Influence of Carbon Source, Nitrogen and PEG on Caffeic Acid Derivatives Production in Callus Cultures of Echinacea purpurea L.

Öz

This study aimed to determine the effects of polyethylene glycol (PEG) as an abiotic elicitor and nutritional factors (different ammonium/nitrate ratios, carbon source and amount) in the culture medium on the production of Caffeic Acid Derivatives (CADs) in callus cultures of Echinacea purpurea L. Petiole and root explants were cultured on MS medium modified in terms of different types (sucrose and maltose) and amounts (sucrose 15, 45, 60 g l-1, and maltose 15, 30, 45, 60 g l-1) of carbon source, different concentrations (5, 10, 15 g l-1) of PEG and ammonium nitrate ratios (0:35, 5:25, 15:15, 35:0 mM). The amounts of CADs in the callus obtained at the end of the 10-week culture period were analysed. In both explant types, the highest amount of CADs were obtained from the medium containing 15 g l-1 sucrose and 15 or 30 g l-1 maltose applications, while the highest amount of CADs was obtained in the medium containing 0:35 mM ammonium/nitrate in nitrogen applications. While the highest amount of CADs in root explant was obtained from the medium containing 10 g l-1 PEG applications, CADs content could not be obtained in petiole explant. As a result, the highest amounts of caftaric, chlorogenic, caffeic, and chicoric acids (respectively, 9.38, 0.71, 0.29, and 34.77 mg g-1) were determined at callus obtained from root explant cultured on MS medium containing 30 g l-1 sucrose and 0:35 mM ammonium/nitrate. In conclusion, optimization of culture conditions and different elicitor applications were made to increase secondary metabolite content in E. purpurea L. under in vitro conditions and the results obtained were presented comparatively.

Anahtar Kelimeler

• Sucrose
• Maltose
• Nitrogen
• PEG
• Caffeic acid derivatives
• Echinacea purpurea L.
• HPLC

Echinacea purpurea L. Kallus Kültürlerinde Karbon Kaynağı, Nitrojen ve PEG'in Kafeik Asit Türevlerinin Üretimine Etkisi

Öz

Bu çalışmada, abiyotik bir elisitör olarak polietilen glikolün (PEG) ve besin ortamındaki farklı beslenme faktörlerinin (farklı amonyum/nitrat oranları, karbon kaynağı ve miktarı) Echinacea purpurea L. kallus kültürlerinde kafeik asit türevlerinin miktarlarına etkisinin belirlenmesi amaçlanmıştır. Petiyol ve kök eksplantları karbon kaynağının farklı türleri (sakkaroz ve maltoz) ve miktarları (sakkaroz 15, 45, 60 g l-1 ve maltoz 15, 30, 45, 60 g l-1), farklı amonyum nitrat oranları (0:35, 5:25, 15:15, 35:0 mM) ve farklı konsantrasyonlarda (5, 10, 15 g l-1) PEG içeren MS besin ortamlarında 10 hafta boyunca kültür alınmış ve kültür süresi sonunda elde edilen kalluslarda kaftarik, klorojenik, kafeik ve kikorik asit miktarları tespit edilmiştir. Her iki eksplant türünde de en yüksek kafeik asit türevlerinin miktarları, karbon kaynağı uygulamalarında, 15 g l-1 sakkaroz ile 15 ve 30 g l-1 maltoz; nitrojen uygulamalarında ise 0:35 mM amonyum/nitrat içeren besin ortamlarında uyarılan kalluslarda tespit edilmiştir. PEG uygulamalarında ise, en yüksek miktarlarda kafeik asit türevleri, 10 g l-1 PEG içeren besin ortamından kök eksplantından uyarılan kalluslarda elde edilirken, petiyol eksplantından elde edilen kallus dokularında kafeik asit türevlerinden hiçbiri tespit edilememiştir. E. purpurea L kallus kültürlerinde, kafeik asit türevlerinin miktarını artırmak için, in vitro şartlar altında, abiyotik elisitör olarak PEG ve farklı beslenme faktörlerinin optimizasyonu araştırıldığı bu çalışmada, elde edilen sonuçlar karşılaştırmalı olarak sunulmuştur. Tüm uygulamalar değerlendirildiğinde; sonuç olarak, en yüksek kaftarik, klorojenik, kafeik ve kikorik asit miktarları (sırasıyla 9.38, 0.71, 0.29 ve 34.77 mg g-1) 0:35 mM amonyum/nitrat ve 30 g l-1 sakkaroz içeren MS ortamında kültüre alınan kök eksplantından elde edilen kallus dokularında tespit edilmiştir.

