Comparative analysis of vitamin E extraction methods in Cleome viscosa L. and the role of key biosynthesis genes
Article Main
Abstract
Vitamin E is a lipid-soluble, potent radical-scavenging antioxidant that inhibits lipid peroxidation, which damages cell membranes, proteins, and DNA in the human body. Currently, it is widely utilized in dietary supplements and cosmetic products and is primarily sourced from plant-derived oils. However, Cleome viscosa L. (Asian spider flower) an ethnobotanical species that is not as widely recognized, could provide a different natural source of tocopherols, especially in the early stages of seed development. The present study aimed to evaluate which extraction methods produced the highest vitamin E contents by comparing different extraction techniques. A method was developed in which different solvents and extraction durations were tested to analyze the vitamin E and antioxidant contents in Cleome viscosa L. seed from 2-5 weeks after fruit set (WAF). Two methods were used, the Soxhlet method and maceration. The highest amount of gamma-tocopherol was present at 5 WAF and a concentration of 1946.78 µg/100 g fresh weight with an IC50 value of 32.02 ± 2.02 µg/ml using the Soxhlet method with ethanol for 6 hours. To gain information on the relationship between vitamin E content and the expression of four genes (VTE1-4) involved in vitamin E biosynthesis was examined. The higher expression of the VTE1 and VTE4 genes from the early seed stage to the mature seed stage. Furthermore, the maximum amount of alpha-tocopherol, 898.44 µg/100 g fresh weight, with an IC50 value of 7.46 ± 2.20 µg/ml, was produced by the maceration method using ethanol solution at 3 WAF, and the VTE4 gene was more highly expressed compared to other tocopherol biosynthesis genes.
Article Details
Article Details
Keywords
Antioxidant activity, Cleome viscosa L., Extraction methods, Gene expression, Vitamin E
References
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Arun, M., Subramanyam, K., Theboral, J., Sivanandhan, G., Rajesh, M., Kapil Dev, G., Jaganath, B., Manickavasagam, M., Girija, S., & Ganapathi, A. (2014). Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars. Applied biochemistry and biotechnology, 172, 1763-1776. https://doi.org/10.1007/s12010-013-0645-9
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Blasi, F., Tringaniello, C.,Verducci, G., & Cossignani, L. (2022). Bioactive minor components of Italian and extra-European hemp seed oils. LWT – Food Science and Technology, 158, 113167. https://doi.org/10.1016/j.lwt.2022.113167
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Chao, Y., Kang, J., Zhang, T., Yang, Q., Gruber, M. Y., & Sun, Y. (2014). Disruption of the homogentisate solanesyltransferase gene results in albino and dwarf pheno types and root, trichome and stomata defects in Arabidopsis thaliana. PLOS ONE, 9(4), e94031. https://doi.org/10.1371/journal.pone.0094031
Ćujić, N., Šavikin, K., Janković, T., Pljevljakušić, D., Zdunić, G., & Ibrić, S. (2016). Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chemistry, 194, 135–142. https://doi.org/10.1016 /j.foodchem.2015.08.008
de Mendiburu, F., & Yaseen, M. (2020). agricolae: Statistical Procedures for Agricultural Research (R package version 1.4.0) [Software]. Retrieved from https://cran.r-project.org/package=agricolae
Dos Santos Freitas, L., Jacques, R. A., Richter, M. F., da Silva, A. L., & Caramão, E. B. (2008). Pressurized liquid extraction of vitamin E from Brazilian grape seed oil. Journal of Chromatography A, 1200(1), 80-83. https://doi.org/10.1016/j.chroma.2008.02.067
Gopalasatheeskumar, K. (2018). Significant role of Soxhlet extraction process in phytochemical research. Mintage Journal of Pharmaceutical & Medical Sciences, 7(1), 43–47. https://doi.org/10.5958/0974-360X.2020.00 490.4
Górnaś, P., Mišina, I., Soliven, A., & Segliņa, D. (2025). Tocopherol and tocotrienol profile in wild St. John’s wort populations in Latvia: Impact of the plant’s aerial parts. Natural Product Research, 1–8. https://doi.org/10.1080/14786419.2025.2477804
Grilo, E. C., Costa, P. N., Gurgel, C. S. S., Beserra, A. F. D. L., Almeida, F. N. D. S., & Dimenstein, R. (2014). Alpha-tocopherol and gamma-tocopherol concentration in vegetable oils. Food Science and Technology, 34, 379-38 5. https://doi.org/ 10.1590/ S0101-20612014005 000 031
Gundev, P., Chauhan, K., & Sachdev, D. (2019). To study the Antioxidative potential of Flaxseed oil at varied temperatures. International Journal of Advanced Scientific Research and Management, 4(4), 409-413.
