Comparative Gas Chromatography-Mass Spectrometry Analysis of Nigella sativa Varieties Romanian, Sudani, Bangladeshi, Hama (Syrian)

Abstract

This study investigates the phytochemical differences between four varieties of Nigella sativa (black seed) which from family Ranunculaceae cultivated in Romania which is native, Sudan, Bangladesh, and Hama (Syria) using gas chromatography-mass spectrometry (GC-MS). The focus is on quantifying key bioactive compounds-thymoquinone, p-cymene, ?-phellandrene, and longifolene-recognized for their therapeutic properties. The analysis reveals significant variation in the concentration of these compounds across the different geographical origins, with the Romanian variety exhibiting the highest thymoquinone content. These findings highlight the impact of geographical origin on the chemical composition and potential therapeutic value of Nigella sativa seeds, offering valuable insights for industrial and pharmaceutical applications. Method and Material: Seeds from each region were collected with unified harvest timing (April, 8 am morning), cleaned, shade-dried, and ground. For extraction, 60 g of seeds per sample were soaked in 400 ml hexane at 23–26°C for three days. The extracts were filtered using nylon filter paper (0.45 µm) and treated with activated charcoal (0.25%) for purity. GC-MS analysis was performed using a Shimadzu GCMS-QP 2010 Plus system, with calibration and validation via standard compounds and the NIST11s.lib spectral library. Linearity and recovery tests confirmed high analytical accuracy (R² > 0.999, recovery 98.3–101.6%, RSD < 1.2%). Results: GC-MS analysis showed clear phytochemical differences between the varieties. The Romanian sample had the highest thymoquinone concentration (27.9%), followed by Hama (Syrian) (16.2%), Sudanese (4.3%), and Bangladeshi (2.2%). Statistical analysis (ANOVA and Kruskal-Wallis) confirmed significant differences between origins (p < 0.001). Toxicity predictions indicated that p-cymene (LD50 = 3 mg/kg) is the most toxic among the analysed compounds, while thymoquinone and others are within safe limits for food and therapeutic use. Conclusion: The study demonstrates that the phytochemical profile of Nigella sativa varies significantly with geographical origin, particularly regarding thymoquinone content. The Romanian variety is richest in thymoquinone, suggesting superior therapeutic potential, while other varieties differ in their dominant compounds. These results underscore the importance of origin in selecting Nigella sativa varieties for medicinal and industrial use and call for further research using advanced analytical methods to optimize economic and health benefits1.

Introduction

Nigella sativa, known for its antioxidant, anti-inflammatory, anticancer, antiasthmatic, and antidiabetic properties, owes much of its medicinal value to active compounds like thymoquinone and p-cymene. This study aims to analyze and compare the phytochemical profiles of Nigella sativa seeds from four geographical origins—Bangladesh, Hama (Syria), Romania (native), and Sudan—using GC-MS (Gas Chromatography-Mass Spectrometry).

Previous research has examined Nigella sativa from regions like Saudi Arabia, India, and Turkey, but there remains a gap in comprehensive phytochemical profiling using advanced analytical techniques across these four origins. This study will assess differences in active compounds such as thymoquinone, p-cymene, α-phellandrene, and longifolene, and evaluate their therapeutic potential and toxicity using computational tools like SuperPred and Protox III.

The research involves careful collection, preparation, and analysis of seed samples, using standardized methods to ensure accuracy and reproducibility. GC-MS data will provide detailed insights into the concentration of key phytochemicals, aiding in identifying the most nutritionally and therapeutically valuable varieties. The study's outcomes will have economic significance by guiding exports and industrial selection of Nigella sativa varieties rich in active compounds, and contribute to better understanding the impact of geography on phytochemical composition.

