Chemical Composition and Antioxidant Activity of Essential Oil and Methanolic Extracts of Ferula microcolea (Boiss.) Boiss (Apiaceae)

Abstract

The essential oils of Ferula microcolea, collected from west Iran were obtained by hydrodistillation during the flowering stage and analyzed by gas chromatography and gas chromatography/mass spectrometry. Under the optimum conditions of analysis, 22 constituents (mainly monoterpen compounds) were identified in Ferula microcolea, representing 93.6% of the oil. The main constituents were α-pinene (27.3%), β-pinene (16.4%), nonanal (8.7%), β-caryophyllene (8.5%), and thymol (6.7%). The samples were also subjected to screening for their possible antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl and β-carotene-linoleic acid assays. In the first case, the free radical-scavenging activity of polar sub-fraction of methanol extract showed to be superior as compared to other extracts (IC₅₀ = 34.3 ± 0.3 μg/ml). Nonpolar sub-fraction of methanol extract exhibited stronger activity than the essential oil. In the case of the linoleic acid system, oxidation of the linoleic acid was effectively inhibited by the polar sub-fraction of methanol extract (86.5 ± 0.9%), while the oil and nonpolar sub-fraction of methanol extract were less effective (55.2 ± 0.4% and 81.5 ± 0.8%, respectively).

Keywords:

• Ferula microcolea
• Antioxidant activity
• Essential oil

INTRODUCTION

Reactive oxygen species (ROS) and free radicals have raised attention over the past decade. ROS, which include free radicals, such as superoxide anion radicals (O₂−), hydroxyl radicals (OH), and non-free-radical species such as H₂O₂ and singlet oxygen (¹O2), are different forms of activated oxygen. These molecules are exacerbating factors in cellular injury and aging process.[1] Free radicals and other ROS are continuously produced in vivo and can also be produced by radiation, chemical reactions, and several redox reactions of various compounds that may contribute to protein oxidation, DNA damage, lipid peroxidation in living tissues, and cells that leads to cancer and cardiovascular diseases. Although almost all organisms are well protected against free-radical damage by enzymes, such as superoxide dismutase and catalase, or by compounds, such as ascorbic acid, tocopherols, and glutathione, these systems are not sufficient to prevent damage entirely.[2]

An increasing number of investigations have been carried out to find antioxidative drugs, which not only prolong the shelf life of food products, but also participate as radical scavengers in living organisms.[3] The use of spices and herbs as antioxidants in processed foods is a promising alternative to the use of synthetic antioxidants. Numerous reports of antioxidative activity of spices have appeared, strongly inspired by an increasing consumer interest in “natural” food additives. Most investigations have been performed using different model systems, and the spices have been evaluated, either as whole spices or as extracts of spices.[4] Major reasons for increasing interest in natural antioxidants are doubts on the safety of the use of synthetic substances (butylatedhydroxytoluene [BHT] and butylatedhydroxyanisole), the antioxidative efficacy of a variety of phytochemicals, the consensus that foods rich in certain phytochemicals can affect the aetiology, and pathology of chronic diseases, the ageing process, and the public conception that natural compounds are innately safer than synthetic compounds and are thus more commercially acceptable.[5]

The genus Ferula comprises perennial herbs distributed from the Mediterranean region to central Asia. Thirty species of genus Ferula are found in Iran, among which 15 are endemic.[6] This genus is phytochemically characterized mainly by coumarins and sesquiterpenes.[7,8] Several species of this genus have been used in traditional medicine for the treatment of various organ disorders. Among different Ferula species that have been used as natural remedies, F. assa-foetida (used as anticonvulsant, carminative, antispasmodic, diuretic, aphrodisiac, antihelmintic, tonic, laxative, alterative, etc.), F. badrakema and F. gummosa (both used as anti-convulsant, tonic, anti-hysteric, decongestant, treatment of neurological disorders, and stomachache), and F. persica (used as laxative, carminative, antihysteric, treatment of lumbago, diabetes, rheumatism, and backache) are most famous.[9–11] Since F. microcolea is used as spice in some foods and also as flavouring powder in yogurt and dough in some parts of Iran (like Lorestan province), so in this research, the constituents of the essential oil of the plant have been investigated as the main reason behind the flavour and antioxidant activity of the plant.