Guo, Y., Li, D., Liu, T., Liao, M., Li, Y., Zhang, W., Liu, Z., & Chen, M. (2022). Effect of overexpression of γ-tocopherol methyltransferase on α-tocopherol and fatty acid accumulation and tolerance to salt stress during seed germination in Brassica napus L. International Journal of Molecular Sciences, 23(24), 15933. https://doi.org/10.3390 /ijms232415933
Hisatomi, E., Matsui, M., Kubota, K., & Kobayashi, A. (2000). Antioxidative activity in the pericarp and seed of Japanese pepper (Xanthoxylum piperitum DC). Journal of Agricultural and Food Chemistry, 48(10), 4924–4928. https://doi.org/10.1021/ jf000540w
Irabor, E. E. I., Onukwugha, P. U., Eze, P. N., & Ogbeide, O. K. (2020). Extraction and characterization of fat-soluble vitamins from oil of Calliandra surrinamensis seed. Journal of Chemical Society of Nigeria, 45(3), 502–507.
Jai-Uean, A., & Sangin, P. (2021). Comparative analysis of tocopherol biosynthesis genes during seed development in the Asian spiderflower, Cleome viscosa (Cleomaceae). Genetics and Molecular Research, 20(2), gmr1882. https://doi.org/10.4238/gmr18802
Jumina, J., Lavendi, W., Singgih, T., Triono, S., Kurniawan, Y. S., & Koketsu, M. (2019). Preparation of monoacylglycerol derivatives from Indonesian edible oil and their antimicrobial assay against Staphylococcus aureus and Escherichia coli. Scientific reports, 9(1), 10941. https://doi.org/10.1038/s41598-019-47373-4
Karunanandaa, B., Qi, Q., Hao, M., Baszis, S. R., Jensen, P. K., Wong, Y. H. H., & Valentin, H. E. (2005). Metabolically engineered oilseed crops with enhanced seed tocopherol. Metabolic Engineering 7(5-6), 384-400. https://doi.org/10.1007/s44372-025-00093-8
Kmiecik, D., Fedko, M., Siger, A., & Kulczyński, B. (2019). Degradation of Tocopherol Molecules and Its Impact on the Polymerization of Triacylglycerols during Heat Treatment of Oil. Molecules, 24(24), 4555. https://doi.org/10.3390/molecules24244555
Lee, Y. Y., Park, H. M., Lee, C. K., Kim, S. L., Hwang, T. Y., Choi, M. S., Kwon, Y. U., Kim, W. H., Kim, S. J., Lee, S. C., & Kim, Y. H. (2012). Comparing extraction methods for the determination of tocopherols and tocotrienols in seeds and germinating seeds of soybean transformed with OsHGGT. Journal of Food Composition and Analysis, 27(1), 70-80. https://doi.org/10.1016/j.jfca.2012.0 3.01 0
Liu, Y., Lv, M., Wang, Y., Wei, J., & Chen, D. (2025). Analytical Strategies for Tocopherols in Vegetable Oils: Advances in Extraction and Detection. Pharmaceuticals, 18(8), 1137. https://doi.org/10.3390/ph18081137
Maarasyid, C., Muhamad, I., & Supriyanto, E. (2014). Potential source and extraction of vitamin E from palm-based oils: a review. Jurnal Teknologi. 69(4), 43-50. https://doi.org/ 10.11113/jt.v69.3172
Mateeva, A., Kondeva-Burdina, M., Peikova, L., Guncheva, S., Zlatkov, A., & Georgieva, M. (2023). Simultaneous analysis of water-soluble and fat-soluble vitamins thorough RP-HPLC/DAD in food supplements and brewer’s yeast. Heliyon, 9(1), https://doi.org/10.1016/j.heliyon.2022.e12706
Núñez, S., Valero, M. S., Mustafa, A. M., Caprioli, G., Maggi, F., Gómez‐Rincón, C., & López, V. (2025). Ultrasound‐assisted extraction versus traditional Soxhlet apparatus for the obtention of polyphenols, carotenoids and tocopherols from Tagetes erecta L. flowers. Journal of the Science of Food and Agriculture, 105(6), 3453–3462. https://doi.org/10.1002/jsfa.1455
Pillai, L. S., & Nair, B. R. (2013). Proximate composition, Mineral elements and Antinutritional factors in Cleome viscosa L. and Cleome burmanni W. & A. (Cleomaceae). International Journal of Pharmaceutics and Pharmaceutical Science, 5(1), 384-387.
