CHARACTERISTICS OF OILS AND NUTRIENT CONTENTS OF NIGELLA SATIVA LINN. AND TRIGONELLA FOENUM-GRAECUM SEEDS

The core objective of this research was to determine the oil characteristics and nutrient contents of Nigella sativa and Trigonella foenum-graecum seeds. Characteristics of seed oils revealed higher degree of unsaturation and as determined by gas liquid chromatography (GLC) reported herein the major unsaturated fatty acids were linoleic acid (52.6% in N. sativa and 42.5% in T. foenum-graecum), followed by oleic acid (23.5% in N. sativa and 20% in T. foenum-graecum), while the main saturated fatty acid was palmitic acid (16% in N. sativa and 10.5% in T. foenum-graecum). Triacylglycerols and neutral lipids were found to be most abounded components recorded to 78.4 and 93.2% for N. sativa and 84.8 and 93.2% for T. foenum-graecum, respectively. The seed oils, therefore, have potential for use as domestic and industrial oils. Compositional analysis revealed that both samples contained considerable amounts of protein (20% in N. sativa and 28% in T. foenum-graecum) and high amount of lipid (37%) in N. sativa seeds. The seeds are shown to be rich sources of potassium, calcium and sodium and other elements. Nutrient information reported herein illustrates the benefits to public health for consumers of these plant seeds.


INTRODUCTION
Nigella sativa L. is an annual herbaceous plant belonging to the Ranunculaceae family [1].It tastes slightly bitter and peppery with a crunchy texture.Seeds are angular, of generally small size (1-5 mg), dark grey or black color.N. sativa seeds are used for edible and medicinal purposes in many countries.They are used as a condiment in bread and other dishes [2,3].They are also used in the preparation of a traditional sweet dish, composed of black cumin paste, which is sweetened with honey or syrup, and in flavoring of foods, especially bakery products and cheese.N. sativa seed oil is considered as one among newer sources of edible oils, thanks to its important role in human nutrition and health [4].On the other hand, Trigonella foenum graecum is an annual herb belonging to the legume family; it is widely grown in India, Egypt, and Middle Eastern countries.T. foenum graecum has historically been utilized mainly as whole seed; it is a potential protein source with high nutritive value [5].The seeds of this ancient herb have been used as both a spice and an herbal remedy in the Middle East, India, and Egypt and slightly shorter time in Europe, China and other parts of the world [6].
Some research works on proximate and fatty acids composition of the seeds of N. sativa and T. foenum-graecum from different origins have been reported.Sultan et al. characterized the indigenous variety of black cumin (Nigella sativa L.) and its fixed and essential oils and concluded that black cumin holds nutraceutical potential against various physiological threats owing to its rich phytochemistry especially due to the presence of thymoquinone, tocopherols, etc [7].Salma et al. determined physicochemical properties of two Nigella seed varieties, having a Tunisian and Iranian origin and results suggested that Nigella seed oil could deserve further consideration and investigation as a potential new multi-purpose product for industrial, cosmetic and pharmaceutical uses [4].Bahman et al. studied the chemical composition of the extracted fixed oil and volatile oil of Nigella sativa L. seeds grown in Iran by GC and GC/MS and identified eight fatty acids (99.5%) and thirty-two compounds (86.7%) in the fixed and volatile oils, respectively [8].Nazar and Tinay determined the proximate composition and physicochemical properties of a protein concentrate prepared from fenugreek (Trigonella Foenum graecum L.) seed.Results showed that fenugreek protein concentrate had high oil absorption capacity, water absorption capacity and bulk density [9].Abdel-Nabey and Damir investigated the changes in some nutrients of fenugreek seeds during water boiling and concluded water boiling of fenugreek seeds for various lengths of time lowers to some extent its nutrients through leaching out into the boiling water or the brew [10].Hemavathy and Prabhakar estimated lipid composition of fenugreek seeds and identified at least five glycolipids and seven phospholipids [11].
Review suggests that comprehensive and systematic studies to create database on oil characteristics and nutrient contents of Nigella sativa and Trigonella foenum-graecum seeds are still limited.It has therefore, decided to make the necessary measurements to characterize seed oils of N. sativa and T. foenum-graecum, including acylglycerol class, lipid class and fatty acid composition and also nutrient contents of their seeds.

