Abstract
The present study was conducted to determine the variation in the content of several plant chemicals, namely hyperforin, hypericin, pseudohypericin, chlorogenic acid, rutin, hyperoside, isoquercetine, kaempferol, quercitrine and quercetine among ten Hypericum orientale L. populations from Northern Turkey. The aerial parts representing a total of 30 individuals were collected at full flowering and dissected into floral, leaf and stem tissues. After dried at room temperature, the plant materials were assayed for chemical contents by HPLC. The populations varied significantly in chemical contents. Among different plant parts, the flowers were found to be the principle organ for hyperforin, hypericin, pseudohypericin and rutin accumulations while the rest of the chemicals were accumulated mainly in leaves in all growing localities. The chemical variation among the populations and plant parts is discussed as being possibly the result of different genetic, environmental and morphological factors.
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Introduction
The genus Hypericum contains approximately 400 different species of annuals, perennials, shrubs and small trees, ranging from very small perennials to trees belonging to Guttiferae family (Robson 2001). The species of this genus have been used as healing agents due to their various medicinal properties for hundred of years. Hypericum species are also used as sedatives, antiseptics, and antispasmodics in Turkish folk medicine. The Hypericum genus is represented in Turkey by 89 species of which 43 are endemic (Davis 1988). Hypericum orientale L. is one of the Turkish species of Hypericum. The stems are 7–45 cm in long, erect or decumbent and sometimes rooting with adventitious roots. Leaves 10–40 mm, oblong or elliptic-oblong to oblanceolate. Sepals unequal, narrowly oblong and ovate or broadly elliptic to obovate. Petals 10–18 mm, entire, without black dotes. Capsule 7–14 mm, ovoid to ovoid-cyclindric (Davis 1988) (Fig. 1).
The major phytomedicinal compounds of Hypericum plants are phloroglucinol derivatives hyperforin and adhyperforin, the naphthodianthrones hypericin and pseudohypericin, the flavonoids hyperoside, rutin, quercitrin, quercetin and biapigenin and the phenylpropanes caffeic acid and chlorogenic acid which possess a wide array of biological properties (Patocka 2003). The pharmacological activities of Hypericum extracts namely, photodynamic, antidepressive and antiviral activities are mainly attributed to the naphthodianthrones, hypericin and pseudohypericin (Bombardelli and Morazzoni 1995). Hyperforin is a prenylated phloroglucinol derivative that consists of a phloroglucinol skeleton with lipophilic isoprene chains. Results from recent studies have indicated hyperforin as the main chemical, responsible for antidepressant effects of Hypericum extracts (Medina et al. 2006). Flavonoids are a group of bioactive compounds present in Hypericum plants. They play an important role in preventing cardiovascular diseases and several kinds of cancer (Chu et al. 2000).
Increased market demand for Hyperici herba has led to intensive studies on the chemistry and biological activities of Hypericum plants. This is especially true for H. perforatum, which is the most common and well-known species. Although numerous investigations have been carried out on the chemical composition of H. perforatum, comparatively few compounds have been reported from other members of Hypericum genus. In previous studies, H. orientale was reported to contain hyperforin, hypericin, psedohypericin, rutin, hyperoside, quercitrin, quercetin, and chlorogenic acid (Cirak et al. 2007a; Smelcerovic et al. 2008). However, no report is available on the chemical variation among wild populations of this species. In the present study, we report our phytochemical investigations of H. orientale populations sampled from ten growing localities in Northern Turkey. Besides, isoquercetine has not yet been detected in this species. Here, we also describe the first occurrence of the chemical in H. orientale.
Materials and methods
Plant materials
The plant materials were described in our previous studies (Cirak et al. 2007a). The species were identified by Dr. Hasan Korkmaz, Faculty of Science and Art, Department of Biology, University of 19 Mayis, Samsun, Turkey.
Experimental procedures
The aerial parts of H. orientale plants representing a total of 30 shoots were collected at full flowering from ten sites of Northern Turkey (Table 1). The sampling sites were not grazed or mown during the plant gathering period. The top of 2/3 plants was harvested between 11:00 a.m. and 01:00 p.m. Conditions on the day of collection were clear and sunny at all sites. Temperatures ranged from 26 to 32°C. After collected, ten individuals were kept as whole plants and the rest were dissected into floral, leaf and stem tissues. The plant materials were dried at room temperature (20 ± 2°C), and subsequently assayed for chemical contents by HPLC.
