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Jan 7, 2010 - ternum was investigated after micropropagation establish- ment and during acclimatization over the phenological development of the plant.
Acta Physiol Plant (2010) 32:675–681 DOI 10.1007/s11738-009-0446-5

ORIGINAL PAPER

Phenolic compounds accumulation in Hypericum ternum propagated in vitro and during plant development acclimatization Amanda Valle Pinhatti • Je´ssica de Matos Nunes • Natasha Maurmann • Luı´s Mauro Gonc¸alves Rosa • Gilsane Lino von Poser • Sandra Beatriz Rech

Received: 23 October 2009 / Revised: 2 December 2009 / Accepted: 28 December 2009 / Published online: 7 January 2010 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2010

Abstract The phenolic compound content of Hypericum ternum was investigated after micropropagation establishment and during acclimatization over the phenological development of the plant. Plantlets cultured in vitro on full Murashige and Skoog medium without growth regulators displayed higher phenolic compound yields, were acclimatized, and field grown. Production of total phenolic compounds as well as hyperoside, chlorogenic acid, quercitrin, guaijaverin, isoquercitrin, and uliginosin B were quantified at vegetative, flowering and fructification stages, and different plant organs (roots, stems, leaves and reproductive parts) showing that reproductive parts at flowering stage and the leaves at fructification stage were the main repository site of secondary metabolites, except for uliginosin B. The stems were the least accumulative organ, while the roots accumulated only hyperoside and uliginosin B. Moreover, the accumulation of most of the flavonoids and uliginosin B in acclimatized plants surpassed the levels found in the wild plant, warranting further research with the species.

Communicated by S. Lewak. A. V. Pinhatti  J. de Matos Nunes  G. L. von Poser  S. B. Rech (&) Faculdade de Farma´cia, UFRGS, Av. Ipiranga, 2752, Porto Alegre 90610-000, Brazil e-mail: [email protected] N. Maurmann Programa de Po´s-Graduac¸a˜o em Biologia Celular e Molecular (PPGBCM/UFRGS), Porto Alegre, Brazil L. M. G. Rosa Departamento de Plantas Forrageiras e Agrometeorologia, Faculdade de Agronomia, UFRGS, Av. Bento Gonc¸alves, 7712, Caixa Postal 15100, Porto Alegre, Brazil

Keywords Field performance  Chlorogenic acid  Flavonoids  Micropropagation  Uliginosin B

Introduction Several species of Hypericum native to south Brazil have been the target of phytochemical studies (von Poser et al. 2006) and the investigation of Hypericum ternum A. St. Hil. led to the isolation of tannins, phenolic acids (Dall’Agnol et al. 2003), and flavonoids (Bernardi et al. 2007) from the leaves, and uliginosin B, a phloroglucinol derivative, from the roots of the plant (Bernardi 2007). The chloroform extract of aerial parts of the plant displayed antifungal activity against standardized strains of clinically important fungi and their clinical isolates (Fenner et al. 2005), whereas the isolated flavonoids showed antioxidant activity (Bernardi et al. 2007). The recent increase in the popularity of plant-based medicines has led to a new segment in horticultural crop production and agriculture. The application of micropropagation for commercial-scale plant production has been well demonstrated in several plant species that contain potentially useful secondary metabolites (Rout et al. 2000). Many of these species, including the genus Hypericum, have shown excellent in vitro performance in terms of the number of explants produced in a short period of time (Murch and Saxena 2006) and an alternative long-term strategy to meet market demands (Bruni and Sacchetti 2009). Given the biological activities demonstrated by H. ternum compounds and that the source of raw material is often only seasonally available, we describe the establishment of in vitro propagation of the species, the evaluation of the micropropagated plant growth on different nutrient

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concentrations and the assessment of total phenolic compound accumulation in vitro, and during plant development acclimatization. Moreover, the variation of chlorogenic acid, hyperoside, isoquercitrin, guaijaverin, quercitrin, and uliginosin B contents of different acclimatized plant parts through its phenological cycle was also evaluated.