Plant materials and chemicals
The seeds of N. sativa and T. foenum-graecum were purchased in April, 2007 from local market in Rajshahi city, Bangladesh.The seeds were dried in sunlight for four consecutive days and then in an electric oven at 40 o C until a constant weight was reached.The seeds were ground to a fine powder, packaged, and stored in a refrigerator at 4 o C prior to the analysis.Solvents were obtained from Merck (Darmstadt, Germany) and BDH (Poole, England).Silica gel (60-120 mesh) and Silica gel (HF 254 ) were products of Merck (Darmstadt, Germany).Esters of fatty acids and bovine serum albumin were from Sigma Chemical Co.(St.Louis, MO, USA).All other chemicals were of analytical grade unless otherwise specified and results were expressed on dry weight basis.

Analysis of N. sativa and T. foenum-graecum seed oils
The oil from the powdered seeds was extracted with light petroleum ether (40-60 o C) in a soxhlet apparatus for about 24 h and the solvent was removed by rotary vacuum evaporator (Buchi Labortechnik AG, Postfach, Switzerland).The percentage of oil content was computed.

Separation of acylglycerols
The oil was separated into mono-, di-and triacylglycerols by IUPAC method no 2.321 [12] using silica gel (60-120 mesh) column chromatography.For quantitative determination of acylglycerol classes, the sample was adsorbed on the top of the column; triacylglycerols were eluted with benzene, diacylglycerols with a mixture of diethyl ether and benzene (1:9, v/v), and monoacylglycerols with diethyl ether.Approximately 1.5-2 mL/min fractions were collected.Elution was monitored by thin layer chromatography (TLC).The percentage of diacylglycerols was calculated by subtracting the weight of free fatty acid (FFA) from the weight of diacylglycerols fraction.

Fractionation of lipids
A total of 598 mg lipid extracted from the seeds by the method of Bligh and Dyer [14], was fractionated into three major lipid groups: neutral lipid, glycolipid, and phospholipid by silica gel column chromatography [15].Neutral lipids were eluted with chloroform, glycolipids with acetone and phospholipids with methanol.Approximately 0.5-1.0mL fractions were collected per minute and elution was monitored by TLC.Solvents were evaporated in vacuum rotary evaporator and percentages of these fractions were determined by gravimetric method.

Fatty acid composition of oil
Fatty acid composition of seed oil was determined as their methyl esters prepared by borontrifluoride methanol complex method [16].A GCD PYE Unicam gas chromatograph (PYE Unicam Ltd., Cambridge, UK) equipped with a flame ionization detector was used to determine the fatty acid methyl esters.Nitrogen carrier gas was used at a flow rate of 30 mL/min.Fatty acids were separated on a 1.8 m × 2 mm i.d.glass column packed with 6% BDS (butanediol succinate polyesters) on solid support, Anakorm ABS (100/120) mesh.Analysis was carried out at isothermal column temperature 190 o C, injector and detector temperatures for all GLC analysis were 240 o C. The peaks were identified by comparison with standard fatty acid methyl esters.

Analysis of N. sativa and T. foenum-graecum seeds
Moisture, ash and crude fiber contents were determined by AOAC methods [17].Lipid content was estimated by the method of Bligh and Dyer [14] using a solvent mixture of chloroform and methanol (2:1 v/v).The micro-Kjeldahl (Buchi Labortechnik AG, Switzerland) method [17] was employed to determine the total nitrogen and protein content was calculated from total nitrogen by using N × 6.25.Water soluble protein was determined by the method of Lowry et al. [18] using bovine serum albumin as the standard.Determination of starch content was based on analytical method outlined elsewhere [19].Total sugar content was determined by colorimetric method [20] and total carbohydrate was calculated by the difference.The samples for mineral analysis were subjected to acid digestion and analyzed following the procedures described by AOAC [17].Phosphorus was determined by vanadomolybdate method while other elements were estimated by using AAS (atomic absorption spectrometer, Pye Unicam model SP9, Cambridge, UK).

RESULTS AND DISCUSSION
The solvent extracts of N. sativa and T. foenum-graecum seeds yielded 32 and 6.4% oil, which are close to the values 28.5% [4] and 7% [9], respectively.Information on detailed characteristics of oil and nutritional composition of seeds from same species are too scanty for meaningful comparisons.

