
|48.||Oil yield (%w/w)||1.55±1.27|
aRI, retention index.
bRA, relative area percentage values are means of three experiments±SD.
|Plant||Thymol (%)||p-cymene (%)||γ-terpinene (%)||Carvacrol (%)||Total (%)|
Values are the average of three experiments±SD. p<0.05.
The GC-MS analysis of M. arvensis essential oil showed the presence of total 28 chemical components in which most are recognized and cited in previous researcher work these compound are given in Table 3. Components with high percentage were: α-Phellandrene (3.22%), Dl-Limonene (1.48%), linalool (2.22%), Linalool (2.22%), cis-Piperitone oxide (3.63%), Menthone (29.42%), neo-Menthol (4.75%), Eucalyptol (6.95%), Isomenthone (10.85%), Menthol (21.35%), and Thymol (1.64%) and some other components with small amount were also identified. Our results agree with that of Souza et al.  they showed the presence of menthol (67.27%) and menthone (13.34%) in highest amount in young leaves of M. arvensis. In another study, researcher showed the presence of 25 different components in the essential oil of M. arvensis in which the higher concentration was observed of menthol and menthone . Some researcher showed that the percentage of chemical component in essential oil can vary in different cultivar of mint and at different growing stage . However literature study showed that essential oil concentration increases at mature stage of M. arvensis . Overall the result of our findings are compatible with those reported in literature.
|7.||6.784||Octyl cyclobutane carboxylate||1284||–||0.33|
aRT, retention time.
bRI, retention indices on BP-20 polar column.
cRI, actual retention indices of columns (Supelcowax-10, HP-20M, CW-20M and BP-20).
dRA, components percentage.
The quantitative result of antimicrobial activity of T. vulgaris essential oil against different bacterial strain are given in Table 4. Results of our study showed that the antimicrobial potential of T. vulgaris essential was concentration dependent by increasing concentration antimicrobial activity also increased. Secondly the effectiveness of essential oil was different against different bacterial strains i.e. high inhibition zone was observed in K. pneumonia, E. coli and S. typhimurium. To compare more thoroughly the effect of T. vulgaris on each microorganism (Figure 5 and Table 4) shows the results of multiple comparisons, at each oil amount, was assumed. Essential oil was checked in different pair to compare with each other. All the pairwise differences showed that they are highly significant (p=0.00) with each other except S. typhimurium and E. coli. The growth of K. pneumoniae, E. faecalis, S. aureus, P. aeruginosa and E. coli was previously recorded along with the efficacy against S. typhimurium, respectively . However, some studies report the inefficiency of thyme essential oil against E. coli, S. aureus and K. pneumonia . Ciprofloxacin and cephalexin was used as positive control. Vast variety of essential oils activity depends on chemical components. Previous studies by the researcher showed a strong relationship of the antimicrobial activities with the presence of phenolic compounds i.e. thymol and γ-terpinene present in essential oil, except p-cymene, which does not show antibacterial efficacy when used alone, but in synergistic with thymol and γ-terpinene it showed effective antibacterial activity.
|Test microorganism||Amount of essential oil (5 μL)||Amount of essential oil (10 μL)||Amount of essential oil (15 μL)||Amount of essential oil (20 μL)|
Effects of thyme oil against bacteria representing by the mean sizes of the inhibitory zones.
The antimicrobial activities of M. arvensis revealed that its essential oil is effective against all bacterial strains as shown in (Table 5 and Figure 6). Our study showed that the menthol essential oil was more effective against microorganism as compared to thyme oil. Even a low concentration of oil was effective to inhibit S. aureus and E. coli. Minimal bactericidal concentration of M. arvensis essential oil, to inhibit growth of different bacteria was, E. coli (5.8±5.7) and S. aureus (1.20±1.8). Like thymol, the researcher relate the antibacterial property of menthol to the presence of terpenoids present in mint family. In our study, the essential oil of M. arvensis was observed rich in terpenoids due to which the essential oil showed strong activity against different bacterial strains. The result of our study was agreed to that of Mickienė et al.  worked on different strains of bacteria i.e. S. aureus, Enterococcus faecium, P. aeruginosa, E. coli, Proteus mirabilis. Horváth and Koščová  worked on the essential oil of M. arvensis against S. aureus also showed that the plant essential oil is effective against microorganisms.
|Series #||Test organism||Amount of essential oil (μL)||% Inhibition (means±SD)|
|Series 1||E. Coli||0.2||(5.8±5.7)|
|Series 2||E. facium||0.2||(50.7±17.8)|
|Series 3||St. aureus||0.2||(1.20±1.8)|
|Series 4||P. aeriginosa||0.2||(38.8±24.7)|
|Series 5||P. mirabilis||0.2||(30.0±18.2)|
|Series 6||C. albicans||0.2||(69.67±32.5)|
These values are average of three experiments±SD. p<0.05.
