Oil Content and Glycerolipids Composition
The oil content of N. sativa L. seeds calculated from the hexane extract on the basis of dry matter weight was of 31.7%. This result was slightly higher than that obtained by Cheikh-Rouhou et al. (2007) who reported an oil content of 28.48% in N. sativa L. seeds from the same location, using the same solvent extraction (hexane). However, they proceeded to the extraction of oil by Soxhlet apparatus during only 8 h, whereas in our case, the extraction time reached 12 h. On the other hand, Atta (2003) reported that the extraction of oil with petroleum ether from the seeds of N. sativa L. originated from Egypt yields 34.78% of crude oil. Nevertheless, this author reported that oil extracted by cold press was lower than that gained by solvent extraction.
In fully ripened seeds, TFAs account for 483.35 mg/g DMW. As shown in Table 1, the main fatty acids detected in N. sativa L. seeds were myristic (C14:0 = 3.2 g/100 g of TFA), palmitic (C16:0 = 12.2 g/100 g of TFA), stearic (C18:0 = 6.3 g/100 g of TFA), oleic (C18:1 = 12.7 g/100 g of TFA) and linoleic (C18:2 = 61.3 g/100 g of TFA), which represents the major fatty acid. It is well known that dietary fats, rich in linoleic acid, prevent cardiovascular disorders such as coronary heart diseases, atherosclerosis and high blood pressure. Also, it was reported that the nutritional value of linoleic acid is because of its metabolism at the tissue levels, which produces the long-chain polyunsaturated fatty acids and prostaglandins (Sayanova et al. 2003).
Table 1. FATTY ACID COMPOSITION (IN PERCENT OF TOTAL FATTY ACIDS) OF TUNISIAN NIGELLA SATIVA L. SEEDS
|Fatty acid||Percent of total fatty acids|
|Myristic (C14:0)||3.2 ± 0.1|
|Palmitic (C16:0)||12.2 ± 0.1|
|Stearic (C18:0)||6.3 ± 0.3|
|Oleic (C18:1)||12.7 ± 0.2|
|Linoleic (C18:2)||61.3 ± 0.4|
|Linolenic (C18:3)||1.5 ± 0.2|
|Arachidic (C20:0)||0.2 ± 0.05|
|Eicosenoic (C20:1)||0.4 ± 0.1|
|Behenic (C22:0)||2.2 ± 0.1|
|Saturated fatty acids||24.1 ± 0.8|
|Unsaturated fatty acids||75.9 ± 0.8|
Other fatty acids present in small amounts in the seeds of N. sativa L. were linolenic (C18:3 = 1.5 g/100 g of TFA), arachidic (C20:0 = 0.2 g/100 g of TFA), eicosenoic (C20:1 = 0.4 g/100 g of TFA) and behenic (C22:0 = 2.2 g/100 g of TFA) acids. In fully ripened seeds, saturated fatty acids represent 24.1% of TFAs, while unsaturated ones form 75.9%. In our study, a few amount of eicosenoic acid was detected (0.4%) in the chloroform–methanol extract and not reported by Babayan et al. (1978). These results are in agreement with those of Cheikh-Rouhou et al. (2007), whereas myristic (C14:0), linolenic (C18:3) and arachidic (C20:0) acids were not detected by Ramadan and Mörsel (2002) in the seeds of N. sativa L. from Turkey. On the other hand, few amounts of myristoleic (C14:1) = 0.18% and lignoceric (C24:0) = 1.08% acids were detected by Saleh Al-Jassir (1992) in N. sativa L. seeds from Saudi Arabia. These two fatty acids were also detected in very few amounts (in trace for myristoleic acid and 0.3% of TFA for lignoceric acid) by Atta (2003) in Egyptian N. sativa L. seeds. This author reported also small rates of palmitoleic acid (C16:1) = 0.7%. Only one study conducted by Cheikh-Rouhou et al. (2007) reported the presence of margaric (C17:0) and margaroleic (C17:1) acids in traces in the seeds of both Tunisian and Iranian N. sativa L. In our study, both of these fatty acids were not detected even in traces, which makes our study in agreement with the majority of previous works. The source of this variability in fatty acid composition may be genetic (plant cultivar, variety grown), environmental, seed quality (maturity, harvesting-caused damage and handling/storage conditions), oil-processing variables or accuracy of detection and quantitative techniques (Ramadan and Mörsel 2002).
Table 2 shows the proportions of NL classes in N. sativa L. seed oil. The data showed that NLs, which represent 93.5% of the total lipids, were mainly formed of TAGs (96%), free fatty acids (FFAs) with 2% and finally, diacylglycerols representing 1.9% of NLs, whereas monoacylglycerols were the less represented with 0.1% of NLs. Nevertheless, these results conflict with those reported by Ramadan and Mörsel (2002) who found that NL profile of N. sativa L. seeds was characterized by exceptionally high levels of FFAs (14.3–16.2% of the total NL). In these studies, the high amounts of FFAs could be the consequence of a high TAG lipolytic activity.
