Effect of elicitors on phenylpropanoid biosynthesis in hairy roots of Prunella vulgaris
DOI:
https://doi.org/10.25081/jp.2026.v18.9950Keywords:
Prunella vulgaris, Hairy root culture, Phenylpropanoids, Abiotic elicitors, Salicylic acid, Copper chloride, Silver nitrateAbstract
Phenylpropanoids are economically important secondary metabolites with strong pharmacological activity substantively, and their pathways often can be adjusted by abiotic elicitors in plant tissue cultures. Silver nitrate, copper chloride, salicylic acid, and hydrogen peroxide affect the phenylpropanoid such as rosmarinic acid (RA) in hairy root (HR) cultures of Prunella vulgaris. Hairy roots (HRs) were developed via Agrobacterium rhizogenes mediated transformation and cultured under dark and agitated conditions. After treatment for 3 days, we used HPLC to quantify four phenylpropanoids, caffeic acid, benzoic acid, rutin, and quercetin from the treatments with different concentrations of elicitors. The most potent elicitor was salicylic acid which increased total phenols by 46.7±1.38 μg/g in control to 95.46±12.63 μg/g at 0.5 mM, largely on account of a nearly threefold increase in caffeic acid (32.15±5.67 μg/g) and a doubled rutin (59.64±6.71 μg/g) level. At 0.5 mM CuCl2, the accumulation of phenols was increased (64.83±2.62 μg/g), due mainly to an increase in rutin levels (45.46±2.11 μg/g) and caffeic acid (13.18±0.04 μg/g). At a concentration of 1mg/L AgNO3, the production of caffeic acid and quercetin was moderately induced (47.01±1.87 μg/g) but suppressed at higher concentrations. The H2O2 treatments demonstrated a low-level elicitation effect with total phenolics maximum of 48.93±1.21 μg/g (100 mM), and a more gradual induction of benzoic acid (1.6±0.16 μg/g) and rutin (32.27±0.16 μg/g). Overall quercetin content decreased in nearly all the treatments and was especially evident at high concentrations of SA and AgNO3, supporting the notion that a metabolic reprogramming with subsequent precursor diversion is occurring. These findings demonstrate that different abiotic elicitors differentially regulate the phenylpropanoid biosynthesis of P. vulgaris HRs, and that SA and CuCl2 were very strong inducers, providing probable strategies to improve the production of medicinally important phenolic compounds.
Downloads
References
Cho, H.-Y., Son, S. Y., Rhee, H. S., Yoon, S.-Y. H., Lee-Parsons, C. W. T., & Park, J. M. (2008). Synergistic effects of sequential treatment with methyl jasmonate, salicylic acid and yeast extract on benzophenanthridine alkaloid accumulation and protein expression in Eschscholtzia californica suspension cultures. Journal of Biotechnology, 135(1), 117-122. https://doi.org/10.1016/j.jbiotec.2008.02.020
Di, P., Zhang, L., Chen, J., Tan, H., Xiao, Y., Dong, X., Zhou, X., & Chen, W. (2013). 13C tracer reveals phenolic acids biosynthesis in hairy root cultures of Salvia miltiorrhiza. ACS Chemical Biology, 8(7), 1537-1548. https://doi.org/10.1021/cb3006962
Dong, J., Wan, G., & Liang, Z. (2010). Accumulation of salicylic acid-induced phenolic compounds and raised activities of secondary metabolic and antioxidative enzymes in Salvia miltiorrhiza cell culture. Journal of Biotechnology, 148(2-3), 99-104. https://doi.org/10.1016/j.jbiotec.2010.05.009
Georgiev, M. I., Pavlov, A. I., & Bley, T. (2007). Hairy root type plant in vitro systems as sources of bioactive substances. Applied Microbiology and Biotechnology, 74, 1175-1185. https://doi.org/10.1007/s00253-007-0856-5
Georgiev, M., Heinrich, M., Kerns, G., Pavlov, A., & Bley, T. (2006). Production of iridoids and phenolics by transformed Harpagophytum procumbens root cultures. Engineering in Life Sciences, 6(6), 593-596. https://doi.org/10.1002/elsc.200620160
Goel, M. K., Mehrotra, S., & Kukreja, A. K. (2011). Elicitor-induced cellular and molecular events are responsible for productivity enhancement in hairy root cultures: An insight study. Applied Biochemistry and Biotechnology, 165, 1342-1355. https://doi.org/10.1007/s12010-011-9351-7
Grzegorczyk, I., Królicka, A., & Wysokińska, H. (2006). Establishment of Salvia officinalis L. hairy root cultures for the production of rosmarinic acid. Zeitschrift für Naturforschung C, 61, 351-356.
