Phenolic Profile of Dark-Grown and Photoperiod-Exposed Hypericum perforatum L. Hairy Root Cultures

Hypericum perforatum L. is a medicinal plant considered as an important natural source of secondary metabolites with a wide range of pharmacological attributes. Hairy roots (HR) were induced from root segments of in vitro grown seedlings from H. perforatum after cocultivation with Agrobacterium rhizogenes A4. Investigations have been made to study the production of phenolic compounds in dark-grown (HR1) and photoperiod-exposed (HR2) cultures. The chromatographic analysis of phenolic acids, flavonols, flavan-3-ols, and xanthones revealed marked differences between HR1 and HR2 cultures. The production of quinic acid, kaempferol, and seven identified xanthones was increased in HR2. Moreover, HR2 showed a capability for de novo biosynthesis of two phenolic acids (3-p-coumaroylquinic acid and 3-feruloylquinic acid), three flavonol glycosides (kaempferol hexoside, hyperoside, and quercetin acetylglycoside), and five xanthones (tetrahydroxy-one-methoxyxanthone, 1,3,5-trihydroxy-6-methoxyxanthone, 1,3,5,6-tetrahydroxy-2-prenylxanthone, paxanthone, and banaxanthone E). On the other side, HR1 cultures were better producers of flavan-3-ols (catechin, epicatechin, and proanthocyanidin dimers) than HR2. This is the first comparative study on phenolic profile of H. perforatum HR cultures grown under dark and photoperiod conditions.


Introduction
Hypericum perforatum L. (St. John's wort) is a traditional medicinal plant with a complex mixture of secondary metabolites. Phenolic compounds as naphthodianthrones, acylphloroglucinols, flavonoids, and xanthones are the main bioactive metabolites commonly described for this plant [1]. In phytomedicine, Hypericum extracts are responsible for a plethora of pharmacological activities including antidepressant, antiviral, antioxidant, anti-inflammatory, and antimicrobial properties [2]. To meet the increasing demand for plants utilized in the pharmaceutical industry, much of the recent research has focussed on the development of new in vitro culture techniques as a useful alternative to improve the yield of bioactive metabolites in plant material.
Agrobacterium rhizogenes-mediated plant transformation represents a convenient experimental system for establishment of hairy roots (HR). Transformed root cultures represent an attractive model system for the production of highvalue secondary metabolites, including pharmaceuticals and other biologically active substances of commercial importance [3]. Namely, HR cultures may synthesize higher levels of secondary metabolites or amounts comparable to those of the intact plant and offer a promising approach to the industrial exploitation of HR for production of novel metabolites [4,5]. Until now, only A. rhizogenes- [6,7] and biolistic-mediated [8] transformation methods have been applied. In recent years, it has been shown that HR are responsive to physical stimuli such as exposure to light which is known to regulate a number of plant developmental processes [9], as well as 2 The Scientific World Journal primary and secondary metabolite production [10]. These findings indicate that the exposure of HR to light leads to alternations in their biosynthetic potentials. Although several studies investigated secondary metabolite production in root cultures [11,12], the capacity of H. perforatum HR to produce phenolic compounds has never been explored.
This study describes the phenolic profile of transformed roots (HR) from H. perforatum transformed with A. rhizogenes strain A4, grown in constant dark (HR1) or in light/dark photoperiod (HR2) conditions. Phenolic compounds in HR were analyzed using high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD) and tandem mass spectrometry (MS ) with electrospray ionization (ESI). All present derivatives of phenolic acids, flavonol glycosides, flavonoid aglycones, flavan-3-ols, and xanthones were identified from corresponding UV and MS spectra and quantified by HPLC-DAD.

Plant Material.
Seeds from H. perforatum were collected from wild plants growing in a natural population in the Pelister National Park at about 1394 m. Voucher specimen (number 060231) of H. perforatum is deposited in the Herbarium at the Faculty of Natural Sciences and Mathematics, University "Ss. Cyril and Methodius, " Skopje, Macedonia. As for a previous study [13], seeds were surface sterilized and in vitro germinated seedlings were maintained in a growth chamber at 25 ± 1 ∘ C under a photoperiod of 16 h light, irradiance at 50 mol ⋅ m 2 ⋅ s −1 , and 50 to 60% relative humidity.