Table 2. GLYCEROLIPID COMPOSITION OF NIGELLA SATIVA L. SEED OIL
| ||g/100 g of NLs||g/100 g of PhLs||g/100 g of GLs|
|MAGs||0.1 ± 0.02||–||–|
|DAGs||1.9 ± 0.04||–||–|
|FFAs||2.0 ± 0.04||–||–|
|TAGs||96.0 ± 0.09||–||–|
|PI||–||9.3 ± 0.03||–|
|PC||–||37.2 ± 0.05||–|
|PG||–||2.1 ± 0.03||–|
|PS||–||12.6 ± 0.04||–|
|PE||–||21.5 ± 0.06||–|
|PA||–||17.3 ± 0.04||–|
|MGDG||–||–||43.7 ± 0.08|
|DGDG||–||–||55.9 ± 0.05|
|SQDG||–||–||0.4 ± 0.02|
Widely distributed in food, the PhLs have both pro- and antioxidant effects (Rathjen and Steinhart 1997; Boyd 2001). Even though they are used as food emulsifiers worldwide and although at the same time, they have a very positive image, their use in functional foods is still limited. Considering the amount of clinical data, there is no doubt that PhL will become a standard ingredient for this rapidly expanding category of food (Schneider 2001). As shown in Table 2, PLs from N. sativa L. seeds account for 6.5% of total lipids (TL) and were constituted of a major percentage of PhLs and a few proportions of GLs representing 0.4 and 0.09%, respectively, of total lipids. PhLs from N. sativa L. seed oil were separated into six classes by TLC and were identified as phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidyl ethanolamine (PE), and phosphatidic acid (PA) by comparing their retention times with those of authentic standards analyzed in the same conditions. PC was the most abundant class with 37.2% of total PhL content followed by PE, PA and PS, respectively. PC and PE together make 58.5% of the total PhL content. As for PG and PI, they were found to be present in insignificant amounts. According to Ramadan and Mörsel (2002), PC and PE together make up to 75% of the total PhLs from the seeds of N. sativa L., which originated from Turkey. On the other hand, these authors have shown that solvent and/or mixtures used in lipid extraction process play an important role in the amount and composition of recovered lipids.
GLs were separated by TLC according to Lepage (1967) into three subclasses: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). The most abundant subclass is DGDG, representing 55.9% of the total GL content, followed by MGDG with 43.7%, while SQDG amount is only 0.4%. The average daily intake of GLs in human has been reported to be 90 mg of MGDG and 220 mg of DGDG (Sugawara and Miyazawa 1999). Therefore, it is noteworthy that black cumin seed oil could be an excellent source of GLs in the human diet.
To our knowledge, there is only one reported study about the TS composition of N. sativa L. seeds (Zeitoun and Neff 1995). In fact, most previous works have described only the free sterol composition of black cumin seeds. As for the extraction of TSs, the most formerly method used is that of Bligh and Dyer (1959), utilizing chloroform and methanol. But more recently, Moreau et al. (2002) demonstrated the deficiency of this method and reported that the mixture chloroform–methanol is unable to extract all sterols contained in the sample. For this reason, we have used, in the present work, the method described by Brenac and Sauvaire (1996). In this method, the authors proceeded to extract lipids by hexane followed by a second extraction with isopropanol–water (70:30, v/v). Applying this method to our samples gave a majority of free sterols (FS) and esterified sterols (ES) and lower amounts of SG and ASG in the hexane extract, while the isopropanol-water extract contained a majority of SG and ASG, and smaller amounts of FS and ES. Comprising these two extracts allowed recovering the totality of sterols contained in the studied sample. On the other hand, before saponification, we have proceeded, in the present work, to a hydrolysis step using H2SO4 (0.18 N in ethanol 95%) according to Grunwald (1970) and permitted the glycosilic forms of sterols (SG and ASG) to hydrolyze, which releases their sterols. Finally, after saponification with KOH (10% in 95% ethanol), the extract contained only free sterols as the sum of sterols coming from the four subclasses (FS + ES + SG + ASG).
According to our results, TSs of N. sativa L. seeds account for 6.931 mg/g of dry matter weight, which is the equivalent of 2.2% of the fixed oil. As shown in Table 3, the GC/MS analyses showed that N. sativa L. sterols were composed of cholesterol representing a minority with 2.2% of TS, campesterol with 10.4%, Δ5-avenasterol forming 2.4%, two nonidentified sterols representing together 5.1%, β-sitosterol and stigmasterol as major sterols representing 60.2 and 19.6%, respectively, of TSs. As for cholesterol, it has been also detected by Atta (2003) in black cumin seeds from Egypt where it represented 7.2% of TSs, whereas this component is not detected by Ramadan and Mörsel (2002) in N. sativa L. seeds from Turkey. However, these authors reported the presence of lanosterol, representing 3.4% of TSs. On the other hand, it is known that the ratio of β-sitosterol/campesterol could be used as an index to identify the purity and the authenticity of oil. In the present investigation, the β-sitosterol/campesterol ratio is of 5.8, which is the same value found by Nergiz and Otles (1993) in black cumin seeds from Turkey.