Materials and methods Plant material Plants of H. ternum A. St. Hil. were collected at full flowering in the city of Sa˜o Francisco de Paula, state of Rio Grande do Sul, Brazil, in the spring of 2003. Voucher specimens were deposited in the herbarium of the Universidade Federal do Rio Grande do Sul (ICN Bordignon 1717). The aerial parts of the plant were thoroughly washed with tap water, surface sterilized in 70% EtOH for 1 min, rinsed twice with sterile-distilled water, immersed in 1.5% sodium hypochlorite for 10 min, and rinsed four times with sterile-distilled water. Culture conditions Shoot tips (0.3–0.5 cm long) were excised and the segments (one per flask) were vertically implanted into 175 mL glass jars with 25 mL of MS modified medium (Murashige and Skoog 1962), named M D (Maurmann et al. 2008), supplemented with 30 g L-1 sucrose (Vetec, Rio de Janeiro, Brazil) without plant growth regulators. The pH was adjusted to 5.8 and the medium was solidified with 6 g L-1 agar (extra pure, Merck). The cultures were maintained at 25 ± 1°C with 16 h light/8 h dark photoperiods (light intensity of 45 lmol m-2 s-1). After 4 weeks of incubation under these conditions, the new shoots formed were separated and transferred to fresh medium for a new round of multiplication. Regenerated plantlets were transferred to the same medium and subcultured every 6 weeks. The analysis of the influence of nutrient concentration in solid medium on in vitro growth was performed by transferring the plantlets to the M D, to full strength MS medium (MS), to half strength MS medium (MS 50), or to diluted MS containing one-quarter of the original concentration of inorganic salts (MS 25). These plants were maintained for three cycles of 6 weeks each, with subculture to the corresponding medium at the end of each cycle, using individual shoot tips as explants, and maintained in the same conditions mentioned earlier. Upon completion of the three passages on the appropriate media for adaptation to the specific medium conditions, growth parameters and total phenolic compounds were evaluated.

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Acclimatization and field growth conditions Plantlets cultured in vitro for 6 weeks in MS medium were transferred to plastic pots containing a sterile mixture of nonfertilized commercial soil and vermiculite (1:2, v/v), covered with transparent plastic sheets, and kept under controlled environmental conditions (25 ± 1°C, 16 h photoperiod, irradiance of 70 lmol m-2 s-1 and irrigation with steriledistilled water once a week) for 30 days. After this period, plants were transplanted to pots (18 cm diameter 9 14 cm high) containing unfertilized commercial soil and grown on open field with irradiance of 2,000 lmol m-2 s-1 at plant level (measured in the middle of sunny days without clouds with a sensor Quantum Li-cor). The pots were randomly rearranged fortnightly to minimize possible positional effects, and the plants were watered as needed. Sample collection and treatment Twelve individuals were harvested at different developmental stages: at vegetative (15 weeks after transplanting to open field conditions), at flowering (after 20 weeks of growth with plants bearing a mixture of buds and flowers at all stages of development, displaying over 50% of open flowers), and at fructification stage (after 25 weeks of plant growth). The material for analysis was collected from whole plants at these developmental stages from four different parts: leaves, stems, roots, and total reproductive parts, when available. Fresh mass was recorded after plant parts excision and their dry mass (DM) recorded after freeze-drying. The mean daytime and nighttime temperatures during the growing periods were 23.1 ± 4.2 and 14.4 ± 3.7°C (15 weeks), 25.5 ± 4.7 and 16.1 ± 3.8°C (20 weeks) and 25.4 ± 4.8 and 16.2 ± 3.9°C (25 weeks), respectively. Determination of total phenol content Crude methanolic extracts were obtained from 0.1 g DM of freeze-dried powdered plant extracted five times at room temperature with 5 mL of methanol, under 20 min sonication and subsequently concentrated to dryness under reduced pressure. Total phenol concentration was determined according to the Folin-Ciocalteu colorimetric method with a slight modification (Singleton and Rossi 1965), using quercetin as the standard. Appropriate dilutions of the samples were oxidized with 0.2 N Folin-Ciocalteu reagent (Merck Darmstadt, Germany, 2 N, diluted tenfold), and after 5 min, the reaction was neutralized with saturated sodium carbonate (75 g L-1). The absorbance of the resulting blue color was measured at 765 nm with an ultraviolet–visible Biospectro SP 220 spectrophotometer after incubation for 30 min at room temperature. Quantification was performed on the basis of the standard curve of