Conclusion

This study comparesbetween Four variations of nigella sativa of different geographical origins and use GCMS analysis study to determine the phytochemical variations among Nigella sativa varieties from Bangladesh, Hama(Syria), and Romania and Sudan , emphasizing the impact of geographical origin on phytochemical compositions that which may effect on therapeutic effects The Romanian variety exhibited the highest thymoquinone content (27.9%), followed by Hama(Syrian) (16.2%) and Sudani was 4.3% and Bangladeshi was (2.2%) sample,When we compare the results of previous research on the Turkish, Saudi, Indian and Moroccan types, it becomes clear that FIG 2 the Turkish type,(Erdo?an, Yilmazer, and Erba? 2020),(Salehi et al. 2019) is the highest 45%, followed by the Indian 29%[9],[11], followed by the Romanian27.9%, followed by the arabia Saudi [14]25.35% , followed by the Bangladeshi 19.1% , followed by the Moroccan 5.69 %[35], followed by the Sudan4.3% , followed by the Syrian Hama 2.2% , establishing a clear link between cultivation in different placesand bioactive compound profiles. The Turkish Nigella sativa variant shows superior thymoquinone content (45%), highlighting its value for pharmaceuticals and food. Caution is advised for Sudanese (45.6% p-cymene) and Indian (41.0% p-cymene) varieties due to toxicity (LD50: 3 mg/kg). Thymoquinone’s safety supports therapeutic applications. Future research should expand to other regions and standardize cultivation/extraction protocols to enhance economic and medicinal potential