Establishment of Hairy Roots.
The wild type Agrobacterium rhizogenes agropine strain A4 (obtained from Institut National de la Recherche Agronomique-INRA, Versailles, France) was used for H. perforatum transformation experiments [14]. Transformation protocol was performed according to Di Guardo et al. [6] with the modifications described in our previous study [15]. Briefly, the HR cultures were induced by A. rhizogenes A4 from root segments of onemonth-old in vitro germinated seedlings from H. perforatum. Transgenic status of the HR was confirmed by PCR analysis using rolB specific primers [15]. Transformed root cultures were maintained by subculturing at one-month intervals on MS/B 5 hormone-free medium. The subculture was carried out at 25 ± 1 ∘ C in the dark (HR1) and under photoperiod (HR2) of 16 h light (50 mol ⋅ m 2 ⋅ s −1 ). One-month-old HR1 and HR2 cultures were harvested (1 g) and then frozen in liquid nitrogen or lyophilized and stored at −80 ∘ C, until analysis.

HPLC/DAD/ESI-
Analysis. Phenolic compounds extraction from freeze-dried lyophilized and powdered root cultures was performed as previously reported by Tusevski et al. [15]. The HPLC system was equipped with an Agilent 1100 series diode array and mass detector in series (Agilent Technologies, Waldbronn, Germany). It consisted of a G1312A binary pump, a G1313A autosampler, a G1322A degasser, and a G1315B photodiode array detector, controlled by Chem-Station software (Agilent, v.08.03). Chromatographic separations were carried out on 150 mm × 4.6 mm, 5 m XDB-C18 Eclipse column (Agilent, USA). The mobile phase consisted of two solvents: water-formic acid (A; 99 : 1, v/v) and methanol (B) in the following gradient program: 90% A and 10% B (0-20 min), 80% A and 20% B (20-30 min), 65% A and 35% B (30-50 min), 50% A and 50% B (50-70 min), and 20% A and 80% B (70-80 min) and continued with 100% B for a further 10 min. Each run was followed by an equilibration period of 10 min. The flow rate was 0.4 mL/min and the injection volume was 10 L. All separations were performed at 38 ∘ C. Formic acid (HCOOH) and methanol (CH 3 OH) were HPLC grade solvents (Sigma-Aldrich, Germany). The HPLC-water was purified by a PURELAB Option-Q system (Elga LabWater, UK). The commercial standards chlorogenic acid, rutin, quercetin, kaempferol, catechin, epicatechin, and xanthone (Sigma-Aldrich, Germany) were used as reference compounds. The reference compounds were dissolved in 80% methanol in water. The concentration of the stock standard solutions was 1 mg ⋅ mL −1 and they were stored at −20 ∘ C. Spectral data from all peaks were accumulated in the range of 190-600 nm, and chromatograms were recorded at 260 nm for xanthones, at 280 nm for flavan-3-ols, at 330 nm for phenolic acids, and at 350 nm for flavonols. Peak areas were used for quantification at wavelengths where each group of phenolic compounds exhibited an absorption maximum. The HPLC system was connected to the Agilent G2445A ion-trap mass spectrometer equipped with electrospray ionization (ESI) system and controlled by LCMSD software (Agilent, v.6.1.). Nitrogen was used as nebulizing gas at a pressurelevel of 65 psi and the flow was adjusted to 12 L ⋅ min −1 . Both the heated capillary and the voltage were maintained at 350 ∘ C and 4 kV, respectively. MS data were acquired in the negative ionization mode. The full scan mass covered the mass range from m/z 100 to 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as a collision gas, with voltage ramping cycle from 0.3 up to 2 V. Maximum accumulation time of the ion trap and the number of MS repetitions to obtain the MS average spectra were set at 300 ms and 3, respectively. Identification of the component peaks was performed by the UV/Vis, MS, and MS 2 spectra and retention times of the abovementioned available standards.

