MORPHOLOGICAL AND CHEMICAL EVALUATION ON HYPERICUM PERFORATUM AND H. MACULATUM IN LITHUANIA

Abstract

An increasing demand for Hyperici herba has caused the evaluation of this genus in all Europe. Hypericum perforatum L. and H. maculatum Cranz are common species in Lithuania. The aim of the investigations was to examine indigenous populations and field accessions of these species in respect to morphological characters and chemical constituents. The results of evaluation revealed high morphological diversity in both species. The t-test comparison showed the difference of characters from wild and field. The content of flavonoids and hypericin varied highly in species, different accessions, and parts of the plant. Significant differences were detected in the mean concentrations of quercetin, hypericin, rutin and hyperoside+isoquercetrin in flowers and leaves of both species. The flavonoid content appeared to be more constant character in both species. The poorer growth and lower mean concentrations of secondary metabolites were discovered in H. maculatum. The germplasm of Hypericum may be a potential source of genetic variation to allow selecting the valuable material for breeding. INTRODUCTION Recently, Hypericum L. has become one of the most worldwide-evaluated genuses. The big interest in the plant has been caused by its supposedly photodynamic, antidepressant, and antiretroviral activities (Bombardelli and Morazzoni, 1995; Gaedcke, 1997, Vitiello, 1999). Holz and Ostrowski (1987) indicated that flavonoids of H. perforatum had a sedative effect. Hyperforin has been recently found to be most relevant to the antidepressant activity of Hypericum (Chatterjee et al., 1998; Laakman et al., 1998). Hypericin shows a significant antiviral and antiretroviral activity (Vlientinck et al., 1998). H. perforatum L. and H. maculatum Cranz are common species in Lithuania. In folk medicine H. perforatum is known as a remedy against 99 diseases. Natural sources of raw material of H. perforatum are sufficient, however, drug manufacturers tend to utilize plants under controlled conditions rather than gathered from the wild. Only one Russian variety ‘Zolotodolinskaja’ has been grown in Lithuania so far. The objective of this study was to assess morphological and chemical interpopulation variability in wild populations and field accessions of Hypericum, and to reveal the most valuable material for further use in breeding. MATERIALS AND METHODS The material of H. perforatum (from 12 sites) and H. maculatum (from 7 sites) was collected during field trips in 1999 in the phase of full flowering. The seeds have been gathered in fall period and sown in the field collection. The evaluation of field accessions has been carried out in 2001 according to the morphometric analysis of phenotype and content of secondary metabolites. The total of 14 morphological and productive characters including height of plants, number of internodes, width and length of inflorescences, dimensions of the leaves, petals, sepals, dry mass of inflorescences, leaves, shoots and raw material have been measured. The spectrophotometric method was used for determination of total amount (percentage) of flavonoids (Tumanova, 1990). Quantitative analysis of flavonoids and hypericin in the ethanolic extracts of flowers and leaves was carried out by modified Proc. XXVI IHC – Future for Medicinal and Aromatic Plants Eds. L.E. Craker et al. Acta Hort. 629, ISHS 2004 Publication supported by Can. Int. Dev. Agency (CIDA) 56 HPLC gradient elution method (Holzl and Ostrowski, 1987). Compounds were identified by means of pure standards. Differences among populations and accessions were tested by one-way analysis of variance (ANOVA) at α = 0.05 level. The Sheffe multiple comparison test was employed to identify significantly homogenous groups among populations. The hierarchical cluster analysis dendrogram of the population productivity was constructed based on agglomerative grouping and the single linkage between groups clustering method using squared Euclidean distances. RESULTS AND DISCUSSION Variability of Morphological and Productivity Characters The one-way analysis of variance (ANOVA) revealed highly significant differences (p<0.05) among H. perforatum populations within all measured characters. Peak values of the F statistic were observed for height of plants, length of sepals, width and length of petals, width of leaves, and length of inflorescences (Table 1). H. maculatum populations were highly different in height of plants, number of internodes, length and of width of petals (Table 2). The most important characters for distinguishing the morphological variants appeared to be the dimensions of leaves. The leaf length/width ratio in different populations of H. perforatum varied from 2.05 to 3.89. The results indicated three morphological variants of H. perforatum: narrow (4:1), intermediate (3:1), and broad (2:1) leaved. Robson (1968) classified H. perforatum into three varieties: var. perforatum with broad leaves, var. angustifolium with narrow leaves, and var. microphyllum with small leaves. According to our results the broad-leaved populations dominated in Lithuania. Two varieties could be accepted – var. perforatum and var. angustifolium. The populations were homogenous in length and width of petals (CV: 12.01 and 14.01 %, respectively) but more heterogenous in dimensions of sepals (CV: 14.89 and 32.34 %, respectively). Results of the Scheffe test showed the existence of 2 homogenous groups in populations of H. maculatum according to the measurement of leaves within the analyzed data set. The first group was comprised of populations with long and broad leaves, while the other group had short and narrow leaves. The majority of plants had flowers with long petals and short sepals. The height of plants, number of internodes, width and length of inflorescences, and weight of raw material are the most important parameters when the productivity is estimated. The height of plants was the most variable parameter among investigated populations of Hypericum. The mean value of height of both species according to the Scheffe test formed 4 statistically different homogeneous groups. The mean value of height in most populations of H. perforatum varied from 45.0 to 50.7 cm, while of H. maculatum from 54.0 to 58.8 cm. The difference between the average number of internodes of H. perforatum and H. maculatum is not big. However, this parameter varies among species populations very much and makes up 3 homogenous groups: few internodes (up to 16), medium number of internodes (17-19), and many internodes (20 and more). According to the length of inflorescences there were 2 homogeneous subsets in populations of both species. The populations of H. perforatum were more homogenous in length of inflorescences than H. maculatum (CV: 16.5-36.3 % and 26.5-56.8 %, respectively). According to these data the cutting height of flowering horizon when being harvested could be less than 30 cm. The width of inflorescences among populations of H. perforatum and H. maculatum varied highly (CV: 21.8-32.1 % and CV: 29.55-74.35 %, respectively). A hierarchical cluster analysis dendrogram provides evidence for the existence of three groups among the studied H. perforatum populations on the basis of productivity characters (Fig. 1). The majority of populations (6) had the lowest herbal mass. Four populations were of mean productivity. The dendrogram manifested the distinct character