Physico-chemical characteristics
As shown in Table 1, specific gravities estimated with N. sativa (0.9071 at 25 o C) and T. foenum-graecum (0.9200 at 25 o C) seed oils were higher than the value 0.8840 at 30 o C for Cucumis prophetarum seed oil reported by Mariod et al. [21].Refractive indices of the oils were found to be 1.4683 for N. sativa and 1.4742 for T. foenum-graecum at 30 o C, being higher than 1.4340 at 40 o C [21] for the Cucumis sativus seed oil.Refractive index of N. sativa is consistent with the reported values 1.4600-1.4700at 40 o C for the same seed oil [4].Iodine values estimated for N. sativus (115) and T. foenum-graecum (112) were consistent with the value 114 for Cucumis sativus seed oil [21].Iodine value of N. sativa was lower than 119 reported by Salma et al. [4] for same seed oil of Tunisian variety.The iodine values obtained in this study indicate that the N. sativus and T. foenum-graecum seed oils contain high level of unsaturated bonds.Therefore, the samples in the present investigation have higher tendency to become rancid by oxidation.The comparatively low saponification value of T. foenum-graecum seed oil (177) as estimated indicates the presence of higher proportion of higher fatty acids than that contained in N. sativus seed oil (204).The investigated saponification value for N. sativus was lower than the values 211-218 mentioned by Salma et al. [4], but similar to 203 mentioned by Atta [1] for the same seed oil.The FFA content (12%) in N. sativus seed oil was similar to the value 11 cited in the literature [1], but much higher than 1.3 for Cucumis sativus [21] and 1.3 for T. foenum-graecum seed oils.Results regarding FFA contents indicate the suitability of the oil sample of T. foenum-graecum for probably edible purpose as it contained significantly lower percentage of FFA than that contained in the sample of N. sativus.The high acidity of oil may be related to the nature of N. sativus seed, akin to many oil-bearing seeds, such as olive, palm and rice bran, contain high acidity oils [22].[21] and 1.0-1.8% in the same oil mentioned by Atta [1], this value being lower than 3.2% in T. foenum-graecum seed oils under present study.The lower percentage of unsaponifiable matters as obtained in the sample of N. sativa points to lower amounts of hydrocarbons, higher alcohols and sterols than that contained in T. foenum-graecum.The peroxide value of N. sativa (12.7 mEq/kg) was within the range of 10.7-13.5 revealed by Atta [1], but much higher than 4.3-5.6 reported by Salma et al. [4] for the same oil.On the other hand, the sample of T. foenum-graecum exhibited peroxide value of 13.7 mEq/kg; being higher than the value 3.5 revealed by Mariod et al. [21] seed oil.The low Reichert-Meissl value as estimated for T. foenum-graecum (0.54) indicate the low content of lower volatile soluble fatty acids than that contained in N. sativus (0.97), and this value is also in agreement with the low saponification value as obtained in T. foenum-graecum.Acetyl values of N. sativus and T. foenum-graecum seed oils were found to have 3.6 and 2.4, respectively.

Acylglycerol and lipid composition
As shown in Table 2, mono-, di-and triacylglycerol contents were accounted to be 3.4, 5.3 and 78.4% for N. sativus and 2.9, 8.2 and 84.8% for T. foenum-graecum seed oils, respectively.The total recovery of acylglycerols in T. foenum-graecum was more than 95% (average) that indicated T. foenum-graecum seed oil contained lower amount of nonocylglycerol than that contained in Mesua ferrea [23] and N. sativus seed oils.Moreover, triacylglycerols content in N. sativus (78.4%) was found to be lower than T. foenum-graecum (84.8%) seed oil, but higher than the values 57.5-63.2%revealed by Atta [1] for the same seed oil.Neutral lipids account for 93.2% in both N. sativus and T. foenum-graecum of total lipids while only 3.2% in N. sativus and 3.4% in T. foenum-graecum glycolipids were detected.Phospholipids make up 2.1% in N. sativus and 2.3% in T. foenum-graecum of total lipids.Results indicated neutral lipids were found to be most abundant component of seed lipid recorded to over 93% of the total weight of the lipid.However, the amounts of glycolipids and phospholipids accounted from N. sativus and T. foenum-graecum seed oils were found to be lower than those for Momordica charantia seed oil; neutral lipids being higher [24].

