An In-Depth Examination into How Genotype, Planting Density, and Time of Sowing Affect Key Phytochemical Constituents in Nigella sativa Seed

Abstract

Nigella sativa, also known as black cumin, is esteemed for its rich reservoir of health-benefitting phytoconstituents nestled within its seeds. The composition of its seeds can be influenced by factors such as genotype diversity and agricultural practices. Understanding these dynamics is important for maximizing the nutritional and medicinal attributes of the seeds. This study investigated how different genotypes, growing densities, and sowing times affect oil yield and phytoconstituents of Nigella seeds in Northern Australia. The aim was to find the optimal combination of these factors to maximize desirable compounds. Our findings revealed variability in oil yield and phytoconstituents among different genotypes, growing densities, and sowing times. No single genotype stood out as having elevated levels of all desired compounds. For instance, genotype AVTKS#5 had high total phenolic content (TPC) and antioxidant capacity, while AVTKS#8 and AVTKS#7 excelled in thymoquinone (TQ) and polyunsaturated fatty acids (PUFAs) and monounsaturated fatty acids (MUFAs), respectively. Planting density had a nuanced impact, with no significant effect on oil yield and CUPRAC values, but higher densities decreased TPC, FRAP, and TQ. Interestingly, seeds cultivated at 20 and 30 plants/m² had higher ratios of MUFAs/SFAs, PUFAs/SFAs, and (MUFAs + PUFAs)/SFAs, indicating the importance of planting density in shaping fatty acid profiles. Sowing times also had a noticeable effect, with late sowing leading to a decrease in oil yield from 19% to 14%. May-sown seeds had higher TPC, FRAP, CUPRAC, and fatty acid ratios, while TQ levels peaked in June-sown seeds. Our study highlighted positive correlations among TPC, FRAP, CUPRAC, and TQ, emphasizing their collective contribution to the nutritional and medicinal potency of Nigella seeds. Fatty acids, on the other hand, showed no significant correlation with these parameters, indicating independent regulation. In summary, our comprehensive analysis provides insights into the factors (genotype and agronomic practice) that shape the phytochemical profile of Nigella seeds, and suggests better genotype, planting density, and time of sowing for the cultivation and quality production.

1. Introduction

Nigella sativa, a member of the Ranunculaceae family, has gained considerable commercial value due to its rich content of essential bioactive phytoconstituents [1]. Among these compounds, thymoquinone (TQ) (2-methyl-5-isopropyl-1,4-benzoquinone) stands out as one of the most crucial and prevalent bioactive substances found in Nigella seeds, contributing significantly to the plant’s medicinal properties [1,2,3,4,5]. Researcher efforts have extensively investigated the diverse molecular pathways through which TQ can potentially address various diseases [2,6,7]. Notably, TQ has demonstrated therapeutic potential in numerous areas, including hepatoprotection [8,9], neuroprotection [10,11], nephroprotection [12], gastroprotection [13], antimicrobial properties [6], and anti-cancer effects [14]. Moreover, studies have examined the synergistic effects of combining TQ with other compounds, revealing additional health benefits [15,16,17,18,19]. For example, research has shown that combining TQ with vitamin E results in heightened antioxidative and free radical scavenging activities compared to TQ alone [15].

Since Nigella is an oilseed crop, it contains a diverse array of fatty acids. Fatty acids are characterized by a straight or branched chain of carbon atoms attached to a carboxyl group (−COOH), with hydrogen atoms attached along the carbon chain. The fatty acids present in Nigella can be classified as saturated, monounsaturated, or polyunsaturated, with chain lengths ranging from C12 to C24 [7,20,21,22,23]. Fatty acids are the major component in N. sativa seeds and play an imperative role in biological systems. It is generally recommended to reduce the consumption of saturated fatty acids (SFAs) due to their association with increased risks of fat accumulation, metabolic disorders, and cardiovascular diseases [24,25]. Conversely, monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have beneficial effects on human health [26]. The consumption of MUFAs has been reported to lower cholesterol levels and reduce the risk of coronary heart disease, while PUFAs are effective in preventing various diseases, including cardiovascular diseases, neurological disorders, diabetes mellitus, and atherosclerosis [27,28,29].

Although N. sativa seeds contain many valuable phytoconstituents, the level of these compounds can be influenced by factors such as genotype and agronomic practices. This variation in composition ultimately determines the quality grade of the seeds. Several studies have reported compositional variation in important phytoconstituents, including fatty acids and TQ, in Nigella seeds from different parts of the world [22,30,31,32]. For example, Ahmad et al. investigated TQ levels in Nigella seeds from various geographical locations, including Sri Lanka, India (Uttar Pradesh, Delhi), Turkey, Egypt, Ethiopia, Saudi Arabia, Pakistan, Syria, Yemen, Afghanistan, and Sudan. They found the highest concentration of TQ, 3.03% (w/w), in seeds from Turkey and the lowest (0.01% (w/w) in seeds from Sudan [30]. This variation has led researchers to screen different sources of Nigella for higher levels of desirable phytoconstituents to identify superior genotypes for future breeding programs. However, there is limited information on the important health-benefiting phytochemical compositional variation in the seeds of N. sativa genotypes [22]. Different genotypes are grown in various locations across Australia, but there is a lack of quantitative data available regarding fatty acids, TQ, antioxidant capacities, and TPC. This data would provide evidence of their therapeutic potential.

Furthermore, N. sativa is found to grow in cool to dry areas with light snowfall and also in warm, humid regions [33]. Although Nigella does well in a range of climates, it prefers cooler growing conditions. The plant appears to prefer sandy loam soils rich in microbial activity, where excess water can drain easily, allowing it to endure in areas of heavy to moderate rainfall [33]. Soils with 60% moisture and a pH of 7.0 to 7.5, accompanied by temperatures between 12 and 18 °C, are found to be best growing conditions for the species [33,34]. Efforts are underway to select suitable sites and cultivate Nigella in various locations around the world to meet the increasing global market demand. However, there is a lack of knowledge regarding appropriate agronomic practices, particularly in terms of the optimal time of sowing and planting density, which may impact the successful qualitative and quantitative production of Nigella seeds. Some studies have been conducted to explore the effects of sowing time and plant density [35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50]. However, most of these studies have focused on plant growth and yield attributes rather than the quality biochemical parameters of the seeds, such as phytochemical analysis. Since the chemical composition of the seeds determines their quality grade and influences their potential health benefits and applications, it is important to explore the effects of sowing time and planting density on the phytochemical characteristics of N. sativa seeds. This exploration could provide valuable insights into optimizing cultivation practices to enhance their medicinal and nutritional value.

Additionally, all previous studies on the effects of sowing time and plant density have been conducted in various parts of the world, including Ethiopia [44], India [39], Greece [46,51], Turkey [48], Iran [35,37,45,50], Egypt [38], Bangladesh [36,41,42,47], and Poland [49], the geographical and environmental conditions of which are different to Australia. Since the optimal time for sowing and plant density varies based on geographical location and environmental conditions, most of these reports are not directly relevant to the Australian conditions. Therefore, this study was initiated with the aim of investigating the fatty acids, TQ, phenolic content, and antioxidant capacity of seed extracts obtained from different genotypes of Nigella. The study included plants grown under different sowing times and planting densities in Central Queensland, Australia, with the goal of identifying the best genotype, sowing time, and planting density for high quality seed production.

2. Materials and Methods

2.1. Chemicals and Reagents

All reagents were of analytical grade and were obtained from ChemSupply (Gillman, SA, Australia) or Sigma-Aldrich (Melbourne, VIC, Australia). Unless stated otherwise, Milli-Q^® water was used to prepare dilutions and conduct chemical analyses. The reagents and solutions were kept in the dark at 4 °C until they were utilized.

2.2. Trial Center and Experimental Material Production

Twelve genotypes of N. sativa seeds (AVTKS#1-AVTKS#12) were acquired from AgriVentis Technologies Pty Ltd. ((https://www.agriventistechnologies.com.au (Macquarie Place, Sydney, NSW, Australia)) and preserved in cold storage at 4 °C on 22 January 2019. The Central Queensland Innovation and Research Precinct (CQIRP), Rockhampton was chosen to grow Nigella plants and study the compositional variation in seeds obtained from different genotypes and sowing times. The geographical coordinates of the precinct were 23°32ʹ S, 150°51ʹ E. The genotypic study seeds were sown at the trial center on 1 May 2022, using a Randomized Complete Block Design (RCBD) with three replications under uniform climatic and soil conditions. The genotype AVTKS#3 was sown on six different dates (1 April, 1 May, 1 June, 1 August, 1 October, and 1 December 2022), also following Randomized Complete Block Design with three replications. Nigella was grown in raised garden beds (2 m × 1 m × 0.40 m) filled with red loam from a sweet potato farm, with controlled drip irrigation until physiological maturity. Soil tests determined the application of NPK (9:2:7) at 83:40:40 Kg NPK/ha based on Verma et al. [52].

Genotype AVTKS#12 was sown at five different densities (10, 20, 30, 40, and 50 plants/m²) with RCBD and five replications on 6 May 2022, at Alton Downs, Rockhampton (23°18′ S, 150°21′ E), using the same NPK dose as the CQIRP trial. The Rockhampton trial involved direct field planting with fortnightly irrigation until crop maturity. Climatic data for Rockhampton’s growing season is in

Table 1. Average monthly climatic conditions of the plant growing season in Rockhampton.

