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The Potential of Natural Resources of Hypericum Maculatum Crantz Growing Wild in Levočské Vrchy (Slovakia)

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The Potential of Natural Resources of Hypericum Maculatum Crantz Growing Wild in Levočské Vrchy (Slovakia) THE POTENTIAL OF NATURAL RESOURCES OF HYPERICUM MACULATUM CRANTZ GROWING WILD IN LEVOČSKÉ VRCHY (SLOVAKIA) Beáta Taylorová Department of Ecology and Environmental Sciences, Faculty of Nature Sciences, Constantine the Philosopher University, 1, Tr. A. Hlinku, 065 01 Nitra, Slovakia Corresponding author: [email protected] Abstract Wild populations of Hypericum maculatum CRANTZ (Hypericaceae) were studied repeatedly in a former Military Training Area Javorina in Levočské vrchy (Slovakia) during the vegetation periods in 2009-2011. The aim of the study was investigation of natural resources of H. maculatum CRANTZ and evaluation of selected production properties - the total yield and active substance levels (total hypericin) in dried Hyperici herba during the five different developmental stages in order to find a potential source for breeding and collection. The plant material was collected from four mountain meadows under two different management regimes. Dried Hyperici herba was extracted to methanol and the content of diantron derivates was determined by the spectrophotometric method (Ph. Eur. 4). Total hypericin content increased with advancing of the stages and rapidly decreased in brown capsules. The maximum hypericin content was recorded during the stage with fully opened flowers (0,16 – 0,29 %) which is principally regarded as the appropriate harvesting time. Fresh and dry weight of plant material was recorded to estimate the biomass at the end of vegetation period. The dry weight varied from 13,34 g/m2 – 102,60 g/m2. The results of evaluation did not show the significant differences in total yield regarding management regime. Key words: Hypericum maculatum CRANTZ , biomass, hypericin, wild populations 1 Introduction In the last few years an increasing interest has been experienced for collection of medicinal plants from wild populations due to an effort to keep pace with growing consumer demand for natural products. Hypericum maculatum CRANTZ is a perennial herb 0,4 – 1.0 m tall, erect or ascending from creeping and rooting base. Stems are always 4-lined with subsidiary lines. Leaves usually without pale glands, venation densely reticulate. Inflorescence rather strict, branches make an angle 25 - 45˚with stem. Sepals are 3.1 – 4.5 x 2.1 – 2.8 mm, entire, broadly elliptic to broadly ovate (ratio length to width 1.3 – 1.9). Petals 9.0 – 11.0 x 3.6 – 5.5 mm, entire, laminar black glands mainly punctiform. Chromosome number 2n = 16 (Mártonfi et al., 1999). The pharmaceutical and therapeutic quality of the Hypericum extracts is highly dependent on the quality of the original herbal material (Schulz et al., 1998). A functional relationship between ontogenetic change and development and between development and accumulation of secondary metabolites has been verified for several medicinal plants. Several groups of secondary metabolites are accumulated by representatives of the genus Hypericum and they are synthesised by various biosynthetic pathways (Mártonfi et al., 1996). The dried herb contains significant amounts of naphthodianthrone structures hypericin and pseudohypericin from which the antidepressant activity is considered to derive. The fresh plant also contains significant levels of protohypericin and pseudohypericin, which are converted into hypericin and pseudohypericin during drying and processing, as a result of irradiation with visible light (Dewick, 2009). Although Hypericum perforatum L. has been used as a herbal remedy for many years, wild populations of Hypericum maculatum CRANTZ have shown to have some secondary metabolites in common. 192 According to Mártonfi et al. (2006) although the probable parent species, diploid H. maculatum CRANTZ is given in several pharmacopoeias together with H. perforatum as component of the drug Herba hyperici, rather little attention was paid to its secondary metabolites and their dynamics. The decrease in number and area of managed hay-meadows over the last century, in combination with the reduction of traditional management and changes in agriculture, threatens the biodiversity connected to these habitats and therefore the research is needed to support management with regard to sustainable development. 