DNA Polymorphism Changes in Bacopa monnieri (L.) Wettst Polyploids Affect Phytochemical and Antioxidant Profiles
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This study aimed to evaluate the morphological, genetic, and phytochemical variations between diploid and colchicine-induced tetraploid Bacopa monnieri plants to explore the effects of polyploidy on plant traits and metabolite synthesis. Tetraploid lines were regenerated and acclimatized for four weeks in ½ HS medium. Morphological traits were measured, and cluster analysis was performed to assess phenotypic divergence. Genetic diversity was examined using RAPD markers, with data analyzed by UPGMA clustering. Phytochemical profiles, including bacosides, phenolics, flavonoids, triterpenoids, and antioxidant activity, were quantified by HPLC and spectrophotometric assays. Correlation analysis further revealed that bacoside accumulation is strongly linked to leaf and node morphological traits rather than overall plant size, suggesting genetic or metabolic regulation beyond simple vegetative growth. In contrast, phenolics, flavonoids, and triterpenoids showed positive correlations with growth traits and antioxidant activity, with triterpenoids exhibiting the strongest free radical scavenging effect. The average polymorphism rate correlated positively with bacoside content but not with other phytochemicals, indicating that genetic diversity plays a major role in bacoside biosynthesis. Results showed that most tetraploids had reduced length and biomass compared to diploids, except line 4-9, which exhibited increased growth and fresh weight. Morphological and genetic cluster analyses consistently identified three distinct groups, with lines 4-9 forming a unique cluster. Tetraploids, especially lines 4-1, 4-5, 4-7, and 4-9, accumulated significantly higher levels of bacosides and secondary metabolites, with line 4-9 yielding the highest bacoside compound (6.10 ± 1.11 mg/plant). Interestingly, some tetraploid lines showed reduced antioxidant activity despite increased phytochemical levels, possibly due to shifts in compound proportions or metabolic regulation. This study demonstrates that colchicine-induced tetraploidy enhances secondary metabolite production and genetic variability in B. monnieri, highlighting polyploid induction as a novel and promising approach to improving medicinal plant quality and diversity.
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[1] Deepak, M., Sangli, G. K., Arun, P. C., & Amit, A. (2005). Quantitative determination of the major saponin mixture bacoside A in Bacopa monnieri by HPLC. Phytochemical Analysis, 16(1), 24–29. doi:10.1002/pca.805.
[2] Deepak, M., & Amit, A. (2013). “Bacoside B” - The need remains for establishing identity. Fitoterapia, 87(1), 7–10. doi:10.1016/j.fitote.2013.03.011.
[3] Christopher, C., Johnson, A. J., Mathew, P. J., & Baby, S. (2017). Elite genotypes of Bacopa monnieri, with high contents of Bacoside A and Bacopaside I, from southern Western Ghats in India. Industrial Crops and Products, 98, 76–81. doi:10.1016/j.indcrop.2017.01.018.
[4] Yen, D. T. H., Cuc, N. T., Tai, B. H., Van Kiem, P., Hoang Duc, M., Nhiem, N. X., Cho, S. H., Jeong, S. B., Seo, Y., & Park, S. J. (2024). Two new triterpenoid glycosides from Bacopa monnieri and their cytotoxic activity. Natural Product Research, 38(7), 1120–1126. doi:10.1080/14786419.2022.2132498.
[5] Pase, M. P., Kean, J., Sarris, J., Neale, C., Scholey, A. B., & Stough, C. (2012). The cognitive-enhancing effects of bacopa monnieri: A systematic review of randomized, controlled human clinical trials. Journal of Alternative and Complementary Medicine, 18(7), 647–652. doi:10.1089/acm.2011.0367.
[6] Mallick, M. N., Akhtar, M. S., Najm, M. Z., Tamboli, E. T., Ahmad, S., & Husain, S. A. (2015). Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line. Journal of Pharmacy and Bioallied Sciences, 7(4), 325–328. doi:10.4103/0975-7406.168038.