Preparation of plant extracts
Air-dried plant material was mechanically ground with a laboratory mill to obtain a homogenous drug powder. Samples of about 0.5 g (weighed with 0.0001 g precision) were extracted in 50 mL of 100% methanol by ultrasonicatation at 40°C for 30 min in a Sonorex Super model RK 225H ultrasonic bath. The prepared extracts were filtered through a membrane filter with pore size of 0.22 μm (Carl Roth GmbH, Karlsruhe, Germany) and kept in a refrigerator until analysis no longer than 3 h. The extracts for naphthodianthrones analysis after ultrasonication were exposure to light for 30 min due to the photoconversion of protohypericin into hypericin and protopseudohypericin into pseudohypericin.
HPLC analysis
A Shimadzu Prominence LC-20A (Shimadzu Europa GmbH, Duisburg, Germany) chromatographic system equipped with two LC-20AD model pumps, a SIL-20AC auto-injector, a thermostat CTO-20AC and a SPD-M20A detector was used for HPLC analysis. Separation of all compounds was carried out using an YMC Pack Pro-C18 (YMC Europe GmbH, Dinslaken, Germany) column (150 mm × 4 mm i.d.; 3 μm particle sizes) with 10 mm guard-precolumn. The mobile phase consists of solvent A [water containing 0.1% trifluoroacetic acid (TFA)] and solvent B (acetonitrile containing 0.1% TFA). The following binary gradient elution program was used: 0–1 min (B 5 → 5%), 1–14 min (B 5 → 20%), 14–20 min (B 20 → 80%), 20–30 min (B 80 → 100%), 30–39 min (B 100 → 100%), 39–39.5 min (B 100 → 5%), 39.5–45 min (B 5–5%). The mobile phase was delivered with a flow rate of 1.0 mL min−1; volume of extract injected was 10 μL. Detection was performed at 210–790 nm wave length range with a constant column temperature at 40°C. The eluted compounds were identified on the basis of their retention time by comparison with retention time of reference standards and also confirmed with UV spectra’s of reference standards in the wavelength ranging from 210 to 790 nm.
The hypericin and pseudohypericin elution program was isocratic. The mobile phase consists of acetonitrile containing 0.1% TFA. Flow rate of mobile phase was 1.1 mL min−1. Ten micro liters of extracts were injected. Detection was recorded at 210–790 nm wave length range with a constant column temperature at 40°C.
The quantification of detected compounds was achieved by external standard method at the maximal absorption on the UV spectra of corresponding compounds: chlorogenic acid, 325 nm; rutin, 353 nm; hyperoside, 353 nm; isoquercetrine, 353; kaempferol, 346 nm; quercetrine, 347 nm; quercetine, 368 nm; hyperforin, 270 nm; hypericin and pseudohypericin, 580 nm wavelength. A six-point calibration curves were obtained with pure standards dissolved in MeOH in the concentration range of 0.2–110 μg/mL. All calibration cures showed good linear regression (r 2 > 0.999) within the test range. Typical HPLC chromatograms of H. orientale extract are shown in Fig. 2. All solvents and standards of reference substances were of HPLC grade and purchased from Roth Chemical Company (Karlsruhe, Germany).
Data analysis
Data for hyperforin, hypericin, pseudohypericin, chlorogenic acid, rutin, hyperoside, isoquercetine, kaempferol, quercitrine and quercetine contents of plant material including whole plant, stem, leaf and flower were objected to ANOVA and significant differences among mean values were tested with the Duncan Multiple Range Test (P < 0.01) by MSTAT statistical software. Mean values of the chemical contents were normalized using x′ = \(\sqrt x + 1 \) transformation before conducting ANOVA, when necessary, because some chemicals were not detected in several cases.