Months	Precipitation (mm)	Humidity (%)	Rainy Days	Average Sun Hours	Min. Temp (°C)	Max. Temp (°C)	Average Temperature (°C)
May	31	65	3	8.4	15.5	24.7	19.9
June	32	67	3	7.7	13.1	22.3	17.4
July	24	63	2	8.1	11.7	22.1	16.7
August	23	61	2	8.6	12.5	23.6	17.9
September	21	62	2	9	15.3	26.6	16.6
October	37	62	4	9	18.2	28.6	22.9
November	54	64	5	9.1	20.1	29.6	24.4
December	95	66	7	9.3	22	30.9	25.9

, sourced from the Australian Government Bureau of Meteorology (http://www.bom.gov.au). These climate conditions are applicable to both the CQIRP and Alton Downs trials. Seeds were grown in three rows, with the central row used for data collection. Plants were harvested at full maturation (170–190 days), air-dried for a week, manually threshed, and cleaned. The seeds were labeled, packed, and stored at 4 °C until phytochemical composition analysis.

2.3. Extraction Protocol

The methanolic extraction protocol developed previously in our laboratory was followed to prepare Nigella seed sample extracts [53]. Briefly, the dried Nigella seeds were ground using a Breville grinder (BCG200) for one minute. Duplicate extractions of 0.5 g of finely powdered samples were prepared. Each 0.5 g sample was mixed with 7 mL of 90% methanol and 10% water in a centrifuge tube, vortexed for 10 s, then shaken on an end-over-end shaker (Ratek RM4) for 60 min at 50 rpm. After centrifugation for 10 min at 1000× g (Heraeus X1 Multifuge, Thermo Fisher Scientific, Melbourne, VIC, Australia, 10 min, 1000× g), the supernatant was collected. The extraction was repeated on the pellet with another 7 mL of 90% methanol, shaken for 20 min. The supernatants were combined, adjusted to 14 mL with 90% methanol, and stored in the dark at 4 °C until analysis.

2.4. Experimental Analysis

2.4.1. Moisture Content

The moisture content of seed samples was determined gravimetrically as described by the ISTA Moisture Committee [54]. Briefly, 3 g of seed powder was uniformly spread within an aluminum tray, then heated to 103 °C in a drying oven (Memmert heating and drying oven, UM400 with natural convection, V: 220 V–50 Hz, Power: 1400 W, Schwabach, Germany) until constant weight was obtained (as estimated from weighing at 30 min intervals). This was achieved in 90–120 min. The weight of powder in the tray was observed stable (the highest difference between the value of last two determination was below 0.0020 g). Moisture content was calculated as follows:

$$Moisture Content \left(\%\right)=\frac{fresh weight of powder-dry weight of powder}{fresh weight of powder}\times 100\%$$

2.4.2. Solid and Oil Yield

The seeds were screw pressed at 60 °C to separate the oil and solid component. First, the seed samples were air-dried directly at 40 °C for 24 h to reduce moisture content below 5%. This initial moisture content was recorded. Then, a specific amount of Milli-Q^® water was added to each seed sample to reach a moisture content of 6%, calculated according to a mass balance equation. The samples were then packed in plastic bags, mixed, and stored in a refrigerator at 4 °C for 24 h [55]. Once all the Nigella seed samples had a similar moisture content, 20 g of seeds were extracted using an oil press machine (an automatic oil extractor pressure, temperature control commercial oil expeller with Voltage: 110 V/220 V, Power: 600 W–1500 W, and Size: 42 × 16 × 32 cm) of Wgwioo brand. The rotating speed of the screw was 58 rpm, and the seed feeding time was 20 g/min. These settings were maintained in order to obtain the oil and solid components (cake and sludge).

The pressed oil was collected in separate 10 mL centrifuge tubes. It was then centrifuged at 3000× g for 20 min. After centrifugation, the sludge accumulated at the bottom of the centrifuge tubes, while the clean oil remained above the sludge. The oils were decanted into new 10 mL centrifuge tubes. N-hexane was used, as suggested by [56], to wash and remove any remaining oil attached to the centrifuge tubes, ensuring that only sludge or sediments were left in the centrifuge.

The solid (sludge, cake, and waste remained in screw press) and oil yield of the extracted seed was estimated following the formula used by previous researchers [57,58]. The results were expressed in % of the dry weight of seeds.

$$Mass ballance equation=\frac{\left(target moisture \%-initial moisture\%\right)}{100}\times \left(seed weight\right)$$

$$Solid yield \%=\left(weight of solid components)/(seed weight used for screw press extraction\right)\times \left(100-moisture\%\right)$$

$$Oil yield\%=\left(weight of oil\right)/\left(seed weight used for screw press extraction\right)\times \left(100-moisture\%\right)$$

2.4.3. Total Phenolic Content (TPC)

The Folin–Ciocalteu method was applied to measure the TPC of the samples [53]. To perform the total phenolic assay, 400 µL of adequately diluted sample extract was added to a centrifuged tube, and then 2 mL of Folin–Ciocalteu reagent diluted in a ratio of 1:10 was added. The tube was vortexed and incubated at ambient temperature in darkness for 10 min. After that, 2 mL of 7.5% sodium carbonate aqueous solution was added, vortexed again, and then incubated at 40 °C for 30 min in a covered water bath. The absorbance reading of the sample was recorded at 760 nm using a UV spectrophotometer (Thermo Scientific Genesys 10S UV–Vis, USA) after measuring the blank using Milli-Q^® water. The TPC of the samples was determined based on the equivalent absorbance of a standard solution of gallic acid within the concentration range of 20 to 100 mg/L. The calibration curves for gallic acid standards exhibited excellent linearity (R² = 0.9967). The derived equation from these curves, y = 0.0095x + 0.0075, was utilized to quantify the total phenolic content in the samples. The outcomes were expressed in milligrams of gallic acid equivalents (GAE) per 100 g of dry sample weight (mg GAE/100 g DW).

2.4.4. Total Antioxidant Capacity Analysis Techniques

The reaction media, redox potentials, and mechanisms of action are different between antioxidant assays. Due to this phenomenon, the determination of total antioxidant capacity with at least two methods has been recommended [59,60,61]. Therefore, the ferric reducing antioxidant power (FRAP) method and cupric reducing antioxidant capacity (CUPRAC) method were selected for antioxidant analysis.

Ferric Reducing Antioxidant Capacity (FRAP) Analysis

The FRAP assay method applied by [53] was followed for this study. Briefly, fresh FRAP reagent was prepared using 300 mM of aqueous sodium acetate buffer, 10 mM of TPTZ (2,4,6-tris(2-pyridyl)-s-triazine), and 20 mM of ferric chloride aqueous solution in a ratio of 10:1:1, respectively. After the preparation of FRAP reagent, 100 µL of the sample was added to a centrifuge tube, and thereafter, 3 mL of FRAP reagent was added, vortexed, and incubated in a covered water bath at 37 °C for 4 min. The absorbance reading at 593 nm was recorded using a spectrophotometer after measuring the blank with Milli-Q^® water. The FRAP value of the samples was determined by assessing the equivalent absorbance of the standard Trolox solution within the 10–150 mg/L range. The calibration curves for Trolox standards demonstrated excellent linearity (R² = 0.9992). The equation derived from these curves, y = 0.0056x + 0.0712, was employed to quantify the total FRAP values in the samples. All results were expressed in milligrams of Trolox equivalents (TE) per 100 g of the dry weight sample (mg TE/100 g DW).

Cupric Reducing Antioxidant Capacity (CUPRAC) Analysis

The CUPRAC assay method applied by [53] was followed for this study. First, 10 mM of aqueous copper (II) chloride, 1 M of aqueous ammonium acetate, and 7.5 mM of neocuproine ethanol solution were prepared. Thereafter, one mL of each reagent including 1 mL of Milli-Q^® water, was added to a centrifuge tube before the addition of the 100 µL of sample extract. The tube was then vortexed and kept in a covered water bath for 30 min at 50 °C. The absorbance reading of the sample was recorded at 450 nm using a spectrophotometer after measuring the blank with Milli-Q^® water. The CUPRAC value for the sample was determined based on the absorbance equivalent of a standard Trolox solution within the 50 to 500 mg/L range. The calibration curves for Trolox standards exhibited excellent linearity (R² = 0.9988). The equation derived from these curves (y = 0.0014x + 0.1686) was then employed to quantify total FRAP values in the samples. All results were expressed in milligrams of Trolox equivalents (TE) per 100 g of the dry weight sample (mg TE/100 g DW).

2.4.5. Quantification of Thymoquinone (TQ)

The determination of TQ in N. sativa seed extract was conducted using High-performance Liquid Chromatography (HPLC). An Agilent 1100 HPLC system was employed, including a G1313A autosampler, G1322A vacuum degasser, G1311A quaternary pump, and G1365B multi-wavelength detector module. TQ was quantified following the conditions used by [62]. Briefly, an Agilent Eclipse XDB-C18 reversed phase column (dimensions: 150 × 4.6 mm; 5 µm particle size) was used. The isocratic mobile phase utilized was composed of water and methanol (40:60, v/v) at a flow rate of 1 mL/min. The injection volume of the sample was 5 µL and analysis was performed at room temperature. The analysis runtime was 10 min, and the UV monitoring of the eluted solutes was carried out at 254 nm for TQ. TQ identification was achieved by comparing retention times and UV spectra of peaks with those of pure standards. Quantitative analysis employed external standardization, measuring peak areas of pure standards prepared in the 10–200 ppm range with methanol. The calibration curves of pure standards demonstrated excellent linearity (R² = 0.9999). The equation derived from the calibration curves (y = 16.386x − 6.4406) was utilized for the quantification of TQ in the samples.