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quercetin (Ivanova et al. 2005), and the results were expressed in milligrams of quercetin equivalents per gram of dry plant mass (QE gDM-1). HPLC estimation of secondary metabolites For uliginosin B determination, 0.05 g DM of freeze-dried powdered material was extracted 15 times at room temperature with 5 mL of n-hexane with 20 min of sonication (Ultrasonic, Sa˜o Paulo, Brazil), whereas chlorogenic acid and flavonoids were quantified in the crude methanolic extracts obtained as described earlier for the determination of total phenol content. All metabolites were analyzed using a Waters HPLC system, comprising a Waters 2487 UV detector and a Waters 600 pump at flow rate of 1 mL min-1. Reversed phase separations were carried out at room temperature using a Waters Nova Pack C18 column adjusted to a guard column Waters Nova Pack C18 60A. Uliginosin B quantification was conducted with 95% CH3CN, 5% H2O, 0.01% TFA as isocratic solvent system and detection performed at 220 nm wavelength. The metabolite identification was obtained by comparison with retention time of pure standard (21.76 min) isolated from underground parts of H. ternum and determined through a calibration curve with concentrations ranging from 2 to 800 lg mL-1. Chlorogenic acid, guaijaverin, hyperoside, isoquercitrin, and quercitrin were analyzed using isocratic elution with 14% CH3CN, 86% H2O, 0.05% TFA and detected at 254 nm. Chlorogenic acid was quantified by a calibration curve of pure standard (Merck) with excellent linearity (r2 = 0.9981) between 3.91 and 2,000 lg mL-1. The flavonoids were expressed as hyperoside mass through a calibration curve with concentrations ranging from 36.25 to 2,320 lg mL-1 and ensured linearity (r2 = 0.9994). Standards purified from H. ternum (Bernardi 2007) with retention times of 3.52 min (chlorogenic acid), 17.80 min (hyperoside), 20.37 min (isoquercitrin), 26.65 min (guaijaverin), and 37.70 min (quercitrin) were used to determine the concentrations of the compounds (expressed as g% DM). Statistical analysis

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and roots on the same medium was observed, eliminating one step of medium transfer in the propagation of these species, thus reducing the total time and cost required for plant regeneration. Similar results were also reported for H. brasiliense (Cardoso and de Oliveira 1996) and H. perforatum (McCoy and Camper 2002). In addition, the species showed an excellent adaptation to the in vitro conditions and was successfully propagated, being subcultured every 6 weeks. Analysis of influence of nutrient concentration in solid medium on in vitro growth The influence of nutrient concentration of MS 25, MS 50, M D, and MS medium on the in vitro growth of H. ternum cultivated for 6 weeks demonstrated distinct effects on the investigated parameters: plantlets grew on all of the media formulations tested (Fig. 1), with higher shoot number observed on full MS medium (7.08 ± 0.99), but longer shoots promoted on diluted medium (MS 25) (9.00 ± 1.22 cm), while plantlets cultivated on MS 50 and M D medium showed higher root number (12 ± 1.83 and 11.2 ± 1.3, respectively), and longer roots (5.03 ± 0.65 cm) were obtained on M D medium. Nevertheless, the variation of fresh mass analyzed was not significant among the different solid media (mean of 299.67 ± 25.66 g). Regarding total phenolic content, plantlets cultured on MS medium showed significant difference (51.07 ± 0.41 QE gDM-1), being approximately twofold higher than plantlets cultured on other tested media. Furthermore, although the increased content of selected micronutrients of M D medium induced root formation and secondary metabolite accumulation in other medicinal plants (Zhao et al. 2005; Maurmann et al. 2008), this was not verified in the present study. The comparison of results of growth and total phenolic compound

15

a

MS 25 a

MS 50 MΔ

a b

10

b

MS

a

In all experiments, the layout was totally randomized. Oneway analysis of variance (ANOVA) was applied with a critical value of P B 0.05.