References

[1] Ahmad, Aftab, Asif Husain, Mohd Mujeeb, Shah Alam Khan, Abul Kalam Najmi, Nasir Ali Siddique, Zoheir A. Damanhouri, and Firoz Anwar. 2013. ‘A Review on Therapeutic Potential of Nigella Sativa: A Miracle Herb’. Asian Pacific Journal of Tropical Biomedicine 3 (5): 337–52. https://doi.org/10.1016/S2221-1691(13)60075-1. [2] Asimi, Sailimuhan, Xin Ren, Min Zhang, Sixuan Li, Lina Guan, Zhenhua Wang, Shan Liang, and Ziyuan Wang. 2022. ‘Fingerprinting of Volatile Organic Compounds for the Geographical Discrimination of Rice Samples from Northeast China’. Foods 11 (12): 1695. https://doi.org/10.3390/foods11121695. [3] Banerjee, Priyanka, Emanuel Kemmler, Mathias Dunkel, and Robert Preissner. 2024. ‘ProTox 3.0: A Webserver for the Prediction of Toxicity of Chemicals’. Nucleic Acids Research 52 (W1): W513–20. https://doi.org/10.1093/nar/gkae303. [4] Centre for Microscopy, Characterisation & Analysis. 2018. ‘Shimadzu GCMS QP2010’. University of Western Australia. https://doi.org/10.26182/H0B2-ZN50. [5] Chetima, Abba, Divine Nde Bup, Fannyuy Kewir, and Abdoul Wahaboua. 2024. ‘Activated Carbons from Open Air and Microwave-Assisted Impregnation of Cotton and Neem Husks Efficiently Decolorize Neutral Cotton Oil’. Heliyon 10 (1): e24060. https://doi.org/10.1016/j.heliyon.2024.e24060. [6] De Vivo, Angela, Andrea Balivo, and Fabrizio Sarghini. 2023. ‘Volatile Compound Analysis to Authenticate the Geographical Origin of Arabica and Robusta Espresso Coffee’. Applied Sciences 13 (9): 5615. https://doi.org/10.3390/app13095615. [7] Erdo?an, Ümit, Mustafa Yilmazer, and Sabri Erba?. 2020. ‘Hydrodistillation of Nigella Sativa Seed and Analysis of Thymoquinone with HPLC and GC-MS’. Bilge International Journal of Science and Technology Research 4 (1): 27–30. https://doi.org/10.30516/bilgesci.688845. [8] Farhan, Nesrain, Nadia Salih, and Jumat Salimon. 2021. ‘Physiochemical Properties of Saudi Nigella Sativa L. (“Black Cumin”) Seed Oil’. OCL 28:11. https://doi.org/10.1051/ocl/2020075. [9] Goyal, Sameer N., Chaitali P. Prajapati, Prashant R. Gore, Chandragouda R. Patil, Umesh B. Mahajan, Charu Sharma, Sandhya P. Talla, and Shreesh K. Ojha. 2017. ‘Therapeutic Potential and Pharmaceutical Development of Thymoquinone: A Multitargeted Molecule of Natural Origin’. Frontiers in Pharmacology 8:656. https://doi.org/10.3389/fphar.2017.00656. [10] Grover, Madhuri, Tapan Behl, Tarun Virmani, Mohit Sanduja, Hafiz A. Makeen, Mohammed Albratty, Hassan A. Alhazmi, Abdulkarim M. Meraya, and Simona Gabriela Bungau. 2022. ‘Exploration of Cytotoxic Potential of Longifolene/Junipene Isolated from Chrysopogon Zizanioides’. Molecules (Basel, Switzerland) 27 (18): 5764. https://doi.org/10.3390/molecules27185764. [11] Ibn-Qaiyim al-?auz?ya, Mu?ammad Ibn-Ab?-Bakr. 2003. Prophetic Medicine: = A?-?ibb an-Nabaw?. 2nd ed. New Delhi: Islamic Book Service. [12] Jha, Avinash Kumar, and Nandan Sit. 2022. ‘Extraction of Bioactive Compounds from Plant Materials Using Combination of Various Novel Methods: A Review’. Trends in Food Science & Technology 119 (January):579–91. https://doi.org/10.1016/j.tifs.2021.11.019. [13] Kabir, Yearul, Yoko Akasaka-Hashimoto, Kikue Kubota, and Michio Komai. 2020. ‘Volatile Compounds of Black Cumin (Nigella Sativa L.) Seeds Cultivated in Bangladesh and India’. Heliyon 6 (10): e05343. https://doi.org/10.1016/j.heliyon.2020.e05343. [14] Kim, Sunghwan, Jie Chen, Tiejun Cheng, Asta Gindulyte, Jia He, Siqian He, Qingliang Li, et al. 2025. ‘PubChem 2025 Update’. Nucleic Acids Research 53 (D1): D1516–25. https://doi.org/10.1093/nar/gkae1059. [15] Li, Tao-Yu, Wan-Li Liang, Yi-Ming Zhao, Wan-Dong Chen, Hong-Xia Zhu, Yuan-Yuan Duan, Han-Bo Zou, Sha-Sha Huang, Xiao-Jun Li, and Wei Kevin Zhang. 2024. ‘Alpha-Pinene-Encapsulated Lipid Nanoparticles Diminished Inflammatory Responses in THP-1 Cells and Imiquimod-Induced Psoriasis-like Skin Injury and Splenomegaly in Mice’. Frontiers in Immunology 15 (October):1390589. https://doi.org/10.3389/fimmu.2024.1390589. [16] Pinheiro-Neto, Flaviano Ribeiro, Everton Moraes Lopes, Boris Timah Acha, Laércio Da Silva Gomes, Willian Amorim Dias, Antonio Carlos Dos Reis Filho, Bianca De Sousa Leal, et al. 2021. ‘?