Effect of Methyl Jasmonate on the Production of Bioactive Compounds in Hairy Root Licorice (Glycyrrhiza glabra)

Allahdou, Maryam; Mehravaran, Leila; Khajeh, Hamideh

doi:10.22059/ijhs.2024.380834.2200

Effect of Methyl Jasmonate on the Production of Bioactive Compounds in Hairy Root Licorice (Glycyrrhiza glabra)

Document Type : Full Paper

Authors

Maryam Allahdou ¹

Leila Mehravaran ²

Hamideh Khajeh ³

¹ Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Zabol, Zabol, Iran

² Department of Plant Breeding and Biotechnology, Agriculture Faculty, University of Zabol, Zabol. Iran

³ Institute of Agricultural Biotechnology, University of Zabol, Zabol. Iran

10.22059/ijhs.2024.380834.2200

Abstract

Considering the low amount of important bioactive compounds such as glycyrrhizin and glycyrrhetinic acid in licorice and their complex biosynthesis, in vitro production of hairy roots is an efficient method for licoric secondary metabolites production. Therefore, this experiment aimed to investigate the efficiency of licorice hairy roots in the presence of Agrobacterium rhizogenes and the effect of methyl jasmonate (MeJA) at 100 µm, as an elicitor, on inducing glycyrrhizin and glycyrrhetinic acid production.. After inoculation, two kinds of the cefotaxime were used to eliminate the bacterium. The hairy roots were then harvested after one, two, three, and five days of elicitation. Overall, The results showed that the amount of glycyrrhizin, glycyrrhetinic acid and total flavonoids decreased in all treatments compared to the control, and the magnitude of this reduction in hairy roots treated with one inoculation of cefotaxime sodium salt and no MeJA was the highest in terms of glycyrrhizin (0.959 times) and glycyrrhetinic acid (0.925 times) production. In addition, the amount of total phenol (31.22 GAE/g D.W) and antioxidant activity (90.85%) was highest in in the hairy roots subjected to MeJA for two days and the control. MeJA had negative or no effect on production of glycyrrhizin and glycyrrhetinic acid, which can be related to type of species, ecotype, and antibiotics as well as the number of subcultures after inoculation, the concentration of the elicitor, and a period of time at which hairy roots exposed to a given elicitor. Based on our findings, cefotaxime is recommended to omit the bacteria from culture media. Overall, our findings could be used as an economically method to increase bioactive compounds, especially glycyrrhizin and glycerethnic acid, at a short time and low cost.

Keywords

Cefotaxime sodium salt

Glycyrrhetinic acid

Glycyrrhizin

Methyl Jasmonate

Subjects

Medicinal plant science

Extended Abstract

Introduction

Licorice, belonging to Fabaceae, is well known for its medicinal properties. Glycyrrhizin and glycyrrhetinic acid are of the most important bioactive compounds mainly accumulated in Licorice roots and stolons. Glycyrrhizin possesses a wide array of pharmacological activities including anti-inflammatory, liver protective, gastric ulcer, antiallergic, and antiviral activities against various RNA or DNA viruses, such as human acquired immunodeficiency virus (HIV) and acute respiratory syndrome. Likewise, glycerhethnic acid has numerous medicinal properties, such as anti-inflammatory, antitumor, and so on. Plants generally contain low amount of above-mentioned compounds; therefore, using hairy roots and eliciting them by an appropriate substance has attracted the attentions in order to produce such compounds (as secondary metabolites) at in vitro condition. Many studies have concentrated on inducting hairy roots production in licorice. Hairy roots generally are able to produce glycyrrhizin. The greatest advantage of hairy roots, as compared to other parts of a plant, is contributed to their biosynthetic capacity for producing secondary metabolites. Recently, the beneficiary effects of methyl jasmonate (MeJA) on inducing secondary metabolites have been documented in different reports, because of its role in the biosynthesis of flavonolignans. This study aimed to investigate the effect of MeJA, as an elicitor, on in vitro production of glycyrrhizin and glycyrrhetinic acid in hairy roots of licorice.