Anahtar Kelimeler

• Sakkaroz
• Maltoz
• Azot
• PEG
• Kafeik asit türevleri
• Echinacea purpurea L.
• HPLC

Kaynakça

1. Aarland, R. C., Bañuelos-Hernández, A. E., Fragoso-Serrano, M., Sierra-Palacios, E. d. C., Díaz de León-Sánchez, F., Pérez-Flores, L. J. and Mendoza-Espinoza, J. A. (2017). Studies on phytochemical, antioxidant, anti-inflammatory, hypoglycaemic and antiproliferative activities of Echinacea purpurea and Echinacea angustifolia extracts. Pharmaceutical Biology, 55(1): 649-656.
2. Ali, M., Abbasi, B. H., Ahmad, N., Ali, S. S., Ali, S. and Ali, G. S. (2016). Sucrose-enhanced biosynthesis of medicinally important antioxidant secondary metabolites in cell suspension cultures of Artemisia absinthium L. Bioprocess and Biosystems Engineering, 39(12): 1945-1954.
3. Bruni, R., Brighenti, V., Caesar, L. K., Bertelli, D., Cech, N. B. and Pellati, F. (2018). Analytical methods for the study of bioactive compounds from medicinally used Echinacea species. Journal of Pharmaceutical and Biomedical Analysis, 160: 443-477.
4. Chiou, S. Y., Sung, J.-M., Huang, P. W. and Lin, S. D. (2017). Antioxidant, antidiabetic, and antihypertensive properties of Echinacea purpurea flower extract and caffeic acid derivatives using in vitro models. Journal of Medicinal Food, 20(2): 171-179.
5. Cui, H.-Y., Baque, M. A., Lee, E. J. and Paek, K. Y. (2013). Scale-up of adventitious root cultures of Echinacea angustifolia in a pilot-scale bioreactor for the production of biomass and caffeic acid derivatives. Plant Biotechnology Reports, 7(3): 297-308.
6. Cui, X. H., Murthy, H. N., Wu, C. H. and Paek, K. Y. (2010). Adventitious root suspension cultures of Hypericum perforatum: effect of nitrogen source on production of biomass and secondary metabolites. In Vitro Cellular and Developmental Biology-Plant, 46(5): 437-444.
7. Demirci, T. (2022). Determination of secondary metabolite production efficiency in Echinacea purpurea callus, shoot, and root in vitro cultures with methyl jasmonate applications. Acta Physiologiae Plantarum, 44(12): 1-12.
8. Elshahawy, O. A., Zeawail, M. E. F., Hamza, M. A., Elateeq, A. A. and Omar, M. A. (2022). Improving the production of total phenolics and flavonoids and the antioxidant capacity of Echinacea purpurea callus through biotic elicitation. Egyptian Journal of Chemistry, 65(12): 137-149.