Prieto, J. M. (2012). Procedure: Preparation of DPPH Radical, and antioxidant scavenging assay. DPPH Microplate protocol, 7-9.
Ribeiro, P. P. C., Silva, D. M. D. L., Dantas, M. M., Ribeiro, K. D. D. S., Dimenstein, R., & Damasceno, K. A. F. D. S. C. (2019). Determination of tocopherols and physicochemical properties of faveleira (Cnidoscolus quercifolius) seed oil extracted using different methods. Food Science and Technology, 39, 280-285. https://doi.org/10.1590/fst.24017
Rizvi, S., Raza, S. T., Ahmed, F., Ahmad, A., Abbas, S., & Mahdi, F. (2014). The role of vitamin E in human health and some diseases. Sultan Qaboos University Medical Journal, 14(2), e157-165.
Rychter, A. M., Hryhorowicz, S., Słomski, R., Dobrowolska, A., & Krela-Kaźmierczak, I. (2022). Antioxidant effects of vitamin E and risk of cardiovascular disease in women with obesity–a narrative review. A narrative review. Clinical Nutrition, 41, 1557-1565 https://doi.org/10.1016/j.clnu.2022.04.032
Tourabi, M., Faiz, K., Ezzouggari, R., Louasté, B., Merzouki, M., Dauelbait, M., ... & Derwich, E. (2025). Optimization of extraction process and solvent polarities to enhance the recovery of phytochemical compounds,
nutritional content, and biofunctional properties of
Mentha longifolia L. extracts. Bioresources and Bioprocessing, 12(1), 24. https://doi.org/10.1186/s40643-025-00859-8
Verwoerd, T. C., Dekker, B. M., & Hoekema, A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research, 17(6), 2362-2362. https://doi.org/ 10.1093/nar/17.6.2362
Wang, S., Hwang, H., Yoon, S., & Choe, E. (2010). Temperature dependence of autoxidation of perilla oil and tocopherol degradation. Journal of Food Science, 75(6), 498-505. https://doi.org/ 10.1111/j.1750-3841.2010.016 81.x
Alves, R. C., Casal, S., & Oliveira, M. B. P. (2009). Determination of vitamin E in coffee beans by HPLC using a micro-extraction method. Food Science and Technology International, 15(1), 57-63. https://doi.org/10.117 7/1082013 208102695
Ara, Z., Waliullah, S., Rastogi, D, Pant, S. (2025). Vitamin E and human health: An update. Glob J Health Sci Res. https://doi.org/10.25259/GJHSR_6_2025
Arun, M., Subramanyam, K., Theboral, J., Sivanandhan, G., Rajesh, M., Kapil Dev, G., Jaganath, B., Manickavasagam, M., Girija, S., & Ganapathi, A. (2014). Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars. Applied biochemistry and biotechnology, 172, 1763-1776. https://doi.org/10.1007/s12010-013-0645-9
Baliyan, S., Mukherjee, R., Priyadarshini, A., Vibhuti, A., Gupta, A., Pandey, R. P., & Chang, C. M. (2022). Determination of antioxidants by DPPH radical scavenging activity and quantitative phytochemical analysis of Ficus religiosa. Molecules, 27(4), 1326. https://doi.org/10.3390/molecules27041326
Blasi, F., Tringaniello, C.,Verducci, G., & Cossignani, L. (2022). Bioactive minor components of Italian and extra-European hemp seed oils. LWT – Food Science and Technology, 158, 113167. https://doi.org/10.1016/j.lwt.2022.113167
Budzianowska, A., Banaś, K., Budzianowski, J., & Kikowska, M. (2025). Antioxidants to Defend Healthy and Youthful Skin Current Trends and Future Directions in Cosmetology. Applied Sciences, 15(5), 2571. https://doi.org/10.3390/app15052571
Chao, Y., Kang, J., Zhang, T., Yang, Q., Gruber, M. Y., & Sun, Y. (2014). Disruption of the homogentisate solanesyltransferase gene results in albino and dwarf pheno types and root, trichome and stomata defects in Arabidopsis thaliana. PLOS ONE, 9(4), e94031. https://doi.org/10.1371/journal.pone.0094031
