Determination of Chemical Composition, Total Phenolic, Antimicrobial, and Antioxidant Activities of Echinophora tenuifolia Essential Oil

Abstract

In this study, the antioxidant and antimicrobial activities, total phenolic content, and essential oil composition of Echinophora tenuifolia L. subsp. sibthorpiana were investigated. The antioxidant activity of investigated essential oil was assessed by ABTS and DPPH assays. DPPH radical scavenging activity expressed by IC₅₀ was 2.84 g/L, whereas the TEAC value determined by ABTS assay was 0.032 g TEAC/kg plant. Total phenol content of essential oil determined by Folin-Ciocalteu method was calculated as 1.32 g GAE/kg plant. The essential oil extracted by hydrodistillation (Clevenger apparatus) was investigated by GC-MS technique and 78 compounds were identified. The main components of essential oils were found to be δ-3-carene (17.93%), p-cymene (8.99%), methyleugenol (16.41%), and α-phellandrene (9.33%). The antimicrobial activity of investigated essential oil was tested using a broth dilution method against 13 bacterial and 2 fungal microorganisms. The lowest minimum inhibitory concentration of essential oil against Bacillus cereus was 62.5 μg/mL while the antifungal activity was greater than 1000 μg/mL for both Candida albicans and Saccharomyces cereviciae. Investigated essential oil has a certain level of antioxidant and antimicrobial effects, which may be attributed to their chemical compounds. The antimicrobial efficiency of essential oil, especially against Bacillus cereus and Staphylocoocus spp., offers its effectiveness to treatment of wound or disease caused by Gram positive bacteria.

Keywords:

• Echinophora tenuifolia
• Essential oil
• Phenolic compounds
• Antioxidant
• Antimicrobial

INTRODUCTION

The genus Echinophora pertains to the Umbelliferae family (subfamily Apioideae tribe Echinophoreae). Chemical compositions of different species of this plant grown in different regions from the Mediterranean eastwards to Afghanistan have been well studied.[1 ⁻ 4] In Iran, these plants are used for various purposes, for example, food seasoning and preserver.[2] The flora of Turkey consists of six species, three of which are endemic.[5] Echinophora tenuifolia L. subsp. sibthorpiana is named locally as ‘çörtük’, ‘çördük’, ‘tarhana out’, and ‘turşu otu’ in Turkish.[6] Fresh or dried Echinophora tenuifolia is used in the treatment of wounds, gastric ulcers, and digestive activities in folk medicine. It is also added to foods, such as soup, meat, dairy products, and meatballs, for enhancing their sensory properties.[5 ⁻ 7]

It has been reported that antibacterial resistance is becoming an increasing health problem due to the fact that human pathogenic microorganisms have developed multiple drug resistance. Preventing microbial growth in foods with novel strategies has become a developing research area that has received much attention in recent years. For example, plant extracts and essential oils have been investigated for their inhibition abilities against pathogenic microorganisms.[8] The essential oils obtained from medicinal plants are commonly used to treat different diseases in folk medicine.[9]

It is well known that the most of medicinal plants are used for their antioxidant properties as well as their antimicrobial potential. Synthetic or natural antioxidants play important roles in inhibition of free radicals and protection of membrane and tissues from oxidative damage.[10] Because of increasing concern about potentially harmful synthetic antioxidants, there is currently much interest on natural sources of antioxidants. Natural antioxidants found in fruits, vegetables, and medicinal plants may prevent cells from damaging effects of hydrogen peroxide and may reduce coronary heart disease risk. Therefore, to find novel and effective sources, the studies about the screening of medicinal plants containing functional compounds on the basis of their antioxidant and antimicrobial properties are very important.[11, 12]

In these scopes, chemical composition, total phenolic, antioxidant, and antimicrobial activities of essential oil from Echinophora tenuifolia grown in the Adiyaman region of Turkey were investigated in the present study. The essential oils were isolated by hydrodistillation using a Clevenger-type apparatus. Chemical composition of essential oil was determined by gas chromatography-mass spectroscopy (GC-MS) technique. The antioxidant activities were determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) methods. The antibacterial activity was tested using a broth dilution method against 13 bacterial and 2 fungal microorganisms.