The antifungal activity of essential oil of samples showed different values i.e. for C. albicans mycelia inhibition (0%, 10%, 63.50%, 72.30%, 88.60%, 90.25% and 94.6%, 97.1%, 97.75%, 99% and 100%) at concentration of 0, 0.7, 1.30, 2.7, 5.5, 10.6, 23, 32, 42, 55, 100 μL mL−1 (Table 6). From our present results it was observed that the antifungal activity was concentration dependent, as with increase concentration the antifungal activity also increases. Our present study are agreed with that of Mickienė et al.  worked on the antimicrobial activity of essential oil obtained from M. arvensis, showed inhibition of C. albicans at higher concentration. These essential oil are not only effective against C. albicans but also against other fungal strain. The literature study revealed the effectiveness of T. vulgaris against Rhizopus oryzae . Quantitative details are shown in Table 6; Figure 7.
|No.||Concentration of oil (μL mL−1)||Inoculum size (mm)||Colony size (mm)||Mean colony size (mm)||Mycelial growth (mm)||% Inhibition of mycelial growth|
MIC=5.5 μL mL−.
Therefore, our study confirm that essential oil of both plants exhibit stronger antimicrobial activity than that of their major constituents or their mixtures, respectively, which suggests synergistic effects of the minor components, but also the importance of all components in relation to the biological activity of essential oils.
Many health issues like cancer and heart diseases are caused by free radicals. Literature study showed that plant extracts containing phenolic compounds, exhibit high antioxidants properties. These chemical constituents protect the body against harmful chemicals produce during metabolism of fats , . In our present study both samples of essential oil were checked for their total phenolic content. Phenolic contents of both samples of T. vulgaris and M. arvensis was expressed as gallic acid equivalent by following the standard curve equation: y=0.027x+0.1784, R2=0.9966. In our finding we observed high phenol content in T. vulgaris (21.6 mg GAE/g dry weight) as compared to M. arvensis values are shown in Table 7.
|Plant||Total phenolic contenta (mg GAE/g DW)||Antioxidant activity (IC50)b (μg/mL)|
aGallic acid (mg/g) dry weight (DW).
p<0.05 values were average of three experiments±SD.
The free radical scavenging activity of these essential oil were assessed with the help of DPPH assay. During the study it was observed that the high content of phenol in T. vulgaris (7.8 μg/mL) lead to the high antioxidant activity as shown in Table 7. So a correlation was observed between radical scavenging capacity and total phenolic content of essential oil. So we can say that the Phenolic compounds play a very important role in scavenging of free radicals. In our finding it was also observed that with increasing concentration the free radical scavenging also increases. Our findings agree with that of Alizadeh  work on the antioxidant activity of T. vulgaris by using DPPH assay. However, in literature study it was observed that the antioxidant activity of plants are not only caused by phenolic constituents but also due to the presence of other secondary metabolites like vitamins, flavonoids and carotenoids . From these results we can suggest that T. vulgaris is a strong antioxidant can be used as natural antioxidants.
Present study showed that plant essential oil are good in their biological activities. Both essential oil was rich in chemical composition and showed the presence of many important chemical constituents. Essential oil of M. arvensis and T. vulgaris showed positive results for all biological activities. However, high antioxidant activity was observed in T. vulgaris which also show a strong correlation to the presence of high phenolic content. As both species have active components due to which they showed positive antimicrobial activities. So, research should be carried on for further biological activities of these oil for betterment of living beings. It is also a suggestion that instead of common hydrodistillation technique an improved technique such as Solvent-free microwave extraction (SFME) should be utilized for extraction of essential oil.
The author is grateful to the Higher Education Commission Pakistan”, “Department of Chemistry’’, Women university of Azad Jammu and Kashmir, Bagh, University of Azad Jammu and Kashmir, Muzaffarabad and COMSATS Institute of Information Technology, Lahore for providing necessary facilities and support.
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