Grzegorczyk-Karolak, I., Krzemińska, M., Grąbkowska, R., Gomulski, J., Żekanowski, C., & Gaweda-Walerych, K. (2024). Accumulation of polyphenols and associated gene expression in hairy roots of Salvia viridis exposed to methyl jasmonate. International Journal of Molecular Sciences, 25(2), 764. https://doi.org/10.3390/ijms25020764
Heredia, J. B., & Cisneros-Zevallos, L. (2009). The effect of exogenous ethylene and methyl jasmonate on PAL activity, phenolic profiles and antioxidant capacity of carrots (Daucus carota) under different wounding intensities. Postharvest Biology and Technology, 51(2), 242-249. https://doi.org/10.1016/j.postharvbio.2008.07.001
Huang, M., Wang, Y., Xu, L., & You, M. (2015). Anti-tumor properties of Prunella vulgaris. Current Pharmacology Reports, 1, 401-419. https://doi.org/10.1007/s40495-015-0038-6
Huang, R.-H., Liu, J.-H., Lu, Y.-M., & Xia, R.-X. (2008). Effect of salicylic acid on the antioxidant system in the pulp of ‘Cara Cara’ navel orange (Citrus sinensis L. Osbeck) at different storage temperatures. Postharvest Biology and Technology, 47(2), 168-175. https://doi.org/10.1016/j.postharvbio.2007.06.018
Jakovljević, D., Warchoł, M., & Skrzypek, E. (2025). Rosmarinic acid as bioactive compound: Molecular and physiological aspects of biosynthesis with future perspectives. Cells, 14(11), 850. https://doi.org/10.3390/cells14110850
Jiang, Y., & Joyce, D. C. (2003). ABA effects on ethylene production, PAL activity, anthocyanin and phenolic contents of strawberry fruit. Plant Growth Regulation, 39, 171-174. https://doi.org/10.1023/A:1022539901044
Kang, S.-M., Jung, H.-Y., Kang, Y.-M., Yun, D.-J., Bahk, J.-D., Yang, J.-K., & Choi, M.-S. (2004). Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora. Plant Science, 166(3), 745-751. https://doi.org/10.1016/j.plantsci.2003.11.022
Kazemi, N., Khavari-Nejad, R. A., Fahimi, H., Saadatmand, S., & Nejad-Sattari, T. (2010). Effects of exogenous salicylic acid and nitric oxide on lipid peroxidation and antioxidant enzyme activities in leaves of Brassica napus L. under nickel stress. Scientia Horticulturae, 126(3), 402-407. https://doi.org/10.1016/j.scienta.2010.07.037
Khalili, M., Hasanloo, T., Kazemi Tabar, S. K., & Rahnama, H. (2009). Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum. Cell Biology International, 33(9), 988-994. https://doi.org/10.1016/j.cellbi.2009.06.003
Kumar, P., Saini, M., Bhushan, S., Warghat, A. R., Pal, T., Malhotra, N., & Sood, A. (2014). Effect of salicylic acid on the activity of PAL and PHB geranyltransferase and shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnst—A medicinally important plant species. Applied Biochemistry and Biotechnology, 173, 248-258. https://doi.org/10.1007/s12010-014-0838-x
Li, B., Wang, B., Li, H., Peng, L., Ru, M., Liang, Z., Yan, X., & Zhu, Y. (2015). Establishment of Salvia castanea Diels f. tomentosa Stib. hairy root cultures and the promotion of tanshinone accumulation and gene expression with Ag+, methyl jasmonate, and yeast extract elicitation. Protoplasma, 253, 87-100. https://doi.org/10.1007/s00709-015-0790-9
Li, W., Koike, K., Asada, Y., Yoshikawa, T., & Nikaido, T. (2005). Rosmarinic acid production by Coleus forskohlii hairy root cultures. Plant Cell, Tissue and Organ Culture, 80, 151-155. https://doi.org/10.1007/s11240-004-9541-x
Ludwig-Müller, J., Jahn, L., Lippert, A., Püschel, J., & Walter, A. (2014). Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications. Biotechnology Advances, 32(6), 1168-1179. https://doi.org/10.1016/j.biotechadv.2014.03.007
Mirjalili, H. M., Fakhr-Tabatabaei, S. M., Bonfill, M., Alizadeh, H., Cusido, R. M., Ghassempour, A., & Palazon, J. (2009). Morphology and withanolide production of Withania coagulans hairy root cultures. Engineering in Life Sciences, 9(3), 197-204. https://doi.org/10.1002/elsc.200800081
Mirmazloum, I., Slavov, A. K., & Marchev, A. S. (2024). The untapped potential of hairy root cultures and their multiple applications. International Journal of Molecular Sciences, 25(23), 12682. https://doi.org/10.3390/ijms252312682