Results
The content of hyperforin, hypericin, pseudohypericin, chlorogenic acid, rutin, hyperoside, isoquercetine, and quercetine in plant materials including whole shoots, stem, leaf and flower tissues varied significantly among populations (P < 0.01) (Table 2). Whereas quercitrin was accumulated in similar levels only in plants from Alacam and Ispir-2 populations and none of analyzed plant materials in the present study produced kaempferol. Hyperforin was detected only in plants from Cakalli, Ispir-2 and Ladik-2 populations and hyperforin content was significantly higher in Ispir-2 population (1.21 mg/g DW). Hypericin and pseudohypericin accumulations occurred only in Alacam, Karapinar, Ispir-1 and Ispir-2 populations. Plants from Ispir-2 population produced the highest amount of both chemicals (1.64 and 3.24 mg/g DW hypericin and pseudohypericin, respectively). Similarly rutin was detectable in the populations of Kizlan, Ladik-1, Nusratli, Karapinar-1, Alacam, and Ispir-2 among which Karapinar-1 produced the highest accumulation level (3.32 mg/g DW). On the contrary, chlorogenic acid, hyperoside, isoquercetine, and quercetine were accumulated in all populations at different levels. Chlorogenic acid accumulation was the highest in Cakalli and Nusratli populations (11.71 and 10.09 mg/g DW) and plants from Ispir-2 population produced the highest amount of hyperoside (16.35 mg/g DW). For isoquercetine, the highest accumulation levels were observed in Nusratli and Ispir-1 populations (4.29 and 3.61 mg/g DW). Similarly, Ispir-2 and Ladik-2 populations produced significantly higher amount of quercetine when compared to the rest of the populations (3.68 and 3.61 mg/g DW).
Stems, leaves and reproductive tissues differed significantly with regard to chemical accumulation (Figs. 3, 4). The highest accumulation level of hyperforin, hypericin, pseudohypericin and rutin was detected in flowers while the rest of the chemicals were accumulated mainly in leaves in all growing localities. As for stems, significantly lower accumulation levels of the secondary metabolites were observed in the organs and they did not produce hyperforin and hypericin.
Discussion
Variations in the levels of bioactive secondary metabolites in populations of Hypericum plants have an important impact on the pharmacological activity of tested extracts (Bergonzi et al. 2001). Hence, investigations of population variability in the content of secondary metabolites from different species of Hypericum have been made over several decades. In Hypericum perforatum, hypericin and pseudohypericin levels in plants from different localities of Australia (Southwell and Bourke 2001), Switzerland (Büter and Büter 2002), USA (Sirvent et al. 2002), Turkey (Cirak et al. 2007b) and Lithuania (Bagdonaite et al. 2010) were investigated. Wild populations of the same species from different growing localities of the world were also investigated for variation in the content of hyperforin (Couceiro et al. 2006), rutin, hyperoside, quercitrin, quercetin, chlorogenic acid and apigenin-7-O-glucoside (Cirak et al. 2007b). In terms of population variability of bioactive substances in other Hypericum species, essential oil components in Hypericum perfoliatum, Hypericum humifusum, Hypericum linarifolium and Hypericum pulchrum were reported to vary significantly with geographic distribution of wild plants (Nogueira et al. 2008). In Hypericum triquetrifolium, significant variations were detected in the content of hypericin, pseudohypericin, hyperforin and several flavonoids as rutin, hyperoside, apigenin-7-O-glucoside, kaempferol, quercitrin, quercetin and amentoflavone among four wild populations from Turkey (Cirak et al. 2011). The previous results indicated the regional distribution of Hypericum plants as an important source of chemical variability. In the present study, similarly, significant differences were detected in chemical accumulations among wild populations of H. orientale. The populations were located in different parts of Northern Turkey and growing sites of them were differed from each other by climatic and geographic factors (Table 1). As a result of these environmental differences, each site has specific ecological conditions and habitat. The chemical variability in the content of evaluated chemicals among populations may be attributed to the different environmental conditions of sampling sites. Especially, it is noteworthy to note that Ispir-2 population produced the highest level of hyperforin, hypericin, pseudohypericin, hyperoside and quercetine and this collection site had the highest elevation (2,450 m) indicating elevation may have a positive effect on secondary metabolite accumulation in H. orientale. However, the present findings also indicate a significant genetic difference/similarity among the populations. For example, Ispir-2 and Ladik-2 populations are separated by a distance of 400 km and have substantially different environment (mean temperature, precipitation, elevation etc.). Hence, they should represent distinct populations. However, both populations produced similar amount of hyperforin, quercetine and isoquercetine. On the contrary, although Kizlan and Ladik-1 are separated by only 10 km and have very similar environment, three- and fourfold differences were detected between the populations in chlorogenic acid and hyperoside contents, respectively. Generally, the observed accumulation levels of hypericins, hyperforin and rutin, hyperoside, quercitrine and quercetine were similar those reported by Smelcerovic et al. (2008) who studied the chemistry of several Hypericum species from Turkey including H. orientale. Similar variations in the content of different phenolics for some other plant species were also reported (Amaral et al. 2010; Dossett et al. 2010). However, it should be noted that it is necessary to perform detailed analyses using biochemical (i.e. allozymes) and molecular (i.e. RFLP, RAPD, AFLP, ISSR) markers to establish the whole range of genetic diversity of populations. Thus, it would be possible to discriminate exactly the effects of genetic and environmental factors on the observed chemical variability among populations. For example, the genetic diversity within and among seven Tunisian natural populations of H. humifusum L., from different geographic regions and bioclimates, was assessed using 11 isozymic polymorphic loci, and 166 RAPD markers by Afef et al. (2012). The authors reported a high genetic variation among Tunisian H. humifusum populations and pointed out the necessity for further studies including the variation of the chemical composition among populations and its relationship with the genetic diversity. Combined phytochemical, morphological and molecular studies are also in progress to evaluate the diversity within and among species of Hypericum (Nürk and Crockett 2011).
