Abstract
Regeneration of transgenic shoots was achieved from Hypericum perforatum L. hairy roots on hormone-free MS/B5 medium for a period of 4 weeks under a photoperiod of 16-h light. A control experiment was set up with root segments obtained from in vitro grown seedlings. Investigations have been made to study the production of phenolic compounds in non transgenic and transgenic shoot cultures. Six groups of phenolic compounds such as phenolic acids, flavonols, flavan-3-ols, naphtodianthrones, phloroglucinols, and xanthones were recorded in the transgenic shoots. Chlorogenic acid was found as the most representative phenolic acid in shoot extracts. With regard to the class of quercetin derivatives in transformed shoots, quercetin 6-C-glucoside usually dominated among the glycosides followed by quercitrin and hyperoside. The analysis of flavan-3-ols in transgenic shoots resulted in the identification of epicatechin and proanthocyanidin dimers. One of the main achievements in this study was considerably enhanced hypericin and pseudohypericin production in transgenic shoots. The concentration of identified naphtodianthrones was about 12-fold higher in transformed shoots compared to control. Chromatographic analysis of phloroglucinols in transgenic shoots resulted in the identification of hyperforin, while its homolog adhyperforin was detected in traces. A twofold higher content of hyperforin was observed in transgenic shoots compared to control. Although mangiferin was found as the main representative xanthone in shoot extracts, several other xanthones identified as γ-mangostin isomers, trihydroxy-1-methoxy-C-prenyl xanthone, garcinone E, and banaxathone E were de novo synthesized in transformed shoots. Therefore, H. perforatum transgenic shoots could be considered as a source for rapid and increased production of naphtodianthrones and other specific phenolic compounds.
Similar content being viewed by others
References
Bais HP, Walker TS, McGrew JJ, Vivanco JM (2002) Factors affecting growth of cell suspension cultures of Hypericum perforatum L. (St. John’s) wort and production of hypericin. In Vitro Cell Dev Biol Plant 38:58–65
Barnes J, Anderson LA, Phillipson JD (2001) St John’s wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. J Pharm Pharmacol 53:583–600
Beerhues L, Berger U (1995) Differential accumulation of xanthones in methyl-jasmonate-and yeast-extract-treated cell cultures of Centaurium erythraea and Centaurium littorale. Planta 197:608–612
Bertoli A, Giovannini A, Ruffoni B, Di Guardo A, Spinelli G, Mazzetti M, Pistelli L (2008) Bioactive constituent production in St. John’s wort in vitro hairy roots. Regenerated plant lines. J Agric Food Chem 56:5078–5082
Bilia AR, Gallori S, Vincieri FF (2002) St. John’s wort and depression: efficacy, safety and tolerability-an update. Life Sci 70:3077–3096
Bombardelli E, Morazzoni P (1995) Hypericum perforatum. Fitoterapia 66:43–68
Briskin DP, Gawienowski MC (2001) Differential effects of light and nitrogen on production of hypericins and leaf glands in Hypericum perforatum. Plant Physiol Biochem 39:1075–1081
Büter B, Orlacchio C, Berger K (1998) Significance of genetic and environmental aspects in the field cultivation of Hypericum perforatum. Planta Med 64:431–437
Butterweck V (2003) Mechanism of action of St John’s wort in depression. CNS Drugs 17:539–562
Čellárová E (2003) Culture and biotechnology of Hypericum. In: Ernst E (ed) Hypericum, the genus Hypericum. Taylor and Francis, New York, pp 65–76
Charchoglyan A, Abrahamyan A, Fujii I, Boubakir Z, Gulder TA, Kutchan TM, Vardapetyan H, Bringmann G, Ebizuka Y, Beerhues L (2007) Differential accumulation of hyperforin and secohyperforin in Hypericum perforatum tissue cultures. Phytochemistry 68:2670–2677
Conceição LFR, Ferreres F, Tavares RM, Dias ACP (2006) Induction of phenolic compounds in Hypericum perforatum L. cells by Colletotrichum gloeosporioides elicitation. Phytochemistry 67:149–155