Materials and Methods

Licorice seeds were initially collected from the Semiram region of Isfahan Province and soked in MS media for germination. Hypocotyls and cotyledons were used as explants for producing bioactive compounds. In this context, Agrobacterium rhizogenes A4 strain was used to induce hairy roots. The two types of antibiotics were also employed to remove bacteria including cefotaxime and cefotaxime sodium salt. After emerging, the 21-day-old roots were immersed in a liquid ½ MS medium for 35 days. In our research, MeJA (100 µM) was used as an elicitor. Hair roots were harvested at one, two, three, and five days after elicitation. In this experiment, numerous traits were measured including glycyrrhizin, glycyrrhetinic acid, total phenol, total flavonoid, and antioxidant activity. In order to analyze the data and draw graphs, SAS (ver. 9.2) and Excel softwares were used, respectively.

Results

The results of this research showed that the content of glycyrrhizin and glycyrrhetinic acid was higher in the control (bacteria eliminated using general cefotaxime and no MeJA). The lower amount of glycyrrhizin and glycyrrhetinic acid was produced using MeJA. The total flavonoid content was lower in all treatments than in the control, but the extent of this decrease was less than the decrease in glycyrrhizin and glycyrrhetinic acid. The roots that were exposed to MeJA for two days contained the highest amount of total phenol, followed by the roots of the control group, as indicated by the results of the mean comparisons. Hairy root in other treatments had the lowest amount of phenolic compounds. Furthermore, the activity of antioxidants declined in all the treatments, except for hairy roots exposed to MeJA for two days as well as control. These roots showed the highest percentage of free radical inhibition as 90.85 and 88.75, respectively, whereas the lowest was recorded in hairy roots treated with MeJA for five days. The percentage of free radical inhibition in hairy roots were derived in media containing sodium salt cefotaxime for bacteria elimination, and without elicitation, as well as those were subjected to Me JA for five day showed 78.9 and 77.34, respectively.

Conclusion

The results of this research showed that the lack or negative effects of MeJA on bioactive compounds, especially glycyrrhizin and glycyrrhetinic acid, are different based on type of species, ecotype, and antibiotic used to remove bacteria as well as the number of sub culture after inoculation, the concentration of given elicitor, and the duration of time subjecting to the elicitor(s). In a vast majority of previous studies, the rate of secondary metabolites produced by elicited hairy roots was lower or approximately close to the amount recorded in our work. Overall, our findings could be used as an economically method to increase bioactive compounds, especially glycyrrhizin and glycerethnic acid, at a short time and low cost.

Author Contributions

Maryam Allahdou contributed to the conception of the study, doing laboratory work, data collection and analysis, interpretation of data, and drafting the manuscript. Leila Mehravaran assisted in the analysis and interpretation of results, and Hamideh Khajeh did laboratory work, data collection, and analysis.

Data Availability Statement

All data supporting the findings of this study are available within the paper and the datasets during the current study are available from the corresponding author on reasonable request.

Acknowledgements

This article is taken from research project number PR-UOZ99-9. The authors of the article thank University of Zabol for the financial support of this research.

Ethical considerations

This article does not contain any studies involving human and animal subjects.

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

REFERENCES

Alcalde, M. A., Perez-Matas, E., Escrich, A., Cusido, R. M., Palazon, J. & Bonfill, M. (2022). Biotic elicitors in adventitious and hairy root cultures: a review from 2010 to 2022. Molecules, 27, 5253. https://doi.org/10.3390/ molecules27165253.

Amagaya, S., Sugishita, E., Ogihara, Y., Ogawa, S., Okada, K. & Aizawa T. (1984). Comparative studies of the stereoisomers of glycirrhithic acid on anti-inflamatory activities. Journal Pharmacobio- Dynamics, 7 (12), 923-928.