2.4.6. Fatty Acids Analysis

Derivatization (Methyl Ester Preparation)

Methyl ester was prepared following the protocol reported by [63]. Briefly, approximately 0.25 g of seed powder was transferred into a centrifuge tube. The tube was then added with 2 mL of the 0.4 M sodium hydroxide (NaOH) reagent solution (prepared with methanol) and incubated in a 55 °C water bath for 1.5 h after vortex. Subsequently, 2 mL of saturated sodium bicarbonate (NaHCO₃), prepared in Milli-Q^® water, and 3 mL of hexane were added into the tube, which was vortexed for 10 s and then centrifuged at 100× g for 10 min. After centrifugation, the hexane layer containing the FAME was separated and washed with 1 mL of Milli-Q^® water twice, and finally the 100 µL of the filtered hexane extract and 900 µL of hexane were combined into a GC glass vial and stored at 4 °C until analysis.

Gas Chromatography

Fatty acid methyl esters were estimated following the GC-MS protocol applied in previous studies [64]. The GC-MS system consisted of a single quadrupole Shimadzu QP2010 Plus system. This system was equipped with an autoinjector/autosampler (AOC-20 i/s) and utilized a Restek FAMEWAX column (30 m length × 0.32 mm ID × 0.25 µm thickness). For the injection, a volume of 0.5 μL was used in split mode, with a split ratio of 10, at an injection temperature of 250 °C. Helium was employed as the carrier gas at a flow rate of 2 mL/min. The temperature in the oven initially started at 195 °C and increased gradually at a rate of 5 °C/min until it reached 240 °C, where it remained constant for 1 min. The total duration of the run was 35 min. Additionally, both the ion source and the mass spectrometer interface were maintained at a temperature of 230 °C throughout the analysis.

For the purpose of exploration, Fatty Acid Methyl Esters (FAMEs) were identified using a scanning mode, where data were collected from 50 to 500 m/z. Subsequently, the identity of FAMEs was confirmed by matching their retention times with those of genuine external standards from the Restek Food Industry FAME Mix (REST-35077). Each fatty acid was quantified based on the calibration curve equation. The calibration range, including the observed calibration equation, linearity, limit of detection (LOD), and limit of quantification (LOQ) of individual fatty acid methyl ester, is illustrated in

Table 2. Quality parameters for the fatty acid methyl esters calibration identified in Nigella seeds.

Compound	Calibration Range (mg/L)	Linearity (R2)	Calibration Equation	σ	LOD (mg/L)	LOQ (mg/L)
Myristic acid, methyl ester	2–200	0.9968	Y = 13,367x − 16,736	67,412.68	16.64	50.43
Pentadecanoic acid	1–100	0.9959	Y = 12,830x – 8431	36,483.59	9.38	28.44
Palmitic acid	3–300	0.9968	Y = 12,841x − 28,151	96,994.24	24.93	75.54
Margaric acid	1–100	0.9972	Y= 12,072x − 7118.4	28,285.37	7.73	23.43
Stearic acid	2–200	0.9961	Y = 12,266x − 21,090	68,546.32	18.44	55.88
Arachidic acid	2–200	0.9957	Y = 11,793x − 20,305	68,610.06	19.20	58.18
Palmitoleic acid	1–100	0.9956	Y = 4944.8x − 3998.8	14,558.54	9.72	29.44
cis-10-Heptadecenoic acid (C17:1 (cis-10)	1–100	0.9961	Y = 5265.4x − 3815.7	14,571.59	9.13	27.67
Oleic acid	2–200	0.9972	Y = 7286.9x − 17,879	34,563.50	15.65	47.43
11-Eicosenoic acid	1–100	0.9966	Y = 5179.9x − 3032.8	13,452.14	8.57	25.97
Linoleic acid	1–100	0.9964	Y = 5454.8x − 4257.7	14,535.96	8.79	26.65
Alpha-Linolenic acid	1–100	0.9962	Y = 5348.5x – 4249	14,724.26	9.08	27.53
11,14-Eicosadienoic acid	1–100	0.995	Y = 5488.8x − 4646.2	17,356.86	10.44	31.62

. The residual standard deviation of regression line (σ) and slope were used to calculate LOD and LOQ, where the formula consisted of 3.3 × (σ/slope) and 10 × (σ/slope), respectively. The value of individual fatty acid was expressed in mg/g of seed.

2.5. Statistical Analysis

The determination of moisture, oil, and solid content (%) including fatty acids was performed on samples (treatment × field replications), but the remaining experiments were conducted with 2 laboratory replications of the samples (treatment × field replications × 2 laboratory replications), and the values were expressed as mean ± standard deviation (SD). Statistical analysis involved the utilization of one-way ANOVA conducted through IBM SPSS software version 28.000 (190). Statistical significance was established where p values < 0.05. Furthermore, the Pearson’s correlation test was employed to elucidate the associations between variables.

3. Results and Discussion

3.1. Moisture Content

The moisture content in the seeds of twelve different Nigella genotypes ranged between 4.8% and 8.1% (

Table 3. Representation of moisture, oil, and solid component; TPC; antioxidant capacity (CUPRAC and FRAP); and TQ in different genotypes of Nigella seeds.

Genotypes	Moisture (%)	Oil Yield (%DW of Seed)	Solid Yield (%DW of Seed)	TPC (mg GAE/100 g DW)	FRAP (mg TE/100 g DW)	CUPRAC (mg TE/100 g DW)	TQ (mg/100 g DW)
AVTKS#1	6.4 ± 0.2 c	17.4 ± 1.7 ab	76.6 ± 1.7 ab	878.4 ± 43.7 de	931.5 ± 83.7 ab	4092.8 ± 250.1 c	675.8 ± 68.1 a
AVTKS#2	5.6 ± 0.3 ab	16.5 ± 1.6 ab	77.5 ± 1.6 ab	722.3 ± 22.9 ab	972.8 ± 80.9 abc	4039.5 ± 208.6 c	740.7 ± 79.7 abc
AVTKS#3	5.8 ± 0.3 abc	17.2 ± 1.6 ab	76.8 ± 1.6 ab	792.1 ± 47.0 bcd	972.0 ± 76.1 abc	3933.1 ± 153.0 c	708.0 ± 67.8 ab
AVTKS#4	8.1 ± 0.5 e	16.6 ± 1.6 ab	77.4 ± 1.6 ab	853.9 ± 61.3 cde	1128.9 ± 78.2 d	4139.3 ± 166.3 c	865.9 ± 53.6 c
AVTKS#5	8.0 ± 0.4 e	17.0 ± 1.2 ab	77.0 ± 1.2 ab	922.5 ± 64.4 e	1138.7 ± 54.6 d	3902.8 ± 154.9 bc	783.3 ± 95.0 abc
AVTKS#6	8.1 ± 0.7 e	16.7 ± 1.7 ab	77.3 ± 1.7 ab	764.2 ± 56.1 bc	1041.6 ± 83.0 bcd	3933.0 ± 182.0 c	733.1 ± 56.9 abc
AVTKS#7	7.6 ± 0.1 de	15.6 ± 1.5 a	78.4 ± 1.5 b	840.1 ± 73.1 cde	1109.4 ± 81.0 cd	4159.0 ± 193.1 c	848.8 ± 64.2 bc
AVTKS#8	6.8 ± 0.3 cd	17.8 ± 1.8 ab	76.2 ± 1.8 ab	839.8 ± 51.3 cde	1070.6 ± 61.6 bcd	4007.1 ± 132.8 c	1118.6 ± 89.3 d
AVTKS#9	6.6 ± 0.3 bcd	15.5 ± 1.5 a	78.5 ± 1.5 b	884.7 ± 51.7 de	983.2 ± 82.2 abc	3796.0 ± 179.7 abc	1066.1 ± 77.3 d
AVTKS#10	4.8 ± 0.4 a	19.3 ± 1.9 ab	74.7 ± 1.9 ab	723.5 ± 36.7 ab	969.4 ± 72.2 abc	4086.9 ± 207.3 c	835.7 ± 75.2 bc
AVTKS#11	5.7 ± 0.3 ab	17.8 ± 0.7 ab	76.2 ± 0.7 ab	647.3 ± 44.8 a	876.8 ± 62.8 a	3487.5 ± 196.0 a	810.1 ± 80.8 abc
AVTKS#12	5.9 ± 0.2 bc	21.0 ± 1.1 b	73.0 ± 1.1 a	660.3 ± 43.3 a	868.7 ± 61.1 a	3538.7 ± 207.8 ab	811.6 ± 75.8 abc

The values of moisture, oil, and solid components are reported as means ± SD of three biological replicate analyses, and the values of other variables are reported as means ± SD of six replicate analyses (n = 3 × 2), where three replications were biological, and the two replications were laboratory replications. Values followed by identical superscript letters along the column are statistically similar.

). Genotypes AVTKS#4, AVTKS#5, and AVTKS#6 had the highest moisture content, while AVTKS#10 had the lowest moisture content.

Additionally, the moisture content in the seeds varied depending on the growing density (10, 20, 30, 40, and 50 plants/m²) and time of sowing (April, May, and June). The moisture content ranged between 7.0% and 7.7% for different growing densities and between 5.8% and 10.5% for different sowing times (

Table 4. Representation of moisture, oil, and solid component; TPC; antioxidant capacity (CUPRAC and FRAP); and TQ in the Nigella seeds according to planting density.

