Journal of Essential Oil Bearing Plants Chemical ...

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Chemical Composition of the Essential Oil of Nigella sativa Seeds Extracted by Microwave Steam Distillation a

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Farid Benkaci-Ali , Rym Akloul , Asma Boukenouche & Edwin De Pauw

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University of Sciences and Technologies Houari Boumediène, Faculty of Chemistry, Laboratory of Organic and Functional Analysis, U.S.T.H.B, B.P. 32 El Alia, Bâb Ezzouar, Algiers, Algeria b

University of Liège, Laboratoire de Spectrométrie de Masse L.S.M, Allée du 6 Août, Bât B6c, 4000 Liège (Sart-Tilman), Belgium Published online: 24 Dec 2013.

To cite this article: Farid Benkaci-Ali, Rym Akloul, Asma Boukenouche & Edwin De Pauw (2013) Chemical Composition of the Essential Oil of Nigella sativa Seeds Extracted by Microwave Steam Distillation, Journal of Essential Oil Bearing Plants, 16:6, 781-794, DOI: 10.1080/0972060X.2013.813275 To link to this article: http://dx.doi.org/10.1080/0972060X.2013.813275

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TEOP 16 (6) 2013 pp 781 - 794

781 ISSN Print: 0972-060X ISSN Online: 0976-5026

Chemical Composition of the Essential Oil of Nigella sativa Seeds Extracted by Microwave Steam Distillation Farid Benkaci-Ali 1*, Rym Akloul 1, Asma Boukenouche 1, Edwin De Pauw 2

Downloaded by [Farid Benkaci-Ali] at 09:35 02 January 2014

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University of Sciences and Technologies Houari Boumediène, Faculty of Chemistry, Laboratory of Organic and Functional Analysis, U.S.T.H.B, B.P. 32 El Alia, Bâb Ezzouar, Algiers, Algeria 2 University of Liège, Laboratoire de Spectrométrie de Masse L.S.M, Allée du 6 Août, Bât B6c, 4000 Liège (Sart-Tilman), Belgium Received 28 May 2012; accepted in revised form 29 November 2012

Abstract: The essential oil of Algerian Nigella sativa seeds, isolated by microwave steam distillation (MSD) with cryogenic grinding of plant material (CG) were analysed by GC and GC-MS. Two procedures have been investigated, the MSD1 (seeds inside of oven apparatus) and MSD2 (seeds outside of oven apparatus). According to the data values reported in this study the effect of cryogrinding and the technique used on the chemical composition of the essential oils is very important. Monoterpene hydrocarbons (51.2-60.2%) were the main group of CG essential oils compared to CLG (classical grinding) essential oils (33.1-47.8 %). Whereas, the ketones formed the main proportion in CLG essential oils (46.1-59.9 %) followed by CG essential oils (32.7-40.8 %). Forty-nine compounds were identified and significant differences in quantities of the major constituents were observed, mainly were thymoquinone (CLG: 42.3-56.1 % and CG: 28.1-36.0 % ), p-cymene (CLG: 23.1-31.5 %, CG: 33.0- 38.0 %), dehydro-sabina ketone (CLG: 3.1- 3.3 %, GC: 4.4-4.5 %), carvacrol (CLG: 1.3-1.4 %, CG: 0.4-1.1 % ) and longifolene (CLG: 1.5-2.1 %, CG:1.3-1.7 % respectively). On the other hand, the N. sativa essential oil was investigated for its antioxidant activities using four different tests then compared with BHT. Results showed that it exhibit a good activity in each antioxidant system with a special attention for β-carotene bleaching test (IC50: 19 to 28 μg/ml) and reducing power (EC50: 8 to 15 μg/ml). Key words: Nigella sativa, Ranunculaceae, composition, Microwave, Steam-distillation, Cryogrinding, GC and GC-MS, Antioxidant activity. Introduction Herbs and spices are invaluable resources, useful in daily life as food additives, flavours, fragrances, pharmaceuticals and colours or directly in medicine. Black seed, the seed of Nigella sativa L. (Ranunculaceae), has been employed for thousands of years as a spice, food preservative and curative remedy for numerous disorders 1,2. The historical tradition of N. sativa seed use in medicine is substantial. N. sativa Linn. (Ranunculaceae) is known to have beneficial *Corresponding author (Farid Benkaci-Ali) E-mail: < [email protected] >

effects on a wide range of diseases, antiasthmatic , antitumor 4, antibacterial 5, anti-inflammatory 6 , gastroprotective 7, antimalarial 8, antihypertensive 9, antidiabetic 10, anti-atherosclerotic 11, protective and antioxidant 12, nutritional 13 and anti-cholesterol 14. Thymoquinone, the main constituent of the essential oil of N. sativa seeds, was capable to also exert beneficial effects on acute gastric ulcer 15 . In addition the thymoquinone and its degradation product the thymohydroquinone have been 3