Ćujić, N., Šavikin, K., Janković, T., Pljevljakušić, D., Zdunić, G., & Ibrić, S. (2016). Optimization of polyphenols extraction from dried chokeberry using maceration as traditional technique. Food Chemistry, 194, 135–142. https://doi.org/10.1016 /j.foodchem.2015.08.008
de Mendiburu, F., & Yaseen, M. (2020). agricolae: Statistical Procedures for Agricultural Research (R package version 1.4.0) [Software]. Retrieved from https://cran.r-project.org/package=agricolae
Dos Santos Freitas, L., Jacques, R. A., Richter, M. F., da Silva, A. L., & Caramão, E. B. (2008). Pressurized liquid extraction of vitamin E from Brazilian grape seed oil. Journal of Chromatography A, 1200(1), 80-83. https://doi.org/10.1016/j.chroma.2008.02.067
Gopalasatheeskumar, K. (2018). Significant role of Soxhlet extraction process in phytochemical research. Mintage Journal of Pharmaceutical & Medical Sciences, 7(1), 43–47. https://doi.org/10.5958/0974-360X.2020.00 490.4
Górnaś, P., Mišina, I., Soliven, A., & Segliņa, D. (2025). Tocopherol and tocotrienol profile in wild St. John’s wort populations in Latvia: Impact of the plant’s aerial parts. Natural Product Research, 1–8. https://doi.org/10.1080/14786419.2025.2477804
Grilo, E. C., Costa, P. N., Gurgel, C. S. S., Beserra, A. F. D. L., Almeida, F. N. D. S., & Dimenstein, R. (2014). Alpha-tocopherol and gamma-tocopherol concentration in vegetable oils. Food Science and Technology, 34, 379-38 5. https://doi.org/ 10.1590/ S0101-20612014005 000 031
Gundev, P., Chauhan, K., & Sachdev, D. (2019). To study the Antioxidative potential of Flaxseed oil at varied temperatures. International Journal of Advanced Scientific Research and Management, 4(4), 409-413.
Guo, Y., Li, D., Liu, T., Liao, M., Li, Y., Zhang, W., Liu, Z., & Chen, M. (2022). Effect of overexpression of γ-tocopherol methyltransferase on α-tocopherol and fatty acid accumulation and tolerance to salt stress during seed germination in Brassica napus L. International Journal of Molecular Sciences, 23(24), 15933. https://doi.org/10.3390 /ijms232415933
Hisatomi, E., Matsui, M., Kubota, K., & Kobayashi, A. (2000). Antioxidative activity in the pericarp and seed of Japanese pepper (Xanthoxylum piperitum DC). Journal of Agricultural and Food Chemistry, 48(10), 4924–4928. https://doi.org/10.1021/ jf000540w
Irabor, E. E. I., Onukwugha, P. U., Eze, P. N., & Ogbeide, O. K. (2020). Extraction and characterization of fat-soluble vitamins from oil of Calliandra surrinamensis seed. Journal of Chemical Society of Nigeria, 45(3), 502–507.
Jai-Uean, A., & Sangin, P. (2021). Comparative analysis of tocopherol biosynthesis genes during seed development in the Asian spiderflower, Cleome viscosa (Cleomaceae). Genetics and Molecular Research, 20(2), gmr1882. https://doi.org/10.4238/gmr18802
Jumina, J., Lavendi, W., Singgih, T., Triono, S., Kurniawan, Y. S., & Koketsu, M. (2019). Preparation of monoacylglycerol derivatives from Indonesian edible oil and their antimicrobial assay against Staphylococcus aureus and Escherichia coli. Scientific reports, 9(1), 10941. https://doi.org/10.1038/s41598-019-47373-4
Karunanandaa, B., Qi, Q., Hao, M., Baszis, S. R., Jensen, P. K., Wong, Y. H. H., & Valentin, H. E. (2005). Metabolically engineered oilseed crops with enhanced seed tocopherol. Metabolic Engineering 7(5-6), 384-400. https://doi.org/10.1007/s44372-025-00093-8
Kmiecik, D., Fedko, M., Siger, A., & Kulczyński, B. (2019). Degradation of Tocopherol Molecules and Its Impact on the Polymerization of Triacylglycerols during Heat Treatment of Oil. Molecules, 24(24), 4555. https://doi.org/10.3390/molecules24244555