Planting Density	Moisture (%)	Oil Yield (%DW of Seed)	Solid Yield (%DW of Seed)	Total Phenolic (mg GAE/100 g DW)	FRAP (mg TE/100 g DW)	CUPRAC (mg TE/100 g DW)	TQ (mg/100 g DW)
10 plants/m2	7.4 ± 0.2 b	16.1 ± 1.2 a	77.9 ± 1.2 a	629.1 ± 61.8 c	722.8 ± 45.2 b	3119.8 ± 245.1 a	703.8 ± 66.7 b
20 plants/m2	7.5 ± 0.1 bc	17.9 ± 1.6 a	76.1 ± 1.6 a	641.3± 63.3 c	714.9± 69.7 b	3109.9 ± 310.0 a	689.3 ± 60.3 b
30 plants/m2	7.7 ± 0.1 c	17.7± 1.6 a	76.3 ± 1.6 a	550.5± 54.3 b	709.4 ± 47.0 ab	3238.1 ± 216.1 a	635.1 ± 60.2 ab
40 plants/m2	7.2 ± 0.1 a	17.6 ± 1.1 a	76.4 ± 1.1 a	520.0± 51.6 b	683.3 ± 36.3 ab	3054.5 ± 291.5 a	629.5 ± 53.6 ab
50 plants/m2	7.0 ± 0.1 a	16.0 ± 2.0 a	78.0 ± 2.0 a	441.8 ± 44.1 a	650.1 ± 23.6 a	2999.5 ± 231.7 a	589.4 ± 54.0 a

The values of moisture, oil, and solid components are reported as means ± SD of five biological replicate analysis, and the values of other variables are reported as means ± SD of ten replicate analyses (n = 5 × 2), where five replications were biological, and the two replications were laboratory replications. Values followed by identical superscript letters along the column are statistically similar.

and

Table 5. Representation of moisture, oil, and solid component; TPC; antioxidant capacity (CUPRAC and FRAP); and TQ in the Nigella seeds according to time of sowing.

Time of Sowing	Moisture (%)	Oil Yield (%DW of Seed)	Solid Yield (%DW of Seed)	TPC (mg GAE/100 g DW)	FRAP (mg TE/100 g DW)	CUPRAC (mg TE/100 g DW)	TQ (mg/100 g DW)
1 April	8.3 ± 0.7 b	18.8 ± 1.8 b	75.2 ± 1.8 a	705.9 ± 46.4 a	905.9 ± 53.4 ab	3649.6 ± 158.0 a	670.0 ± 55.2 a
1 May	5.8 ± 0.3 a	17.2 ± 1.6 ab	76.8 ± 1.6 ab	792.1 ± 47.0 b	972.0 ± 76.1 b	3933.1 ± 153.0 b	708.0 ± 67.8 ab
1 June	10.5 ± 1.2 c	14.2 ± 1.2 a	79.8 ± 1.2 b	760.1 ± 44.2 ab	863.0 ± 74.5 a	3570.4 ± 183.9 a	834.2 ± 72.1 b

The values of moisture, oil, and solid components are reported as means ± SD of three biological replicate analyses, and the values of other variables are reported as means ± SD of six replicate analyses (n = 3 × 2), where three replications were biological, and the two replications were laboratory replications. Values followed by identical superscript letters along the column are statistically similar.

). Seeds obtained from the May planting season and two planting densities (40 and 50 plants/m²) had the lowest moisture content. In contrast, seeds from the June planting season and 30 plants/m² planting density had the highest moisture content. Our study observed less difference of moisture content between the seeds sourced from different planting densities but a higher difference of moisture content between the seeds sourced from different times of sowing. Since the seeds sourced from different times of sowing had different harvesting times, the reason for the higher difference in moisture content between the seeds might be because of harvesting time. Because many researchers have extensively reported harvesting time as one of the major factors responsible for moisture content in seed [65,66,67,68].

The seed moisture content values of different Nigella treatments and genotypes obtained in this study were consistent with values reported in the literature, where moisture levels of Nigella seeds range between 3 and 10% [39,69,70,71,72]. The moisture content in oilseeds, such as Nigella, can have significant implications for their viability, quality, storage, and processing [73,74,75,76]. Some researchers have reported the significant impacts of moisture content on the physical and engineering properties of Nigella seeds, such as density, porosity, and frictional properties, which are crucial in the design of post-harvest equipment [75,77,78]. Furthermore, moisture content in the seeds has been found to affect the mechanical pressed oil yield and the transfer ratio of chemical compounds from the seed to the oil [76]. Excess moisture can negatively affect the quality of the oil extracted from the seeds, increasing the likelihood of oil acidity, and ultimately leading to a decrease in shelf life. Therefore, it is crucial to meticulously monitor and manage moisture levels during harvesting, drying, storage, and transportation to enhance seed quality and reduce the chances of spoilage or degradation.

3.2. Screw-Pressed Oil and Solid Component

The present study revealed that the oil and solid yield of Nigella was considerably affected by different genotypes including times of sowing and planting densities (Table 3, Table 4 and Table 5). Table 3 provides the quantification of screw-pressed oil and solid components in various genotypes of Nigella seeds. The oil content ranged from 15.5 to 21.0%, with the highest yield in genotype AVTKS#12 and the lowest in genotype AVTKS#9. The solid component (cake, sludge, and waste) ranged from 73.0 to 78.5%, with the highest yield in genotype AVTKS#9 and the lowest in genotype AVTKS#12.

Furthermore, the seeds obtained from the August, October, and December sowings were insufficient for further investigation due to the lower survival rate of plants. Therefore, results are presented only for the April, May, and June sowing dates (Table 5). Notably, there was a substantial difference in screw-pressed oil content (14.2–18.8%) among seeds obtained from different seasons. The highest oil yield was from April sowings, while the lowest was from June sowings. Similarly, the solid component varied across seasons, with the highest in June sowings (79.8%) and the lowest in April sowings (75.2%). Moreover, the screw-pressed oil yield and solid components of Nigella seeds grown in five different planting densities ranged from 16.0 to 17.9% and 76.1 to 78.0%, respectively (Table 4). However, no statistically significant differences were observed among the sample plots in terms of oil yield and solid component. The different levels of effect of genotypes [20,31,79] and agricultural practices, including planting densities and sowing times [20,80,81], on oil yield have also been reported.

There were no data in the current literature on screw-pressed Nigella oil and solid content obtained following similar parameters used in this study for comparison. However, the mechanically cold-pressed (pressed at 25 °C) oil yields of Nigella seeds from different locations worldwide have been investigated by a few researchers, with reported oil yields ranging between 20 and 30% (

Table 6. Oil yield of the different sources of N. sativa while using mechanical pressed extraction method.

Country	Method	Oil Yield%	Reference
Australia	Screw pressed method at 60 °C	14–21	Present study
Saudi Arabia	Screwless cold press method	21.73	[82,83]
Thailand	Single screw cold press method	27.38	[84]
Morocco	Screwless cold press method	27	[85]
Egypt	Hydraulic press and screw press method	20.5 and 21.1	[86]
Egypt	Hydraulic press	24.76	[87]
Turkey	Screw cold press method	20.1–30.7	[79]
China	Domestic cold press oil expeller	29.1	[88]

). Theoretically, oil yield increases with the increase in the screw press machine’s temperature up to a certain point. Therefore, the results obtained in this study seem slightly lower because the temperature applied for this study was 60 °C, while other past researchers used ambient temperature.

The lower value of oil content in the present study might be due to several factors, including the extraction method. In addition to screw press heating temperature, other parameters in the screw press extraction method, such as moisture content in the seed, pressure, pressing time, nozzle size, diameter of the saft screw, and rotational speed, can directly affect the oil yield [76,89,90]. For example, Sakdasri et al. [76] purchased Nigella seeds from the local market in China and obtained oil through the screw press method, considering the different parameters like feeding rates (36, 45, 54 g/min), moisture content (6, 12, 18%) of seeds, and heating temperature (40, 55, 70 °C), to study the effect of these parameters on oil yield. They observed oil content in the range of 8.18–31.67% [76]. Moreover, Deli et al. [89] purchased Nigella seeds from the Indian market; pressed seeds at 60 °C with different combinations using nozzle size (6, 10 and 12 mm), shaft screw diameters (8 and 11 mm), and screw press machine speeds (21, 54, 65 and 98 rpm); and recorded oil yield ranging between 8.73 and 22.27%.

3.3. TPC and Antioxidant Capacity

The TPC varied among the different Nigella genotypes studied, ranging from 647.3 to 922.5 mg GAE/100 g (Table 3). Genotype AVTKS#5 exhibited the highest TPC at 922.5 mg GAE/100 g, followed by AVTKS#9 and AVTKS#1 with values of 884.7 and 878.4 mg GAE/100 g, respectively. Conversely, genotype AVTKS#11 had the lowest TPC at 647.3 mg GAE/100 g, followed by AVTKS#12 at 660.3 mg GAE/100 g. Additionally, the FRAP values ranged from 868.7 to 1138.7 mg TE/100 g across different genotypes (Table 3). Genotype AVTKS#5 displayed the highest FRAP value at 1138.7 mg TE/100 g, followed by AVTKS#4 and AVTKS#7 at 1128.9 and 1109.4 mg TE/100 g, respectively. Conversely, genotype AVTKS#12 had the lowest FRAP value at 868.7 mg TE/100 g, followed by AVTKS#11 and AVTKS#1 at 876.8 and 931.5 mg TE/100 g, respectively. Moreover, the CUPRAC values ranged from 3487.5 to 4159.0 mg TE/100 g (Table 3). Genotype AVTKS#7 exhibited the highest CUPRAC value at 4159.0 mg TE/100 g, followed by AVTKS#4, AVTKS#1, AVTKS#10, AVTKS#2, AVTKS#8, AVTKS#3, and AVTKS#6 at 4139.3, 4092.8, 4086.9, 4039.5, 4007.1, 3933.1, and 3933.0 mg TE/100 g, respectively. Conversely, genotype AVTKS#11 had the lowest CUPRAC value at 3487.5 mg TE/100 g, followed by AVTKS#12 at 3538.7 mg TE/100 g. Overall, the results indicate that AVTKS#5 demonstrated high TPC and antioxidant capacity, while AVTKS#11 and AVTKS#12 showed low TPC and antioxidant capacity compared to other genotypes.