MATERIAL AND METHODS

Plant Material and Extraction of Essential Oil

The leaves of Echinophora tenuifolia L. subsp. sibthorpiana were collected during the flowering season in July 2010 from the Adiyaman region of Turkey. Collected plant material was dried under the shadow at room temperature. The plant sample was identified by Dr. Emel Yigit from the Department of Biology, Inonu University. A total of 100 g of air-dried plant material were subjected to hydrodistillation for 4 h with 500 mL of distilled water using a Clevenger type apparatus. The essential oil obtained was collected and dried over anhydrous sodium sulfate and stored in sealed glass at −18 ± 0.5°C until use.

ABTS Assay

ABTS (2.2′-azinobis-[3-ethylbenzthiazoline-6-sulphonic acid]; Fluka, Steinheim, Germany) radical cation (ABTS⁺) solution was produced by reacting 7.0 mM ABTS stock solution with 2.45 mM (final concentration) potassium per sulfate in the dark for 16 h.[13] The resulting solution was diluted with methanol by adjusting the absorbance to 0.700 ± 0.020 at 734 nm. A total of 50 μL of essential oil dissolved in 2950 μL methanol (stock solution) was used in ABTS assay. A diluted ABTS solution (1.9 mL) was added to methanolic solution (100 μL) and the absorbance was measured after 6 min at 734 nm. The activities of standard Trolox were estimated within the range of the dose–response curve of trolox and expressed as the trolox equivalent antioxidant capacity (TEAC). The results were expressed as mg TEAC in g/kg plant. Each assay was performed in triplicate.

DPPH Assay

DPPH (1,1-diphenyl-2-picrylhydrazyl; Fluka, Steinheim, Germany) radical scavenging activity of Echinophora tenuifolia essential oil was evaluated according to a modified version of the method described by Brand-Williams.[14] Briefly, 50 μL of essential oil dissolved in 1000 μL methanol (stock solution) was used in DPPH assay. To evaluate scavenging capacity, methanolic solution of essential oil in various volumes (50, 100, 125, 150, 200, 250 μL) were placed in test tubes and adjusted to 300 μL with methanol and 2700 μL fresh methanolic solution of DPPH was added. The decrease in absorbance at 517 nm related to the color decrease was determined at t = 0 and after t = 90 min for all samples. The inhibition of the DPPH radicals expressed as a percentage was calculated according to the following equation:

where A ₀ and A_t represent the absorbance of the reaction mixtures measured at t = 0 and after 90 min, respectively. Using a graph on which was plotted the percentage inhibition against essential oil concentration in the reaction mixtures; it was possible to determine the sample concentration that provided 50% inhibition (IC₅₀). A lower IC₅₀ value corresponds to a higher antioxidant activity of essential oil. In order to evaluate the sensitivity of DPPH assay, different concentrations of trolox standard were used. Each assay was performed in triplicate.

Total Phenols Assay

The amount of total phenols in the essential oil was determined spectrophotometrically according to the Folin-Ciocalteu method based on the procedures described by Velioglu et al.[15] with slight modifications. Briefly, 20 μL of extract (essential oil diluted in methanol 1:60) was mixed with 1 mL of Folin-Ciocalteu's phenol reagent (Merck, Darmstadt, Germany). After 3 min, 1 mL of saturated sodium carbonate solution was added to the mixture and adjusted to 980 μL with distilled water. The reaction mixture was kept in the dark for 120 min after which the absorbance was read at 725 nm. Gallic acid was used for constructing the standard curve. The result was expressed as gallic acid equivalents (GAEs) in g/kg plant.

