Hairy Roots of Hypericum Perforatum L.: a Promising System for Xanthone Production

Cent. Eur. J. Biol. • 8(10) • 2013 • 1010-1022 DOI: 10.2478/s11535-013-0224-7 Central European Journal of Biology Hairy roots of Hypericum perforatum L.: a promising system for xanthone production Research Article Oliver Tusevski1, Jasmina Petreska Stanoeva2, Marina Stefova2, Dzoko Kungulovski3, Natalija Atanasova Pancevska3, Nikola Sekulovski4, Saso Panov4, Sonja Gadzovska Simic1,* 1Department of Plant Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 2Department of Analytical Chemistry, Institute of Chemistry, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 3Department of Microbiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 4Department of Molecular Biology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia Received 22 April 2013; Accepted 03 June 2013 Abstract: Hypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) ofHypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identification of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones. Keywords: Agrobacterium rhizogenes A4 • Phenolic acids • Flavonol glycosides • Flavan-3-ols • Flavonoid aglycones • Xanthones © Versita Sp. z o.o. naphthodianthrones and phloroglucinols are 1. Introduction distributed in the aerial parts of the plant, whereas Hypericum perforatum L. (Saint John’s wort) is a xanthones are mainly produced in the roots [2]. medicinal plant considered as an important natural Flavonol derivatives, naphthodianthrones and source of secondary metabolites with a wide phloroglucinols are used for the treatment of mild range of pharmacological attributes. It contains and moderate depression [3]. Xanthones are a naphthodianthrones, acylphloroglucinols, flavonoids, class of polyphenolics that exhibit well-documented biflavones, phenylpropanes, xanthones and an pharmacological properties, such as monoamine essential oil rich in sesquiterpenes [1]. Flavonoids, oxidase inhibition, and antioxidant, antimicrobial, * E-mail: [email protected] 1010 O. Tusevski et al. cytotoxic and hepatoprotective activity [4]. To meet 2. Experimental Procedures the increasing demand for Hypericum utilized in the pharmaceutical industry [5], the emphasis in recent 2.1 Plant material research has been focused on the development of Seeds from H. perforatum were collected from wild new in vitro culture techniques as a useful alternative plants growing in a natural population in the National to improve the yield of bioactive metabolites in plant Park Pelister at about 1394 m. Voucher specimen material. number (060231) of H. perforatum is deposited in Plant genetic transformation offers an opportunity the Herbarium at the Faculty of Natural Sciences and to introduce new qualities into medicinal and aromatic Mathematics, University “Ss. Cyril and Methodius”- plants. Agrobacterium rhizogenes-mediated hairy root Skopje, Republic of Macedonia (MKNH). As for a (HR) cultures represent an attractive experimental previous study [15], seeds were washed with 70% system for the production of high-value secondary ethanol for 30 sec, surface sterilized with 1% NaOCl metabolites, including pharmaceuticals and other for 15 min, rinsed 3 times in sterile deionized water biologically active substances of commercial and cultured on MS macro and oligoelements [16], B5 importance [6,7]. Namely, HR cultures may synthesize vitamin solution [17], supplemented with 3% sucrose higher levels of secondary metabolites or amounts and solidified with 0.7% agar. No growth regulator was comparable to those of the intact plant and offer added. The medium was adjusted to pH 5.8 before a promising approach to the production of novel autoclaving (20 min at 120°C). In vitro cultures were metabolites [8]. The first step towards the application of maintained in a growth chamber at 25±1°C under a transformation procedures to few Hypericum species photoperiod of 16 h light, irradiance at 50 mmol m2 s-1 has been encountered. Until now, only A. rhizogenes- and 50 to 60% relative humidity. [9-11] and biolistic-mediated [12] transformation methods have been applied. Wild agropine strain 2.2 Preparation of Agrobacterium rhizogenes A. rhizogenes ATCC 15834 was used in the first A4 suspension successful transformation of H. Perforatum [9]. Also, The wild type Agrobacterium rhizogenes agropine an efficient transformation protocol of this species was strain A4 (obtained from INRA, Versailles, France) was reported with A. rhizogenes A4M70GUS [10]. Recently, used for H. perforatum transformation experiments two other Hypericum species (H. tomentosum and H. [18]. The procedure for A. rhizogenes A4 culture tetrapterum) were successfully transformed with A. preparation was based on the method of Di Guardo rhizogenes ATCC 15834 and A4 [11]. HR cultures of et al., [9] with the following modifications. H. perforatum exhibited high potential for spontaneous A. rhizogenes A4 was grown on nutrient agar medium regeneration into whole transgenic plants [9,10]. (15 g l-1 peptone, 3 g l-1 beef extract, 5 g l-1 NaCl, 0.3 g l-1 -1 Selected Hypericum HR regenerated plants have been KH2PO4 and 15 g l agar). The suspension culture was evaluated for their bioactive secondary metabolites prepared by growing a single bacterial colony in 10 ml [9,13,14]. However, no study has been published of nutrient broth medium at 28ºC with continuous rotary on the identification and quantification of secondary shaking (120 rpm) for 24 h. Subsequently, 1 ml of the metabolites in H. perforatum HR cultures. bacterial suspension was transferred into 9 ml fresh nutrient The objectives of this study were to establish an broth medium and maintained under similar conditions efficient A. rhizogenes A4-mediated transformation for 12 h or until bacterial concentration of approximately system that would result in the rapid formation of HR 4.2x109 colony-forming units (CFU) per ml medium was cultures for the purposes of studying the production achieved. Overnight-grown bacterial suspension was and accumulation of bioactive compounds. Phenolic diluted 1:20 (v/v) in sterile water (0.1 absorbance at compounds in the control roots and transformed 660 nm) and used for transformation protocol. HR were analyzed using high-performance liquid chromatography (HPLC) coupled with diode-array 2.3 Transformation protocol and establishment detection (DAD) for routine analysis and tandem of hairy roots mass spectrometry (MSn) with electrospray A. rhizogenes A4-mediated transformation protocol was ionization (ESI) as a more sophisticated means performed by Di Guardo et al., [9] with the following for identifying phenolic compounds. All present modifications. Root segments (1-2 cm) without apical derivatives of phenolic acids, flavonol glycosides, tip were excised from 4 week-old in vitro seedlings flavonoid aglycones, flavan-3-ols and xanthones and gently wounded with a sterile lancet blade. Root were identified from corresponding UV and MS explants were soaked for 15 min in bacterial suspension spectra and quantified by HPLC-DAD. and blotted on sterile filter paper. Control root explants 1011 Xanthone production in Hypericum perforatum hairy roots were soaked in sterile distilled water. Fifty root explants were: 95ºC for 5 min (initial denaturation), 35 cycles of were used in each treatment and this experiment was 95ºC for 30 sec, 64ºC for 1 min and 72ºC for 1 min and repeated three times. Infected and control explants a final extension at 72ºC for 7 min. PCR amplification were than placed on MS/B5 hormone-free medium in products were analysed by electrophoretic separation on the dark at 25±1°C. After 2 days, the explants were 2% (w/v) agarose gel in TE buffer (40 mM Tris acetate, transferred to hormone-free medium supplemented with 1 mM EDTA, pH 8.3) and were detected by fluorescence 200 mg l-1 cefotaxime. The transformation frequency under UV light after staining with ethidium bromide. was calculated in percentage ((final number of explants forming HR/initial number of infected explants) x100) 2.5 HPLC/DAD/ESI-MSn analysis

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