© 2013, Har Krishan Bhalla & Sons


Farid Benkaci-Ali et al., / TEOP 16 (6) 2013 781 - 794 reported to have an antibacterial activity and benefic interaction with some antibiotics 16. In this paper, the potential of the MSD1 (microwave steam distillation, seeds exposed radiations) and MSD2 (microwave steam distillation, seeds not exposed to radiations) techniques have been studied by using the classical and the cryogenic grinding, as the current techniques and commercial situation call for research into new extracts and new extraction techniques. The fundamental principle of cryogenic grinding (cryogrinding) for herbal medicine is similar to that of grinding methods for conventional materials. While using the liquid nitrogen or liquid air as the cryogen, all of these thermosensitive herbal medicines can be ground below their brittle temperature. The colour and other properties of the products of cryogrinding will not be changed and the flavour and nutrition of the herbal medicines will not be lost. In fact, the compositions are very complex, containing aromatics of high volatility, oils and fats, which are easily oxidized. Considering the success of the culture of spices, particularly N. sativa seed in the arid areas (Adrar, Southwest of Algeria), it is useful to investigate the chemical composition and the biological activities of this seed according to the extraction technique and the grinding mode. We have applied MSD1, MSD2, MSD1-CG and MSDE-CG techniques to extract essential oils from aerial parts (seeds) of Algerian (Adrar) N. sativa L. belonging to the family Ranunculaceae which is a highly advanced and homogeneous family, largely used in food preparation and medicine. We make appropriate comparisons in term of extraction yields and rates, essential oil composition, and energy consumption. In addition, the other aim of this work was to study the antioxidant activity against several pathogenic microorganisms according the techniques used. Material and methods Plant material Mature black cumin (Nigella sativa L.) seeds were cultivated and collected from Adrar area (Southwest of Algeria). The initial moisture of

782

these seeds was 8 %. They were stored at 4°C until extraction. Nigella Seeds (60 g) were milled in an electric heavy-duty grinder (speed: 20,000 rpm) for 20 s, to pass 180-250 μm screens (Ika Werke standard model Germany) and then subjected immediately to oil extraction. The cryogenic grinding was carried out by adding about 80 ml of the liquid nitrogen at -196°C to seeds (60 g) and subjected immediately to grinding in the same conditions as the classical grinding. The average size of seeds is < to 100 μm. MSD apparatus and procedure The microwave extraction process was carried out in a microwave laboratory oven 17-19, at atmospheric pressure. The microwave power was fixed at 600W (non-focused radiation). The plant material samples was submitted to MSD using a simple column (Diameter ϕ = 3 cm, Height h=15 cm) containing the ground seeds (figures 1-a and b) coupled to a Clevenger apparatus 20 and treated with 300 ml water for 7 min (until no more essential oil was obtained). The plant material is in the first technique inside of the microwave apparatus MSD1 (Fig.1-a) exposed to radiations. Whereas in the second method, the seeds are placed out of the oven (MSD2) (Fig.1-b). The recovered essential oil was dried over anhydrous sodium sulphate and stored at -4°C. GC and GC-MS analysis GC analysis of the essential oils were carried out on a 6890 series instrument (Agilent Technologies, Palo Alto, CA, USA), equipped with FID and a HP-5 fused silica capillary column (30 m x 0.25 mm (internal diameter), film thickness 0.25 μm). The temperature was programmed from 60-245°C at 5°C/min; held isothermal at 60°C for 8 min and at 250°C for 10 min. The injector and detector temperature are respectively kept at 280 and 300°C; sample injection volume, 0.2 μL; splitless. The carrier gas was nitrogen at a flow rate of 0.5 mL/min (0.03 MPa). GC-MS (70 eV) data were measured on the same gas chromatograph coupled with MSD