-Phellandrene Exhibits Antinociceptive and Tumor-Reducing Effects in a Mouse Model of Oncologic Pain’. Toxicology and Applied Pharmacology 418 (May):115497. https://doi.org/10.1016/j.taap.2021.115497. [17] Radice, Matteo, Andrea Durofil, Raissa Buzzi, Erika Baldini, Amaury Pérez Martínez, Laura Scalvenzi, and Stefano Manfredini. 2022. ‘Alpha-Phellandrene and Alpha-Phellandrene-Rich Essential Oils: A Systematic Review of Biological Activities, Pharmaceutical and Food Applications’. Life (Basel, Switzerland) 12 (10): 1602. https://doi.org/10.3390/life12101602. [18] Rahim, Muhammad Abdul, Aurbab Shoukat, Waseem Khalid, Afaf Ejaz, Nizwa Itrat, Iqra Majeed, Hyrije Koraqi, et al. 2022. ‘A Narrative Review on Various Oil Extraction Methods, Encapsulation Processes, Fatty Acid Profiles, Oxidative Stability, and Medicinal Properties of Black Seed (Nigella Sativa)’. Foods 11 (18): 2826. https://doi.org/10.3390/foods11182826. [19] Ramalho, Theresa, Maria Pacheco De Oliveira, Ana Lima, Claudio Bezerra-Santos, and Marcia Piuvezam. 2015. ‘Gamma-Terpinene Modulates Acute Inflammatory Response in Mice’. Planta Medica 81 (14): 1248–54. https://doi.org/10.1055/s-0035-1546169. [20] Ravi, Y., Irene P. Vethamoni, Shailendra N. Saxena, S. Velmurugan, V. P. Santanakrishnan, M. Raveendran, Himanshu Bariya, and Mistry Harsh. 2023. ‘Guesstimate of Thymoquinone Diversity in Nigella Sativa L. Genotypes and Elite Varieties Collected from Indian States Using HPTLC Technique’. Open Life Sciences 18 (1): 20220536. https://doi.org/10.1515/biol-2022-0536. [21] Salas-Oropeza, Judith, Manuel Jimenez-Estrada, Armando Perez-Torres, Andres Eliu Castell-Rodriguez, Rodolfo Becerril-Millan, Marco Aurelio Rodriguez-Monroy, Katia Jarquin-Yañez, and Maria Margarita Canales-Martinez. 2021. ‘Wound Healing Activity of ?-Pinene and ?-Phellandrene’. Molecules 26 (9): 2488. https://doi.org/10.3390/molecules26092488. [22] Salehi, Bahare, Shashi Upadhyay, Ilkay Erdogan Orhan, Arun Kumar Jugran, Sumali L D Jayaweera, Daniel A Dias, Farukh Sharopov, et al. 2019. ‘Therapeutic Potential of ?- and ?-Pinene: A Miracle Gift of Nature’. Biomolecules 9 (11): 738. https://doi.org/10.3390/biom9110738. [23] Seethalakshmi, Souprayen, Ravishankar Sarumathi, and Chandrasekaran Sankaranarayanan. 2024. ‘Effect of Alpha-Phellandrene on Glucose Uptake and Adipogenesis in Insulin Resistant 3T3-L1 Adipocytes: An in Vitro and in Silico Approach’. Asian Journal of Biological and Life Sciences 12 (3): 603–10. https://doi.org/10.5530/ajbls.2023.12.79. [24] Share, J. B. 1976. ‘Review of Drug Treatment for Down’s Syndrome Persons’. American Journal of Mental Deficiency 80 (4): 388–93. [25] Shaukat, Arslan, Arsalan Zaidi, Haseeb Anwar, and Nadeem Kizilbash. 2023. ‘Mechanism of the Antidiabetic Action of Nigella Sativa and Thymoquinone: A Review’. Frontiers in Nutrition 10 (September):1126272. https://doi.org/10.3389/fnut.2023.1126272. [26] Stein, Stephen E., and Donald R. Scott. 1994. ‘Optimization and Testing of Mass Spectral Library Search Algorithms for Compound Identification’. Journal of the American Society for Mass Spectrometry 5 (9): 859–66. https://doi.org/10.1016/1044-0305(94)87009-8. [27] Tiji, Salima, Yahya Rokni, Ouijdane Benayad, Nassima Laaraj, Abdeslam Asehraou, and Mostafa Mimouni. 2021. ‘Chemical Composition Related to Antimicrobial Activity of Moroccan Nigella Sativa L. Extracts and Isolated Fractions’. Edited by Manzoor A. Rather. Evidence-Based Complementary and Alternative Medicine 2021 (October):1–14. https://doi.org/10.1155/2021/8308050. [28] Y, Ravi, Irene Vethamoni Periyanadar, Shailendra Nath Saxena, Raveendran Muthurajan, Velmurugan Sundararajan, Santhanakrishnan Vichangal Pridiuldi, Sumer Singh Meena, et al. 2024. ‘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’. PeerJ 12 (March):e17177. https://doi.org/10.7717/peerj.17177.

Copyright

Copyright © 2025 Mohamed A Farag Fannosh, Md Gousuddin, Dr. Sreemoy. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.