Composition, In Vitro Antioxidant a

Composition, In Vitro Antioxidant and Antimicrobial Activities of Essential Oil and Oleoresins Obtained from Black Cumin Seeds (Nigella sativa L.)
Sunita Singh, 1 S. S. Das, 1 G. Singh, 1 ,* Carola Schuff, 2 Marina P. de Lampasona, 2 and César A. N. Catalán 2
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Abstract
Gas chromatography-mass spectrometry (GC-MS) analysis revealed the major components in black cumin essential oils which were thymoquinone (37.6%) followed by p-cymene (31.2%), α-thujene (5.6%), thymohydroquinone (3.4%), and longifolene (2.0%), whereas the oleoresins extracted in different solvents contain linoleic acid as a major component. The antioxidant activity of essential oil and oleoresins was evaluated against linseed oil system at 200 ppm concentration by peroxide value, thiobarbituric acid value, ferric thiocyanate, ferrous ion chelating activity, and 1,1-diphenyl-2-picrylhydrazyl radical scavenging methods. The essential oil and ethyl acetate oleoresin were found to be better than synthetic antioxidants. The total phenol contents (gallic acid equivalents, mg GAE per g) in black cumin essential oil, ethyl acetate, ethanol, and n-hexane oleoresins were calculated as 11.47 ± 0.05, 10.88 ± 0.9, 9.68 ± 0.06, and 8.33 ± 0.01, respectively, by Folin-Ciocalteau method. The essential oil showed up to 90% zone inhibition against Fusarium moniliforme in inverted petri plate method. Using agar well diffusion method for evaluating antibacterial activity, the essential oil was found to be highly effective against Gram-positive bacteria.

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1. Introduction
Preservation of food degradation, mainly by oxidation processes or by microorganism activity, during production, storage, and marketing is an important issue in the food industry. There is currently a large interest in substituting synthetic food preservatives and synthetic antioxidants for substance that can be marketed as natural. Synthetic antioxidants such as gallates, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butyl hydroquinone (TBHQ) were the first preservatives designed for widespread industrial use. However, some physical properties of BHA and BHT, such as their high volatility and instability at elevated temperatures, strict legislation on the use of synthetic food additives, and consumer preferences, have shifted the attention of manufacturers from synthetic to natural antioxidant [1]. It is well known that most spices possess a wide range of biological and pharmacological activities.

Black cumin (Nigella sativa L.) belonging to family Ranunculaceae is a spice that has been used for decades for both culinary and medicinal purposes. It is also used as a natural remedy for asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness, and influenza [2]. The seeds are known to be carminative, stimulant, and diuretic [3]. The essential oil from the seeds of this herbaceous plant has been found to contain high concentrations of thymoquinone and its related compounds such as thymol and dithymoquinone, which have been implicated in the prevention of inflammation [4], antioxidant activities [5], such as quenching reactive oxygen species, antimicrobial activity [6], and anticarcinogenic and antiulcer activity [2].

The present paper deals with the chemistry and antioxidative and antimicrobial behavior of essential oil and oleoresins (extracted in ethanol, ethyl acetate, and n-hexane) of black cumin seeds.

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2. Materials and Methods
The seeds of black cumin were purchased from the local market of Gorakhpur, Uttar Pradesh, India. A voucher specimen was deposited at the herbarium of the Faculty of Science, DDU Gorakhpur University.

2.1. Reagents

Thiobarbituric acid (TBA), 1,1′-diphenyl-2-picrylhydrazyl radical (DPPH), and linoleic acid are of Acros (New Jersey, USA); butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate (PG) are of S D Fine Chemicals Ltd., Mumbai, India. Folin-Ciocalteu reagent and gallic acid were from Qualigens Chemicals Ltd., Mumbai, India, and Qualikems Chemicals Ltd., New Delhi, India, respectively. Tween 20 and ferrozine were from Merck Pvt. Ltd., Mumbai, India. Ampicillin was purchased from Ranbaxy Fine Chemicals (New Delhi), India. Crude linseed oil was obtained from local oil mill in Gorakhpur. All solvents used were of analytical grade.

2.2. Sample Extraction

Powdered seeds of black cumin (250 g) were subjected to hydrodistillation in Clevenger apparatus for 3 h according to the method recommended by European Pharmacopoeia, [7]. A volatile oil with light orange characteristic odour was obtained with yield of 0.9%. It was dried over anhydrous sodium sulphate and the sample was stored at 4°C before use.

Oleoresins were obtained by extracting 30 g of powdered spice with 300 mL of various solvents (ethanol, ethyl acetate, and n-hexane) for 3 h in Soxhlet extractor. Evaporation of the solvents at reduced pressure gave viscous extracts. The oleoresins were stored in freezer until further use.