Baek, S., Ho, T.-T., Lee, H., Jung, G., Kim, Y. E., Jeong, C.-S. & Young Park, S. (2020). Enhanced biosynthesis of triterpenoids in Centella asiatica hairy root culture by precursor feeding and elicitation. Plant Biotechnology Reports, 14, 45–53. https://doi: 10.1007/ s11816-019-00573-w.

Baenas, N., García-Viguera, C. & Moreno, D. A. (2014). Elicitation: a tool for enriching the bioactive composition of foods. Molecules, 19, 13541-13563. https://doi: 10.3390/molecules190913541.

Castellanos-Arévalo, A. P., Estrada-Luna, A. A., Cabrera-Ponce, J. L., Valencia-Lozano, E., Herrera-Ubaldo, H., de Folter., S. …& Délano-Frier, J. P. (2020). Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths. Plant Cell Reports, 39(9), 1143-1160. https://doi: 10.1007/s00299-020-02553-9.

Chang, C. C., Yang, M. H., Wen, H. M. & Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food Drug Analysis, 10, 3. https://doi.org/10.38212/2224-6614.2748.

Cheruvathur, M. K. & Thomas, T. D. (2014). Effect of plant growth regulators and elicitors on rhinacanthin accumulation in hairy root cultures of Rhinacanthus nasutus (L.) Kurz. Plant Cell, Tissue and Organ Culture, 118, 169-177. https://doi: 10.1007/ s11240-014-0473-9.

Ding, H., Deng, W., Ding, L., Ye, X., Yin, S. & Huang, W. (2020). Glycyrrhetinic acid and its derivatives as potential alternative medicine to relieve symptoms in nonhospitalized COVID-19 patients. Journal of Medicinal Virology, 92 (10), 2200-2204. https://doi: 10.1002/jmv.26064.

Gai, Q-Y., Jiao, J., Wang, X., Zang, Y-P., Niu, L-L. & Fu, Y-J. (2019). Elicitation of Isatis tinctoria L. hairy root cultures by salicylic acid and methyl jasmonate for the enhanced production of pharmacologically active alkaloids and flavonoids. Plant Cell, Tissue and Organ Culture, 137, 77–86. https://doi: 10.1007/s11240-018- 01553-8.

Ghahraman, A. (1999). Basic botany: anatomy and morphology, University of Tehran Press, pp. 539.

Giri, C. C. & Zaheer, M. (2016). Chemical elicitors versus secondary metabolite production in vitro using plant cell, tissue and organ cultures: recent trends and a sky eye view appraisal. Plant Cell, Tissue and Organ Culture, 126, 1-18. https://doi: 10.1007/s11240-016-0985-6.

Gundlach, H., Muller, M. J., Kutchan, T. M., & Zenk, M. H. (1992). Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proceeding of the National Academy of Sciences of the USA, 89(6), 2389-2393.

Häkkinen, S. T., Moyano, E., Cusidó, R. M. & Oksman-Caldentey, K-M. (2016). Exploring the metabolic stability of engineered hairy roots after 16 years maintenance. Frontiers in Plant Science, 7, 1486. https://doi: 10.3389/fpls.2016.01486.

Ho, T. T., Lee, J. D., Ahn, M. S., Kim, S. W. & Park, S. Y. (2018). Enhanced production of phenolic compounds in hairy root cultures of Polygonum multiflorum and its metabolite discrimination using HPLC and FT-IR methods. Applied Microbiology and Biotechnology, 102, 9563-9575. https://doi.org/10.1007/s00253-018-9359-9.

Huang, D., Ou, B. & Prior, R. L. (2005). The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6), 1841-1856. https://doi.org/10.1021/jf030723c.

Hussain, M. J., Abbas, Y., Nazli, N., Fatima, S., Drouet, S., Hano, C. & Abbasi, B. H. (2022). Root cultures, a boon for the production of valuable compounds: a comparative review. Plants, 11(3), 439. https://doi:10.3390/plants11030439.