MATERIALS AND METHODS

Plant Material

The aerial parts of Ferula microcolea were collected during flowering stage (June 15, 2009) from Lorestan Province, southwest Iran. The voucher specimen have been deposited in the national Herbarium of Iran (TARI). Collected plant materials were dried in the shade.

Solvents and Chemicals

2,2-Diphenyl-1-picrylhydrazyl (DPPH, 95%), β-carotene, linoleic acid, BHT, quercetin, and gallic acid were procured from Sigma–Aldrich Chemie (Steinheim, Germany). Analytical-grade methanol, ethanol, and dimethyl sulphoxide (DMSO); HPLC-grade chloroform; standard Folin–Ciocalteu'sphenol reagent; anhydrous sodium sulphate; sodium carbonate; aluminiumtrichloride; and Tween 40 were obtained from Merck (Darmstadt, Germany). Ultrapure water was used for the experiments.

Isolation of the Essential Oils

Dry aerial parts (100 g) of Ferula microcolea were subjected to the hydrodistillation of 2.5 h, using a Cleavenger-type apparatus, according to the method recommended by the European Pharmacopia[12] to produce oils. The obtained essential oils were dried over anhydrous sodium sulphate and stored at +4°C until tested and analysed.

Preparation of Methanol Extracts

The air-dried and finely ground samples were extracted by using the method described previously.[13] Briefly, the sample weighing about 100 g were extracted in a Soxhlet with methanol (MeOH) at 60°C for 6 h. The extracts were then filtered and concentrated in vacuo at 45°C yielding a waxy material. The resulting extracts were suspended in water and partitioned with chloroform (CHCl₃) to obtain water-soluble (polar) and water-insoluble (nonpolar, chloroformic) sub-fractions. Extracts were concentrated, dried and kept in the dark at +4 C until tested.

Analysis of the Oils

Flame ionization detector (FID)-gas chromatography (GC) was carried out using a Hewlett-Packard 6890 with an HP-5 capillary column (phenyl methyl siloxane, 25 m × 0.25 mm i.d., 0.25 μm film thickness); carrier gas He; split ratio of 1:25, and a flam ionization detector. The temperature progamme consisted of 60°C (2 min) rising to 240°C at 4°C/min, injector temperature of 250°C, and detector temperature of 260°C. GC-mass spectrometry (MS) was performed using Hewlett-Packard 6859 with quadrupole detector, on an HP-5 column (see GC), operating at 70 eV ionization energy, using the same temperature program and carrier gas as above. Retention indices were calculated by using retention times of n-alkanes that were injected after the oils at the same chromatographic conditions according to Van Den Dool method.[14]

Identification of the components was based on comparison of their mass spectra with those of internal Wiley GC-MS specteral library, or with published mass spectra and those described by Adams and others.[15]

Antioxidant Activity

DPPH assay

The DPPH assay usually involves hydrogen atom transfer reaction but, based on kinetic data, an electron transfer mechanism has also been suggested for this assay. Radical-scavenging activities (RSA) of Ferula microcolea essential oil and extracts were determined using a published DPPH RSA assay method[16] with minor modifications. Briefly, stock solutions (10 mg/ml each) of the essential oils, extracts, and the synthetic standard antioxidant BHT were prepared in methanol. Dilutions are made to obtain concentrations ranging from 1 to 5 × 10⁻¹⁰ mg/ml. Diluted solutions (2 ml each) were mixed with 2 ml of freshly prepared 80 μg/ml DPPH methanol solution and allowed to stand for 30 min in the dark at room temperature for any reaction to take place. Ultraviolet (UV) absorbancies of these solutions were recorded on a spectrometer (Cintra 6, GBC, Dandenong, Australia) at 517 nm using a blank containing the same concentration of oils or extracts or BHT without DPPH. Inhibition of free radical DPPH in percent (I%) was calculated as follows:

where A_blank is the absorbance of the control reaction (containing all reagents except the test compound), and A_sample is the absorbance of the test compound. The sample concentration providing 50% inhibition (IC₅₀) was calculated by plotting inhibition percentages against concentrations of the sample. All tests were carried out in triplicate, and IC₅₀ values were reported as means ± SD of triplicates.