[7] Sushma, Sahu, M. R., Murugan, N. A., & Mondal, A. C. (2024). Amelioration of Amyloid-β Induced Alzheimer’s Disease by Bacopa monnieri through Modulation of Mitochondrial Dysfunction and GSK-3β/Wnt/β-Catenin Signaling. Molecular Nutrition and Food Research, 68(13), 2300245. doi:10.1002/mnfr.202300245.
[8] Uabundit, N., Wattanathorn, J., Mucimapura, S., & Ingkaninan, K. (2010). Cognitive enhancement and neuroprotective effects of Bacopa monnieri in Alzheimer’s disease model. Journal of Ethnopharmacology, 127(1), 26–31. doi:10.1016/j.jep.2009.09.056.
[9] Kamkaew, N., Paracha, T. U., Ingkaninan, K., Waranuch, N., & Chootip, K. (2019). Vasodilatory Effects and Mechanisms of Action of Bacopa monnieri Active Compounds on Rat Mesenteric Arteries. Molecules, 24(12), 2243. doi:10.3390/molecules24122243.
[10] Valotto Neto, L. J., Reverete de Araujo, M., Moretti Junior, R. C., Mendes Machado, N., Joshi, R. K., dos Santos Buglio, D., Barbalho Lamas, C., Direito, R., Fornari Laurindo, L., Tanaka, M., & Barbalho, S. M. (2024). Investigating the Neuroprotective and Cognitive-Enhancing Effects of Bacopa monnieri: A Systematic Review Focused on Inflammation, Oxidative Stress, Mitochondrial Dysfunction, and Apoptosis. Antioxidants, 13(4), 393. doi:10.3390/antiox13040393.
[11] Sangeet, S., & Khan, A. (2025). Bacopa monnieri phytochemicals as promising BACE1 inhibitors for Alzheimer’s disease therapy. Scientific Reports, 15(1), 13504. doi:10.1038/s41598-025-92644-y.
[12] Jeena, G. S., Kumar, S., Bharti, S., Singh, N., Joshi, A., Lahane, V., Meghani, R., Yadav, A. K., Shukla, S., Tripathi, V., & Shukla, R. K. (2025). Engineering Bacopa monnieri for improved bacoside content and its neurological evaluation. Applied Microbiology and Biotechnology, 109(1), 83. doi:10.1007/s00253-025-13453-x.
[13] Singh, V., Jain, N., & Shri, R. (2021). Effect of abiotic factors on bacoside A content, acetylcholinesterase inhibitory and antioxidant activities of Bacopa monnieri (L.) Wettst. Journal of Applied Pharmaceutical Science. doi:10.7324/JAPS.2021.11s107.
[14] Kumari, A., Kumar, A., Mandal, S., Roy, P., & Sircar, D. (2023). A metabolic reprogramming in Bacopa monnieri plants induced by methyl-jasmonate and enhanced biosynthesis of triterpene saponins. Industrial Crops and Products, 204(Part A), 117241. doi:10.1016/j.indcrop.2023.117241.
[15] Banyai, W., Sangthong, R., Karaket, N., Inthima, P., Mii, M., & Supaibulwatana, K. (2010). Overproduction of artemisinin in tetraploid Artemisia annua L. Plant Biotechnology, 27(5), 427–433. doi:10.5511/plantbiotechnology.10.0726a.
[16] Inthima, P., & Sujipuli, K. (2019). Improvement of growth and bacoside production in Bacopa monnieri through induced autotetraploidy with colchicine. PeerJ, 2019(10), 7966. doi:10.7717/peerj.7966.
[17] Madani, H., Hosseini, B., Karimzadeh, G., & Rahimi, A. (2019). Enhanced thebaine and noscapine production and modulated gene expression of tyrosine/dopa decarboxylase and salutaridinol 7-O-acetyltransferase genes in induced autotetraploid seedlings of Papaver bracteatum Lindl. Acta Physiologiae Plantarum, 41(12), 194. doi:10.1007/s11738-019-2984-9.