2.3. Chemical Characterization

2.3.1. Gas Chromatography-Mass Spectrometry (GC-MS)
Analysis of the volatile oils and oleoresins was run on a Hewlett Packard (6890) GC-Ms system coupled to a quadruple mass spectrometer (model HP 5973) with a capillary column of HP-5MS (5% phenyl methylsiloxane; length = 30 m, inner diameter = 0.25 mm, and film thickness = 0.25 μm). GC-MS interphase, ion source, and selective mass detector temperatures were maintained at 280°C, 230°C, and 150°C, respectively. Carrier gas used was helium with a flow rate of 1.0 mL min−1. The oven temperature was programmed as follows.

For essential oil: at 60°C for 1 min then increased from 60 to 185°C at the rate of 1.5°C min−1 and held at the rate of 9°C min−1 and held at 275°C for 2 min.

For oleoresin: 60°C for zero min then increased from 60 to 300°C at the rate of 1.5°C min−1 and held at the rate of 5°C min−1 and held at 300°C for 10 min.

2.4. Identification of Components

Most of the components were identified on the basis of comparison of their retention indices and mass spectra with published data [6, 8, 9], and computer matching was done with the Wiley 275 and National Institute of Standards Technology libraries provided with the computer controlling GC-MS systems. The retention indices were calculated using a homologous series of n-alkanes C8–C18 and C8–C22 for essential oil and oleoresins, respectively, which are reported in Tables ​Tables11 and ​and22.

Table 1
Table 1
Chemical composition of essential oil obtained from black cumin seeds analyzed by GC-MS.
Table 2
Table 2
Chemical composition of oleoresins obtained from black cumin (Nigella sativa L.) seeds in different solvents analysed by GC-MS.
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3. Antioxidant Activity
The antioxidant activity is system dependent and according to the method adopted and lipid system used as substrate. Hence, different methods have been adopted in order to assess antioxidative potential of black cumin oil and its oleoresins are as follows.

3.1. Chelating Activity on Ferrous Ions

The chelating activity of the aqueous and ethanolic extract on ferrous ions (Fe2+) was measured according to the method described by Decker and Welch [10]. Aliquots of 1 mL of different concentrations of the samples were mixed with 3.7 mL of deionized water. The mixture was incubated with FeCl2 (2 mM, 0.1 mL). After incubation the reaction was initiated by addition of ferrozine (5 mM and 0.2 mL) for 10 min at room temperature, and then the absorbance was measured at 562 nm in a spectrophotometer. A lower absorbance indicates a higher chelating power. The chelating activity of the extract on Fe2+ was compared with that of EDTA that was used as positive control. Chelating activity was calculated using the following formula:

Chelating  activity(%)  =[1−(Absorbance  of  sampleAbsorbance  of  control)]×100.
(1)
3.2. Scavenging Effect on DPPH

The DPPH assay constitutes a quick and low cost method, which has frequently been used for the evaluation of the antioxidative potential of various natural products, [11]. Due to its odd electron, DPPH gives a strong absorption band at 517 nm (deep violet colour). In the presence of a free radical scavenger, this electron becomes paired, resulting in the absorption loss and consecutive stoichiometric decolorization with respect to the number of electron acquired. The absorbance change produced by this reaction is assessed to evaluate the antioxidant potential of the test sample. 5, 10, 15, and 20 μL of the sample were added to 5 mL of 0.004% methanol solution of DPPH. After a 30 min incubation period at room temperature, the absorbance was read against a blank at 515 nm. All determination was performed in triplicate and results were performed in triplicate and results are reported as scavenging effect (%) versus concentration in Figure 2.

Figure 2
Figure 2
Scavenging effect (%) of black cumin oil and its oleoresins on DPPH radical.
3.3. Estimation of Total Phenolic Content (TPC)

TPC were determined using the Folin-Ciocalteu reagent method described by Singleton and Rossi [12]. Gallic acid stock solution (1000 μg mL−1) was prepared by dissolving 100 mg of gallic acid in 100 mL of ethanol. Various dilutions of standard gallic acid were prepared from this stock solution. Calibration curve (Figure 3) was plotted by mixing 1 mL aliquots of 10–100 μg mL−1 of gallic acid solutions with 5.0 mL of Folin-Ciocalteu reagent (diluted tenfold) and 4.0 mL of sodium carbonate solution (75 g L−1). The absorbance was measured after 30 min at 20°C at 765 nm.

Figure 3
Figure 3
Calibration curve of gallic acid.
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4. Evaluation of Antioxidant Activity for Linseed Oil System
For present investigation, crude linseed oil, having initial peroxide value 5.2 meq kg−1, was taken to assess the antioxidant