Results and discussion In vitro cultures establishment Hypericum ternum was easily propagated in M D medium without growth regulators and the formation of both shoots

ab 5

c

b a

bc c c

ab

a

b

0 Shoots number

Shoots length (cm)

Roots number

Roots length (cm)

Fig. 1 Effect of media concentration on growth and development of H. ternum plantlets cultured in vitro for 6 weeks. Values are mean of three different experiments ± SE, and different letters indicate significant differences at P B 0.05 (Tukey test)

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accumulation indicated the election of MS medium for further experiments. Acclimatization and secondary metabolite accumulation Under the culture conditions used, plant growth appeared rather optimal, none of the plants died during the experimental period and field-grown plants displayed a mean height of 25–30 cm. Concerning the analysis of total phenolic compounds, yields were affected by the developmental stage and assessed acclimatized plant parts (Fig. 2). The evaluation demonstrated that the reproductive parts accumulated the highest levels of total phenolic compounds at flowering stage (157.47 ± 1.27 QE gDM-1), decreasing the concentration at fructification. On the other hand, the leaves accumulated higher levels of the metabolites at fructification (115.68 ± 10.49 QE gDM-1) and in the roots and stems at vegetative stage (Fig. 2). In addition, the analysis of the wild plant collected at full flowering revealed the accumulation of 135.14 ± 1.27 and 131.48 ± 1.12 QE gDM-1 of total phenolic compounds in the vegetative and reproductive parts, respectively. The analysis of isolated compounds previously identified in leaves and roots of H. ternum (Dall’Agnol et al. 2003; Bernardi 2007; Bernardi et al. 2007) demonstrated that the field-grown acclimatized plants accumulated all searched metabolites frequently found in Hypericum species (Ma´rtonfi et al. 2006; C ¸ irak et al. 2007; Sagratini et al. 2008). Nevertheless, the naphthodianthrones hypericin and pseudohypericin, which together with the phloroglucinols hyperforin and adhyperforin are the major phytochemical

160 Vegetative a ab

Flowering Fructification

b

QE (gDM)

-1

120

80

a a

a

40

b

b

c 0 Leaves

Reprodutive Parts

Stems

Roots

Fig. 2 Total phenolic compounds accumulated in various tissues of H. ternum field-grown acclimatized plants. Vertical bars are mean ± SE of 12 samples of plants harvested at each ontogenic stage. Different letters indicate significant differences at P B 0.05 (Tukey test)

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constituents of H. perforatum, were not detected in H. ternum (data not shown) and other south Brazilian species (Ferraz et al. 2002). Considering that the hypericin content was found to be positively correlated with leaf dark gland density, it can be speculated that higher density of dark gland on plant tissues means higher hypericin content (C¸irak et al. 2006). Moreover, the absence of this metabolite in the Brazilian species is consistent with the absence of these glands observed in species from Trigynobrathys section (Robson 1981). Chlorogenic acid was found in all parts and at all developmental stages except for the roots of the acclimatized plants, with the leaves being the main source of the metabolite and highest level detected at fructification stage, while in the stems and reproductive parts the metabolite was quantified in higher concentration at flowering stage (Table 1). The leaves have also been reported to be the main source of the metabolite in H. origanifolium (C¸irak et al. 2007). Hyperoside was found in all parts of the acclimatized plants (Table 1) and its distribution pattern was dependent on the plant developmental stage. The greatest concentrations were found in the leaves during fructification, while in the stems and reproductive parts the higher contents were detected at the vegetative and flowering stage, respectively. Moreover, the accumulation of the metabolite displayed the same pattern of distribution reported for H. origanifolium (C¸irak et al. 2007) and is the major metabolite accumulated in some species from central Italy (Sagratini et al. 2008). Guaijaverin displayed the same pattern of distribution of hyperoside, except for the roots, where the metabolite was not detected (Table 1). Quercitrin and isoquercitrin were not detected in the roots of the acclimatized plants and the former was always higher in all plant parts throughout the analyzed period (Table 1). The highest level of uliginosin B was quantified in the reproductive parts at fructification (0.68 ± 0.08 g% DM), whereas the metabolite was detected in the roots at a constant concentration throughout the course of ontogenesis. On the other hand, this compound was not detected in the stems of the plants, being present in the leaves only at vegetative stage (Table 1). Furthermore, the analysis of the wild plant (collected at full flowering) revealed the absence of the metabolite in the flowers and vegetative parts. It is interesting to point out that in the study of the distribution of bioactive substances from H. brasiliense during plant growth the highest content of isouliginosin B was observed in the roots at the flowering stage, while the metabolite was not detected in the shoot of the plants (Abreu et al. 2004). However, it was previously identified in leaves of a population of H. brasiliense from a different source (Rocha