9. Erkoyuncu, M. T. and Yorgancilar, M. (2021). Optimization of callus cultures at Echinacea purpurea L. for the amount of caffeic acid derivatives. Electronic Journal of Biotechnology, 51: 17-27. https://doi.org/10.1016/j.ejbt.2021.02.003
10. Khan, T., Abbasi, B. H., Zeb, A. and Ali, G. S. (2018). Carbohydrate-induced biomass accumulation and elicitation of secondary metabolites in callus cultures of Fagonia indica. Industrial Crops and Products, 126: 168-176.
11. Kim, S. I., Choi, H. K., Kim, J. H., Lee, H. S. and Hong, S. S. (2001). Effect of osmotic pressure on paclitaxel production in suspension cell cultures of Taxus chinensis. Enzyme and Microbial Technology, 28(2-3): 202-209.
12. Kretzschmar, F. S., Oliveira, C. J. and Braga, M. R. (2007). Differential sugar uptake by cell suspension cultures of Rudgea jasminoides, a tropical woody Rubiaceae. In Vitro Cellular and Developmental Biology-Plant, 43(1), 71-78.
13. Lee, Y., Lee, D. E., Lee, H. S., Kim, S. K., Lee, W. S., Kim, S. H. and Kim, M. W. (2011). Influence of auxins, cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (Morus alba L.). Plant Cell, Tissue and Organ Culture, 105(1): 9-19.
14. Lemcoff, J. H., Ling, F. and Neumann, P. M. (2006). Short episodes of water stress increase barley root resistance to radial shrinkage in a dehydrating environment. Physiologia Plantarum, 127(4): 603-611.
15. Lin, Z., Neamati, N., Zhao, H., Kiryu, Y., Turpin, J. A., Aberham, C. and Pannecouque, C. (1999). Chicoric acid analogues as HIV-1 integrase inhibitors. Journal of Medicinal Chemistry, 42(8): 1401-1414.
16. Liu, C. Z., Abbasi, B. H., Gao, M., Murch, S. J. and Saxena, P. K. (2006). Caffeic acid derivatives production by hairy root cultures of Echinacea purpurea. Journal of Agricultural and Food Chemistry, 54(22): 8456-8460.
17. Liu, C. Z. and Cheng, X. Y. (2008). Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of Cistanche deserticola by osmotic stress. Plant Cell Reports, 27(2): 357-362.
18. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3): 473-497.
19. Murthy, H. N., Kim, Y. S., Park, S. Y. and Paek, K. Y. (2014). Biotechnological production of caffeic acid derivatives from cell and organ cultures of Echinacea species. Applied Microbiology and Biotechnology, 98(18): 7707-7717.
20. Pavlik, M., Vacek, J., Klejdus, B. and Kubáň, V. (2007). Hypericin and hyperforin production in St. John's wort in vitro culture: influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens. Journal of Agricultural and Food Chemistry, 55(15): 6147-6153.
21. Pehlivan, E. C., Kunter, B. and Royandazagh, S. D. (2017). Choise of explant material and media for in vitro callus regeneration in Sultana grape cultivar (Vitis vinifera L.). Journal of Tekirdag Agricultural Faculty, The Special Issue of 2nd International Balkan Agriculture Congress, 30-34.
22. Pleschka, S., Stein, M., Schoop, R. and Hudson, J. B. (2009). Anti-viral properties and mode of action of standardized Echinacea purpurea extract against highly pathogenic avian influenza virus (H5N1, H7N7) and swine-origin H1N1 (S-OIV). Virology Journal, 6(1): 1-9.
23. Rady, M. R., Aboul-Enein, A. M. and Ibrahim, M. M. (2018). Active compounds and biological activity of in vitro cultures of some Echinacea purpurea varieties. Bulletin of the National Research Centre, 42(1): 1-12.
24. Rajasekaran, D., Palombo, E. A., Chia Yeo, T., Lim Siok Ley, D., Lee Tu, C., Malherbe, F. and Grollo, L. (2013). Identification of traditional medicinal plant extracts with novel anti-influenza activity. Plos One, 8(11): e79293.