Farid Benkaci-Ali et al., / TEOP 16 (6) 2013 781 - 794

783

Figure 1. a-Microwave steam distillation, seeds exposed to radiations, b- Microwave steam distillation, seeds not exposed to radiations 5973. MS source temperature at 230°C; MS quadrapole temperature at 150°C; interface temperature at 280°C; mass scan, 50-550 amu; carrier gas He at a flow rate of 1.0 mL/min. The solvent delay time was 4 min. Oven temperature progression, column operating conditions, injection mode, carrier gas conditions and injector temperature were similar to GC ones. The volume injection for all samples was 1 μl (1 % in hexane in splitless). The retention index was calculated using a homologous series of n-alkanes C7-C28. Compounds were identified by comparison of their retention indices and massspectra with the data given in the literature, National Institute of Standards and Technology (NIST). The comparison of the retention indices (RI) of the volatile oil constituents compared with those of the published index data 18,21 confirmed the identification. Analyses were performed at least three times, and the mean values were reported. Antioxidant activity 1-Diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical-scavenging activity The effect of the tested essential oils on DPPH

degradation was estimated according to the method described by Hanato et al. 22 And Espin et al. 23 with small modifications. Each volatile oil was diluted in pure methanol at different concentrations, and then 2 ml were added to 0.5 ml of a 0.2 mmol/l DPPH methanolic solution. The mixture was shaken vigorously and left standing at room temperature for 30 min. The absorbance of the resulting solution was then measured at 517 nm measured after 30 min. The antiradical activity (three replicates) was expressed as IC50 (μg/ml), the antiradical dose required to cause a 50 % inhibition. A lower IC50 value corresponds to a higher antioxidant activity of essential oil 24. The ability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect (%)=[(A0 -A1) x 100]/A0 (1)

Where A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. Superoxide anion scavenging activity was assessed using the method described by Duh et al. 25. The reaction mixture contained 0.2 ml of


Farid Benkaci-Ali et al., / TEOP 16 (6) 2013 781 - 794 essential oil has different concentration, 0.2 ml of 60 mM PMS stock solution, 0.2 ml of 677 mM NADH, and 0.2 ml of 144 mM NBT, all in phosphate buffer (0.1 mol/l, pH 7.4). After incubation at ambient temperature for 7 min, the absorbance was read at 560 nm against a blank. The value of the antioxidant activity was based on IC50. The IC50 index value was defined as the amount of antioxidant necessary to reduce the generation of superoxide radical anions by 50 %. The IC50 values (three replicates per treatment) were expressed as μg/ml. As for DPPH, a lower IC50 value corresponds to a higher antioxidant activity of plant extract. The inhibition percentage of superoxide anion generation was calculated using the following formula: Superoxide (%) = [(A0 -A1) x 100]/A0

(2)

where A0 and A1 having the same meaning as in Equation (1). Reducing power The ability of the extracts to reduce Fe3+ was assayed by the method of Oyaizu 26, 1 ml of Nigella sativa essential oil were mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1 % K3Fe(CN)6. After incubation during 50°C for 25 min, 2.5 ml of 10 % trichloroacetic acid was added and the mixture was centrifuged at 650 g for 10 min. Then, 2.5 ml of the upper layer was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1 % aqueous FeCl3. The absorbance was measured at 700 nm. The average absorbance values were plotted against concentration and a linear regression analysis was carried out. Increased absorbance of the reaction mixture indicated increased reducing power. EC50 value

784

(mg/ml) is the effective concentration at which the absorbance was 0.5 for reducing power. Ascorbic acid was used as positive control. Results Extraction Table 1 lists the yields, extraction times and energy consumption of the MSD1, MSD1-CG, MSD2, and MSD2-CG essential oils compared to HD and HDAM essential oils (respectively isolated by conventional hydrodistillation and hydrodistillation assisted by microwave previously studied 27. The higher values were attributed to the seeds from MSD1-CG (1.2 %) followed by MSD2-CG (0.9 %), MSD1 (0.9 %) and MSD2 (0.9 %). These values are widely higher than those obtained by some authors 28-31 (0.2 to 1.3 %). All oils (dark yellow color) are characterized by strong odor, especially those isolated by the MSD1-CG and MSD2-CG techniques, which were relatively fresher. They tend to crystallize partially at a lower temperature (-8°C) after 72 to 96 hours of freezing, and acquire a lightly greenish and black-colored crystalline structure probably due to the thymoquinone. The cryogrinding process allows extracting more amount of essential oil compared to the classical grinding. In addition the major quantity of oil was obtained during the two first minutes. As shown in table 1, an extraction time of 7 min (MSD) provides high yields compared to those obtained by means of HD (2.5 h) or HDAM (10 min) 27. As noted previously, the reduced cost of extraction is clearly advantageous for the MSD method, particularly with cryogrinding in terms of time, energy and speed to reach the extraction temperature.