Fatty acid composition
The fatty acid patterns (Table 3) of N. sativus seed oil were qualitatively similar to those of other plants; the essential fatty acid linoleic (52.6%) being the major fatty acid followed by oleic acid (23.5%).Linolenic acid was detected in small amount in the sample.Also, it was noted that N. sativus oil contained mainly unsaturated fatty acids (78.4%), while saturated fatty acids detected were only 21.6%.Major saturated fatty acid was palmitic (16%).The most prominent feature of the fatty acid composition in N. sativus seed oil was the high amount of linoleic acid, being slightly higher than that reported by Atta [1] and Salma et al. [4] for the same oil.On the other hand, T. foenum-graecum seed oil contained higher amount of linoleic (42.5%) while linolenic acid, oleic acid and palmitic acid contents were found to be 18, 20 and 10.5%, respectively.Besides these fatty acids, the oil also contained small amounts of stearic acid (6.5%) and arachidic acid (2%).Moreover, the amount of linoleic acid detected in both plant seed oils, was comparable to many seed oils such as Cucurbita maxima (43-50.3%),Cucurbita argyrosperma (35.6-45.3%)and Cucurbita argyrosperma (56%) reported by Applequist et al. [25]; it is likely to satisfy the essential fatty acid requirement for humans.The nutritional value of linoleic acid is due to its metabolism at tissue levels, which produce the long chain polyunsaturated fatty acids and prostaglandins [26].Vegetable oils high in unsaturated fatty acids have been well documented to provide numerous health benefits [27]; therefore, incorporation of N. sativus and T. foenum-graecum seed oils into the diet would be salubrious.Palmitic acid, which was the highest in the saturated acid profile in the present investigation, may be the precursor for higher fatty acids.The percentages of the fatty acids evaluated in N. sativus and T. foenum-graecum seed oils were slightly different from the previously reported works mentioned above, that might be due to the genetic factors and environmental conditions during fruit development and maturity [28].42.5 ± 1 Linoleinic acid (C18:3) 18 ± 0.2 Arachidic acid (C20:0) 2 ± 0.1