Statistical Analysis.
The experiments were independently repeated twice under the same conditions and all analyses were performed in triplicate. Secondary metabolite contents were expressed as mg ⋅ 100 g −1 dry weight (DW). Standard deviation of mean value was shown as ± S.D. The statistical analyses including calculations of means and standard deviations were performed applying Excel (Microsoft Office, 2007).

Results and Discussion
3.1. Establishment of Hairy Roots. As previously reported [15], H. perforatum HR were initiated by inoculation of root The Scientific World Journal 3 Table 1: Retention times, UV, and mass spectral data of phenolic acids, flavonols, and flavan-3-ols in Hypericum perforatum dark-grown (HR1) and photoperiod-exposed (HR2) hairy root culture extracts a . explants with A. rhizogenes A4. On the basis of culture conditions, selected dark-grown (HR1) and photoperiod-exposed (HR2) cultures showed differences in the morphology. Darkgrown hairy root cultures were thinner and whitish in colour showing rapid plagiotropic growth with active branching and vigorous production of elongated lateral roots. Present results confirmed that transformed roots of H. perforatum had characteristic traits of HR previously described by Tepfer [16]. In contrast, HR2 cultures began to turn pale green after 7 days of culture and continued to acquire green coloration during the course of subsequent growth period. Moreover, HR2 appeared intense greenish-brown after one month of culture. It was seen that the growth of HR was generally most vigorous between the 3rd and 4th weeks of the cultivation period (1 month), but their growth declined after the 5th week due to the nutrient depletion. For HPLC analysis, onemonth-old HR cultures were further evaluated.

HPLC/DAD/ESI-Analysis.
The HPLC/DAD/ESI-MS technique was used to analyse the phenolic profile of H. perforatum HR1 and HR2 cultures. Four groups of phenolic compounds such as phenolic acids, flavonols, flavan-3-ols, and xanthones were recorded in HR cultures (Tables 1 and  2). The identification of phenolic compounds (Tables 1 and 2, Figure 1) was based on the typical UV/Vis spectral data and LC/MS in the negative ionization mode [M-H] − with the subsequent MS 2 , MS 3 , and MS 4 analysis for further identification with reference to similar data previously reported [15,[17][18][19][20][21][22][23][24][25][26]. The HPLC analysis of phenolic compounds revealed marked differences between HR1 and HR2 cultures (Tables 1 and 2, Figure 1). ion at m/z 173 was also observed. Compound F1 was identified as quinic acid, taking into account its MS fragmentation pattern and the literature data [17]. Quinic acid (F1) was the only detectable phenolic acid in both HR cultures. A 1.4-fold increase of quinic acid was observed in HR2 compared to HR1 cultures.