Cui X-H, Chakrabarty D, Lee E-J, Paek K-Y (2010) Production of adventitious roots and secondary metabolite by Hypericum perforatum L. in a bioreactor. Bioresour Technol 101:4708–4716
Cuyckens F, Rozenberg R, Hoffmann E, Claeys M (2001) Structure characterization of flavonoid O-diglycosides by positive and negative nano-electrospray ionization ion trap mas spectrometry. J Mass Spectrom 36:1203–1210
Di Guardo A, Čellárová E, Koperdáková J, Pistelli L, Ruffoni B, Allavena A, Giovannini A (2003) Hairy roots induction and plant regeneration in Hypericum perforatum L. J Genet Breed 57:269–278
Dias ACP, Seabra RM, Andrade PB, Fernandes-Ferreira M (1999) The development and evaluation of a HPLC-DAD method for the analysis of the phenolic fractions from in vivo and in vitro biomass of Hypericum species. J Liq Chromatogr Relat Technol 22:215–227
Dias ACP, Seabra RM, Andrade PB, Ferreres F, Fernandes-Ferreira M (2000) Xanthone biosynthesis and accumulation in calli and suspended cells of Hypericum androsaemum. Plant Sci 150:93–101
Erdelmeier CAJ, Koch E, Hoerr R (2000) Hypericum perforatum-St. John’s wort chemical, pharmacological and clinical aspects. Stud Nat Prod Chem 22:643–716
Ferreres F, Andrade PB, Valentao P, Gil-Izquierdo A (2008) Further knowledge on barley (Hordeum vulgare L.) leaves O-glycosyl-C-glycosyl flavones by liquid chromatography–UV diode-array detection–electrospray ionization mass spectrometry. J Chromatogr A 1182:56–64
Floryanowicz-Czekalska K, Wysokińska H (2000) Transgenic shoots and plants as a source of natural phytochemical products. Acta Soc Bot Pol 69:131–136
Franklin G, Oliveira M, Dias ACP (2007) Production of transgenic Hypericum perforatum plants via particle bombardment-mediated transformation of novel organogenic cell suspension cultures. Plant Sci 172:1193–1203
Franklin G, Conceição LFR, Kombrink E, Dias ACP (2009) Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress. Phytochemistry 70:60–68
Gadzovska S, Maury S, Ounnar S, Righezza M, Kascakova S, Refregiers M, Spasenoski M, Joseph C, Hagège D (2005) Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures. Plant Physiol Biochem 43:591–601
Gadzovska S, Maury S, Delaunay A, Spasenoski M, Joseph C, Hagège D (2007) Jasmonic acid elicitation of Hypericum perforatum L. cell suspensions and effects on the production of phenylpropanoids and naphtodianthrones. Plant Cell Tissue Organ Cult 89:1–13
Gadzovska S, Maury S, Delaunay A, Spasenoski M, Hagège D, Courtois D, Joseph C (2013) The influence of salicylic acid elicitation of shoots, callus, and cell suspension cultures on production of naphtodianthrones and phenylpropanoids in Hypericum perforatum L. Plant Cell Tissue Organ Cult 113:25–39
Gadzovska-Simic S, Tusevski O, Antevski S, Atanasova-Pancevska N, Petreska J, Stefova M, Kungulovski D, Spasenoski M (2012) Secondary metabolite production in Hypericum perforatum L. cell suspensions upon elicitation with fungal mycelia from Aspergillus flavus. Arch Biol Sci 64:113–121
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures soybean root cells. Exp Cell Res 50:148–151
Guillon S, Trémouillaux-Guiller J, Pati PK, Rideau M, Gantet P (2006) Hairy root research: recent scenario and exciting prospects. Curr Opin Plant Biol 9:341–346
Karppinen K, Hokkanen J, Tolonen A, Mattila S, Hohtola A (2007) Biosynthesis of hyperforin and adhyperforin from amino acid precursors in shoot cultures of Hypericum perforatum. Phytochemistry 68:1038–1045
Kartnig T, Göbel I, Heydel B (1996) Production of hypericin, pseudohypericin and flavonoids in cell cultures of various Hypericum species and their chemotypes. Planta Med 62:51–53
Karuppusamy S (2009) A review on trends in production of secondary metabolites from higher plants by in vitro tissue, organ and cell cultures. J Med Plants Res 3:1222–1239
Kirakosyan A, Hayashi H, Inoue K, Charchoglyan A, Vardapetyan H (2000) Stimulation of the production of hypericins by mannan in Hypericum perforatum shoot cultures. Phytochemistry 53:345–348
