Identification, validation and quantification of thymoquinone in conjunction with assessment of bioactive possessions and GC-MS profiling of pharmaceutically valuable crop Nigella (Nigella sativa L.) varieties

Chemicals

The standard thymoquinone and all solvents utilized were procured from Sigma–Aldrich (St. Louis, MO, USA), ensuring the highest available purity.

Plant material

Seeds from nationally released varieties of Nigella sativa L., for commercial cultivation, along with their sources of collection and respective characteristics, are outlined in Table 1. The collected seed samples underwent a thorough washing in double-distilled water and were subsequently dried under ambient conditions. For further analysis, precisely weighed 50 g seed samples were ground into a powder using a Malavasi mill (Bologna, Italy). Particular attention was paid to prevent overheating during the grinding process, and the resulting particle sizes fell within the range of 250–425 µm.

Extraction from Nigella seeds by Soxhlet apparatus

The extraction from seeds of various Nigella sativa L., varieties was performed using a Soxhlet apparatus. In this process, 15 g of powdered, dry seeds were immersed in 50 ml of absolute n-hexane at 65 °C for 6 h, incorporating minor modifications. Subsequently, the extract underwent rotary flash evaporation under reduced pressure to eliminate solvent residue. The resulting oily extract was then collected and stored under refrigerated conditions for further analysis. To quantify the efficiency of the extraction process, the extraction yield was calculated as a percentage of the raw material. This was achieved using the formula outlined by Zhang, Bi & Liu (2007).

$$\mathrm{E}\mathrm{x}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}{\mathrm{W}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}\mathrm{X}100.}$$

Identification of thymoquinone via thin-layer chromatography

TLC plates (Merck, Darmstadt, Germany) characterized by silica gel 60G and dimensions of (4.5 mm × 10 mm) were employed for the identification of TQ. The extracted oil was spotted on the TLC plate and kept for development of the chromatograms. The development system was made up of n-hexane:ethyl acetate (8:2 v/v), which produced a strong and well-defined band for the metabolite and the identity was validated by comparing the bands of standard thymoquinone with those of studied sample extracts based on the visibility of bands under ultraviolet light.

HPLC analysis

Standard calibration

A TQ stock solution with a concentration of 1.0 mg ml⁻¹ was prepared by dissolving 1.0 mg of TQ in HPLC-grade methanol, and the volume was adjusted to 1 ml. The solution underwent sonication at 37 °C for 10 min. Prior to HPLC analysis, all dilutions were meticulously made stepwise from the main stock in methanol, followed by filtration through a 0.22 µm membrane filter. Seven reference points for the TQ calibration curve (0.05, 0.1, 0.2, 1, 2.5, 5, 10 µg/mL) in methanol were established and are illustrated in Fig. 1. The calibration curves were generated using TQ (1.0 mg ml⁻¹) through linear least square regression, plotting the analysis concentration against the peak area. The lowest concentration on the calibration curve represents the lower limit of quantification (LLOQ), crucial for determining accurate and precise findings.

Antioxidant activity

Statistical analysis

The mean and standard deviation were computed, and a graph was generated. Additionally, MS Excel (Microsoft, Redmond, WA, USA) was employed for ANOVA calculations.

Percentage yield of extract in released varieties

Using n-hexane as the solvent, extracts of Nigella sativa L., were prepared from five distinct varieties to investigate and screen the most suitable plant type for thymoquinone concerning consumer value. As presented in Table 2, the percentage yields varied from 4.32% to 5.82%. The highest yield was observed in Ajmer Nigella-20 (5.82 ± 0.03%), while the variety Azad kalonji exhibited the lowest yield (4.32 ± 0.03%). Similar results were seen in a study conducted by Gupta et al. (2021), in which the methanol extract demonstrated an optimal yield. A yield study usually reveals a better foundation for bioactive compound extraction when more amounts of a particular solvent are used. Methanol extract was utilized to assess the quantity of thymoquinone in a specific variety of the plant, Nigella sativa L., HPLC analysis was conducted to determine the maximum accumulation of thymoquinone in the released variety of the plant. Prior to thymoquinone quantification, a standard curve was constructed using a stock solution of 1 mg ml⁻¹, generating a linear calibration curve with a concentration range of 0.05 to 10 µg ml⁻¹. The average coefficient of determination (R²) was found to be 0.998. The method’s validation included assessments of linearity, accuracy, robustness, precision, and data selectivity. The resulting chromatograms are illustrated in Fig. 2. The percentage composition varied among the varieties, with Ajmer Nigella-20 recording the maximum percentage composition (0.20 ± 0.07%), and Azad Kalonji reporting the minimum (0.11 ± 0.17%). Phytochemicals produced by a specific plant type could be the cause of fluctuations in yield, leading to corresponding changes in the percentage yield. This suggests that Ajmer Nigella-20 is the prominent source of thymoquinone among the studied varieties. The observed variation in thymoquinone quantity is likely linked to the varietal genetic makeup, responding significantly to the given environment and involving physiological and biochemical phenomena that influence the levels of phytochemicals in the crop plant variety. The distinct quantities of thymoquinone in different varieties imply varied thymoquinone expression, highlighting a connection between percentage yield and phytochemical ingredient makeup. According to research, as the yield increases, so does the percentage composition. Therefore, the percentage yield can be considered a relevant factor for the bioactive ingredients included in the extract.