Kandoudi, W., Tavaszi-Sárosi, S. & Németh-Zámboriné, E. (2023). Inducing the production of secondary metabolites by foliar application of methyl jasmonate in peppermint. Plants, 12, 2339. https://doi.org/10.3390/plants12122339.

Kim Y., Wyslouzil B. E . & Weathers P. J. (2002). Secondary metabolism of hairy root cultures in bioreactors. In Vitro Cellular & Developmental Biology – Plant. 38, 1-10. https://doi.org/10.1079/IVP2001243.

Kowalska, A. & Kalinowska-Lis, U. (2019). 18β-Glycyrrhetinic acid: its core biological properties and dermatological applications. International Journal of Cosmetic Science, 41(4), 325-331. https://doi: 10.1111/ics.12548.

Kuzovkina, I. N., Guseva, A. V., Kovács, D., Szöke, É. & Vdovitchenko, M. Y. (2005). Flavones in genetically transformed Scutellaria baicalensis roots and induction of their synthesis by elicitation with methyl jasmonate. Russian Journal of Plant Physiology, 52, 77-82. https://doi.org/10.1007/s11183-005-0012-y.

Li, J. Y., Cao, H. Y., Liu, P., Cheng, G. H. & Sun, M. Y. (2014). Glycyrrhizic acid in the treatment of liver diseases: literature review. Biomedical Research International, 872139. https://doi: 10.1155/2014/872139.

Mehrotra, S., Kukreja, A.K., Khanuja, S.P.S. & Mishra, B.N. (2008). Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor. Electronic Journal of Biotechnology, 11(2), 69-75. http://dx.doi.org/10.4067/S0717-34582008000200009.

Moradi, N., Sadat Noori, S. A., Fadavi, A., Mortazavian, S. M. M. & Pakdin Parizi, A. (2021). Analysis efficiency of Iranian Ajowan ecotypes on hairy root production by different agrobacterium rhizogenesis strains. Iranian Journal of Genetics and Plant Breeding, 10(1), 117-127. http://doi:10. IJGPB.2022.17488.1325.

Nasrollahi, V., Mirzaie-asl, A., Piri, K., Nazeri, S. & Mehrabi, R. (2014). The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponins in liquorice (Glycyrrhiza glabra). Phytochemistry, 103, 32-37. http://doi: 10.1016/j.phytochem.2014.03.004.

Qu, X. & Christ, B.J. (2007). In vitro culture of the obligate parasite Spongospora subterranea (Cercozoa; plasmodiophorida) associated with root-inducing transferred- DNA transformed potato hairy roots. Journal of Eukaryotic Microbiology, 54(6), 465-467. http://doi: 10.1111/j.1550- 408.2007.00289.x.

Ruiz-May, E., De-la- Peña, C., Galaz- Ávalos, M., Lei, Z., Watson, B. S., Sumner, L. W., Loyola-Vargas, V. M., (2011). Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism inCatharanthus roseus (L.) G. hairy roots, Plant and Cell Physiology, 52(8), 1401-1421. https://doi.org/10.1093/pcp/pcr086.

Sa´nchez-Sampedro, M. A., Fernandez-Tarrago, J., Corchete, P., (2005). Yeast extract and methyl jasmonate induced silymarin production in cell culture of Silybum marianum (L.) Gaertn. Journal of Biotechnology, 119(1), 60-69.

Sarkar, J., Misra, A. & Banerjee, N. (2020). Genetic transfection, hairy root induction and solasodine accumulation in elicited hairy root clone of Solanum erianthum D. Don. Journal of Biotechnology, 323, 238-245. http://doi: 10.1016/j.jbiotec.2020.09.002.