Lee, Y. Y., Park, H. M., Lee, C. K., Kim, S. L., Hwang, T. Y., Choi, M. S., Kwon, Y. U., Kim, W. H., Kim, S. J., Lee, S. C., & Kim, Y. H. (2012). Comparing extraction methods for the determination of tocopherols and tocotrienols in seeds and germinating seeds of soybean transformed with OsHGGT. Journal of Food Composition and Analysis, 27(1), 70-80. https://doi.org/10.1016/j.jfca.2012.0 3.01 0
Liu, Y., Lv, M., Wang, Y., Wei, J., & Chen, D. (2025). Analytical Strategies for Tocopherols in Vegetable Oils: Advances in Extraction and Detection. Pharmaceuticals, 18(8), 1137. https://doi.org/10.3390/ph18081137
Maarasyid, C., Muhamad, I., & Supriyanto, E. (2014). Potential source and extraction of vitamin E from palm-based oils: a review. Jurnal Teknologi. 69(4), 43-50. https://doi.org/ 10.11113/jt.v69.3172
Mateeva, A., Kondeva-Burdina, M., Peikova, L., Guncheva, S., Zlatkov, A., & Georgieva, M. (2023). Simultaneous analysis of water-soluble and fat-soluble vitamins thorough RP-HPLC/DAD in food supplements and brewer’s yeast. Heliyon, 9(1), https://doi.org/10.1016/j.heliyon.2022.e12706
Núñez, S., Valero, M. S., Mustafa, A. M., Caprioli, G., Maggi, F., Gómez‐Rincón, C., & López, V. (2025). Ultrasound‐assisted extraction versus traditional Soxhlet apparatus for the obtention of polyphenols, carotenoids and tocopherols from Tagetes erecta L. flowers. Journal of the Science of Food and Agriculture, 105(6), 3453–3462. https://doi.org/10.1002/jsfa.1455
Pillai, L. S., & Nair, B. R. (2013). Proximate composition, Mineral elements and Antinutritional factors in Cleome viscosa L. and Cleome burmanni W. & A. (Cleomaceae). International Journal of Pharmaceutics and Pharmaceutical Science, 5(1), 384-387.
Prieto, J. M. (2012). Procedure: Preparation of DPPH Radical, and antioxidant scavenging assay. DPPH Microplate protocol, 7-9.
Ribeiro, P. P. C., Silva, D. M. D. L., Dantas, M. M., Ribeiro, K. D. D. S., Dimenstein, R., & Damasceno, K. A. F. D. S. C. (2019). Determination of tocopherols and physicochemical properties of faveleira (Cnidoscolus quercifolius) seed oil extracted using different methods. Food Science and Technology, 39, 280-285. https://doi.org/10.1590/fst.24017
Rizvi, S., Raza, S. T., Ahmed, F., Ahmad, A., Abbas, S., & Mahdi, F. (2014). The role of vitamin E in human health and some diseases. Sultan Qaboos University Medical Journal, 14(2), e157-165.
Rychter, A. M., Hryhorowicz, S., Słomski, R., Dobrowolska, A., & Krela-Kaźmierczak, I. (2022). Antioxidant effects of vitamin E and risk of cardiovascular disease in women with obesity–a narrative review. A narrative review. Clinical Nutrition, 41, 1557-1565 https://doi.org/10.1016/j.clnu.2022.04.032
Tourabi, M., Faiz, K., Ezzouggari, R., Louasté, B., Merzouki, M., Dauelbait, M., ... & Derwich, E. (2025). Optimization of extraction process and solvent polarities to enhance the recovery of phytochemical compounds,
nutritional content, and biofunctional properties of
Mentha longifolia L. extracts. Bioresources and Bioprocessing, 12(1), 24. https://doi.org/10.1186/s40643-025-00859-8
Verwoerd, T. C., Dekker, B. M., & Hoekema, A. (1989). A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Research, 17(6), 2362-2362. https://doi.org/ 10.1093/nar/17.6.2362
Wang, S., Hwang, H., Yoon, S., & Choe, E. (2010). Temperature dependence of autoxidation of perilla oil and tocopherol degradation. Journal of Food Science, 75(6), 498-505. https://doi.org/ 10.1111/j.1750-3841.2010.016 81.x
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Comparative analysis of vitamin E extraction methods in Cleome viscosa L. and the role of key biosynthesis genes. (2025). Journal of Applied and Natural Science, 17(3), 1393-1400. https://doi.org/10.31018/jans.v17i3.6846