Phenolic Acids. Compound
Two peaks, 3-p-coumaroylquinic acid (F3) and 3feruloylquinic acid (F5), were detected only in HR2 cultures with identical UV spectra characterized by absorption band at 314 nm. Compounds F3 and F5 were readily distinguished by their cinnamic acid-derived MS 2 base peaks at m/z 163 and at m/z 193, respectively. Quinic acid is the most important component as a key intermediate in the biosynthesis of aromatic compounds. The condensation between quinic acid and caffeic acid leads to the formation of chlorogenic acid in the shikimic acid pathway [27]. Chlorogenic acid is an 4 The Scientific World Journal Table 2: Retention times, UV, and mass spectral data of xanthones in Hypericum perforatum dark-grown (HR1) and photoperiod-exposed (HR2) hairy root culture extracts a . Peak no.
Compounds The Scientific World Journal 5 The compound F11 was identified as kaempferol derivative with glycosylation in position 3 according to its UVspectra (256, 266, 350 nm). The MS and MS 2 spectra were consistent with the presence of a hexose residue and confirm the kaempferol aglycone. Therefore, this compound was identified as kaempferol hexoside. Compound With regard to the class of flavonol glycosides, our results showed that both HR cultures had capability to produce quercetin and kaempferol derivatives. However, there is no available study for the potential of H. perforatum root cultures to produce flavonol derivatives. Several differences can be pointed out when comparing the composition of flavonol glycosides in HR cultures with those of H. perforatum in vitro cultures. In our previous work [31,32], we indicated that H. perforatum cells, calli, and shoots demonstrate a considerable potential for producing quercetin, isoquercitrin, and quercitrin upon elicitation with jasmonic acid and salicylic acid. The LC-MS screening of twelve H. perforatum HR transgenic plants showed a large variability in the content of rutin, hyperoside, quercetrin, and quercetin [29]. Moreover, the abovementioned flavonol glycosides had been identified in H. perforatum regenerated plantlets [33] and H. undulatum shoot cultures [34].
The HPLC-MS analysis of flavonoid aglycones in HR cultures resulted in the identification of kaempferol (F15). Its molecular ion at m/z 285 corresponded to that of kaempferol. The identification was made by comparing its UV and MS spectra to analytical standards and literature data [28]. A 1.6fold increase of kaempferol was observed in HR2 compared to HR1 cultures.  [22], and were recognized as proanthocyanidin dimers. Dark-grown HR were better producers of catechins and proanthocyanidin dimers than HR2. Literature data about the production of catechin derivatives in in vitro cultures of H. perforatum are scarce. In our previous work [35], we indicated that H. perforatum root cultures may be considered as a promising source of proanthocyanidin dimers. Nevertheless, catechin, epicatechin, and proanthocyanidin dimers had been previously identified in shoots and calli of H. erectum [36] and H. undulatum shoot cultures [34].
Among the twenty-five identified xanthones, seven (X6, X7, X12, X14, X15, X18, and X28) were upregulated in HR2 compared to HR1 cultures. Moreover, five xanthones (X8, X10, X20, X21, and X30) were synthesized only in HR2 cultures. Recent studies showed that Hypericum in vitro cultures have the potential to accumulate xanthones and their production can be manipulated by the hormonal supplementation [28], or/and by the culture type [33]. It is probable that phytohormones either facilitate or hamper the expression and activity of specific xanthone enzymes that influence xanthone accumulation in H. perforatum callus [33], cells [28], and root cultures [12]. Namely, Tocci et al. [12] suggested that root cultures grow continuously on nutrient media supplemented with auxins, but sometimes repetitive subcultures may induce loss of morphogenetic potential, resulting in poor or negligible secondary metabolite production. On the other hand, our results showed that H. perforatum HR cultures successfully grow on hormonefree media and represent a continuous source for high-level xanthone production.
Recent study showed that the phenolic biosynthesis and flavonoids formation are light-dependent processes [41]. Moreover, changes in light intensity are capable of inducing the production of flavonoids and total phenolics in plants [42]. Therefore, de novo biosynthesis and accumulation of phenolic acids, flavonols, and xanthones in HR2 cultures are not surprising since considerable evidence now shows that many of the enzymes in the phenylpropanoid/flavonoid pathway could be upregulated by light. In addition, Abbasi et al. [43] demonstrated light-stimulated accumulation of phenolic acids and phenylalanine ammonia lyase (PAL) activity in Echinacea purpurea HR cultures. Considering results from our study, we could hypothesize that shifting the dark-grown HR to photoperiod might induce a short-term "light-stress" response. In this view, the presence of light could induce a variety of responses along with metabolic changes that directly or indirectly trigger a "later" increase in xanthone accumulation. On the other hand, our results showed that photoperiod has an inhibitory effect on the accumulation of flavan-3-ols in HR2 cultures. Possible reasons for downregulation of flavan-3-ols could be due to the activation of their catabolism and/or reaction to unidentified products that exist in photoperiod-exposed cultures. Therefore, photoregulation of phenolic compounds biosynthesis in H. perforatum HR may offer additional advantages of quantitative and qualitative improvements of these medicinally important metabolites.

Conclusions
In conclusion, H. perforatum HR cultures provided a promising system for the production of various groups of phenolic compounds. Distinct phenolic profile between dark-grown and photoperiod-exposed HR cultures was shown as detailed for the first time. HR cultures grown under photoperiod can be proposed as a useful source for accumulation of phenolic acids and flavonols, while dark-adapted HR represent an alternative tool for flavan-3-ol production. More importantly, both HR cultures synthesized and stored significant quantities of xanthones. The use of the results reported here might contribute to further study on photoregulation and optimal control of secondary metabolite production in H. perforatum HR cultures. Polymerase chain reaction.