Essential Oil Composition and Antioxidant Activity of Calamintha officinalis Moench

The essential oils are aromatic compounds usually used in medical, food and cosmetic industries. They contain more than 200 diverse compounds, mostly made of monoterpene and Sesquiterpenes hydrocarbons and some branched chemicals such as esters, alcohols, aliphatic aldehydes and ketones. Essential oils are normally produced in fragrant herbs.1 Eos have antioxidant and microbicide properties.2,3 The herbs from Calamintha sp. are used to add pleasant flavor to the food, due to their characteristic tastes. They are also used in a number of therapeutic applications having anticough and antipyretic properties.4 Calamintha officinalis Moench (COM) from Lamiaceae family is very similar to the common mints not only in its appearance but also in terms of its aroma. It is, therefore, commonly used as an alternative to mint for usage in different drinks.5 The antioxidant activity of plant extracts and essential oils is of special importance owing to their positive physiological activities on human cells and their potential ability to substitute synthetic antioxidants.6 Its essential oil is used in baking as flavoring agent and to add a desirable taste to some pharmacological. It is also consumed for its spicing, sudorific and microbicide effects.7 In recent years, numerous researchers have investigated the chemical components in the essential oil of Calamintha species from diverse sources. The purpose of the present study was to obtain the main components of COM plant collected from north area of Iran (Gilan, Lahijan). This was then compared to essential oil composition of COM from other origins. On the other hand, we also aimed to assess the enzymatic antioxidant activity in extracts obtained from the plant’s leaves.


Introduction
The essential oils are aromatic compounds usually used in medical, food and cosmetic industries.They contain more than 200 diverse compounds, mostly made of monoterpene and Sesquiterpenes hydrocarbons and some branched chemicals such as esters, alcohols, aliphatic aldehydes and ketones.Essential oils are normally produced in fragrant herbs. 1 Eos have antioxidant and microbicide properties. 2,3he herbs from Calamintha sp. are used to add pleasant flavor to the food, due to their characteristic tastes.They are also used in a number of therapeutic applications having anticough and antipyretic properties. 4alamintha officinalis Moench (COM) from Lamiaceae family is very similar to the common mints not only in its appearance but also in terms of its aroma.It is, therefore, commonly used as an alternative to mint for usage in different drinks. 5The antioxidant activity of plant extracts and essential oils is of special importance owing to their positive physiological activities on human cells and their potential ability to substitute synthetic antioxidants. 6ts essential oil is used in baking as flavoring agent and to add a desirable taste to some pharmacological.It is also consumed for its spicing, sudorific and microbicide effects. 7n recent years, numerous researchers have investigated the chemical components in the essential oil of Calamintha species from diverse sources.The purpose of the present study was to obtain the main components of COM plant collected from north area of Iran (Gilan, Lahijan).This was then compared to essential oil composition of COM from other origins.On the other hand, we also aimed to assess the enzymatic antioxidant activity in extracts obtained from the plant's leaves.

Plant Samples
The plant, COM was collected from northern Iran (Guilan, Lahijan).The fresh leaves were washed with water thoroughly and dried at 40°C.The leaves were then crushed into small pieces and kept frozen for later experiments.

Preparation of Plant Extract
Frozen leaves (1 g of fresh mass) were ground in liquid nitrogen and extracted with a cool extraction buffer 3 mL (50 mM potassium phosphate, pH 7.5).The extract was centrifuged for 30 min at 12 000 rpm at 4°Cand the resulting supernatants was used as crude extract. 8PH Free Radical-Scavenging Activity DPPH Free radical scavenging activity was estimated by determining the scavenging activity of the essential oil by Burcul with some modification.9 Superoxide Radical Scavenging Activity One hundred microliters of the plant extract was added to 3 mL of a reaction mixture and mixed thoroughly.The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 2 µM riboflavin, 0.1 mM EDTA and 75 µM NBT.A blank was made of the reaction mixture without enzyme and NBT and the control contained reaction mixture without enzyme.The tubes containing solutions were subjected to 400 W bulbs (4×100 W bulbs) for 15 minutes and the absorbance was read instantly at 560 nm. 10 The percent scavenging of the superoxide radical was calculated using the following equation: % scavenging = (1−A e /A 0 ) ×100 Where, A 0 is the absorbance without sample and Ae is absorbance with sample. 11