25. Ram, M., Prasad, K., Singh, S., Hada, B. and Kumar, S. (2013). Influence of salicylic acid and methyl jasmonate elicitation on anthocyanin production in callus cultures of Rosa hybrida L. Plant Cell, Tissue and Organ Culture, 113(3): 459-467.
26. Ramezannezhad, R., Aghdasi, M., and Fatemi, M. (2019). Enhanced production of cichoric acid in cell suspension culture of Echinacea purpurea by silver nanoparticle elicitation. Plant Cell, Tissue and Organ Culture, 139(2): 261-273.
27. Rao, S. R. and Ravishankar, G. (2002). Plant cell cultures: chemical factories of secondary metabolites. Biotechnology Advances, 20(2): 101-153.
28. Romero, F. R., Delate, K., Kraus, G. A., Solco, A. K., Murphy, P. A. and Hannapel, D. J. (2009). Alkamide production from hairy root cultures of Echinacea. In Vitro Cellular and Developmental Biology-Plant, 45(5): 599.
29. Royandazagh, S. D. and Pehlivan, E. C. (2016). In vitro micropropagation of Lilium candidum L. and alkaloids. Journal of Tekirdag Agricultural Faculty, 13(3): 100-110.
30. Shahrajabian, M. H., Sun, W. and Cheng, Q. (2020). Traditional herbal medicine for the prevention and treatment of cold and flu in the autumn of 2020, overlapped with COVID-19. Natural Product Communications, 15(8): 1431.
31. Sharif, K. O. M., Tufekci, E. F., Ustaoglu, B., Altunoglu, Y. C., Zengin, G., Llorent-Martínez, E. and Baloglu, M. C. (2021). Anticancer and biological properties of leaf and flower extracts of Echinacea purpurea (L.) Moench. Food Bioscience, 41: 101005.
32. Smetanska, I. (2008). Production of Secondary Metabolites Using Plant Cell Cultures. In Food Biotechnology (pp. 187-228): Springer.
33. Sökmen, A. and Gürel, E. (2002). Secondary Metabolite Production (TR, Trans.). In M. Babaoğlu, E. Gurel, and S. Ozcan (Eds.), Plant Biotechnology, Tissue Culture and Application (2. ed., Vol. 1, pp. 211-261). Konya: Selçuk University Press, Turkey.
34. Suan See, K., Bhatt, A. and Lai Keng, C. (2011). Effect of sucrose and methyl jasmonate on biomass and anthocyanin production in cell suspension culture of Melastoma malabathricum (Melastomaceae). Revista de Biologia Tropical, 59(2): 597-606.
35. Taha, H., Abd, E. R. ve Maharik, N. (2009). In vitro production of caffic acid derivatives in calli and regenerate cultures of Echinacea angustifolia Dc and Echinacea pallida Nutt, Journal of Applied Sciences Research, 5(1): 13-20.
36. Tanur Erkoyuncu, M. and Yorgancılar, M. (2015). Plant Tissue Culture forthe Production of Secondary Metabolites. Selcuk Journal of Agriculture and Food Sciences, 2(1): 66-76.
37. Thygesen, L., Thulin, J., Mortensen, A., Skibsted, L. H. and Molgaard, P. (2007). Antioxidant activity of cichoric acid and alkamides from Echinacea purpurea, alone and in combination. Food Chemistry, 101(1): 74-81.
38. Veliky, I. and Rose, D. (1973). Nitrate and ammonium as nitrogen nutrients for plant cell cultures. Canadian Journal of Botany, 51(10): 1837-1844.
39. Wang, D., Du, F., Liu, H. and Liang, Z. (2010). Drought stress increases iridoid glycosides biosynthesis in the roots of Scrophularia ningpoensis seedlings. Journal of Medicinal Plants Research, 4(24): 2691-2699.
40. Wu, C. H., Dewir, Y. H., Hahn, E. J. and Paek, K. Y. (2006). Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots of Echinacea angustifolia. Journal of Plant Biology, 49(3): 193.
41. Yamaner, O. and Erdag, B. (2013). Effects of sucrose and polyethylene glycol on hypericins content in Hypericum adenotrichum. Eurasian Journal of Biosciences, 7: 101-110.
42. Zobayed, S., Afreen, F. and Kozai, T. (2007). Phytochemical and physiological changes in the leaves of St. John's wort plants under a water stress condition. Environmental and Experimental Botany, 59(2): 109-116.