Kirakosyan A, Sirvent TM, Gibson DM, Kaufman PB (2004) The production of hypericins and hyperforin by in vitro cultures of St. John’s wort (Hypericum perforatum). Biotechnol Appl Biochem 39:71–81
Klejdus B, Kováčik J, Babula P (2013) PAL inhibitor evokes different responses in two Hypericum species. Plant Physiol Biochem 63:82–88
Komarovská H, Giovannini A, Košuth J, Čellárová E (2009) Agrobacterium rhizogenes-mediated transformation of Hypericum tomentosum L. and Hypericum tetrapterum Fries. Z Naturforsch C 64:864–868
Koperdáková J, Katkovèinová Z, Košuth J, Giovannini A, Čellárová E (2009a) Morphogenetic response to plant growth regulators in transformed and untransformed Hypericum perforatum L. clones. Acta Biol Crac Ser Bot 51:61–70
Koperdáková J, Komarovská H, Košuth J, Giovannini A, Čellárová E (2009b) Characterization of hairy root-phenotype in transgenic Hypericum perforatum L. clones. Acta Physiol Plant 31:351–358
Košuth J, Koperdáková J, Tolonen A, Hohtola A, Čellárová E (2003) The content of hypericins and phloroglucinols in Hypericum perforatum L. seedlings at early stage of development. Plant Sci 165:515–521
Liu XN, Zhang XQ, Sun JS (2007a) Effects of cytokinins and elicitors on the production of hypericins and hyperforin metabolites in Hypericum sampsonii and Hypericum perforatum. Plant Growth Regul 53:207–214
Liu XN, Zhang XQ, Zhang SX, Sun JS (2007b) Regulation of metabolite production by precursors and elicitors in liquid cultures of Hypericum perforatum. Plant Cell Tissue Organ Cult 91:1–7
Medina MA, Martínez-Poveda B, Amores-Sánchez MI, Quesada AR (2006) Hyperforin: more than an antidepressant bioactive compound? Life Sci 79:105–111
Mehrotra S, Goel MK, Rahman LU, Kukreja AK (2013) Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina. Plant Cell Tissue Organ Cult 114:31–38
Mertens-Talcott SU, Percival SS (2005) Ellagic acid and quercetin interact synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle arrest in human leukemia cells. Cancer Lett 218:141–151
Miketova P, Schram KH, Whitney JL, Kerns EH, Valcic S, Timmermann BN, Volk KJ (1998) Mass spectrometry of selected components of biological interest in green tea extracts. J Nat Prod 61:461–467
Mulinacci N, Giaccherini C, Santamaria A, Caniato R, Ferrari F, Valletta A, Vincieri F, Pasqua G (2008) Anthocyanins and xanthones in the calli and regenerated shoots of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh. Plant Physiol Biochem 46:414–420
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Phys Plant 15:473–497
Murch SJ, Saxena PK (2006) St. John’s wort (Hypericum perforatum L.): challenges and strategies for production of chemically-consistent plants. Can J Plant Sci 86:765–771
Nurulain TZ (2006) Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications. Life Sci 78:2073–2080
Odabas MS, Radusiene J, Camas N, Janulis V, Ivanauskas L, Cirak C (2009) The quantitative effects of temperature and light intensity on hyperforin and hypericins accumulation in Hypericum perforatum L. J Med Plants Res 3:519–525
Pappeti A, Daglia M, Aceti C, Sordelli B, Spini V, Carazzone C, Gazzani G (2008) Hydroxycinnamic acid derivatives occurring in Cichorium endivia vegetables. J Pharm Biomed Anal 48:472–476
Pasqua G, Avato P, Monacelli B, Santamaria AR, Argentieri MP (2003) Metabolites in cell suspension cultures, calli, and in vitro regenerated organs of Hypericum perforatum cv. Topas. Plant Sci 165:977–982
Pavlík M, Vacek J, Klejdus B, Kuban V (2007) Hypericin and hyperforin production in St. John’s wort in vitro culture: influence of saccharose, polyethylene glycol, methyl jasmonate, and Agrobacterium tumefaciens. J Agric Food Chem 55:6147–6153
Pinhatti AV, de Matos Nunes J, Maurmann N, Rosa LMG, von Poser GL, Rech SB (2010) Phenolic compounds accumulation in Hypericum ternum propagated in vitro and during plant development acclimatization. Acta Physiol Plant 32:675–681
Piovan A, Filippini R, Caniato R, Borsarini A, Maleci LB, Cappelletti EM (2004) Detection of hypericins in the ‘red glands’ of Hypericum elodes by ESI–MS/MS. Phytochemistry 65:411–414