Gas Chromatography-Mass Spectrometry Analysis

Chromatographic analysis was performed in a GC (Shimadzu GC–2010)-MS (Shimadzu QP–2010) (Shimadzu Corp., Kyoto, Japan) system fitted with a DB-Wax (60 m, 0.25 mm inner diameter, 0.25 μm film thickness [J&W Scientific, Folsom, CA, USA]). The GC oven temperature was programmed to 40°C for 2 min, subsequently at 3°C/min up to 245°C, and then held isothermal for 15 min. Other operating conditions were as follows: The injector temperature, 250°C; split ratio, 1:100; injection volume, 1 μL of 1% solution (diluted in hexane). Helium was used as a carrier gas with a constant column flow rate of 1 mL/min. The mass detector operated in electron impact (EI)-mode at 70 eV in a range of 15 to 210 amu. The identification of the volatile compounds was performed by calculation of retention indices (RI) of each compound by using n-alkane series from C10 to C26 (Labor Dr. Ehrenstorfer-Schafers, Augsburg, Germany) under the same conditions. The tentative identifications were based on comparing mass spectra of unknown compounds with those in Wiley 7 (7th edition) and NIST/WILEY mass spectral library. The RI values were also compared with those described in literature determined under the same conditions for matching the compounds. Three analyses were performed for each sample.

Antimicrobial Assay

Test microorganisms

In the antimicrobial assay, 13 bacterial and 2 fungal microorganisms were used as test microorganisms. Microorganisms were provided by Refik Saydam National Public Health Agency (RS) and Department of Clinical Microbiology Faculty of Medicine at Inonu University (ML). The bacterial cultures were Staphylococcus aureus (RS No: 1020/06008), Staphylococcus warneri (RS No: 95052, American Type Culture Collection (ATCC) No: 27836), Staphylococcus hominis (RSHC No: 869, ATCC No: 27844), Staphylococcus epidermidis (RSHC No: 95), Bacillus cereus (RS No: 869), Escherichia coli (RS No: 347/01003), Enterococcus faecalis (ML), Streptococcus spp. (ML), MRSA (ML), Shigella flexneri (RS No: 184), Enterobacter spp. (ML), Salmonella spp. (ML), Klebsiella spp. (ML), and the fungal cultures were Candida albicans (ATCC No: 90028) and Saccharomyces cerevisiae (RS No: 08022).

In this assay, nutrient agar (Merck, Darmstadt, Germany) was used for bacterial cultures at +4°C while sabouroud dextrose agar (Merck) was used as the media to maintain yeast cultures at 37°C until use. The overnight cultures of all microorganisms were prepared in nutrient broth at 37°C (Merck) for bacterial strains and in sabouroud dextrose broth (Merck) at 35°C for yeast strains.

Minimum inhibitory concentration (MIC)

In antimicrobial assay, the broth dilution method, which is developed by Andrews,[16] was used in determination of antimicrobial properties. MIC values of essential oil for each microorganism were described as the lowest concentration providing inhibition. Antibacterial standards and stock solution of essential oil were prepared using sterile Mueller Hinton Broth (MHB) containing 10% dimethyl sulfoxide (DMSO). Two-fold serial dilutions were prepared from essential oil stock solution from 1000 to 3.9 μg/mL with sterile MHB in test tubes and then a total of 100 μL overnight culture of each microorganism (approximately 10⁸ CFU/mL [using McFarland No. 0.5]) was added. Separately, the test tube containing MHB and overnight culture of each microorganism was exerted as the negative control. Ampicilin and gentamicin (Sigma-Aldrich, Steinheim, Germany) were used as antibacterial standards against all bacteria. After incubation at 37°C for 24 and 48 h for bacterial and yeast strains, respectively, p-iodonitrotetrazolium violet (INT; Sigma-Aldrich) was used for the determination of the MIC. A total of 0.2 mg/mL INT was added to the test tube. After further incubation, bacterial growth was indicated by the red color of the INT formazan produced.[17]

RESULTS AND DISCUSSION

Antioxidant Activity

To assess the antioxidant activity or radical scavenging capacity of the plant extracts, several methods have been used extensively. The most common and reliable methods are DPPH and ABTS, which reflect the free radical-scavenging ability for lipophilic radicals[18] and antioxidant activity for both lipophilic and hydrophilic radicals,[19] respectively. The result of DPPH and ABTS assays was calculated and summarized in Table 1. For DPPH assay, essential oil was further diluted with 60-fold methanol and then IC₅₀ value was estimated as 2.84 g/L. The antioxidant capacity, based on the ABTS assay was found to be 0.032 g TEAC/kg. The scavenging capacity of Echinophora tenuifolia might be due to hydroxyl groups existing in the phenolic compounds’ chemical structure that can provide the necessary compound as a radical scavenger.