The results obtained in this study align with values reported by some researchers. For example, Mani et al. [62] studied the methanolic seed extracts of nine different Nigella genotypes grown in Central Queensland, Australia, reporting their TPC in the range of 794–1126 mg GAE/100 g DW. However, other researchers have reported both higher and lower values of TPC for various Nigella seed samples. For instance, Haron et al. [91] collected Nigella seeds from Yemen, Iran, and Malaysia, prepared their methanolic extract, and observed TPC in the range between 1619 and 3084 mg GAE/100 g. Thippeswamy and Naidu [92], meanwhile, studied TPC in the methanolic seed extract of Nigella sourced from India and observed an average value of 410 mg GAE/100 g. Thilakarathne et al. [93] studied the methanolic seed extract of Nigella obtained from Ethiopia and India, reporting 543 and 437 mg GAE/100 g, respectively. Rababah et al. [94] also reported 426 mg GAE/100 g TPC in the methanolic seed extract of Nigella obtained from Amman, Jordan, in the Mediterranean region. Interestingly, Sen et al. [95] prepared methanolic seed extracts of Nigella sourced from six different regions of Turkey and observed TPCs of ≤292 mg GAE/100 g, which is more than two times lower than the values reported in this study.

The FRAP values determined in this investigation closely align with those documented by Mani et al. [62], who recorded FRAP values of methanolic seed extracts ranging from 532 to 805 mg TE/100 g when examining nine distinct Nigella genotypes in Central Queensland, Australia. However, certain researchers have noted lower FRAP values. For instance, Kamiloglu et al. [96] documented a value of 182 mg TE/100 g in 80% methanolic seed extract of Nigella sourced from Turkey.

Only a handful of researchers have reported CUPRAC values for Nigella seed extracts. For instance, Kamiloglu et al. [96] reported 2260 mg TE/100 g, which is approximately two times lower than the values obtained in the present study. On the other hand, Toma et al. [97] documented 355 mg TE/100 g, which is more than ten times lower than our values. These findings underscore the variability in CUPRAC values observed in Nigella derived from diverse origins and sources.

Furthermore, the TPC, FRAP, and CUPRAC values in the seeds obtained from five different planting densities (10, 20, 30, 40, and 50 plants/m²) showed ranges of 441.8–641.3 mg GAE/100 g, 650.1–722.8 mg TE/100 g, and 2999.5–3238.1 mg TE/100 g, respectively (Table 4 or Figure A1). The highest value of TPC (641.3 mg GAE/100 g), FRAP (722.8 mg TE/100 g), and CUPRAC (3238.1 mg TE/100 g) was observed in the seeds obtained from plots with 20 plants/m², 10 plants/m², and 30 plants/m² plots, respectively. It is also worth noting in the table that the results of 20 plants/m² were statistically similar with those plots which showed highest value of FRAP and CUPRAC. Furthermore, the lowest value of TPC (441.8 mg GAE/100 g), FRAP (650.1 mg TE/100 g), and CUPRAC (2999.5 mg TE/100 g) was observed in the seeds grown in plots with 50 plants/m². Overall, TPC and FRAP showed a decreasing trend with the increasing plant density, with 10 or 20 plants/m² plant density showing higher values of TPC and antioxidant capacity, and 50 plants/m² with lower values.

No information on the effect of planting density on the health-promoting phytoconstituents was found in the literature to compare with the results of this study. However, several studies conducted worldwide have examined the impact of planting density on Nigella, specifically focusing on growth development and yield [36,37,38,40,48]. These studies have consistently shown that the planting density has a significant influence on the growth development and yield characteristics of the plant. Most of these studies have reported that lower planting densities result in improved growth development and yield parameters [38,40]. For instance, Kadi et al. [38] investigated the effect of different plant densities (20, 40, and 80 plants/plot) on the seed and oil yield of Nigella and found that the lowest planting density resulted in highest values for both parameters. In line with these findings, the current study also observed higher levels of TPC and antioxidant capacity in seeds obtained from lower planting densities, suggesting a positive correlation between plant growth, yield, and the availability of important bioactive phytoconstituents.

In addition, the TPC, FRAP, and CUPRAC values in the seeds obtained from three different sowing times (April, May, and June) showed ranges of 705.9–792.1 mg GAE/100 g, 863.0–972.0 mg TE/100 g, and 3570.4–3933.1 mg TE/100 g, respectively (Table 5). The highest values of TPC (792.1 mg GAE/100 g), FRAP (972.0 mg TE/100 g), and CUPRAC (3933.1 mg TE/100 g) were found in the seeds obtained from May sowing. On the other hand, the lowest value of TPC (705.9 mg GAE/100 g) was observed in the seeds obtained from April sowing, and the lowest value of FRAP (863.0 mg TE/100 g) and CUPRAC (3570.4 mg TE/100 g) were observed in the seeds obtained from June sowing.

Although no literature reports were found that provided information about the effect of sowing time on health-benefitting phytoconstituents for comparison with the present study, some studies have been conducted worldwide in the past to understand the effect of sowing time on Nigella plants, specifically the parameters related to growth development and yield [39,43,80,98,99,100,101,102,103,104,105,106,107]. These studies have shown a considerable effect of sowing time on the growth and yield attributes of the plant. Most of the reports have shown better results in the seeds obtained from earlier sowing (between the last month of autumn and the starting month of winter) of Nigella [80,103]. For example, Kizil et al. [103] grew Nigella seeds in two different seasons, winter (November) and spring (March), maintaining a 30 cm row distance in Turkey. They analyzed plant growth parameters, seed yield, oil yield, and non-volatile and volatile content in the oil of harvested seeds, and observed their highest value in the seeds grown in the winter season [103]. Since March, April, and May are the autumn season in Australia, and the present study also shows better performance of TPC and antioxidant capacity in the last month of autumn (May), our result shows a positive correlation of TPC and antioxidant capacity with the better performance of growth and yield attributes of the plant.

3.4. TQ Composition

In all the HPLC chromatograms derived from the analysis of methanolic seed extracts, the predominant peak identified was TQ. A chromatogram of genotype AVTKS#9 has been shown in Figure 1 as an example, where the highest peak area with blue color indicates TQ. The concentration of TQ ranged from 675.8 to 1118.6 mg/100 g (Table 3). Genotype AVTKS#8 exhibited the highest TQ concentration at 1118.6 mg/100 g, followed by AVTKS#9 at 1066.1 mg/100 g. Conversely, AVTKS#1 displayed the lowest concentration at 675.8 mg/100 g, followed by AVTKS#3 at 708.0 mg/100 g. Similar findings have been documented by several researchers. Belete and Dagne [108] reported a TQ concentration of 1000 mg/100 g in the methanolic seed extract of Nigella from Ethiopia. Foudah et al. [109] examined the TQ concentration in methanolic extracts of Nigella seeds from six different countries (Saudi Arabia, Egypt, Jordan, Palestine, Syria, and India), recording values ranging from 651 to 1076 mg/100 g. Ravi et al. [110] also reported a TQ concentration of 900 mg/100 g in the methanolic seed extract of Nigella from India. Furthermore, Mani et al. [62] documented TQ levels ranging from 896 to 1728 mg/100 g while investigating methanolic seed extracts of nine different genotypes of Nigella grown in Central Queensland, Australia. Some authors have also reported lower TQ content compared to our study. For instance, Aziz et al. [111] analyzed the methanolic seed extracts of Nigella from India and Kuwait, reporting TQ levels ranging from 10 to 29 mg/100 g. Ravi et al. [112] further examined methanolic extracts of five nationally released varieties and 35 unique collections of Nigella seeds gathered across India, observing TQ levels ranging from 0 to 248 mg/100 g.

Furthermore, the concentration of TQ in the seeds grown in different planting densities ranged between 589.4 and 703.8 mg/100 g, where the highest TQ (703.8 mg/100 g) was observed in the seeds grown in 10 plants/m² planting density and the lowest value of TQ (589.4 mg/100 g) was observed in the seeds grown in 50 plants/m² planting density (Table 4 or Figure A1). Additionally, the concentration of TQ in the seeds grown under different times of sowing ranged between 670.0 and 834.2 mg/100 g, where the highest value of TQ (834.2 mg/100 g) was found in the seeds obtained from June sowing and the lowest value of TQ (670.0 mg/100 g) was observed in the seeds obtained from April sowing (Table 5). There is limited research on the effect of time of sowing and growing density on TQ levels. Giridhar [113] studied the effect of sowing densities (25, 33.3 and 50 plants/m²) on TQ in India and for the first time reported the higher concentration of TQ in the seeds grown earlier at 25 plants/m² plant density, which is consistent with the trend observed in this study where lower densities (10 and 20 plants/m²) yielded higher TQ level. Although this study confirms that there is a higher level of TQ when planting densities are lower and sowing is completed in June, further research is necessary to refine these findings and create comprehensive guidelines for Nigella cultivation.

3.5. Fatty Acids

The composition of fatty acids in Nigella seeds obtained from different genotypes, planting densities, and sowing times has been illustrated in

Table 7. Composition of fatty acids in different genotypes of N. sativa.