Table 1. Yields, extraction time and energy consumption of N. sativa seeds essential compared to those obtained in literature

Time extraction (min) Yields (%) Energy consumption/g of essential oils (KWh)

MSD-1

MSD1-CG

MSD-2

MSD2-CG

7 0.9 0.086

7 1.2 0.057

7 0.9 0.080

7 0.9 0.077

HD 27 HDAM 27 250 0.2 1.75

10 0.20 0.14


Farid Benkaci-Ali et al., / TEOP 16 (6) 2013 781 - 794 Chemical composition Analysis of the oils by GC and GC-MS revealed 49 compounds (All identified) in the N. Sativa seed oils samples (Table 2). Substantially higher amounts of oxygenated compounds and lower amounts of monoterpene hydrocarbons are present in the essential oils of the seeds extracted by MSD1 and MSD2 in comparison with MSD1CG and MSD2-CG. Monoterpene hydrocarbons are less valuable than oxygenated compounds in terms of their contribution to the fragrance of the essential oil. Conversely, the oxygenated compounds are highly odoriferous and, hence, the most valuable. The greater proportion of oxygenated compounds in the MSD and MSD-CG essential oils is probably due to the diminution of thermal and hydrolytic effects, compared with HDAM and particularly with HD22 which uses a large quantity of water and is time and energy consuming. The unidentified pick reported by many authors (m/z (% rel.int.) 125 (100), 153 (88), 85 (85), 72 (82), 93 (80), 121 (42), 81 (74), 136 (37), 168 (2)) (27, 32) was tentitatively elucidated as the dehydrosabina ketone present in all samples studied. The MSD1 oils could be distinguished from the MSD2 oils by their richness in ketones (59.9 % MSD1 vs. 46.1 MSD2) and alcohols (4.2 % MSD1 vs 2.9 % MSD2). Whereas, MSD2 oils could be also differentiated from the MSD1 oils by their greater richness in monoterpene hydrocarbons (33.1 % MSD1 vs. 47.8 % MSD2). The sesquiterpene hydrocarbon percentages are practically close (2.9 % MSD1 vs. 2.6 % MSD2). The same observations were noted when we used the cryogrinding with high percentage in ketones (40.8 % MSD1-CG vs 32.7 % MSD2-CG) and of alcohols (4.8 % MSD1-CG vs 2.1 % MSD2CG) by MSD1-CG compared to MSD2-CG and high percentage of monoterpene hydrocarbons (51.2 % MSD1-CG vs 60.2 % MSD2-CG) in MSD2-CG compared to MSD1-CG. Consequently, the best and the most natural oils as MSD1-CG and MSD2-CG are richer in monoterpene hydrocarbons than other oils MSD1-CG and MSD2-CG. The MSD1-CG oils could be also differentiated