Seki, H., Sawai, S., Ohyama, K., Mizutani, M. & Muranaka T. (2011). Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin. The Plant Cell, 23, 4112-4123. https://doi.org/10.1105/tpc.110.082685.

Sivanandhan, G., Dev, K. G., Jeyaraj, M., Rajesh, M., Arjunan, A., Muthuselvam, M., Manickavasagam, M. & Ganapathi, A. (2013). Increased production of withanolide A, withanone and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell, Tissue and Organ Culture, 114, 121-129. https://doi.org/10.1007/s11240-013-0297-z.

Spollansky, T., Pitta-Alvarez, S. & Giulietti, A. (2000). Effect of jasmonic acid and aluminum on production of tropane alkaloids in hairy root cultures of Brugmansia candida. Electronic Journal of Biotechnology. 3(1), 72-75.

Srivastava, M., Singh, G., Sharma, S., Shukla, S. & Misra, P. (2019). Elicitation Enhanced the Yield of Glycyrrhizin and Antioxidant Activities in Hairy Root Cultures of Glycyrrhiza glabra L. Journal of Plant Growth Regulation, 38, 373-384. https:// doi: 10.1007/s00344-018-9847-2.

Syklowska-Baranek, K., Pietrosiuk, A., Kokoszka, A. & Furmanowa, M. (2009). Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii. Journal of Plant Physiology, 166(17), 1950–1954. https:// doi: 10.1016/j.jplph.2009.05.001.

Tenea, G.N, Calin A., Gavrila, L. & Cucu N. (2008). Manipulation of root biomass and biosynthethic potential of Glycirrhiza glabra L. plants by Agrobacterium rhizogenes mediated transformation. Roumanian Biotechnological Letters, 13, 3922-3932.

Thwe, A., Arasu, M., Li, X., Park, CH., Kim, Sj., Al-Dhabi, NA. & Park, S.U. (2016). Effect of different Agrobacterium rhizogenes strains on hairy root induction and phenylpropanoid biosynthesis in tartary buckwheat (Fagopyrum tataricum Gaertn). Frontiers in Microbiology, 7, 1-10. https://doi.org/10.3389/fmicb.2016.00318

Torkamani, M., Jafari, M., Abbaspour, N., Heidary, R. & Safaie, N. (2014). Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation. Open Life Science, 9, 853-863. https://doi.org/10.2478/s11535-014-0320-3.

Wang M., Qin, Y-Y., Wei, N-N., Xue, H-Y. & Dai, W-S. (2023). Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis. Frontiers in Plant Science, 14, 1293374. http://dx.doi.org/10.3389/fpls.2023.1293374

Wojdyło, A., Oszmiański, J. & Czemerys, R. (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chemistry, 105, 940-949. https:// doi. org/ 10. 1016/j. foodc hem. 2007. 04. 038.

Wongwicha, W., Tanaka, H., Shoyama, Y. & Putalun, W. (2011). Methyl jasmonate elicitation enhances glycyrrhizin production in Glycyrrhiza inflata hairy roots cultures. Zeitschrift fur Naturforschung, 66c, 423 - 428. https://doi: 10.1515/znc-2011-7-815.

Yordanova, Z. P., Georgiev, M. I. (2017). Cell factories. In T. Brian, B.G. Murray & D.J. Murphy (Eds.), Encyclopedia of Applied Plant Sciences, (pp. 72–76), vol II, Second edition. Academic Press. https://doi.org/10.1016/B978-0-12-394807-6.00158-1.

Zhao, J., Davis, LC. & Verpoorte, R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23(4), 283-333. https:// doi: 10.1016/j.biotechadv.2005.01.003.

Zhou, F., Wu, G., Cai, D., Xu, B.,Yan, M. …& Lei, H.M. (2019). Synthesis and biological activity of glycyrrhetinic acid derivatives as antitumor agents. European Journal of Medicine Chemistry, 178, 623-635. https://doi: 10.1016/j.ejmech.2019.06.029.