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%),
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 [
Black cumin (
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.
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.
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.
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, [
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.
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
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.
Most of the components were identified on the basis of comparison of their retention indices and mass spectra with published data [
Chemical composition of essential oil obtained from black cumin seeds analyzed by GC-MS.
Compounds | %MS | RI# | Identification |
---|---|---|---|
|
5.6 | 919 | MS, RI, co-GC |
|
1.4 | 928 | MS, RI, co-GC |
Sabinene | 0.8 | 967 | MS, RI, co-GC |
|
1.7 | 973 | MS, RI, co-GC |
|
0.1 | 1003 | MS, RI |
|
0.2 | 1012 | MS, RI, co-GC |
|
31.4 | 1019 | MS, RI, co-GC |
Limonene | 1.0 | 1024 | MS, RI, co-GC |
1,8-Cineole | 0.1 | 1025 | MS, RI, co-GC |
|
0.2 | 1050 | MS, RI, co-GC |
|
0.1 | 1101 | MS, RI |
Unidentified B* | 6.8 | 1113 | − |
Terpinen-4-ol | 1.0 | 1172 | MS, RI, co-GC |
|
Trace | 1179 | MS, RI |
|
Trace | 1189 | MS, RI, co-GC |
Cuminal | Trace | 1240 | MS, RI |
Carvone | Trace | 1241 | MS, RI |
Thymoquinone | 37.6 | 1248 | MS, RI |
|
0.1 | 1258 | MS, RI |
Bornyl acetate | 0.2 | 1285 | MS, RI |
Thymol | 0.2 | 1289 | MS, RI |
Carvacrol | 1.4 | 1295 | MS, RI |
|
0.5 | 1353 | MS, RI |
Longifolene | 2.0 | 1405 | MS, RI |
Thymohydroquinone | 3.4 | 1559 | MS, RI |
10-epi- |
0.3 | 1625 | MS, RI |
|
0.5 | 1652 | MS, RI |
|
0.4 | 1655 | MS, RI |
|
|||
Total | 90.2% |
Trace < 0.05%; #the retention index was calculated using a homologous series of n-alkanes C8–C18;
Chemical composition of oleoresins obtained from black cumin (
Compounds | M1 | M2 | M3 | RI# | Identification |
---|---|---|---|---|---|
|
Trace | 0.4 | 0.6 | 919 | MS, RI, co-GC |
|
0.9 | 2.8 | 2.2 | 1019 | MS, RI, co-GC |
Unidentified A | 0.5 | 0.5 | 0.4 | 1092 | − |
Thymoquinone | 5.7 | 6.1 | 3.7 | 1248 | MS, RI |
Carvacrol | 0.4 | Trace | Trace | 1295 | MS, RI, co-GC |
|
Trace | Trace | Trace | 1353 | MS, RI |
Longifolene | 0.5 | 0.6 | 0.3 | 1405 | MS, RI |
Thymohydroquinone | 2.5 | 1.6 | 0.5 | 1559 | MS, RI |
Palmitic acid, ethyl ester | 2.8 | Trace | Trace | 1979 | MS, co-GC |
Linoleic acid, methyl ester | 0.6 | 0.5 | 0.5 | − | MS, co-GC |
Linoleic acid, ethyl ester | 11.6 | Trace | 0.6 | − | MS, co-GC |
Oleic acid, ethyl ester | 4.6 | Trace | 0.2 | − | MS, co-GC |
Oleic acid | 0.3 | Trace | 0.2 | − | MS, co-GC |
Linoleic acid | 33.0 | 43.9 | 27.7 | − | MS, |
Linoleic acid, butyl ester | 0.9 | 5.7 | 16.0 | − | MS |
Oleic acid, butyl ester | 1.2 | 4.5 | 7.3 | − | MS |
Glyceryl palmitate | 3.7 | 1.6 | 2.3 | − | MS |
Glyceryl linoleate | 27.7 | 21.9 | 23.1 | − | MS |
Sitosterol | Trace | 1.3 | Trace | − | MS, co-GC |
|
|||||
Total | 96.4% | 90.9% | 85.2% |
Trace < 0.05; #the retention index was calculated using a homologous series of n-alkanes C8–C20;
Percentages were obtained from electronic integration measurements using selective mass detector.
M1: ethanol oleoresin; M2: ethyl acetate oleoresin; M3: n-hexane oleoresin.
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.
The chelating activity of the aqueous and ethanolic extract on ferrous ions (Fe2+) was measured according to the method described by Decker and Welch [
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, [
TPC were determined using the Folin-Ciocalteu reagent method described by Singleton and Rossi [
For present investigation, crude linseed oil, having initial peroxide value 5.2 meq kg−1, was taken to assess the antioxidant activity of black cumin oil and its oleoresins. This oil is most frequently used edible oil in central Europe and is rather unstable because of the presence of substantial amount of linoleic acid. The antioxidant activity of volatile oil and extract was examined by comparing the activity of known antioxidants such as PG, BHT, and BHA by the following peroxide value and thiobarbituric acid value methods.
For measuring the peroxide value (PV), a modified oven test was used [
TBA value of different samples was determined according to the method previously reported [
Antioxidant activity of black cumin oil and its oleoresins was compared to synthetic standards according to the ferric thiocyanate method in linoleic acid emulsion [
The antifungal activity of the essential oil and extract against various pathogenic fungi,
The essential oil and extract were individually tested against a panel of microorganisms using agar well diffusion method [
For the essential oil or oleoresin, three samples were prepared for assays of every antioxidant and antimicrobial attribute. The data are presented as mean (standard deviation of three determinations (data are not shown). Statistical analyses were performed using a one-way analysis of variance [
Careful and detailed interpretations of the experimental GC-MS data (EM fragmentation, retention indices) were carried out which permitted identification of a large number of components in essential oil and oleoresins (Tables
From Table
The ferrous ion (Fe2+) chelating effect of black cumin oil and its different oleoresins is presented in Figure
Chelating effect of black cumin oil and its different oleoresins.