Fatty Acids	Fatty Acid (mg/g of Seed) in the Seeds of Different Nigella Genotypes
	AVTKS#1	AVTKS#2	AVTKS#3	AVTKS#4	AVTKS#5	AVTKS#6	AVTKS#7	AVTKS#8	AVTKS#9	AVTKS#10	AVTKS#11	AVTKS#12
Saturated Fatty Acids (SFAs)
C14:0	0.3 ± 0.1 a	0.4 ± 0.0 b	0.4 ± 0.0 b	0.4 ± 0.0 ab	0.4 ± 0.0 ab	0.4 ± 0.0 ab	0.4 ± 0.0 b	0.4 ± 0.0 b	0.4 ± 0.0 b	0.4 ± 0.0 b	0.4 ± 0.0 b	0.3 ± 0.0 ab
C15:0	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a
C16:0	14.8 ± 1.2 ab	17.3 ± 0.5 b	16.8 ± 0.3 ab	15.0 ± 1.1 ab	14.4 ± 1.1 ab	15.5 ± 0.9 ab	17.5 ± 0.7 b	16.8 ± 0.5 ab	16.5 ± 0.6 ab	16.4 ± 0.5 ab	15.8 ± 1.3 ab	13.7 ± 1.1 a
C17:0	0.1 ± 0.0 a	0.2 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a
C18:0	2.1 ± 0.2 a	3.1 ± 0.2 c	3.1 ± 0.1 c	2.6 ± 0.2 abc	2.5 ± 0.2 ab	2.8 ± 0.1 bc	3.0 ± 0.1 bc	3.0 ± 0.1 bc	3.0 ± 0.1 bc	2.9 ± 0.1 bc	2.9 ± 0.2 bc	3.1 ± 0.2 bc
C20:0	0.3 ± 0.0 ab	0.4 ± 0.0 c	0.4 ± 0.0 bc	0.3 ± 0.0 abc	0.3 ± 0.0 ab	0.4 ± 0.0 abc	0.4 ± 0.0 abc	0.4 ± 0.0 bc	0.4 ± 0.0 abc	0.4 ± 0.0 abc	0.3 ± 0.0 abc	0.3 ± 0.0 a
Total SFAs	17.7 ± 1.4 ab	21.5 ± 0.7 b	21.0 ± 0.4 ab	18.6 ± 1.4 ab	17.8 ± 1.4 ab	19.3 ± 1.0 ab	21.6 ± 0.8 b	20.8 ± 0.5 ab	20.5 ± 0.7 ab	20.3 ± 0.6 ab	19.7 ± 1.5 ab	17.6 ± 1.4 a
Monounsaturated Fatty Acids (MUFAs)
C16:1 (cis-9)	0.3 ± 0.0 abc	0.4 ± 0.0 d	0.4 ± 0.0 bcd	0.3 ± 0.0 abcd	0.3 ± 0.0 a	0.3 ± 0.0 abcd	0.4 ± 0.0 cd	0.4 ± 0.0 bcd	0.3 ± 0.0 abcd	0.4 ± 0.0 cd	0.3 ± 0.0 abcd	0.3 ± 0.0 ab
C17:1 (cis-10)	0.1 ± 0.0 ab	0.1 ± 0.0 bc	0.1 ± 0.0 bc	0.1 ± 0.0 abc	0.1 ± 0.0 a	0.1 ± 0.0 bc	0.1 ± 0.0 bc	0.1 ± 0.0 c	0.1 ± 0.0 bc	0.1 ± 0.0 bc	0.1 ± 0.0 bc	0.1 ± 0.0 abc
C18:1	12.1 ± 0.5 a	15.6 ± 0.5 d	14.8 ± 0.2 bcd	13.6 ± 0.3 abcd	12.8 ± 0.9 ab	14.4 ± 0.7 bcd	15.3 ± 0.6 cd	14.9 ± 0.3 bcd	15.3 ± 0.6 d	14.3 ± 0.4 bcd	13.2 ± 0.6 abc	11.7 ± 0.9 a
C20:1 (cis-11)	0.4 ± 0.0 a	0.5 ± 0.0 c	0.4 ± 0.0 abc	0.4 ± 0.0 ab	0.4 ± 0.0 ab	0.4 ± 0.0 ab	0.4 ± 0.0 bc	0.4 ± 0.0 abc	0.4 ± 0.0 bc	0.4 ± 0.0 abc	0.4 ± 0.0 ab	0.4 ± 0.0 ab
Total MUFAs	12.8 ± 0.6 a	16.6 ± 0.6 d	15.7 ± 0.2 bcd	14.4 ± 0.4 abcd	13.6 ± 0.9 ab	15.2 ± 0.8 bcd	16.2 ± 0.7 cd	15.8 ± 0.4 bcd	16.2 ± 0.6 cd	15.3 ± 0.5 bcd	14.0 ± 0.7 abc	12.4 ± 0.9 a
Polyunsaturated Fatty Acids (PUFAs)
C18:2	109.4 ± 8.0 bcd	120.1 ± 7.6 cd	119.1 ± 8.3 cd	115.3 ± 5.3 bcd	107.9 ± 7.5 bc	115.9 ± 4.8 bcd	130.9 ± 3.9 d	126.6 ± 3.0 cd	123.1 ± 5.6 cd	124.6 ± 3.8 cd	96.1 ± 8.6 ab	80.1 ± 5.9 a
C18:3(cis 9,12,15)	0.3 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.4 ± 0.0 a	0.3 ± 0.0 a
C20:2 (cis-11,14)	2.3 ± 0.1 a	3.7 ± 0.2 d	3.4 ± 0.0 cd	3.2 ± 0.2 bcd	3.0 ± 0.2 bc	3.2 ± 0.2 bcd	3.7 ± 0.1 d	3.6 ± 0.2 d	3.6 ± 0.1 cd	3.4 ± 0.1 cd	2.9 ± 0.2 ab	2.3 ± 0.1 a
Total PUFAs	112.1 ± 8.2 bcd	124.2 ± 7.6 cd	123.0 ± 8.3 cd	118.9 ± 5.6 bcd	111.3 ± 7.7 bc	119.5 ± 5.0 bcd	135.0 ± 4.0 d	130.6 ± 3.2 cd	127.0 ± 5.6 cd	128.5 ± 4.0 cd	99.3 ± 8.8 ab	82.8 ± 5.8 a
Total MUFAs + PUFAs	124.9 ± 8.6 bc	140.8 ± 7.8 cd	138.6 ± 8.3 cd	133.3 ± 5.9 bcd	124.9 ± 8.6 bc	134.7 ± 5.7 bcd	151.2 ± 4.6 d	146.4 ± 3.5 cd	143.2 ± 6.2 cd	143.7 ± 4.4 cd	113.4 ± 9.2 ab	95.3 ± 6.6 ab
MUFAs/SFAs	0.7 ± 0.1 a	0.8 ± 0.0 a	0.7 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.7 ± 0.0 a	0.7 ± 0.0 a
PUFAs/SFAs	6.3 ± 0.2 c	5.8 ± 0.4 bc	5.9 ± 0.3 c	6.4 ± 0.2 c	6.2 ± 0.1 c	6.2 ± 0.1 c	6.3 ± 0.1 c	6.3 ± 0.1 c	6.2 ± 0.1 c	6.3 ± 0.0 c	5.1 ± 0.3 ab	4.7 ± 0.2 a
MUFAs +PUFAs/SFAs	7.1 ± 0.3 b	6.6 ± 0.4 b	6.6 ± 0.3 b	7.2 ± 0.2 b	7.0 ± 0.1 b	7.0 ± 0.1 b	7.0 ± 0.1 b	7.0 ± 0.1 b	7.0 ± 0.1 b	7.1 ± 0.0 b	5.8 ± 0.3 a	5.4 ± 0.3 a

The values of individual fatty acids are reported as means ± SD of three biological replicate analyses. Values followed by identical superscript letters along the row are statistically similar. Abbreviations: C14:0, myristic acid; C15:0, pentadecanoic acid; C16:0, palmitic acid; C17:0, margaric acid; C18:0, stearic acid; C20:0, arachidic acid; C16:1(cis-9), palmitoleic acid; C17:1 (cis-10), heptadecenoic acid; C18:1, oleic acid; C20:1 (cis-11), eicosenoic acid; C18:2, linoleic acid; C18:3 (cis-9,12,15), alpha-linolenic acid; C20:2 (cis-11,14), eicosadienoic acid.

,

Table 8. Composition of fatty acids in the Nigella seeds according to planting density.

Name of Fatty Acids	Fatty Acid (mg/g of Seed) in Nigella Different Planting Densities
	10 Plants/m2	20 Plants/m2	30 Plants/m2	40 Plants/m2	50 Plants/m2
Saturated Fatty Acids (SFAs)
C14:0	0.3 ± 0.0 a	0.3 ± 0.0 b	0.3 ± 0.0 a	0.3 ± 0.0 a	0.2 ± 0.0 a
C15:0	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a
C16:0	12.0 ± 0.4 a	11.9 ± 0.5 a	11.9 ± 0.3 a	12.4 ± 0.5 a	12.9 ± 0.8 a
C17:0	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a	0.1 ± 0.0 a
C18:0	3.5 ± 0.2 a	3.6 ± 0.2 a	3.0 ± 0.2 a	3.3 ± 0.2 ab	3.1 ± 0.2 a
C20:0	0.2 ± 0.0 a	0.2 ± 0.0 a	0.3 ± 0.0 a	0.3 ± 0.0 a	0.2 ± 0.0 a
Total SFAs	16.3 ± 0.4 a	16.3 ± 0.5 a	15.6 ± 0.2 a	16.4 ± 0.4 a	16.6 ± 0.9 a
Monounsaturated Fatty Acids (MUFAs)
C16:1 (cis-9)	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a
C17:1 (cis-10)	1.4 ± 0.1 a	1.3 ± 0.1 a	1.3 ± 0.1 a	1.3 ± 0.1 a	1.3 ± 0.0 a
C18:1	18.0 ± 1.3 a	19.0 ± 1.5 a	17.8 ± 2.3 a	17.5 ± 0.8 a	15.9 ± 1.4 a
C20:1 (cis-11)	0.2 ± 0.0 ab	0.3 ± 0.0 b	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a
Total MUFAs	19.8 ± 1.2 a	20.7 ± 1.4 a	19.5 ± 2.3 a	19.2 ± 0.8 a	17.6 ± 1.3 a
Polyunsaturated Fatty Acids (PUFAs)
C18:2	81.5 ± 4.0 ab	86.5 ± 4.9 b	82.8 ± 2.6 ab	78.1 ± 1.9 a	77.3 ± 4.0 a
C18:3(cis 9,12,15)	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a
C20:2 (cis-11,14)	2.1 ± 0.1 a	2.2 ± 0.1 a	2.1 ± 0.0 a	2.1 ± 0.1 a	2.1 ± 0.1 a
Total PUFAs	83.8 ± 4.0 ab	89.0 ± 4.7 b	85.1 ± 2.6 ab	80.4 ± 1.9 a	79.5 ± 4.0 a
Total MUFAs + PUFAs	103.6 ± 4.7 ab	109.7 ± 5.2 b	104.6 ± 3.2 ab	99.6 ± 2.2 a	97.1 ± 4.7 a
Ratio MUFAs/SFAs	1.2 ± 0.1 ab	1.3 ± 0.1 b	1.3 ± 0.1 ab	1.2 ± 0.0 ab	1.1 ± 0.1 a
Ratio PUFAs/SFAs	5.2 ± 0.3 ab	5.5 ± 0.2 b	5.5 ± 0.2 b	4.9 ± 0.2 a	4.8 ± 0.3 a
Ratio MUFAs +PUFAs/SFAs	6.4 ± 0.4 ab	6.7 ± 0.2 b	6.7 ± 0.2 b	6.1 ± 0.2 a	5.9 ± 0.3 a