The localization of the various types of secretory structures in which biologically active substances are synthesized and/or accumulated varies among plant tissues, and the levels of phytomedicinal compounds present in a particular Hypericum tissue depends on the relative abundance of these secretory structures on the harvested material. In the present study, flowers were found to be main organs for the accumulations of hyperforin, hypericin, pseudohypericin and rutin while chlorogenic acid, hyperoside, isoquercetine, kaempferol, quercitrin and quercetin were accumulated mainly in leaves. Similarly, floral parts had the highest level of hyperforin, hypericin and pseudohypericin in H. perforatum (Sirvent et al. 2002) and leaves were superior over generative tissues with respect to chlorogenic adic and hyperoside accumulation in H. origanifolium and H. perfoliatum (Cirak et al. 2007c, d).
Results from the present study indicate that H. orientale accumulates lower concentrations of hyperforin, hypericin, rutin, quercetin and quercitrine, comparable concentrations of pseudohypericin and higher concentrations of chlorogenic acid and hyperoside when compared to H. perforatum, a well known and commercial source of the compounds examined (Table 3). In taxonomic point of view, besides, H. orientale belongs to the section Crossophyllum Spach. (Robson 2001). To author’s knowledge only one other member of the section, H. adenotrichum Spach. had already been investigated for the presence of main secondary metabolites (Cirak et al. 2009). The comparison of our results with our previously published report revealed that both members of the section have similar array of constituents and contain hyperforin, hypericin, pseudohypericin, chlorogenic acid, rutin, hyperoside, quercitrine and quercetine. Among the chemicals, hypericins have a proven taxonomic value for the infrageneric classification of the genus Hypericum. Because they are not found in species from the primitive sections and seem to be specific only for the taxa of phylogenetically more advanced sections (Robson 2001). Hence, detection of hypericin as well as hyperforin and the phenolics examided in H. orientale in the present study supports the taxonomic position of the section Crossophyllum within the genus Hypericum.
Conclusions
Results of the present study indicate a significant variation in the chemical contents of H. orientale from Turkish populations. It is interesting to note that we did not detect hypericin in this species, represented by only one wild population in our previous study (Cirak et al. 2007a). Hence, regional distribution of this medicinal plant may be an important source of chemical variability and should be considered while optimizing the processing methodology of wild-harvested plant material. Further studies including molecular studies on the wild populations and the relationship between their genetic profiles and secondary metabolite contents are necessary to make more substantial conclusions. Besides, in the present study, the accumulation of isoquercetine in this species was reported by us for the first time. Such kind of data could also be useful for elucidation of the chemotaxonomical significance of the corresponding compounds and phytochemical evaluation of H. orientale.
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Communicated by M. J. Reigosa.
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Cirak, C., Radusiene, J., Stanius, Z. et al. Secondary metabolites of Hypericum orientale L. growing in Turkey: variation among populations and plant parts. Acta Physiol Plant 34, 1313–1320 (2012). https://doi.org/10.1007/s11738-012-0928-8
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DOI: https://doi.org/10.1007/s11738-012-0928-8