Catalase Assay
The catalase (CAT) reaction mixture (3 mL) contained 50 mM phosphate buffer (pH 7.0), 15 mM H 2 O 2 and 0.1 ml of the plant extract.Reaction started as soon as the extract was added to the reaction mixture.Alterations in absorbance of the reaction mixture at 240 nm were then recorded every 20 seconds.One unit of CAT activity was defined as an absorbance change of 0.01 unit.min - . 12eparation of Essential Oil Dried leaves of the plant (50 g) were hydrodistilled for 3 hours, by a Clevenger-type apparatus.The yield of essential oil was 1% (w/w).The essential oil was poured out, dehydrated using anhydrous sodium sulfate and stored at low temperature. 13olation and Analysis of Essential Oil by GC-MS: Gas chromatography-mass spectrometry (GC-MS) analysis was performed by Hewlett-Packard (HP-6890), with a crosslinked 5% phenyl dimethyl siloxane HP-5MS capillary column (dimensions, 30 m × 0.25 mm), the carrier gas was helium with a flow rate of 1 mL/min.The column temperature was set from 60°C to 250°C at a rate of 6°C/min and the injector and detector (FID) temperature at 250°C.The injected volume was 0.1 μL of the oil with split ratio of 1/30 with an ionizing voltage of 70 eV.
The percentage of the essential oil composition was determined by retention indices.Retention indices were defined by retention time for n-alkanes that were injected after the essential oil in the same chromatographic conditions.The components were identified by comparing with retention indices found in literature and through comparison of their mass spectra with issued mass spectra data. 14Kovats retention indices (KI) were then calculated using the following formula: Where tʹ n and tʹ n+1 are retention times of the reference n-alkane hydrocarbons eluting instantly before and after compound "X, " and tʹ x is the retention time of compound "X.". 15 Statistical Analysis Each experiment was repeated at least three times and the statistical analysis were performed using SPSS version 22 statistical software.

Ethical Considerations
No human or animal samples were used in this study.

Superoxide Radical Scavenging Activity
Superoxide anions produce active free radicals that can react with biological macromolecules causing tissue injury.Rapid detection of superoxide is highly important, as the lipid peroxidation begins quite instantly.The superoxide anion has a key role in the creation of other free radicals including hydrogen peroxide, hydroxyl radical, and singlet oxygen, which cause oxidative injury in lipids, proteins, and DNA. 16sing the following relationship, the activity of COM extract to scavenge superoxide anion was found to be 89.8%.The results obtained from this part of research indicated the high scavenging activity of the COM extract.% scavenging = (1−A e /A 0 ) ×100 Catalase Assay CAT converts H 2 O 2 to nontoxic compounds water and oxygen and, therefore, arise in CAT activity could have a role in the defense of the plants against destroying effect of H 2 O 2 .Specific catalase activity of COM extract was found to be 97 U/min/mg protein.In this study, the activity of CAT showed a significant increase during time (Figure 1).

Identification of Essential Oil Components
The yield of the essential oil obtained from the leaves of COM extracted by hydro-distillation was 1.0% (w/w).Identification of the essential oil components were done by GC-MS. Figure 2 is the chromatogram of essential oil from C. officinalis.Fortyone components were isolated, constituting 23.09% of the total oil.However, only 11 components of the oil were identified in our laboratory (Table 1).The major constituents of this oil were trans-caryophyllene (8.55%), isomenthol (2.98%), tetrahydrolinalyl acetate (2.96%), and pinene (2.24%).