β-Carotene/linoleic acid bleaching assay

In this assay, antioxidant activity was determined by measuring the inhibition of volatile organic compounds and conjugated dienehydroperoxides arising from linoleic acid oxidation. The method described by Miraliakbari and Shahidi was used with slight modifications.[17] A stock solution of β-carotene and linoleic acid was prepared with 0.5 mg of β-carotene in 1 ml chloroform, 25 μl of linoleic acid, and 200 mg Tween 40. The chloroform was evaporated under vacuum and 100 ml of aerated distilled water was then added to the residue. The samples (2 g/l) were dissolved in DMSO, and 350 μl of each sample solution was added to 2.5 ml of the above mixture in test tubes. The test tubes were incubated in a hot water bath at 50°C for 2 h, together with two blanks, one contained the antioxidant BHT as a positive control, and the other contained the same volume of DMSO instead of the extracts. The test tube with BHT maintained its yellow colour during the incubation period. The absorbancies were measured at 470 nm on a UV spectrometer. Antioxidant activities (inhibition percentage, I%) of the samples were calculated using the following equation:

Where A _{-β -carotene after 2 h assay} is the absorbance of β-carotene after 2 h assay remaining in the samples, and A _{initial β -carotene} is the absorbance of β-carotene at the beginning of the experiments. All tests were carried out in triplicate, and inhibition percentages were reported as means ± SD of triplicates.

Assay for Total Phenolics

Many natural molecules, especially those produced in the plant kingdom, have at least one benzene ring with a hydroxyl functional group in their skeleton. Such compounds are collectively known as phenolic compounds and, due to their hydrogen or single electron donating potentials, usually play important rules in the antioxidant activity of the plant extracts. Total phenolic constituents of the polar and nonpolar subfractions of methanol extracts of Ferula microcolea were determined by literature methods involving Folin–Ciocalteu reagent and gallic acid standard.[18] Solutions of the extracts (0.1 ml each) containing 1000 μg of the extracts were taken individually in volumetric flasks, 46 ml of distilled water and 1 ml Folin–Ciocalteu reagent were added, and the flasks were thoroughly shaken. After 3 min, 3 ml of 2% Na₂CO₃ solution was added and the mixtures were allowed to stand for 2 h with intermittent shaking. Absorbencies were measured at 760 nm. The same procedure was repeated for all the standard gallic acid solutions (0–1000 mg/0.1 ml) and a standard curve obtained with the following equation:

Total phenols of the extract, as gallic acid equivalents, were determined by using the absorbance of the extract measured at 760 nm as input to the standard curve and the equation. All tests were carried out in triplicate, and phenolic contents as gallic acid equivalents were reported as means ± SD of triplicate determinations.

Total Flavonoid Content (TFC)

TFC was determined using the Dowd method as adapted by Arvouet-Grand et al.[19] Briefly, 1 ml of 2% aluminum trichloride (AlCl₃) in methanol was mixed with the same volume of the extracts (2000 μg). Absorption readings at 415 nm were taken after 10 min against a blank sample consisting of a 1 ml extract solution with 1 ml methanol without AlCl₃. The concentrations of flavonoid compounds were calculated according to the following equation that was obtained from the standard quercetin graph:

RESULTS AND DISCUSSION

Chemical Composition of the Essential Oils

Air-dried herbal parts of Ferula microcolea were subjected to hydrodistillation using a Clevenger apparatus and the pale yellow-colored essential oil was obtained (yield 1.1% v/w). The results obtained by GC–MS analysis of the essential oil of the plant are presented in Table 1. Twenty-two compounds were identified, representing 93.6% of the total oil. The oil profile exhibits α-pinene (27.3%) as the main compound, additionally, other major compounds were β-pinene (16.4%), nonanal (8.7%), β-caryophyllene (8.5%), and thymol (6.7%).The volatile oil contained 18 monoterpenoids (70.8%), 4 sesquiterpenoids (14.2%), and 1 alkyl aldehyde (8.7%).