785

from the MSD2-CG oils by their greater richness in sesquiterpene hydrocarbons (2.7 % vs 1.9 % respectively). Notable amounts of sesquiterpene hydrocarbons were identified in the oils essentially in MSD1 (2.9 %) and MSD1-CG (2.7 %) oils dominated mainly by longifolene, αlongipinene and Z-β-bisabolene. Whereas, a few amount of aldehydes was isolated in many oils not exceeding 0.1 %. On the other hand, no trace of diterpene hydrocarbons was detected in our oils compared to the conventional hydrodistillation HD and HDAM 19,27, 28, where a moderate amount of the diterpene hydrocarbons (pimaradiene) was identified (Table 2). Globally, the cryogenic grinding accelerates the extraction phenomena, enhances the global essential oil yields and considerably reduces thermal reactions as the transformation of thymoquinone (TQ) to thymohydroquinone (THQ) as reported in other works 31. Indeed, the longer extraction techniques as HD 32 or solvent extraction procedure followed by steam distillation (SESD) 30 showed a high amount of thymohydroquinone varying from 6.10 to 12.2 % with HD and from 19.7 to 53.6 % with SESD. Whereas only a few amount of THQ was detected in MSD essential oils not exceeding 0.1 %. The main components in MSD1, MSD1-CG, MSD2 and MSD2-CG oils were thymoquinone (56.1 %, 36.0 %, 42.3 % and 28.1 % respectively), p-cymene (23.1 %, 33.0 %, 31.5 % and 38.0 % respectively), dehydrosabina ketone (3.1 %, 4.4 %, 3.3 % and 4.5 % respectively) and carvacrol (1.3 %, 1.1 %, 1.4 % and 0.4 % respectively) followed by longifolene (1.5 %, 1.7 %, 2.1 % and 1.3 % respectively). As reported in the literature, the valuable constituents that have beneficial chemical effects were principally thymoquinone 11,15,16, p-cymene 33 , carvacrol 34, 4-terpineol 28 (Table 3). They represent a global amount of 81.1 % of ground seed in MSD1, 70.9 % in MSD1-CG, 75.7 % in MSD2 and 66.6 % in MSD2-CG. Furthermore, it is interesting to note that the previously characterized dimmer dithymoquinone (or nigellone) 32 was not detected in all oils seen by the authors, as confirmed by Burits and Bucar 28. The two methods of steam distillation assisted

RI ref 930 939 952 954 957 961 975 975 979 990 1002 1017 1024 1031 1029 1042 1050 1059 1062 1070 1088 1089 1098 1114 1120 1121 1134 1137 1138 1155

Compounds

α-Thujene α-Pinene α-Fenchene Camphene 2,4(10)-Thujadien Benzaldehyde trans-Pinane Sabinene β-Pinene β-Myrcene α-Phelandrene α-Terpinene p-Cymene 1,8-Cineole Limonene Benzene acetaldehyde (E)-β-Ocimene γ-Terpinene +(o)-Tolualdehyde ti cis-Sabinene hydrate Terpinolene p-Cymenene trans-Sabinene hydrate 2-Isopropyl -5-mthyl-(2-Z)-hexanal Dehydro-sabina ketone ti β-Fenchol cis-Limonene oxide Mentha-2.8-diene-1-ol-cisCamphor Karahanaenone

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

932 937 949 955 956 958 973 977 979 991 1005 1013 1030 1034 1035 1046 1050 1058 1058 1068 1088 1091 1097 1116 1124 1124 1131 1135 1147 1154

RI 5.2 1.1 tr tr 0.7 1.4 tr tr tr 23.1 0.4 1.0 tr tr 0.3 tr tr tr 0.5 3.1 0.1 0.1 -

MSD1 9.8 2.1 tr tr 0.3 3.3 tr tr 0.1 0.33 0.4 1.2 tr tr 1.0 tr tr tr tr 0.8 0.1 4.4 0.1 0.1 -

MSD1-CG 7.6 1.6 0.1 tr 1.0 2.1 tr 0.1 0.2 31.5 0.4 1.7 tr tr 1.2 tr tr 0.1 0.2 0.1 3.3 0.1 -

MSD2 7.7 7.6 0.1 0.1 tr 2.5 2.0 0.1 tr tr 38.0 0.3 1.1 tr 2.0 1.2 tr tr tr 0.1 4.5 tr -

MSD2-CG 4.1 tr tr tr 1.2 2.3 tr 0.4 32.0 0.4 1.2 0.2 0.2 2.2 0.1 -

0.6 2.0

HDAM 27

0.7 0.1 tr 0.4 0.1 0.1 7.2 0.4 0.4 0.8 5.9 0.1

HD 27

Table 2. Chemical composition of Algerian N. sativa seeds essential oils extracted by different techniques


Farid Benkaci-Ali et al., / TEOP 16 (6) 2013 781 - 794 786

RI ref 1159 1171 1177 1182 1193 1194 1205 1205 1235 1242 1252 1253 1266 1257 1258 1270 1284 1287 1288 1286 1291 1299 1314 1352 1359 1365 1376 1390 1401 1401

Compounds

β-Pinene oxyde Umbellulone 4-Terpineol p-Cymen-8-ol cis-Piperitol Myrtenol trans-Piperitol p-Cymen-9-ol Thymol methyl ether ti Carvone Thymoquinone trans-Sabinene hydrate acetate (E)-Cinnamaldehyde Linalool acetate 3-Methylcatechol Geranial (E)-Anethole p-Cymen-7-ol Bornyl acetate Thymol 2-Undecanone Carvacrol (E,E)-2,4-Decadienal α-Longipinene Eugenol Neryl acetate Copaene iso-longifolene Dodecenal Methyl eugenol

No.