Piperopoulos G, Lotz R, Wixforth A, Schimierer T, Zeller K (1997) Determination of naphtodianthrones in plant extracts from Hypericum perforatum L. by liquid chromatography–electrospray mass spectrometry. J Chromatogr B 695:309–319
Rainha N, Koci K, Coelho AV, Lima E, Baptista J, Fernandes-Ferreira M (2013) HPLC–UV–ESI–MS analysis of phenolic compounds and antioxidant properties of Hypericum undulatum shoot cultures and wild-growing plants. Phytochemistry 86:83–91
Sarris J, Fava M, Schweitzer I, Mischoulon D (2012) St John’s wort (Hypericum perforatum) versus sertraline and placebo in major depressive disorder: continuation data from a 26-week RCT. Pharmacopsychiatry 45:275–278
Silva BA, Ferreres F, Malva JO, Dias ACP (2005) Phytochemical and antioxidant characterization of Hypericum perforatum alcoholic extracts. Food Chem 90:157–167
Sirvent T, Gibson D (2002) Induction of hypericins and hyperforin in Hypericum perforatum L. in response to biotic and chemical elicitors. Physiol Mol Plant Pathol 60:311–320
Soelberg J, Jørgensen LB, Jäger AK (2007) Hyperforin accumulates in the translucent glands of Hypericum perforatum. Ann Bot 99:1097–1100
Solomon D, Adams J, Graves N (2013) Economic evaluation of St. John’s wort (Hypericum perforatum) for the treatment of mild to moderate depression. J Affect Disord 148:228–234
Sooriamuthu S, Varghese RJ, Bayyapureddy A, John SST, Narayanan R (2013) Light-induced production of antidepressant compounds in etiolated shoot cultures of Hypericum hookerianum Wight & Arn. (Hypericaceae). Plant Cell Tissue Organ Cult 115:169–178
Tocci N, D’Auria FD, Simonetti G, Panella S, Palamara AT, Pasqua G (2012) A three-step culture system to increase the xanthone production and antifungal activity of Hypericum perforatum subsp. angustifolium in vitro roots. Plant Physiol Biochem 57:54–58
Tolonen A, Uusitalo J, Hohtola A, Jalonen J (2002) Determination of naphthodianthrones and phloroglucinols from Hypericum perforatum extracts by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 16:396–402
Tusevski O, Petreska Stanoeva J, Stefova M, Kungulovski D, Atanasova Pancevska N, Sekulovski N, Panov S, Gadzovska Simic S (2013) Hairy roots of Hypericum perforatum L.: a promising system for xanthone production. Cent Eur J Biol 8:1010–1022
Varghese RJ, Sooriamuthu S (2013) Differences in hypericin synthesis between experimentally induced seedling shoot cultures of Hypericum hookerianum Wight & Arn. Plant Biotechnol Rep 7:511–518
Vinterhalter B, Ninković S, Cingel A, Vinterhalter D (2006) Shoot and root culture of Hypericum perforatum L. transformed with Agrobacterium rhizogenes A4M70GUS. Biol Plant 50:767–770
Walker TS, Bais HP, Vivanco JM (2002) Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L. (St. John’s wort). Phytochemistry 60:289–293
Yazaki K, Okuda T (1990) Procyaninins in callus and multiple shoots of Hypericum erectum. Planta Med 56:490–491
Zhang Y, Shi P, Qu H, Cheng Y (2007) Characterization of phenolic compounds in Erigeron breviscapus by liquid chromatography coupled to electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 21:2971–2984
Zobayed SMA, Murch SJ, Rupasinghe HPV, Saxena PK (2003) Elevated carbon supply altered hypericin and hyperforin contents of St. John’s wort (Hypericum perforatum L.) grown in bioreactors. Plant Cell Tissue Organ Cult 75:143–149
Zobayed SMA, Afreen F, Goto E, Kozai T (2006) Plant–environment interactions: accumulation of hypericin in dark glands of Hypericum perforatum. Ann Bot 98:793–804
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by M. H. Walter.
Rights and permissions
About this article
Cite this article
Tusevski, O., Petreska Stanoeva, J., Stefova, M. et al. Identification and quantification of phenolic compounds in Hypericum perforatum L. transgenic shoots. Acta Physiol Plant 36, 2555–2569 (2014). https://doi.org/10.1007/s11738-014-1627-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11738-014-1627-4