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Table 1 Ontogenetic changes in chlorogenic acid, guaijaverin, hyperoside, isoquercitrin, quercitrin, and uliginosin B contents (g% DM) of roots, stems, leaves and reproductive tissues in acclimatized plants and wild harvested Hypericum ternum Roots

Stems

na

na

Leaves

Reproductive tissues

Chlorogenic acid Wild plant

3.33 ± 0.17b b

3.75 ± 0.15a

c

Vegetative

nd

0.15 ± 0.003

2.40 ± 0.22



Flowering

nd

0.27 ± 0.002a

2.73 ± 0.3ab

1.30 ± 0.06b

nd

b

0.17 ± 0.006

4.37 ± 0.1

Wild plant Vegetative

na nd

na 0.19 ± 0.03a

0.12 ± 0.013c 0.47 ± 0.09b

0.13 ± 0.015b –

Flowering

nd

0.087 ± 0.02b

0.43 ± 0.013b

0.23 ± 0.04a

nd

b

Fructification

a

0.55 ± 0.09c

Guaijaverin

Fructification

0.085 ± 0.02

a

0.98 ± 0.08

0.18 ± 0.02ab

0.78 ± 0.018c

0.93 ± 0.002b

Hyperoside Wild plant

na

na b

a

b

Vegetative

0.04 ± 0.019

0.81 ± 0.09

3.84 ± 0.6

Flowering

0.14 ± 0.038a

0.40 ± 0.009b

3.29 ± 0.07b

Fructification

a

b

0.15 ± 0.009

0.47 ± 0.09

na

na

– 1.33 ± 0.1a

a

0.83 ± 0.08b

4.67 ± 0.16

Isoquercitrin Wild plant

0.49 ± 0.013b c

b

0.63 ± 0.013a

Vegetative

nd

0.085 ± 0.019

0.46 ± 0.011



Flowering

nd

0.27 ± 0.013a

0.6 ± 0.063b

0.45 ± 0.03b

nd

b

0.17 ± 0.014

0.79 ± 0.093

0.11 ± 0.005c

Wild plant Vegetative

na nd

na 0.36 ± 0.015b

1.24 ± 0.02b 1.55 ± 0.15a

1.05 ± 0.045a –

Flowering

nd

0.63 ± 0.05a

1.57 ± 0.049a

0.58 ± 0.017b

Fructification

a

Quercitrin

Fructification

b

a

nd

0.07 ± 0.009

1.79 ± 0.14

0.16 ± 0.026c

na

na

nd

0.005 ± 0.001c

Uliginosin B Wild plant Vegetative

0.21 ± 0.09

nd

0.15 ± 0.024



Flowering

0.29 ± 0.11

nd

nd

0.47 ± 0.07b

Fructification

0.25 ± 0.1

nd

nd

0.68 ± 0.08a

Values are mean ± SE of 12 samples and different letters within columns for each compound and plant part differ significantly at the level of P B 0.05 (Tukey test) na not analyzed, nd not detected