Table 1 Chemical composition of essential oil of Ferula microcolea

NO	Compounds	RI	% Composition	NO	Compounds	RI	% Composition
1	α-Pinene	939	27.3	12	Borneol	1166	3.3
2	Camphene	946	0.9	13	Terpinene-4-ol	1177	0.8
3	Sabinene	970	0.3	14	α-Terpineol	1189	0.8
4	β-Pinene	980	16.4	15	Pulegone	1238	0.4
5	α-Phellenderene	1005	0.3	16	Bornyl acetate	1285	1.7
6	1,8-Cineole	1033	3.2	17	Thymol	1290	6.7
7	trans-Ocimene	1048	1.2	18	Bornyl acetare	1303	0.8
8	γ-Terpinene	1062	0.3	19	β-Caryophyllene	1418	8.5
9	Linalool	1099	4.9	20	Germacrene-D	1448	1.2
10	Nonanal	1109	8.7	21	Spathulenol	1577	2.7
11	Comphor	1143	1.4	22	Caryophyllene oxide	1581	1.8
RI: retention indicies (kovats index) on HP-5MS column.
% Composition: relative percentages of the compounds were obtained electronically from FID area percent data.

α-Pinene (19.2%), nonane (13.2%), and β-pinene (13.0%) have been previously reported as the main components of the essential oil of F. microcolea collected from Tehran province,[20] which resembles to results of the present study. In 2005, Rustaiyan et al. identified 42 compounds in the essential oil of aerial parts of Ferula macrocolea (Boiss.) with β-pinene (15.9%), α-pinene (10.4%), and β-caryophyllene (8.6%) as the dominant components,[21] which is also similar to oil composition of F. microcolea in this study.

Different Ferula species share some similarities in their volatile components, but there are many compositional differences. Their essential oils can be classified based on their compositional differences. The most prominent measure that can be applied to categorize Ferula oils is the presence of sulfur-containing compounds. The essential oils obtained from F. assa-foetida, F. fukanensis, F. latisecta, F. persica, and F. sinkiangensis contained sulfur compounds. On the other hand, other oils were devoid of these compounds among their identified components. sec-Butyl-(Z)-propenyl disulfide and secbutyl-(E)-propenyl disulfide were found to be the most prevalent sulfur-containing compounds in the essential oils of some Ferula species. The terpenoid compounds were almost the most abundant components of Ferula oils. The most frequent terpenoid compounds that occurred as main components in the essential oils were α-pinene, β-pinene, myrcene, and limonene (among monoterpene hydrocarbons); linalool, γ-terpineol, and neryl acetate (among oxygenated monoterpenes); β-caryophyllene, germacrene B, germacrene D, and δ-cadinene (among sesquiterpene hydrocarbons); and caryophyllene oxide,α-cadinol, guaiol, and spathulenol (among oxygenated sesquiterpenes). Despite the existing reports and considering the total number of identified Ferula species (more than 170), there are still many species uninvestigated. Therefore, conducting future studies on the chemical composition and particularly biological activities of uninvestigated Ferula oils is greatly recommended.[22] Based on the results obtained from the present study, it is concluded that the essential oil of F. microcolea can be classified under non-sulfur containing compounds category.