The values of individual fatty acids are reported as means ± SD of five biological replicate analyses. Values followed by identical superscript letters along the row are statistically similar.

and

Table 9. Composition of fatty acids in the Nigella seeds according to time of sowing.

Name of Fatty Acids	Fatty Acid (mg/g of Seed) in Different Time of Sowing
	1 April	1 May	1 June
Saturated Fatty Acids (SFAs)
C14:0	0.2 ± 0.0 a	0.4 ± 0.0 b	0.2 ± 0.0 a
C15:0	0.1 ± 0.0 a	0.1 ± 0.0 b	0.1 ± 0.0 a
C16:0	12.3 ± 0.5 a	16.8 ± 0.3 b	11.4 ± 0.4 a
C17:0	0.1 ± 0.0 a	0.1 ± 0.0 b	0.1 ± 0.0 a
C18:0	3.3 ± 0.3 b	3.1 ± 0.1 b	2.5 ± 0.1 a
C20:0	0.3 ± 0.0 a	0.4 ± 0.0 b	0.2 ± 0.0 a
Total SFAs	16.3 ± 0.8 b	21.0 ± 0.4 c	14.5 ± 0.4 a
Monounsaturated Fatty Acids (MUFAs)
C16:1 (cis-9)	0.2 ± 0.0 a	0.4 ± 0.0 b	0.2 ± 0.0 a
C17:1 (cis-10)	1.4 ± 0.1 c	0.1 ± 0.0 a	1.1 ± 0.1 b
C18:1	18.6 ± 1.5 b	14.8 ± 0.2 a	13.0 ± 0.5 a
C20:1 (cis-11)	0.2 ± 0.0 a	0.4 ± 0.0 b	0.2 ± 0.0 a
Total MUFAs	20.4 ± 1.5 b	15.7 ± 0.2 a	14.4 ± 0.5 a
Polyunsaturated Fatty Acids (PUFAs)
C18:2	107.9 ± 4.2 a	119.1 ± 8.3 a	112.6 ± 7.1 a
C18:3(cis 9,12,15)	0.2 ± 0.0 a	0.4 ± 0.0 b	0.2 ± 0.0 a
C20:2 (cis-11,14)	1.4 ± 0.1 b	3.4 ± 0.0 c	1.1 ± 0.0 a
Total PUFAs	109.5 ± 4.3 a	123.0 ± 8.3 a	113.9 ± 7.1 a
Total MUFAs + PUFAs	129.9 ± 5.3 a	138.6 ± 8.3 a	128.4 ± 7.6 a
Ratio MUFAs/SFAs	1.3 ± 0.1 b	0.7 ± 0.0 a	1.0 ± 0.0 a
Ratio PUFAs/SFAs	6.7 ± 0.1 ab	5.9 ± 0.3 a	7.8 ± 0.5 b
Ratio MUFAs +PUFAs/SFAs	8.0 ± 0.2 b	6.6 ± 0.3 a	8.8 ± 0.6 b

The values of individual fatty acids are reported as means ± SD of three biological replicate analyses. Values followed by identical superscript letters along the row are statistically similar.

. As shown in the Tables, all samples contained a total of 13 fatty acids (six SFAs, four MUFAs, and three PUFAs). The major fatty acids were C16:0 and C18:0 from the SFA, C18:2, and C20:2 (cis-11,14) from PUFA, and C18:1 from MUFA categories. Fatty acids with a contribution less than or equal to ≤1 mg/g of seed were observed in other fatty acids, representing four fatty acids from SFA (C14:0, C15:0, C17:0, C20:0), three fatty acids from MUFA (C16:1 (cis-9), C17:1 (cis-10), and C20:1 (cis-11)), and one fatty acid from PUFA (C18:3 (cis9,12,15) categories. There were no specific reports of fatty acid composition in seeds found in the literature. However, many studies of fatty acids have been conducted in Nigella oil, and the reports confirm the presence of those aforementioned fatty acids in Nigella seeds [31,80,114].

As can be seen in Table 7, Table 8 and Table 9, the range of SFA, MUFA, and PUFA was 14.5–21.6, 12.4–20.7, and 79.5–135.0 mg/g of seed, respectively, among the genotypes and different agricultural practices. This clearly indicates significant impacts of genotypes and agricultural practices on the fatty acid composition of Nigella seeds. Many authors have reported the value of SFA and unsaturated fatty acid (UFA) in Nigella oil samples, and their results also showed the lowest concentration of SFAs (6.5–24.1% of total fatty acids) and the highest concentration of UFAs (75.9–93.5% of total fatty acids) in Nigella oil [22,85,115,116].

Furthermore, the ratios of SFA, MUFA and PUFA inform the quality of seeds in terms of human consumption and are therefore important tools for selecting the best sources for further breeding programs [27,31]. The higher the ratio of MUFAs/SFAs, PUFAs/SFAs, and (MUFAs + PUFAs)/SFAs, the lower the SFAs but higher the concentration of UFAs in the seeds. In the present study, the range of fatty acid ratios MUFAs/SFAs, PUFAs/SFAs, and (MUFAs + PUFAs)/SFAs was observed between 0.74 and 1.25, 4.70 and 7.84, and 5.46 and 8.84, respectively. A similar range of ratios was observed by previous authors while investigating Nigella oil [31].

Furthermore, the range of SFA, MUFA, and PUFA among different genotypes was 17.6–21.6, 12.4–16.6, and 82.8–135.0 mg/g of seed, respectively (Table 7). The highest value of SFA and PUFA was observed in genotype AVTKS#7, while the value of MUFA was highest in AVTKS#2. Conversely, the lowest value of SFA, PUFA, and MUFA was observed in genotype AVTKS#12. The value of MUFA and PUFA combined was highest (151.2 mg/g) in the seeds of genotypes AVTKS#7, and the lowest (95.3 mg/g) in the seeds of genotype AVTKS#12. The MUFAs/SFAs, PUFAs/SFAs, and (MUFAs + PUFAs)/SFAs ratios were observed to be 0.7–0.8, 4.7–6.4, and 5.4–7.2, respectively. The (MUFAs + PUFAs)/SFAs ratio was observed to be highest in AVTKS#4, and the lowest ratio was observed in AVTKS#12.

Moreover, the range of SFA, MUFA, and PUFA in the Nigella seeds grown at different densities was observed to be 15.6–16.6, 17.6–20.7, and 79.5–89.0 mg/g of seed, respectively (Table 8). The highest SFA was in the seeds grown at a density of 50 plants/m², while the highest MUFA and PUFA were observed in the seeds grown at a density of 20 plants/m². The lowest value of SFA was observed in the seeds grown at a density of 30 plants/m², but the MUFA and PUFA were lowest in the seeds grown at a density of 50 plants/m². The value of MUFA and PUFA altogether was highest (109.7 mg/g) in the seeds grown at a density of 20 plants/m² and lowest (97.1 mg/g) in the seeds grown at a density of 50 plants/m².

In addition, the MUFAs/SFAs, PUFAs/SFAs, and (MUFAs +PUFAs)/SFAs ratios were observed to be 1.1–1.3, 4.8–5.5, and 5.9–6.7, respectively. The (MUFAs +PUFAs)/SFAs ratio was highest in the seeds grown at a density of 20 plants/m² and lowest in the seeds grown at a density of 50 plants/m².

Furthermore, the range of SFA, MUFA, and PUFA in the Nigella grown at different times were observed to be 14.5–21.0, 14.4–20.4, and 113.9–123.0 mg/g of seed, respectively (Table 9). The highest value of SFA and PUFA was observed to be present in the seeds grown on 1 May. However, the value of SFA was lowest in the seeds grown on 1 June, and the value of PUFA was lowest in the seeds grown on 1 April. The MUFA was highest in the seeds grown on 1 April and the lowest in the seeds grown on 1 June. The value of MUFA and PUFA altogether was highest (138.6 mg/g) in the seeds grown on 1 May and lowest (128.4 mg/g) in the seeds grown on 1 June.