[18] Javadian, N., Karimzadeh, G., Sharifi, M., Moieni, A., & Behmanesh, M. (2017). In vitro polyploidy induction: changes in morphology, podophyllotoxin biosynthesis, and expression of the related genes in Linum album (Linaceae). Planta, 245(6), 1165–1178. doi:10.1007/s00425-017-2671-2.
[19] Bharati, R., Gupta, A., Novy, P., Severová, L., Šrédl, K., Žiarovská, J., & Fernández-Cusimamani, E. (2023). Synthetic polyploid induction influences morphological, physiological, and photosynthetic characteristics in Melissa officinalis L. Frontiers in Plant Science, 14, 1332428. doi:10.3389/fpls.2023.1332428.
[20] Corneillie, S., De Storme, N., Van Acker, R., Fangel, J. U., De Bruyne, M., De Rycke, R., Geelen, D., Willats, W. G. T., Vanholme, B., & Boerjan, W. (2019). Polyploidy affects plant growth and alters cell wall composition. Plant Physiology, 179(1), 74–87. doi:10.1104/pp.18.00967.
[21] Babu, K. N., Sheeja, T. E., Minoo, D., Rajesh, M. K., Samsudeen, K., Suraby, E. J., & Kumar, I. P. V. (2020). Random Amplified Polymorphic DNA (RAPD) and Derived Techniques. Molecular Plant Taxonomy, 219–247. doi:10.1007/978-1-0716-0997-2_13.
[22] Stefańska, I., Kwiecień, E., Górzyńska, M., Sałamaszyńska-Guz, A., & Rzewuska, M. (2022). RAPD-PCR-Based Fingerprinting Method as a Tool for Epidemiological Analysis of Trueperella pyogenes Infections. Pathogens, 11(5), 562. doi:10.3390/pathogens11050562.
[23] Abuhena, M., Al-Rashid, J., & Azad, A. K. (2024). Analysis of genetic variability among regional mango varieties grown in Rajshahi district using RAPD markers. Ecological Genetics and Genomics, 32, 100258. doi:10.1016/j.egg.2024.100258.
[24] Chau, T. P., Samdani, M. S., Fathima H, A., Jhanani, G. K., Sathiyamoorthi, E., & Lee, J. (2024). Metal accumulation and genetic adaptation of Oryza sativa to Cadmiun and Chromium heavy metal stress: A hydroponic and RAPD analyses. Environmental Research, 242, 117793. doi:10.1016/j.envres.2023.117793.
[25] Agrawal, G. K., Pandey, R. N., & Agrawal, V. P. (1992). Isolation of DNA from Cheorospondias asillaris leaves. Biotechnology and Biodiversity Letters, 2(1), 19-24.
[26] Nei, M., & Li, W. H. (1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences of the United States of America, 76(10), 5269–5273. doi:10.1073/pnas.76.10.5269.
[27] Nopparat, J., Sujipuli, K., Ratanasut, K., Weerawatanakorn, M., Prasarnpun, S., Thongbai, B., Laothaworn, W., & Inthima, P. (2024). Exploring the excellence of commercial Brahmi products from Thai online markets: Unraveling phytochemical contents, antioxidant properties and DNA damage protection. Heliyon, 10(2), 24509. doi:10.1016/j.heliyon.2024.e24509.
[28] Govarthanan, M., Rajinikanth, R., Kamala-Kannan, S., & Selvankumar, T. (2015). A comparative study on bioactive constituents between wild and in vitro propagated Centella asiatica. Journal of Genetic Engineering and Biotechnology, 13(1), 25–29. doi:10.1016/j.jgeb.2014.12.003.
