Hydrodistillation of Nigella Sativa Seed and Analysis of Thymoquinone with HPLC and GC-MS

N. sativa seeds, commonly known as black seeds, are for headaches in traditional medicine by many Asian, Middle Eastern and Far Eastern countries. It is used to treat cough, abdominal pain, diarrhea, asthma, rheumatism and other diseases. The seeds contain both fixed and essential oils, proteins, alkaloids and saponin. Much of the biological activity of the seeds has been shown to be due to thymoquinone, the major component of the essential oil, but which is also present in the fixed oil. The essential oil of black cumin seeds, Nigella sativa L., was tested for a possible antioxidant activity. In this study, we measured the amount of thymoquinone compound in distilled water taken from aromatic water. The proportion of thymoquinone passing into the aromatic water show that it is necessary to consume the correct amount of water and essential oil, which is significantly measurable by GC MS and HPLC. Thymoquinone was dedected 790 ppm in aromatic water and 45.78% in essential oil. Sufficient analytical work was performed with this method and the results were shown.


Plant Material
N. sativa seeds were collected from Turkey Isparta region. After harvesting, all seeds were kept in the dark at +4 o C until extraction.

Essential oil extraction
The extraction procedure of the essential oil is as follows; The extract (fixed oil) obtained from the N. sativa seeds by cold press method was applied hydrodistillation using Clevenger type apparatus (Isparta University of Applied Sciences-Industrial Crops Laboratory). 50 g of the fixed oil obtained by the cold press method was dissolved in 500 mL of water which contains 10% of Tween 80 (5g) according to oil weight and the final volume was completed to 1 L. 50 grams of fixed oil in 1 L of water ,was hydrodistilled for 2.5 hours. The essential oil obtained with dark yellow color and pungent odor was dried with anhydrous sodium sulfate and kept in the dark at +4 o C until use. Aromatic water, along with essential oil from hydrodistilation products, was also collected for future analysis.

HPLC analysis of thymoquinone
The thymoquinone analysis of the black seed aromatic water was performed according to the modified HPLC method of developed by Selin et al. (2017).
For quantity analysis; standard thymoquinone solutions were prepared at concentrations of 62.5 125, 250, 500 and 1000 ppm. The areas of all these concentrations were used for calibration (r 2 =0.9995). Aromatic water was injected directly. Samples, a diode detector (DAD detector (Lambdamax = 278nm), an auto sampler (SIL -10AD vp), a vacuum degasser (DGU-14A), system controlled (SCL-10Avp) and a pump (LC-10ADvp). Chromatographic separations were carried out on AgilentEclipse XDB-C18 column (250x4.60 mm, 5 µm) The column temperature was 30 o C. 3% acetic acid, Methanol (50:50) was selected as mobile phase and the flow rate was 0.8. The injection volume is 20µL for each sample and standard The analysis time is 30 minutes and the detection wavelength is set to 278 nm for thymoquinone.

Essential oil components analysis with GC-MS
Essential oil analysis of volatile components were performed on a Shimadzu GCMS-QP2010 SE (Japan) model with Support Rx-5Sil MS capillary column (30 m x 0.25 mm, film thickness 0.25 µm). GC analysis were performed under the following conditions. Carrier gas 1mL / min. helium with flow. The split ratio is 1:10. After 1 minute at 60 o C, the temperature program reaches 250 o C with an increase of 4 o C per minute and was kept at 250 o C for 15 minutes. The mass spectra were taken at 70 eV. 970 microlitre hexan is added over 30 microliters of pure essential oil. 1 microliter is injected from the capped vial. Identification of components with mass spectra data according to mass library was used NIST, WILEY also thymoquinone reference standart was used. % areas of essential oil showed that thymoquinone was the major volatile component.

RESULTS AND DISCUSSION
Fixed oil obtained from N. sativa seeds by cold press had a green color, pungent aromatic odor. From the fixed oil, dark yellow, pungent essential oil was obtained by hydrodistillation method according to the above method. In conventional hydrodistilation methods, plant materials such as leaves, flowers, seeds, roots and so on are used. In our study fixed oil obtained from N. Sativa seeds was dissolved in water with Tween 80 surfactant and then hydrodistillation was performed. The purpose of using Tween 80 is to minimize the problems in the fixed oil-water mixture during hydrodistillation and to ensure a homogeneous mixture of constant oil and water. In similar conditions, a trace of essential oil was obtained in the hydrodistilation of black seed oil without surfactant. On the other hand 2.1% essential oil was obtained from the fixed oil obtained from the N. Sativa seed and collected for analysis in aromatic water. In a similar study, yellow essential oil was obtained by hydrodistilation method from fixed oil obtained by hexane extraction ( Nickavar et al. 2003). The main pharmacologically active compound in the structure of N. Sativa essential oil is thymoquinone. Determination of thymoquinone content in aromatic water according to HPLC method was performed as above. Standard, aromatic water chromatograms are given in Figure 2, Figure 3 respectively. The amounts of thymoquinone in the investigated sample are also given in Table 1.   Thymoquinone was determined by HPLC analysis of the aromatic water. In a similar study, 10 different black seed seeds and oil obtained from herbal extracts from Ankara were examined by HPLC according to the amount of thymoquinone they contain (Selin et al. 2017). Due to the presence of thymoquinone in aromatic water is likely. Solubility studies showed that thymoquinone had the same solubility in commonly used solvents such as 0.1N HCl and PBS (pH 5; 7.4 and 9.0) (Goyal et al. 2017) thought to be used.

CONCLUSIONS
In our study, 2.1% essential oil was obtained in hydrodistilation of black seed oil in the presence of surfactant (Tween 80). We used HPLC for the determination of the amount of thymoquinone in aromatic water, and GC-MS was used in the analysis of Nigella essential oil components. According to the analysis results thymoquinone was dedected 790±12 ppm in aromatic water and 45.78% in essential oil. In this original study, especially; It has been shown that Thymoquinone can be dissolved in aromatic water by distillation method. According to its benefits of Thymoquinone , this water can be used as drinking or other applications such as food and cosmetic. There are not much data for water solubility of Thymoquinone. With this study, we think that aromatic water can be contributed by the food, cosmetic and drug industry.