Table 3. STEROL PATTERN OF BLACK CUMIN SEED OILS
|Sterols (mg/g in oil)|
| β-Sitosterol||13.24 ± 0.05|
| Stigmasterol||4.31 ± 0.03|
| Campesterol||2.28 ± 0.04|
| Cholesterol||0.48 ± 0.02|
| Δ5-Avenasterol||0.52 ± 0.02|
| Unknown a||0.68 ± 0.03|
| Unknown b||0.44 ± 0.01|
|Sterolic forms (g/100 g of total sterol)|
| FS||36.1 ± 0.07|
| ES||51.2 ± 0.09|
| Steryl glycosides||7.8 ± 0.07|
| Acylated steryl glycosides||4.9 ± 0.05|
As shown in Table 3, N. sativa L. sterols were mainly present in the esterified form with 51.2% of the TSs (3.122 mg/g of dry matter weight). In addition, free sterols accounted for 36.1% (2.201 mg/g of dry matter weight) and a few amounts of sterols were distributed between the SG and the ASG, representing together 12.7% of the TSs (0.775 mg/g of dry matter weight).
The predominance of the esterified and the free forms in seeds has been also reported by Katayama and Katoh (1973) during the ripening of soybean seeds. Davis and Poneleit (1974) revealed in corn seeds high proportions of free sterols at the beginning of seed development, which decreased during ripening in favor of esterified sterols probably by the activation of the acyl glycerol: sterol acyltransferase.
The optimization of the extraction yield of the volatiles contained in the oleoresin by the technique of dynamic headspace showed that 3 h is the time that permits the extraction of the maximum number of compounds, which is equivalent to 18 volatiles illustrated in Table 4. These compounds belong to four chemical classes: monoterpenic hydrocarbons forming 86.3% of the total volatiles with p-cymene as the major compound (53.1%) followed by ocimene (18.5%); monoterpenic alcohols representing 7.1% of the total volatiles and with octen-3-ol as the major compound (6.5%); terpenic ethers represented only by 1,8-cineole with a percentage of 1.9%; and monoterpenic phenols represented only by thymol with a rate of 1.8% of total compounds.
Table 4. AROMA COMPOSITION OF THE OLEORESIN EXTRACTED FROM NIGELLA SATIVA L. SEEDS
|Volatile compounds||N°||Retention indexes in HP Innowax capillary column||% of total aroma compounds|
| α-Pinene||1||1,009.10||1.4 ± 0.03|
| α-Thujene||2||1,015.63||7.2 ± 0.04|
| β-Pinene||3||1,088.35||1.8 ± 0.03|
| Sabinene||4||1,107.89||0.7 ± 0.03|
| α-Phellandrene||5||1,150.60||0.1 ± 0.02|
| Myrcene||6||1,165.37||2.1 ± 0.04|
| Limonene||7||1,187.62||0.1 ± 0.01|
| Ocimene||8||1,236.16||18.5 ± 0.05|
| γ-Terpinene||9||1,241.12||1.2 ± 0.02|
| p-Cymene||10||1,263.77||53.1 ± 0.07|
| Terpinolene||11||1,269.07||0.1 ± 0.01|
| Octen-3-ol||12||1,281.69||6.5 ± 0.03|
| Linalool||13||1,562.52||0.1 ± 0.02|
| Terpinene-4-ol||14||1,590.07||0.4 ± 0.01|
| α-Terpineol||15||1,698.13||0.1 ± 0.01|
| 1,8-Cineole||16||1,183.08||1.9 ± 0.03|
| Thymol||17||>2,000||1.8 ± 0.02|
|Nonidentified|| || ||2.9 ± 0.02|
Most of these compounds have been identified by D'Antuono et al. (2002) in the essential oil of black cumin originated from Morrocco and extracted by hydrodistillation. Also, these authors have mentioned that p-cymene is the major compound. On the other hand, a less important percentage of p-cymene (14%) was detected by Nickavar et al. (2003) in the essential oil of Iranian N. sativa L. extracted by steam distillation of the oleoresin. These authors reported that trans-anethole was the major component representing 38.3% of the total aroma.
These results confirm that N. sativa essential oil is a good source of bioactive compounds such as p-cymene, limonene, α-pinene, linalool and thymol, which were considered as powerful bactericides (Knobloch et al. 1989). These results may justify and support the utilization of this plant in traditional medicine for the treatment of certain infections.