Antioxidant Activity

The antioxidant activity may be due to different mechanisms, such as prevention of chain initiation, decomposition of peroxides, prevention of continued hydrogen abstraction, free radical scavenging, reducing capacity, and binding of transition metal ion catalysts.[23] It is thus important that for evaluating the effectiveness of antioxidants, several analytical methods and different substrates are used. The methods chosen are the most commonly used for the determination of antioxidant activities of plant extracts. In the β-carotene-linoleic acid system, β-carotene undergoes a rapid discoloration in the absence of an antioxidant. The presence of an antioxidant such as phenolics can hinder the extent of β-carotene destruction by “neutralizing” the linoleate free radical and any other free radicals formed within the system.[24] Table 2 depicts the inhibition of β-carotene bleaching by the essential oil and extracts of F. microcolea, and by the positive control (BHT). Using the β-carotene/linoleic acid method, essential oil and various extracts of F. microcolea showed different patterns of antioxidant activities. As can be seen from Table 2, the most active one was the polar sub-fraction of methanol extract with 86.5 ± 0.9% antioxidant activity. This is closely followed by nonpolar sub-fraction of methanol extract (81.5 ± 0.8%).

Table 2 Antioxidative capacities, phenolic and flavonoid content of the essential oils and methanol extracts of Ferula microcolea ^a

	Test system
Plant oils, methanol extracts, and controls	DPPH IC50 (μg/ml)	β-Carotene/linoleic acid (% inhibition rate)	Phenolic content (GEs/mg extract) ^b	Flavonoid content (μg QEs/mg extract) ^c
Essential oil	253.1 ± 2.2	55.2 ± 0.4	ND	ND
Polar sub-fraction of methanol extract	34.3 ± 0.3	86.5 ± 0.9	211.4 ± 1.9	37.6 ± 0.4
Nonpolar sub-fraction of methanol extract	61.9 ± 0.5	81.5 ± 0.8	50.1 ± 0.4	19.4 ± 0.3
BHT	17.9 ± 0.3	95.7 ± 0.1.0	ND	ND
^aResults are means of three different experiments.
^bGEs: gallic acid equivalents.
^cQEs: quercetin equivalents.
ND: not determined.

The reduction ability of DPPH radicals' formation was determined by the decrease in its absorbance at 517 nm induced by antioxidants. The effect of antioxidants on DPPH radical scavenging is thought to be due to their hydrogen donating ability. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule.[25] In DPPH assay, the strongest free RSA was exhibited by synthetic antioxidant BHT (17.9 ± 0.3 μg/ml). This is followed by polar sub-fraction of methanol extract and nonpolar sub-fraction of methanol extract (34.3 ± 0.3 and 61.9 ± 0.5 μg/ml, respectively). These activities can be related to their phenolic and flavonoid contents which were estimated as 211.4 ± 1.9 (GEs/mg extract) and 37.6 ± 0 (μg QEs/mg extract) (Table 2). As far as the literature survey could ascertain, antioxidant activity of F. microcolea has not previously been reported.

Assays for Total Phenolics and Flavonoids

Based on the absorbance values of the various extract solutions, reacted with Folin–Ciocalteu reagent and compared with the standard solutions of gallic acid equivalents as described above, results of the colorimetric analysis of total phenolics are given in Table 2. As can be seen from the table, data obtained from the total phenolic assay supports the key role of phenolic compounds in free radical scavenging and/or reducing systems. As expected, the amount of the total phenolics is the highest in polar sub-fraction of methanol extract 211.4 ± 1.9 (GEs/mg extract). It is extremely important to point out that there is a positive correlation between antioxidant activity potential and the amount of phenolic compounds of the extracts. On the other hand, polar sub-fraction of methanol extract has been found to be rich in flavonoids with a value of 37.6 ± 0 (μg QEs/mg extract).

CONCLUSION

The results of the present work indicated that antioxidant activity of the methanol extracts of Ferula microcolea is higher than the essential oil. The methanol extracts of Ferula microcolea may be an alternative additive in foods, pharmaceuticals, and cosmetic preparations instead of more toxic synthetic antioxidants. But, further studies are needed for better clarifying the cytotoxicity and other biological properties of this extracts. According to literature data, this is the first study on the antioxidant activity of the essential oil and extracts of Ferula microcolea indicating good to moderate antioxidant activity for the plant. These results encourage complementary and more in-depth studies on the chemical composition of the plant extracts with the aim of separation and structure elucidation of their active components and also evaluation of biological activity of each compound separately.