[29] Shraim, A. M., Ahmed, T. A., Rahman, M. M., & Hijji, Y. M. (2021). Determination of total flavonoid content by aluminum chloride assay: A critical evaluation. Lwt, 150, 111932. doi:10.1016/j.lwt.2021.111932.
[30] Wei, L., Zhang, W., Yin, L., Yan, F., Xu, Y., & Chen, F. (2015). Extraction optimization of total triterpenoids from jatropha curcas leaves using response surface methodology and evaluations of their antimicrobial and antioxidant capacities. Electronic Journal of Biotechnology, 18(2), 88–95. doi:10.1016/j.ejbt.2014.12.005.
[31] Laywisadkul, S., Weerawatanakorn, M., Maneerattanarungrojn, C., & Sujipuli, K. (2017). Investigating the antioxidant and preventing DNA-damage properties of various honeys in Phitsanulok province. Food and Applied Bioscience Journal, 5(2), 93–103. doi:10.14456/fabj.2017.8.
[32] Simko, I., & Zhao, R. (2023). Phenotypic characterization, plant growth and development, genome methylation, and mineral elements composition of neotetraploid lettuce (Lactuca sativa L.). Frontiers in Plant Science, 14, 1296660. doi:10.3389/fpls.2023.1296660.
[33] Liu, Y., Huang, X., Gao, X., Zhang, X., Huang, H., Li, W., & Zhang, Y. (2025). Tetraploidization Altered Phenotypic Traits and Metabolite Profile of Java Ginseng (Talinum paniculatum (Jacq.) Gaertn.). Plants, 14(3), 480. doi:10.3390/plants14030480.
[34] Zhang, X., Chen, K., Wang, W., Liu, G., Yang, C., & Jiang, J. (2022). Differences in Leaf Morphology and Related Gene Expression between Diploid and Tetraploid Birch (Betula pendula). International Journal of Molecular Sciences, 23(21), 12966. doi:10.3390/ijms232112966.
[35] Nkongolo, K. K., Michael, P., & Gratton, W. S. (2002). Identification and characterization of RAPD markers inferring genetic relationships among pine species. Genome, 45(1), 51–58. doi:10.1139/g01-121.
[36] Tikendra, L., Potshangbam, A. M., Dey, A., Devi, T. R., Sahoo, M. R., & Nongdam, P. (2021). RAPD, ISSR, and SCoT markers based genetic stability assessment of micropropagated Dendrobium fimbriatum Lindl. var. oculatum Hk. f.- an important endangered orchid. Physiology and Molecular Biology of Plants, 27(2), 341–357. doi:10.1007/s12298-021-00939-x.
[37] Saha, S., Adhikari, S., Dey, T., & Ghosh, P. (2016). RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1. Meta Gene, 7, 7–15. doi:10.1016/j.mgene.2015.10.004.
[38] Choi, T. Y., & Lee, S. R. (2021). A review of intraspecific genetic diversity on wild plants in Korea estimated from varying nuclear DNA markers. Journal of Asia-Pacific Biodiversity, 14(4), 622–627. doi:10.1016/j.japb.2021.08.004.
[39] Li, S., Lin, Y., Pei, H., Zhang, J., Zhang, J., & Luo, J. (2020). Variations in colchicine-induced autotetraploid plants of Lilium davidii var. unicolor. Plant Cell, Tissue and Organ Culture, 141(3), 479–488. doi:10.1007/s11240-020-01805-6.
[40] Fu, Y., Ren, J., Guo, R., Lu, X., Lei, Y., & Xu, W. (2025). In vitro induction and molecular and cellular identification of mutant plants of Lilium leichtlinii var. Maximowiczii (Regel) Baker by colchicine. Ornamental Plant Research, 5, 14. doi:10.48130/opr-0025-0012.
[41] Weiss, H., & Maluszynska, J. (2001). Chromosomal rearrangement in autotetraploid plants of Arabidopsis thaliana. Hereditas, 133(3), 255–261. doi:10.1111/j.1601-5223.2000.00255.x.