The MUFAs/SFAs, PUFAs/SFAs, and (MUFAs + PUFAs)/SFAs ratios were observed in the range of 0.7–1.3, 5.9–7.8, and 6.6–8.8, respectively. The (MUFAs +PUFAs)/SFAs ratio was highest in the seeds grown on 1 June and lowest in the seeds grown on 1 May. Few researchers have studied the fatty acid composition in Nigella oil obtained from different growing densities and sowing times and they have also reported significant variation in the fatty acid composition. Safaei et al. [80] planted Nigella seeds in two different seasons, autumn (November) and spring (April), in Iran and observed a significant decrease in fatty acid composition in the oil obtained from the seeds sown in spring. For example, the representation of major fatty acids, linoleic, oleic, and palmitic acids, was 91.19% in the autumn-sown seed oil while it was only 89.97% in the spring-sown seed oil [80]. Furthermore, Kizil [40] and Kizil et al. [103] investigated the effect of planting space (20, 30, 40, and 50 cm raw space) and time sowing (winter and spring) on different quantitative and qualitative parameters, including fatty acid composition of Nigella seeds in Turkey, and concluded that 30–40 cm planting space and winter planting were the optimal conditions.

3.6. Correlation of Different Variables

To investigate potential correlations between various phytochemical constituents (TPC, antioxidant capacity, TQ, and different individual fatty acids), Pearson’s R correlation analysis was conducted, and the results are shown in

Table 10. Correlations among different variables.

	Oil	TPC	FRAP	CUPRAC	TQ	C14:0	C15:0	C16:0	C17:0	C18:0	C20:0	C16:1	C17:1	C18:1	C20:1	C18:2	C18:3	C20:2	Σ SFA	Σ MUFA
TPC	0.049
FRAP	0.187	0.819 ***
CUPRAC	−0.060	0.784 ***	0.794 ***
TQ	−0.013	0.543 ***	0.561 ***	0.435 ***
C14:0	0.074	0.167	0.231	0.202	−0.374 ***
C15:0	0.056	0.043	0.159	0.099	−0.424 ***	0.925 ***
C16:0	0.020	0.124	0.101	0.131	−0.475 ***	0.906 ***	0.894 ***
C17:0	0.052	0.127	0.061	0.109	−0.546 ***	0.883 ***	0.886 ***	0.899 ***
C18:0	0.070	−0.055	0.223	0.174	0.359 ***	−0.059	−0.167	−0.175	−0.323 ***
C20:0	0.082	0.149	0.108	0.126	−0.531 ***	0.921 ***	0.918 ***	0.932 ***	0.958 ***	−0.291 **
C16:1	0.085	0.161	0.159	0.186	−0.507 ***	0.931 ***	0.908 ***	0.944 ***	0.932 ***	−0.218	0.974 ***
C17:1	−0.081	−0.141	−0.069	−0.088	0.570 ***	−0.837 ***	−0.840 ***	−0.836 ***	−0.933 ***	0.486 ***	−0.942 ***	−0.914 ***
C18:1	−0.064	0.092	0.154	0.113	0.593 ***	−0.321 ***	−0.403 ***	−0.373 ***	−0.538 ***	0.742 ***	−0.511 ***	−0.464 ***	0.682 ***
C20:1	0.063	0.200	0.172	0.153	−0.472 ***	0.940 ***	0.870 ***	0.928 ***	0.920 ***	−0.184	0.957 ***	0.970 ***	−0.902 ***	−0.428 ***
C18:2	−0.109	0.018	0.042	−0.003	−0.401 ***	0.731 ***	0.750 ***	0.786 ***	0.770 ***	−0.306 **	0.768 ***	0.763 ***	−0.706 ***	−0.335 ***	0.742 ***
C18:3	0.105	0.161	0.139	0.154	−0.503 ***	0.939 ***	0.895 ***	0.923 ***	0.946 ***	−0.265 **	0.970 ***	0.968 ***	−0.937 ***	−0.496 ***	0.963 ***	0.746 ***
C20:2	−0.003	0.276 **	0.249 **	0.217	−0.333 ***	0.922 ***	0.857 ***	0.902 ***	0.845 ***	−0.097	0.904 ***	0.915 ***	−0.815 ***	−0.290 **	0.927 ***	0.678 ***	0.911 ***
Σ SFA	0.037	0.118	0.142	0.166	−0.424 ***	0.919 ***	0.886 ***	0.986 ***	0.865 ***	−0.012	0.905 ***	0.928 ***	−0.776 ***	−0.260 **	0.918 ***	0.750 ***	0.900 ***	0.905 ***
Σ MUFA	−0.069	0.062	0.130	0.093	0.611 ***	−0.399 ***	−0.475 ***	−0.445 ***	−0.611 ***	0.741 ***	−0.587 ***	−0.539 ***	0.753 ***	0.994 ***	−0.503 ***	−0.395 ***	−0.572 ***	−0.369 ***	−0.333 ***
Σ PUFA	−0.105	0.028	0.051	0.007	−0.405 ***	0.748 ***	0.764 ***	0.801 ***	0.783 ***	−0.302 **	0.784 ***	0.779 ***	−0.720 ***	−0.338 ***	0.759 ***	1.000 ***	0.763 ***	0.700 ***	0.766 ***	−0.399 ***

*** correlation is significant at the 0.01 level; ** correlation is significant at the 0.05 level; the following sample size of the variables were used to study the correlation: oil% and fatty acids (n = 67), TPC, antioxidant capacity and TQ (n = 134); three colors scale, red, white, and green, was used to represent the combination between −1, 0, and 1, respectively.

. As observed in the Table, there was a significant positive correlation (r = 0.784–0.819, p < 0.01) among TPC, FRAP, and CUPRAC, with varying strengths. Several other authors have also reported a positive correlation among TPC and antioxidant capacity when studying Nigella seeds from different sources [97,117]. This supports the potential contribution of phenolic compounds in Nigella seeds to their antioxidant properties.

Furthermore, the present study showed a moderate positive correlation of TQ with TPC, CUPRAC, and FRAP (r= 0.435–0.561, p < 0.01). This indicates a lesser contribution of TQ to antioxidant properties. To validate this, we further prepared different concentrations of pure TQ compound to test and understand its relationship to TPC and antioxidant capacity (CUPRAC and FRAP), and the results are shown in

Table 11. TPC and antioxidant capacity values in different concentration of TQ.

TQ (mg/L)	TPC (GAE mg/L)	FRAP (TE mg/L)	CUPRAC (TE mg/L)
10	9.9 ± 0.3 a	4.0 ± 0.3 a	2.4 ± 0.4 a
20	10.7 ± 0.1 ab	4.7 ± 0.3 ab	7.0 ± 0.6 ab
50	12.1 ± 0.3 ab	5.8 ± 0.5 b	10.3 ± 1.0 b
100	13.4 ± 0.8 bc	7.3 ± 0.6 c	20.3 ± 1.9 c
200	15.8 ± 1.5 cd	9.9 ± 0.5 d	42.0 ± 3.1 d
250	17.9 ± 1.7 d	11.3 ± 0.7 d	55.8 ± 5.4 e

The values are reported as means ± SD of three replicate analyses. Values followed by similar letters along the column are statistically similar.

. As observed, there was a slight increment in the values of TPC and antioxidant capacity with the increment of TQ concentration; however, the values were not directly proportional to the concentration, following the Beer–Lambert Law. Since TQ has been previously reported to have strong antioxidant potential by many researchers [2,118,119], further exploration is needed.

The fatty acids, on the other hand, did not show any significant correlation with TPC, FRAP, and CUPRAC, except for C20:2 (cis-11,14), which was positively correlated with TPC and FRAP (r = 0.249–0.276, p < 0.05). Diverse results have been reported in the literature when dealing with different plant sources [64,120,121,122]. However, our results are coherent with several reports, including Channaoui et al. [122] and Hoyos et al. [64], who have reported no significant correlation of fatty acids to TPC and antioxidant capacity while studying Rapeseed and Sesame seeds, respectively.

Additionally, it is also worth noting that PUFA showed a weak negative correlation with MUFA (r= −0.399, p < 0.05). A significant negative correlation between PUFA and MUFA has also been reported by Czerniak et al. while studying Rapeseed varieties [120]. Furthermore, PUFA was positively correlated with SFA (r = 0.700, p < 0.05), but MUFA was negatively correlated with SFA (r = 0.333, p < 0.05). Channaoui et al. have reported similar results in their study [122]. Overall, our study supports the idea of increasing the SFA and PUFA, decreasing the MUFA. However few researchers have reported different correlations among SFA, PUFA, and MUFA than our findings [123]. Therefore, further research is needed to confirm these findings across diverse populations and environmental conditions.

4. Conclusions

The present study observed a significant impact of genotypes, planting densities, and times of sowing in phytochemical composition of Nigella seeds. No single genotype or planting density or sowing time exhibited the highest levels of all desired compounds. For instance, genotype AVTKS#5 showed a relatively higher concentration of TPC and FRAP, while CUPRAC and TQ were higher in AVTKS#7 and AVTKS#8, respectively. However, considering the overall results on oil yield, TPC, antioxidant capacity, TQ, and fatty acids, genotype AVTKS#5 displayed superior performance. Furthermore, a planting density of 20 plants/m² showed overall better performance in terms of oil yield, TPC, antioxidant capacity, TQ content, unsaturated fatty acids, and the fatty acids ratio. Besides, planting on the 1 May exhibited overall better results in terms of oil yield, TPC, antioxidant capacity, and TQ content. However, seeds with better fatty acid profiles were obtained from sowing on the 1 June or April.

These findings underscore the importance of considering multiple factors, including genotype, planting density, and sowing time, to optimize the production of Nigella seeds with desirable compounds.