Discussion
In this paper, CAT and superoxide anion radical scavenging activity of COM extract along with GC-MS analysis of its http://www.biotechrep.irJ Appl Biotechnol Rep, Volume 5, Issue 2, 2018 57 essential oil were evaluated.According to our literature survey, it has been reported that although C. officinalis yields comparatively high volume of essential oil with a number of useful pharmacological active compounds, some of them are not still explored pharmacologically.It has also been reported that, similar to other members of mint family, the essential oil of C. officinalis preferably used in baking industry as a flavoring agent.The essential oil also improves the taste and aroma of some medicinal products.It is also usually used for its spicing, sudorific, and microbicide effects. 7he ecological variants, such as temperature, relative moisture, and sun shine period, have an absolute effect on the leaves of COM. 7 According to GC-MS results, the major component of the C. officinalis oil was trans-caryophyllene (8.55%), a bicyclic sesquiterpene, known for anti-inflammatory property. 17It has been reported that trans-caryophyllene possess many pharmacological effects including strong antimicrobial property 18 and ataractic activity. 19ouchra et al reported 1, 8-cineole (36.6%), pulegone (17.9%) and limonene (9.2%) as major constituents. 20n agreement with our results, a number of compounds have been identified and reported in C. officinalis oil. 21owever, the major constituents of their oil were carvone (46.7%) and pulegone (22.1%).Burzo et al from Romani reported p-menthone (28.29), pulegone (48.75) as the main components. 4esearchers in Iran have identified thirty-four components in the oil of C. officinalis.The major constituents of their oil were β-bisabolene (9.9%), germacrene D (7.6%), β-bourbonene (7.4%) and piperitenone (5.3%). 22 report from Egypt has shown a number of various compounds including carvone (38.7%), neo-dihydrocarveol (9.9%), dihydrocarveol acetate (7.6%), dihydrocarveol (6.9%), 1,8 cineole (6.4%), cis-carvyl acetate (6.1%), and pulegone (4.1%) as the major components of their essential oil. 7he result of our study on Calamintha revealed the existence of a specific component of the essential oil (trans-caryophyllene) that has not been reported by other researchers.It has been reported that trans-caryophyllene possesses strong antimicrobial property. 18It is expected, therefore, that our essential oil should exhibit antibacterial activity.This is the remaining part of research that should be explored and reported later.However, antimicrobial property of C. officinalis essential oil has been reported by other researchers. 23he free radical scavenging activity of essential oil was determined by DPPH method.The results have indicated that these essential oil possess a low inhibitory activity (21%) relative to plant extract.
We also measured CAT and SOD activities in COM extract, a line of research that has not been reported for this plant in the literature.
CAT is a major antioxidant defense enzyme that primarily catalyses the decomposition of H 2 O 2 to H 2 O and O 2 .The CAT activity of COM extract was significantly increased during time.
Oxygen derived free radicals, such as the superoxide anion and hydroxyl radical are cytotoxic and promote tissue injury.Antioxidants act as a major defense against radical mediated toxicity by protecting from the damages caused by free radicals.Furthermore, although medicinal plants are   used as 'antioxidants' in traditional medicine, their claimed therapeutic properties could be due, in part, to their capacity for scavenging oxygen free radicals. 17We found that the free radical scavenging activity of the COM extract was quite considerable.
Results of our experiment confirmed that COM extract can protect human cells against oxidative damage due to the activity of these enzymes, and it is suggested that strong antioxidant properties of COM can be used for therapeutic or pharmaceutical applications in future.
In this study, the essential oils of C. officinalis were extracted by hydrodistillation.It was observed that the plant contains large amount of essential oil and only 11 components of the oil were identified in our research.Trans-caryophyllene (8.55%) was the main constituent of this oil.The compound is known for its anti-inflammatory and antimicrobial properties.In addition to the antioxidant property that was confirmed in this study, the major component of its essential oil was found to be an antimicrobial agent.It was, therefore, concluded that based on the results of this study, the essential oil of COM from northern Iran could be considered as a reasonable candidate for food and pharmaceutical industries.

Conclusions
The result of our study on Calamintha revealed the existence of a specific component of the essential oil (trans-caryophyllene) that has not been reported by other researchers.Results of our experiment confirmed that COM extract can protect human cells against oxidative damage due to the activity of these enzymes, and it is suggested that strong antioxidant properties of COM can be used for therapeutic or pharmaceutical applications in future.

Figure 1 .
Figure 1.Change in Absorbance of CAT During Time.

Table 1 .
Main Components Identified in the Essential Oil of COM Abbreviations: KI: kovats retention indices, RT: retention time.