31 32 33 34 35 36 37 38 49 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

table 2. (continued).

1160 1169 1178 1183 1195 1198 1206 1206 1236 1240 1260 1265 1266 1268 1270 1273 1286 1288 1290 1292 1294 1305 1317 1356 1363 1367 1375 1392 1395 1402

RI 0.4 0.8 1.1 tr 36.0 0.1 tr tr 0.1 0.1 1.1 0.4 0.4 tr -

0.5 0.8 56.1 0.1 tr tr tr 0.1 1.3 0.8 0.3 tr -

MSD1-CG

0.2

MSD1

0.5 tr 42.3 0.1 0.1 0.2 0.1 0.1 1.4 0.2 0.5 tr -

0.2

MSD2


0.5 0.8 28.1 tr tr tr 0.1 0.1 0.4 0.3 0.3 tr -

0.3

MSD2-CG 0.3 8.9 2.1 0.2 3.1 0.3 21.8 0.1 0.7 0.1 0.7 0.1 12.9 0.1 0.7 0.1 tr tr

HD 27

1.2 2.0 0.1 3.3 0.3 0.1 24.6 0.1 0.3 0.3 0.1 6.0 2.2 0.1 tr 0.1 -

HDAM 27


RI ref 1400 1407 1404 1408 1419 1433 1480 1504 1509 1515 1521 1522 1524 1529 1555 1563 1611 1671 1677 1683 1726 1941 1959

Compounds

β-Longipinene Longifolene (Z)-Caryophyllene Dodecanal (E)-Caryophyllene γ-Elemene ar-Curcumene 2-Tridecanone β-Bisabolene Z-γ-Bisabolene 6-Methyl-α-(E)-ionone Eugenol acetate δ-Cadinene Citronellyl n-butyrate Thymohydroquinone (Z)-Isoeugenol acetate Tetradecanal β-Bisabolol 5-Neo-cedranol α-Bisabolol iso-Longifolol Tetradecanoic acid Isopropyl myristate Pimaradiene n-Hexadecanoic acid Monoterpene hydrocarbons Sesquiterpene hydrocarbons Alcohols Ketones

No.

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

table 2. (continued).

1407 1410 1412 1409 1424 1435 1487 1497 1505 1516 1522 1526 1521 1530 1557 1565 1606 1672 1678 1682 1733 1769 1823 1931 1962

RI tr 1.5 tr 0.3 tr 0.3 0.1 tr 0.6 tr 33.1 2.9 4.2 59.9

MSD1 tr 1.7 tr 0.4 tr 0.2 tr tr 0.4 tr 51.2 2.7 4.8 40.8

MSD1-CG tr 2.1 tr 0.1 0.1 0.1 0.1 0.1 0.4 tr 47.8 2.6 2.9 46.1

MSD2


tr 1.3 tr 0.1 tr 0.2 tr tr 0.1 tr 60.2 1.9 2.1 32.7

MSD2-CG 1.8 tr 0.1 0.1 0.2 0.4 0.1 0.1 12.2 0.2 0.1 0.3 0.2 0.2 0.1 0.1 0.1 2.5 9.8 2.8 42.1 25.1

HD 27 6.0 0.1 0.2 0.3 0.2 0.3 0.2 0.2 1.1 0.2 0.3 1.1 0.1 0.3 1.3 42.1 8.9 15.1 25.5

HDAM 27


0.5 0.8 2.7 0.2 1.0 8.8 RI ref: Retention indices of reference 21 RI: calculated retention indices in this work tr = trace, less than 0.1%, ti: Tentitatively identified, (-): not detected, MSD1: Steam distillation assisted by microwave technique 1 classical grinding of Nigella S. Seeds MSD1-CG: Steam distillation assisted by microwave technique 1 with cryogrinding of Nigella S. seeds MSD2: Steam distillation assisted by microwave technique 2 classical grinding of Nigella S. seeds MSD2-CG: Steam distillation assisted by microwave technique 2 with cryogrinding of Nigella S. Seeds