2 Material and Methods 2.1 Study sites and management Four wooded-meadows of varying conditions (Tab. 1) with 20 x 20 m permanent plots situated in a former Military Training Area Javorina in the Levočské vrchy, East Slovakia were selected for field studies during vegetation periods in 2009-2011. The meadows situated in L2 and L5 have a similar long management history with the components of traditional management - raking, mowing for hay, mulching and aftermath grazing. L1 and L3 represent semi-natural meadows with long no management history. Tab. 1 General properties of studied sites of Hypericum maculatum, CRANTZ in Levočské vrchy, Slovakia (Taylorová, 2012) Altitude Site No Site / habitat Longitude Latitude (m.a.s.l.) Hadia lúka / native grassland, L1 N 49˚ 06′ 36″ E 20˚ 30′ 16″ 776 meadow at the edge of the wood Kacvink / semi-natural grassland, L2 N 49˚ 05′ 46″ E 20˚ 31′ 60″ 833 hay-meadow with mowing, grazing Hradiská dolina / native grassland, L3 N 49˚ 04′ 16″ E 20˚ 32′ 24″ 819 meadow by the small lake Dvorce / semi-natural grassland, L5 hay-meadow with mowing, raking, N 49˚ 05′ 03″ E 20˚ 33′ 45″ 927 grazing 2.2 Plant material Samples were collected at each site in a marked out plot of 20 x 20 m. Within each plot five permanent 0,5 x 0,5 m quadrates were randomly selected. Hyperici herba was obtained from Hypericum maculatum CRANTZ by random selection of plants in permanent plots in five different ontogenesis stages (vegetative stage, stage of closed green buds, beginning of blooming, stage with fully opened flowers and stage of brown capsules), air-dried and analysed for hypericin content. At the end of each vegetation period plants from all 20 permanent quadrates were collected and the fresh and dry weight was recorded to estimate the biomass. 2.3 Quantitative determination of hypericin content Air-dried leaves and flowers were extracted in methanol. The determination of the total hypericin content in plant materials was carried out by spectrophotometric method at λ=590 nm. A Shimadzu UV-1800 apparatus (Japan) was used for the measurements. Three determinations were made from each sample and the mean value was calculated. 193 2.4 Quantitative determination of biomass production The above and under-ground fresh and dry biomass weight was recorded straight after the collection using the scale KERN EMB 500-K1. The dry biomass weight was recorded after air-drying process. 2.5 Data analyses Calculations of results were done in Excel program. Together 20 samples each year were analysed for hypericin and three determinations were made from each sample, the mean value was calculated for each ontogenetic stage. The biomass was calculated for each site as the mean from 5 quadrates and estimated in g.m-2. 3 Results and discussion Hypericin and pseudohypericin from the group of naphthodianthrones are known in many Hypericum taxa and they are present in the leaves and flowers of H. maculatum CRANTZ. Generally, the total hypericin contents increased with advanced generative stages and rapidly decreased in the stage of brown capsules with no black glands (Tab. 2). The quality and quantity of active substances of medicinal plants under various ontogenesis stages are different (Mártonfi, Repčák, 1994, Stefanovits-Bányai et al., 2002). According to earlier experiences (Büter, 2002) employing the correct developmental stage i.e., keeping the appropriate harvesting time, is imperative for the production of a Hyperici herba drug with a satisfying hypericin content. In harmony with the literature data published by Repčák and Mártonfi (1997) we have found that the generative parts (closed green buds, beginning of blooming, stage with fully opened flowers) of the plants contain more hypericin (0.05 – 0.29%) than the vegetative parts (0.03 – 0.18%). Tab. 2 Changes of total hypericin content (%) in different developmental stages in wild populations of Hypericum maculatum CRANTZ, Levočské vrchy, Slovakia (Taylorová, 2012) Developmental stages vegetative closed green beginning of fully opened brown Site Year stage buds blooming flowers capsules 2009 0,115 0,122 0,146 0,172 0,029 L1 2010 0,133 0,185 0,221 0,265 0,122 2011 0,087 0,129 0,153 0,176 0,038 2009 0,108 0,112 0,155 0,178 0,027 L2 2010 0,122 0,227 0,239 0,178 0,063 2011 0,057 0,064 0,168 0,277 0,029 2009 0,085 0,137 0,092 0,161 0,022 L3 2010 0,183 0,190 0,273 0,290 0,053 2011 0,027 0,048 0,200 0,279 0,015 2009 0,085 0,129 0,133 0,174 0,043 L5 2010 0,165 0,199 0,255 0,266 0,122 2011 0,056 0,074 0,176 0,263 0,034 Although according to the European Pharmacopoeia 6.0, Hyperici herba are dried flowering tops of Hypericum perforatum, L. harvested during flowering time and the minimum content of total hypericin in the drug has to be 0.08%, with the average values of 194 total hypericin in the stage with fully opened flowers, H. maculatum CRANTZ growing wild in Levočské vrchy can be considered as a potential source. The average values of hypericin were higher in the first vegetation cycle in plant material collected from meadows with traditional management. In accordance with Maškova et al. (2009) H. maculatum seems to be promoted by mulching and slightly suppressed by mowing and leaving the meadow fallow. We experienced the heterogeneity of yields (Tab. 3) due to individual differences in the number of growing plants. Limits of herba masses (above-ground fresh biomass) in population averages, experienced by one cutting per year were as follows: 36,06 - 118,08 g/m2 in the first, 34,03 - 161,97 g/m2 in the second and 46,90 - 178,90 g/m2 in the third vegetation cycle.
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