Table 1 ABTS radical cation DPPH radical scavenging capacity (IC₅₀) and total phenol content of Echinophora tenuifolia essential oil

DPPH (IC50) (g/L)	ABTS (g TEAC/kg plant)	Total phenols (g GAE/kg plant)
2.84 ± 0.07	0.032 ± 0.001	1.32 ± 0.09

Echinophora tenuifolia was recorded for the first time by Cakilcioglu et al.[20] In a previous study,[21] the antioxidant activity of some plant extracts (chloroform, acetone, ethanol, and water), including Echinophora tenuifolia, were assessed by lipid peroxidation and DPPH assay. In this study, high antioxidant power was reported for different solvent extracts of Echinophora tenuifolia. However, antioxidant properties of methanolic extracts were not taken into account and assessed in this study. So, the antioxidant capacity and total phenolic content of methanolic extracts from Echinophora tenuifolia were investigated by DPPH and ABTS assays in the current study. Our result of antioxidant activity based on the DPPH is also in agreement with the previous findings for different plants previously reported. For example, in the study of Pourmorad et al.[22] on antioxidant activity (IC₅₀) determined by DPPH assay in five Iranian medicinal plants changed from 0.01 to 2.03 mg/mL.

Phenolic compounds are the most important antioxidant plant components and are frequently investigated in many medicinal plants and vegetables for screening their antioxidant behaviors.[23] Total phenolic content of essential oil determined by Folin-Ciocalteu phenol reagent was found to be 1.32 g GAE/kg. Compared to the total phenolic contents of some plants[24] in dry basis, such as cereals (0.2–1.3 mg GAE/g), vegetables (0.4–6.6 mg GAE/g), and berries (12.4–50.8 mg GAE/g), a higher level was found in the analyzed sample.

Table 2 Essential oil compounds of Echinophora tenuifolia

Compound	RI	Area, %	Compound	RI	Area, %
Terpenes			Alcohols
α-Pinene	998	0.98	Eucalyptol	1219	0.39
α-Thujene	1002	0.86	3-Hexen-1-ol. (Z)-	1385	0.07
Cantharene	1016	0.69	Linalool	1524	0.77
α-Fenchene	1045	0.03	1-Terpinenol	1536	0.65
Camphene	1056	0.11	4-Terpineol	1556	0.44
2,4,6-Octatriene	1068	0.09	1-Terpineol	1568	0.79
1,3,5-Octatriene (E.Z)	1102	0.02	α-Terpineol	1604	0.25
β-Pinene	1111	0.16	α-Phellandren-8-ol	1625	7.19
Sabinene	1123	0.95	Pinocarveol (E)-	1672	1.79
Verbenene	1134	0.12	Sum		12.33
δ-3-Carene	1159	17.93	Ketones
β-Myrcene	1167	0.62	2-Propanone	821	0.03
α-Phellandrene	1174	9.33	1-Penten-3-one	991	0.01
Cymene	1181	2.15	2-Methyl-3-octanone	1322	0.03
α-Terpinene	1184	0.23	6-Methyl-5-hepten-2-one	1339	0.02
Dehydrocineole	1197	0.14	E-3-Octen-2-one	1411	0.07
Sylvestrene	1200	0.31	Hexahydrofarnesyl acetone	2033	0.44
α-Limonene	1204	2.55	Eucarvone	1617	2.84
β-Phellandrene	1214	2.82	Sum		3.44
Cineole	1216	0.20	Aldehydes
1,5,8-p-Menthatriene	1224	0.26	Propanal	811	0.01
β-Ocymene	1235	0.39	Butanal	872	0.01
γ-Terpinen	1249	0.43	2-Methylbutanal	883	0.06
β-cis-Ocimene	1253	0.30	β-Methylbutanal	886	0.02
p-Cymene	1277	8.99	Pentanal	944	0.01
Terpinolene	1281	0.41	2.4 Pentadienal	1074	0.11
α-Terpinolen	1287	1.10	Octanal	1291	0.02
1,3,8-p-Menthatriene	1399	0.21	(E)-2-Heptenal	1327	0.03
Limonene oxide	1457	1.55	Benzaldehyde	1514	0.43
Epoxyterpinolene	1474	0.15	Myrtanal	1597	1.05
Umbellulon	1594	1.64	Phellandral	1636	0.39
Verbenone	1668	1.35	Sum		2.12
Phellandrene epoxide	1717	3.12	Esters
Sum		60.21	2-Methylbutyl 2-methylbutanoate	1283	0.04
Benzeneoids			Solusterol (isopentyl ester)	1297	0.04
Toluene	1017	0.35	3-Hexen-1-ol. benzoate (Z)-	2041	0.27
Xylene	1141	0.03	Sum		0.35
o-Allyltoluene	1431	0.88	Furanoids
Isocrotylbenzene	1440	0.88	Furan. 2-methyl-	851	0.01
Methyleugenol	1928	16.41	Furan. 2-ethyl-	917	0.07
Thymol	2117	2.41	2-Pentylfurane	1232	0.19
o-Cumenol	2125	0.30	3-(4-Methyl-3-pentenyl) furan	1420	0.03
Sum		21.26	Sum		0.30