Pandey, R., Krishnasamy, V., Kumaravadivel, N., & Rajamani, K. (2014). Establishment of hairy root culture and production of secondary metabolites in Coleus (Coleus forskohlii). Journal of Medicinal Plants Research, 8(1), 58-62. https://doi.org/10.5897/JMPR12.1182
Park, C. H., Park, H. W., Yeo, H. J., Jung, D. H., Jeon, K. S., Kim, T. J., Kim, J. K., & Park, S. U. (2022). Combined transcriptome and metabolome analysis and evaluation of antioxidant and antibacterial activities in white, pink, and violet flowers of Angelica gigas. Industrial Crops and Products, 188, 115605. https://doi.org/10.1016/j.indcrop.2022.115605
Park, C. H., Park, Y. E., Yeo, H. J., Park, N. I., & Park, S. U. (2021). Effect of light and dark on the phenolic compound accumulation in Tartary buckwheat hairy roots overexpressing ZmLC. International Journal of Molecular Sciences, 22(9), 4702. https://doi.org/10.3390/ijms22094702
Petersen, M., & Simmonds, M. S. J. (2003). Rosmarinic acid. Phytochemistry, 62(2), 121-125. https://doi.org/10.1016/S0031-9422(02)00513-7
Psotová, J., Kolář, M., Soušek, J., Švagera, Z., Vičar, J., & Ulrichová, J. (2003). Biological activities of Prunella vulgaris extract. Phytotherapy Research, 17(9), 1082-1087. https://doi.org/10.1002/ptr.1324
Ru, M., An, Y., Wang, K., Peng, L., Li, B., Bai, Z., Wang, B., & Liang, Z. (2016). Prunella vulgaris L. hairy roots: Culture, growth, and elicitation by ethephon and salicylic acid. Engineering in Life Sciences, 16(5), 494-502. https://doi.org/10.1002/elsc.201600001
Şahin, S., Ari, F., Demir, C., & Ulukaya, E. (2014). Isolation of major phenolic compounds from the extracts of Prunella L. species grown in Turkey and their antioxidant and cytotoxic activities. Journal of Food Biochemistry, 38(2), 248-257. https://doi.org/10.1111/jfbc.12043
Shanks, J. V., & Morgan, J. (1999). Plant ‘hairy root’ culture. Current Opinion in Biotechnology, 10(2), 151-155. https://doi.org/10.1016/S0958-1669(99)80026-3
Sharma, P., Padh, H., & Shrivastava, N. (2013). Hairy root cultures: A suitable biological system for studying secondary metabolic pathways in plants. Engineering in Life Sciences, 13(1), 62-75. https://doi.org/10.1002/elsc.201200030
Srivastava, S., & Srivastava, A. K. (2007). Hairy root culture for mass-production of high-value secondary metabolites. Critical Reviews in Biotechnology, 27(1), 29-43. https://doi.org/10.1080/07388550601173918
Tada, H., Murakami, Y., Omoto, T., Shimomura, K., & Ishimaru, K. (1996). Rosmarinic acid and related phenolics in hairy root cultures of Ocimum basilicum. Phytochemistry, 42(2), 431-434. https://doi.org/10.1016/0031-9422(96)00005-2
Xiao, Y., Gao, S., Di, P., Chen, J., Chen, W., & Zhang, L. (2009). Methyl jasmonate dramatically enhances the accumulation of phenolic acids in Salvia miltiorrhiza hairy root cultures. Physiologia Plantarum, 137(1), 1-9. https://doi.org/10.1111/j.1399-3054.2009.01257.x
Xu, H.-X., Lee, S. H. S., Lee, S. F., White, R. L., & Blay, J. (1999). Isolation and characterization of an anti-HSV polysaccharide from Prunella vulgaris. Antiviral Research, 44(1), 43-54. https://doi.org/10.1016/S0166-3542(99)00053-4
Yin, H., Kjaer, A., Fretté, X. C., Du, Y., Christensen, L. P., Jensen, M., & Grevsen, K. (2012). Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L. Process Biochemistry, 47(11), 1559-1562. https://doi.org/10.1016/j.procbio.2011.12.020
Zhang, S., Li, H., Liang, X., Yan, Y., Xia, P., Jia, Y., & Liang, Z. (2015). Enhanced production of phenolic acids in Salvia miltiorrhiza hairy root cultures by combining the RNAi-mediated silencing of chalcone synthase gene with salicylic acid treatment. Biochemical Engineering Journal, 103, 185-192. https://doi.org/10.1016/j.bej.2015.07.019
Zhao, J., Davis, L. C., & Verpoorte, R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23(4), 283-333. https://doi.org/10.1016/j.biotechadv.2005.01.003
Published
How to Cite
Issue
Section
Copyright (c) 2026 Kihyun Kim, Bao Van Nguyen, Md Romij Uddin, Chanung Park, Ramaraj Sathasivam, Sang Un Park
This work is licensed under a Creative Commons Attribution 4.0 International License.
.