Nutritional composition
Nutritive composition of N. sativus and T. foenum-graecum seeds were determined and the results are shown in Table 4.It is found that moisture content (4.2%) of N. sativus was lower than the reported value of 7% for the same source [1] and also lower than 5.6% for Cucumis sativus seed [29] and 8.1% estimated herein for T. foenum-graecum seed.Knowledge of moisture content is important to determine a product that can be stored for a long time without the probability of being attacked by bacterial and fungal agents that could alter the quality through decomposition [30].Total lipid contents (37% for N. sativus and 12% for T. foenumgraecum) as estimated, were higher than the reported value 31.8% for N. sativus [31] and 7.1% for T. foenum-graecum seeds [9].The ash content of N. sativus (4%) was close to reported values of 3.7% [1] and 4.2% [7] for the same source.T. foenum-graecum seed contained 2.6% ash; being lower than the reported value 3.3% [9] for the same seed.Total protein content of N. sativus seed was found to be 20% in which 4.5% of it was water soluble, and this value for total protein was similar to 20.8% as quantified by Atta [1], but lower than 28.6% [29] for Cucumis sativus seeds.On the other hand, the total protein in T. foenum-graecum seed was 28% in which 10.7% of it was water soluble; this quantity for total protein was similar to 28.4% reported elsewhere for the same source [9].The present results reveal that both seed samples are qualified as protein-rich to satisfy the protein needs of consuming population.Starch contents of N. sativus and T. foenum-graecum were determined to be 4.1 and 4.8%, being lower with respect to 23.3% for Cucumis melo var.inodorus and 19.0 % for Cucumis melo hybrid AF-522 seeds [32].Crude fiber content in N. sativus (5.1%) was lower than 6.0% reported for the same seed sample [7].T. foenum-graecum seed contained crude fiber 4.7% that was lower than the reported value 9.3% [9].There is evidence that crude fiber has a number of beneficial effects related to its indigestibility in the small intestine [33].Total sugar content was estimated as 1% and carbohydrate content as 30% from N. sativus seeds.Carbohydrate content in N. sativus is slightly lower than the value 33.7% reported by Atta [1], but higher than 19.8% for Cucumis melo var.inodorus seeds [32].T. foenum-graecum seed contained total sugar 1.7% and carbohydrate 45 %.The carbohydrate content was slightly lower than the 47.4% reported by Nazar and Tinay [9].The most challenging aspect of providing trace elements in plant-based material is to obtain a sufficient concentration for the supplement to be ingested without consuming large quantities of plant tissue.With regard to minerals, N. sativus seeds, on which we report herein (Table 5), appeared to contain useful quantities of calcium (611 mg/100 g) and copper (3.8 mg/100 g); these amounts being higher compared to the corresponding values 570.0 and 2.6 mg/100 g reported in the literature [7].T. foenum-graecum seed contained 226 mg/100 g calcium and 5.4 mg/100 g copper.Calcium content was lower than 234 mg/100 g, but copper content being higher than 0.94 mg/100 g in T. foenum-graecum reported elsewhere [34].The calcium content makes the N. sativus seed flour attractive as a natural source of calcium supplementation for pregnant and lactating women, as well as for children and the elderly people.Iron contents in N. sativus (10.2 mg/100 g) and T. foenum-graecum (11.6 mg/100 g) seeds were close to the reported values 9.7 mg/100 g for N. sativus [7] and 10.2 mg/100 g for T. foenum-graecum [10].Thus, N. sativus and T. foenum-graecum seeds could contribute significant amounts of iron in the diet.Iron is an essential microelement for haemoglobin formation, normal functioning of the central nervous system and in the oxidation of carbohydrate, protein and fats [35].The high iron content in seed makes it a good source of iron particularly for menstruating and lactating women.The use of the seeds in soup may be encouraged.
N. sativus contained 6.4 mg/100 g zinc that was similar to 6.2 mg/100 g reported by Sultan et al. [7] whereas zinc content in T. foenum-graecum (4.4 mg/100 g) was lower than the value 5.4 mg/100 g [34], but higher than 2.3 mg/100 g [10] reported elsewhere.Potassium content in N. sativus (702 mg/100 g) was lower than the literature value, 808 mg/100 g [7].On the other hand, T. foenum-graecum seeds contained 1080 mg/100 g potassium; being higher than the value found in Detarium microcarpum seeds [36].Potassium is the most abundant element in all the plant parts analyzed.Magnesium and phosphorus contents in N. sativus seeds were found to have 85.2 and 108 mg/100 g, respectively.The corresponding values in T. foenum-graecum seeds were 78.4 and 200 mg/100 g.
Magnesium and phosphorus contents in N. sativus were lower compared to the reported values 265.0 and 543.0 mg/100 g [7].Magnesium content in T. foenum-graecum was lower compared to the previously reported value 188.0 mg/100 g [10].Magnesium is an important element in connection with circulatory diseases and calcium metabolism in bone [37].Phosphorus is related to calcium for bones, teeth and muscles growth and maintenance [38].Sodium contents in N. sativus and T. foenum-graecum were found to be 280 and 290 mg/100 g respectively; these values are higher than 17.6 mg/100 g for N. sativus seeds reported by Sultan et al. [7], but lower than 438.6 mg/100 g for Detarium microcarpum seeds [36].N. sativus and T. foenum-graecum seeds contained 1.4 and 1.6 mg/100 g manganese.The present value of manganese content in N. sativus was lower than 8.5 mg/100 g reported by Sultan et al. [7], but that in T. foenum-graecum being close or similar to 1.5 mg/100 g observed by Abdel-Nabey and Damir [10] and 1.6 mg/100 g observed by Kan et al. [34].
From the results, it becomes evident that N. sativus and T. foenum-graecum seeds could be considered as a good source of some important macro and micro elements.The mineral contents in N. sativus and T. foenum-graecum seeds have shown to a certain extent similar sequences in the order K > Ca > Na > P > Mg > Fe > Zn > Cu > Mn and K > Na > Ca > P > Mg > Fe > Cu > Zn > Mn, respectively.The contents were found to be different in some elements from what has been reported in the literatures as stated above.Such variation in nutrient contents may be related to the variations of cultivated regions, storage conditions and maturity stage.It may also be due to geographical and climatic differences where the sample seeds had been grown [1].

CONCLUSIONS
From the quality point of view, N. sativus and T. foenum-graecum seed oils are comparable to other oils and can be utilized in the paint, varnish and ink industries and also recommended for human consumption after properly refining.They constitute a good alternative source of essential fatty acids compared with common vegetable oils and could contribute to the overall dietary intake.On the other hand, in terms of both quantity and quality, these seeds are potentially attractive source of protein, lipid (only for N. sativus) and some common minerals that appear to have a very positive effect on human health.Nutrient information would be critical to the success of efforts to promote the wider use of indigenous plant foods as part of a broader program aimed at educating local populations with regard to the nutritional benefits of the many cultivated plant foods that exist in their environment.