Chemical Composition of the Essential Oil

Fruits, vegetables, and medicinal herbs contain a wide variety of bioactive molecules, such as phenolic compounds, terpenoid, and some other endogenous metabolites, that are rich in antioxidant and antimicrobial activities.[20 ⁻ 25] In this respect, a great interest has been given to characterization of the compounds in essential oils of the medicinal plants. The essential oil of Echinophora tenuifolia extracted by a hydrodistillation method showed a light yellow color and was obtained in a yield of 0.77%. The compounds and relative percentages were determined by GC-MS analysis and results were summarized in Table 2. A total of 74 components were identified representing approximately 98% of the total essential oil. The investigated essential oil was characterized by the occurrence of terpenes, mainly δ-3-carene (17.93%), α-phellandrene (9.33%), and p-cymene (8.99%) representing 60.21% of the total compounds in the essential oil. Our results are in agreement with those obtained by Rahimi-Nasrabadi et al.[3] who investigated chemical composition of essential oil from Echinophora platyloba, which is a different species of the same plant grown in Iran. Methyleugenol, a phenilpropanoid derivative,[4] was also presented in the essential oil with a high percentage (16.41%) after terpenes. According to the previous studies, methyleugenol (28.6%),[25] α-phellandrene (43.8%),[25] and δ-3-carene (36.6%)[26] were the most abundant compounds in essential oils of Echinophora tenuifolia. The minor constituents of the essential oil were found to be alcohols (12.33%), ketones (3.44%), aldehydes (2.12%), esters (0.35%), and furanoids (0.30%).

Telci et al.[5] reported only 45 components in essential oil of the same plant, whereas 74 components were identified in our study. Compared to this study, the diversity of the compounds and their percentage were found to be quite different. As stated previously,[27] analysis method, environmental conditions, climate, location, seasonal factors, and development stage may affect essential oil compositions of medicinal plants.

Table 3 MIC values of essential oils of Echinophora tenuifolia

		PC (μg/mL)
Microorganisms	EO (μg/mL)	Ampicilin	Gentamicin	NC
Gram positive bacteria
Staphylococcus aureus	125	3.12	1.5	+
Staphylococcus hominis	250	3.12	1.5	+
Staphylococcus warneri	250	3.12	>100	+
Staphylococcus epidermidis	125	3.12	3.1	+
Bacillus cereus	62.5	6.24	1.5	+
Enterococcus feacalis	500	6.24	>100	+
Streptococcus	500	3.12	25	+
Gram negative bacteria
Enterobacter	1000	3.12	6.2	+
Salmonella	1000	6.24	50	+
Shigella flexneri	1000	>100	12.5	+
Klebsiella	>1000	>100	12.5	+
Escherichia coli	>1000	>100	12.5	+
Yeast
Candida albicans	>1000	nt	nt	+
Saccharomyces cerevisiae	>1000	nt	nt	+
EO: essential oil; PC: positive control; NC: negative control; +: growth; nt: not test.