The GC-MS profiling of n-hexane extract from varieties of Nigella sativa L., revealed the presence of several peaks (Figs. 3–7). The chromatogram peaks were interpreted using the GC-MS library’s spectrum of known components database, and Table 3 lists the identified compounds along with their peak area, retention time, molecular formula, and molecular weight. The GC-MS profiling showed the presence of 11 major compounds in the studied varieties, with p-cymene, longifolene, and myristic acid identified as the major chemical compounds present in the oil. These results are consistent with Khalid & Shedeed (2016), where GC-MS analysis of Nigella sativa L. showed the existence of sixteen different compounds, with p-cymene, α-thujene, and γ-terpinene identified as major compounds. p-Cymene, also known as p-isopropyl toluene, is a naturally occurring alkyl-substituted aromatic chemical found in essential oils of various medicinal and aromatic plants, as well as several edible plants. Studies have revealed that p-cymene has antioxidant, anti-inflammatory, antiparasitic, antidiabetic, antiviral, anticancer, antibacterial, and antifungal effects (Harish et al., 2023). Additionally, p-cymene has been associated with analgesic, antinociceptive, immunomodulatory, vasorelaxant, and neuroprotective properties (Verruck et al., 2019). Myristic acid, another major compound identified, plays a direct role in post-translational protein modifications and mechanisms that regulate essential metabolic processes in the human body (Legrand & Rioux, 2015). Modest intake of myristic acid has been associated with increased levels of long-chain omega-3 fatty acids in plasma phospholipids, potentially improving cardiovascular health markers in humans (Dabadie et al., 2005). This discussion highlights that the oil from Nigella sativa L., has potential applications not only as an edible oil but also due to its pharmaceutical properties.

Table 3 presents the concentration of total phenolic compounds in the studied varieties, assessed using the Folin–Ciocalteau procedure and quantified as gallic acid equivalents. The results indicate that Ajmer nigella-20 had the highest concentration of total phenolic compounds (31.85 ± 0.97 mg GAE/g seed) among the Nigella varieties, while the lowest was observed in NDBC-10 (30.18 ± 0.65 mg GAE g⁻¹ seed). These findings align with those of Saxena et al. (2017), who also determined the overall concentration of phenolic compounds in this plant, with an average phenol concentration of 164 mg GAE ml⁻¹ in nigella genotypes. Regarding total flavonoids content, the highest was recorded in Ajmer Nigella-20 (7.31 ± 0.11 mg QE g⁻¹), while the lowest was in NDBC-10 (7.31 ± 0.11 mg QE g⁻¹). This finding is in line with earlier research by Ahmad et al. (2014), where a methanolic extract of Nigella sativa L. seeds reported 1.40 ± 0.29 mg g⁻¹ of total flavonoids. It also corresponds with reports by Saxena et al. (2017), who stated a flavonoid content of 818 mg QE ml⁻¹.

The total antioxidant activity was highest in Ajmer Nigella-20 (76.18 ± 1.78%) and lowest in Pant Krishna (72.18 ± 1.95%), as measured using the DPPH free radical test, indicating varying levels of antioxidants in the studied varieties (Table 1). The results highlight the high antioxidant activity of Nigella seed oil, consistent with findings in the report by Gupta et al. (2021), where the highest antioxidant activity was reported in the ethanolic extract of the flowering bud. The observed differences in thymoquinone levels among the studied varieties were accompanied by variations in total antioxidant activity, suggesting a connection between thymoquinone quantity and total antioxidant activity. Furthermore, the antioxidant potential of nigella varieties in the DPPH test showed a linear proportional relationship with their total phenolic components. Antioxidant activity increased in direct proportion to polyphenol content, indicating a positive linear association between percent antioxidant activity and total phenolic compounds. The presence of phenolic and flavonoid content is known to enhance antioxidant action. Thymoquinone, carvacrol, anethole, and 4-terpineol, all found in Nigella essential and fixed oil, exhibit significant radical scavenging capacity (Badary et al., 2003).