Scavenging effect (%) of black cumin oil and its oleoresins on DPPH radical.
Calibration curve of gallic acid.
Inhibitory effect of black cumin oil and its oleoresins on the primary oxidation of linseed oil measured using peroxide value method.
DPPH• is a stable radical showing a maximum absorbance at 515 nm. In DPPH• assay, the antioxidant was able to reduce the stable radical DPPH to the yellow-colored diphenylpicrylhydrazone. The method is based on the reduction of DPPH• in alcoholic solution in the presence of a hydrogen-donating antioxidant due to formation of the nonradical form DPPH-H in the reaction. DPPH• is usually used as a reagent to evaluate free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The disappearance of the DPPH radical absorption at 515 nm by the action of antioxidants is taken as a measure of antioxidant activity. The scavenging effects of black cumin oil and oleoresins on DPPH radical linearly increased as concentration increased from 5 to 20
The amount of total phenols was determined with Folin-Ciocalteu reagent. Gallic acid was used as standard compound. The absorbance for various dilutions of gallic acid with Folin-Ciocalteu reagent and sodium carbonate was obtained and found standard curve equation:
The changes of PV in linseed oil of all investigated samples are presented in Figure PG > Black cumin oil > Ethyl acetate oleo. > BHT > BHA > EtOH oleo. > n-hexane oleo. > Control.
Simultaneously with the measurements of PV, changes in secondary product such as malondialdehyde, the compound used as an indicator of lipid peroxidation was measured by TBA values (Figure
Inhibitory effect of black cumin oil and its oleoresins on the primary oxidation of linseed oil measured using TBA value method.
Inhibitory effect of black cumin oil and its oleoresins on the primary oxidation of linoleic acid system measured using ferric thiocyanate method.
The FTC method was used to measure the amount of peroxides at the primary stage of linoleic acid peroxidation (Figure
Antioxidant activities of essential oils and oleoresins may be related to the diverse compounds present in them including terpenes, sesquiterpenes, and phenolic acids, which act in various ways such as inhibition of peroxidation, scavenging the radicals, and chelating the metal ions. The main constituents of black cumin oil were thymoquinone (37.6%) and p-cymene (31.4%) with minor amounts of longifolene, carvacrol, and thymohydroquinone which were responsible for the antioxidant activity of black cumin oil [
Using inverted petri plate technique (Table
(a) Antifungal investigations of black cumin oil and its oleoresins (% zone inhibitiona) using inverted petriplate method. (b) Antifungal investigations of black cumin oil and its oleoresins (% zone inhibition) using food poisoned method.
Mycelial zone inhibition at different dosesa of sample (%) | ||||||
---|---|---|---|---|---|---|
Samples | Doses ( |
AN | AF | FM | FG | PV |
Black cumin oil | 5 |
|
|
|
|
|
10 |
|
|
|
|
|
|
EtOH oleoresin | 5 |
|
|
|
|
|
10 |
|
|
|
|
|
|
n-Hexane oleoresin | 5 |
|
|
|
|
|
10 |
|
|
|
|
|
|
Ethyl acetate oleoresin | 5 |
|
|
|
|
|
10 |
|
|
|
|
|
AN:
(−): no inhibition.
Mycelial zone inhibitiona at different doses of sample (%) | ||||||
---|---|---|---|---|---|---|
Samples | Doses (ppm) | AN | AF | FM | FG | PV |
Black cumin oil | 5 |
|
|
|
|
|
10 |
|
|
|
|
|
|
Ethanol oleoresin | 5 |
|
|
− |
|
− |
10 |
|
|
|
|
− | |
n-Hexane oleoresin | 5 |
|
|
− |
|
− |
10 |
|
|
− |
|
− | |
Ethyl acetate oleoresin | 5 |
|
|
− |
|
− |
10 |
|
|
|
|
− |
AN:
(−): no inhibition.
Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method.
Diameter of inhibition zone (mma) | ||||||
---|---|---|---|---|---|---|
Samples | Doses (ppmb) | BS | BC | SA | EC | PA |
Black cumin oil | 1000 | ++ |
|
++ | − |
|
3000 | ++ | ++ | ++ |
|
|
|
Ethanol oleoresin | 1000 |
|
− |
|
− | − |
3000 |
|
− |
|
− | − | |
n-Hexane oleoresin | 1000 |
|
− |
|
− | − |
3000 |
|
− |
|
− | − | |
Ethyl acetate oleoresin | 1000 |
|
− |
|
− | − |
3000 |
|
− |
|
− | − | |
Ampicillin | 1000 |
|
− |
|
− | − |
3000 |
|
− |
|
− | − |
bDMSO was used as solvent.
BS:
The antibacterial investigations were undertaken using agar well diffusion method (Table
The results obtained in antimicrobial investigations of black cumin oil and oleoresins were in good agreement with the previous reported work [
Seeds of black cumin seem to possess magical properties and have been worked out extensively. This study revealed that black cumin essential oil and its oleoresins constitute a good alternative source of essential fatty acids compared with common vegetable oil. The present results showed that essential oil and oleoresins of black cumin exhibited higher antioxidant activity than synthetic antioxidants. These findings could be used to prepare multipurpose products for pharmaceutical applications and its usage as dietary source of antioxidant should be considered largely for alleviating and ameliorating diseases.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to the Head of Department of Chemistry, DDU Gorakhpur, University, Gorakhpur for providing laboratory facilities. The financial support from UGC to Sunita Singh (JRF) and Emeritus Fellow to Dr. Gurdip Singh is also acknowledged.