Antimicrobial Activities

Antimicrobial properties of essential oil, Echinophora tenuifolia, were evaluated by a broth dilution method against eight Gram positive bacteria, five Gram negative bacteria, and two yeasts. It was considered that if the extracts displayed a MIC value less than 100 μg/mL, the antimicrobial activity was good; from 100 to 500 μg/mL, the antimicrobial activity was moderate; from 500 to 1000 μg/mL, the antimicrobial activity was weak; over 1000 μg/mL, the extract was considered inactive.[20] Based on the results given in Table 3, the most sensitive microorganism tested was Bacillus cereus with a MIC value of 62.5 μg/mL. The MIC values for the other Gram positive bacterial strains ranged from 125 to 500 μg/mL. On the other hand, it was determined that essential oil presented a weak antibacterial activity against the Gram negative strains, such as Shigella flexneri, which was inhibited at the concentration of 1000 μg/mL. MIC value for Escherichia coli and Klebsiella spp. was greater than 1000 μg/mL. MIC value for yeasts, namely Candida albicans and Saccharomyces cerevisiae, were also greater than 1000 μg/mL.

The MIC results showed that the essential oil was more effective on Gram positive strains than on Gram negative strains and yeasts. Many studies about antimicrobial capacity of medicinal plants against both bacterial strains and yeasts determined that Gram positive bacterial strains were more sensitive than Gram negative strains and yeasts.[28 ⁻ 30] A possible reason of this sensitivity could be explained by the cell membrane of Gram positive bacteria having lipophilic ends of lipoteichoic acids, thus hydrophobic compounds more easily penetrate from cell membrane of the Gram positive strain than the Gram negative strains.[31] Furthermore, type and size of effective molecules, which are found in medicinal plants, can affect antimicrobial capacity.[32] Also, minor components could contribute to antimicrobial properties of essential oil as well as major components. Cosentino et al.[30] reported that synergistic or antagonistic effects between minor components and other active components may affect antimicrobial capacity of essential oils.

In our study, δ-3-carene, p-cymene, methyleugenol, and α-phellandrene were defined as the major compounds in investigated essential oil. Pichette et al.[33] determined that δ-3-carene, which is a constituent of the Abies balsamea essential oil, was active against Staphylococcus aureus, while it was inactive against Escherichia coli. In accordance with this study, examined essential oil including the same major compounds did not show any activity against Escherichia coli and Klebsiella. In contrast to our findings, in a previous study,[34] an inhibitory effect was observed with thyme and basil essential oils, including mainly p-cymene, which exhibited an antimicrobial activity against Shigella flexneri. Compared to previous findings,[35] a contradictory result for the antimicrobial effect of methyleugenol against Escherichia coli was also observed in our study. Methyleugenol has been reported as an effective antibacterial agent[35, 36] and a major compound of the essential oil investigated here. These inconsistent results may be explained by the inadequate concentrations of these compounds in examined essential oils.

CONCLUSIONS

There are several reports on the usage of the plants belonging to Echinophora family as an additive in foods, such as tarhana, pickles, dairy products, and meatballs. The amounts of plants added range from 0.5 to 1.5% in traditional applications.[6] These herbs are mainly used traditionally for their flavoring properties. Furthermore, it is accepted that this amount could prevent the oxidative and microbial deteriorations. In light of these assessments, Echinophora tenuifolia could be used in foods for providing pleasant flavor and its protective properties. In conclusion, investigated essential oil has a certain level of antioxidant and antimicrobial effects, which may be attributed to their chemical compounds. The investigated essential oil was found to be composed of mainly terpenes including δ-3-carene, α-phellandrene, and p-cymene in higher levels. The antimicrobial efficiency of essential oil Echinophora tenuifolia, especially against Bacillus cereus and Staphylocoocus spp., offer its effectiveness to treatment of wound or disease caused by Gram positive bacterial strains in folk medicine. This study revealed important antioxidant and antimicrobial properties of methanolic extracts from Echinophora tenuifolia, which could be attractive to the food or pharmaceutical industry. The data about the influence of new essential oil compounds, in particular on their functional behaviors, could provide useful information for production of novel food or the prevention and treatment of various human diseases.