et al. 1995), and such variation might be explained by several factors, from endogenous regulation of physiological processes to environmental characteristics. Although the reproductive parts displayed higher yields of phenolic compounds during flowering stage in relation to the leaves (Fig. 2), the total amount of the individual quantified products is smaller (Table 1). This could be due to the presence of other compounds such as I3,II8-biapigenin and the quercetin derivatives (quercetin 3-methyl ether, quercetin 3,7-dimethyl ether), occurring as free aglycone, previously isolated from this plant (Bernardi et al. 2007) and not quantified in this work. This hypothesis is corroborated by the study of C ¸ irak et al. (2007) in which

quercetin, as free aglycone, was found as the main component in the flowers of H. origanifolium. The field performance of acclimatized plants demonstrates superior accumulation than the wild plants of hyperoside and guaijaverin (in all plant parts), chlorogenic acid, quercitrin and isoquercitrin (in the leaves) and uliginosin B (in the reproductive parts), the leaf tissues being, despite the collection period, accumulators of higher amounts of all metabolites, except for uliginosin B. Nevertheless, reproductive parts accumulated inferior levels of chlorogenic acid, quercitrin, and isoquercitrin throughout the cultivation period, with higher levels at flowering stage (Table 1).

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Plants can interact with stressful environments by physiological adaptation, alter the biochemical profile of the plant tissues, and produce a diverse array of secondary metabolites (Briskin and Gawienowski 2001; Abreu and Mazzafera 2005; Couceiro et al. 2006). Furthermore, other possible reasons for the variation between acclimatized and wild plants include the cultivation season (Southwell and Bourke 2001) and contamination by insects and pathogens (Zobayed and Saxena 2004). Bu¨ter et al. (1998) studied several accessions of H. perforatum cultivated in three different localities over two consecutive years and observed significant content variations of hyperforin, hypericin, pseudohypericin, quercetin, rutin, and other compounds in flowers. The authors suggested that climatic conditions or different physiological stages might account for the variation of the secondary metabolites. The accumulation of phenolic compounds is a carefully controlled process with both the levels of secondary metabolites and the composition of the phenolic pool varying considerably between organisms, tissues, developmental stages and, in relation to environmental conditions (Winkel-Shirley 2002; Koes et al. 2005). Moreover, understanding the regulatory and biochemical mechanisms that control the types and amounts of phenolic compounds synthesized under different conditions continues to be a high priority for research aiming crop plants that could overproduce antioxidant phenolics. To highlight, Grace et al. (1998) reported a higher chlorogenic acid content in the leaves of Mahonia repens under highlight conditions during the winter than in the leaves of plants under high light during the summer. In another study, Cle´ et al. (2008) studied the relationship between phenolic accumulation and UV-susceptibility in transgenic tomato plants with altered hydroxycinnamoyl CoA quinate transferase (HQT) expression. Overall, increased chlorogenic acid accumulation was associated with increased UV protection. However, the genetic manipulation of HQT expression also resulted in more complex alterations in the profiles of the phenolics. Environmental stresses have also been demonstrated to induce health-promoting phytochemicals in lettuce, including chicoric acid and chlorogenic acid (Oh et al. 2008). Additional field tests of H. ternum acclimatized plants are in progress to evaluate the effect of biotic and abiotic stresses on both overall phenolic accumulation and the specific phenolic profile to optimize the production of pharmacologically desired compounds.

Conclusions The micropropagation of H. ternum can supply uniform acclimatized plants with accumulation of all the analyzed

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secondary metabolites present in the wild plant. Based on ontogenetic and morphogenetic changes in the content of chlorogenic acid, guaijaverin, hyperoside, isoquercitrin, quercitrin, and uliginosin B, it can be concluded that there is a close relationship between chemical content of plant parts and development stages during phenological cycle of this species. Among different tissues, leaves of the plants harvested during fructification produced significantly higher amounts of all metabolites, except for uliginosin B, quantified in higher yield in the reproductive parts also during fructification. The present results might also be useful to obtain enhanced concentration of these compounds through further agronomical and technological approaches.

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