[42] Tammu, R. M., Nuringtyas, T. R., & Daryono, B. S. (2021). Colchicine effects on the ploidy level and morphological characters of Katokkon pepper (Capsicum annuum L.) from North Toraja, Indonesia. Journal of Genetic Engineering and Biotechnology, 19(1). doi:10.1186/s43141-021-00131-4.
[43] Abdoli, M., Moieni, A., & Naghdi Badi, H. (2013). Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.). Acta Physiologiae Plantarum, 35(7), 2075–2083. doi:10.1007/s11738-013-1242-9.
[44] Pansuksan, K., Sangthong, R., Nakamura, I., Mii, M., & Supaibulwatana, K. (2014). Tetraploid induction of Mitracarpus hirtus L. by colchicine and its characterization including antibacterial activity. Plant Cell, Tissue and Organ Culture, 117(3), 381–391. doi:10.1007/s11240-014-0447-y.
[45] Dudonné, S., Vitrac, X., Coutiére, P., Woillez, M., & Mérillon, J. M. (2009). Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. Journal of Agricultural and Food Chemistry, 57(5), 1768–1774. doi:10.1021/jf803011r.
[46] Adebiyi, O. E., Olayemi, F. O., Ning-Hua, T., & Guang-Zhi, Z. (2017). In vitro antioxidant activity, total phenolic and flavonoid contents of ethanol extract of stem and leaf of Grewia carpinifolia. Beni-Suef University Journal of Basic and Applied Sciences, 6(1), 10–14. doi:10.1016/j.bjbas.2016.12.003.
[47] Muflihah, Y. M., Gollavelli, G., & Ling, Y. C. (2021). Correlation study of antioxidant activity with phenolic and flavonoid compounds in 12 indonesian indigenous herbs. Antioxidants, 10(10), 1530. doi:10.3390/antiox10101530.
[48] Saeed, N., Khan, M. R., & Shabbir, M. (2012). Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complementary and Alternative Medicine, 12, 221. doi:10.1186/1472-6882-12-221.
[49] Xie, Z., Huang, H., Zhao, Y., Shi, H., Wang, S., Wang, T. T. Y., Chen, P., & Yu, L. (2012). Chemical composition and anti-proliferative and anti-inflammatory effects of the leaf and whole-plant samples of diploid and tetraploid Gynostemma pentaphyllum (Thunb.) Makino. Food Chemistry, 132(1), 125–133. doi:10.1016/j.foodchem.2011.10.043.
[50] Comai, L. (2005). The advantages and disadvantages of being polyploid. Nature Reviews Genetics, 6(11), 836–846. doi:10.1038/nrg1711.
[51] Soltis, P. S., & Soltis, D. E. (2000). The role of genetic and genomic attributes in the success of polyploids. Proceedings of the National Academy of Sciences of the United States of America, 97(13), 7051–7057. doi:10.1073/pnas.97.13.7051.
[52] Naik, P. M., Manohar, S. H., Praveen, N., Upadhya, V., & Murthy, H. N. (2012). Evaluation of bacoside a content in different accessions and various organs of bacopa monnieri (L.) wettst. Journal of Herbs, Spices & Medicinal Plants, 18(4), 387–395. doi:10.1080/10496475.2012.725456.
[53] Rawat, S., Jugran, A. K., Bhatt, I. D., & Rawal, R. S. (2018). Influence of the growth phenophases on the phenolic composition and anti-oxidant properties of Roscoea procera Wall. in western Himalaya. Journal of Food Science and Technology, 55(2), 578–585. doi:10.1007/s13197-017-2967-z.
[54] Karati, D., Mukherjee, S., & Roy, S. (2025). The antioxidant potential of bacoside and its derivatives in Alzheimer’s disease: The molecular mechanistic paths and therapeutic prospects. Toxicology Reports, 14, 101945. doi:10.1016/j.toxrep.2025.101945.
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