Secondary metabolites of Hypericum scabrum and Hypericum bupleuroides

Abstract

The genus Hypericum (Guttiferae) is a source of biologically active secondary metabolites, notably hyperforin, hypericins and various phenolics. In the present study the presence of the phloroglucinol derivative hyperforin, the naphthodianthrones hypericin and pseudohypericin, the phenylpropane chlorogenic acid and the flavonoids such as rutin, hyperoside, apigenin-7-O-glucoside, kaempferol, quercitrin, quercetin and amentoflavone were investigated in two Turkish species of Hypericum, namely, Hypericum scabrum L. and Hypericum bupleuroides Gris. The aerial parts representing a total of 30 individuals were collected at full flowering and dissected into floral, leaf and stem tissues. After being dried at room temperature, the plant materials were assayed for secondary metabolite concentrations by HPLC. All of the secondary metabolites examined were detected in both species at various levels depending on plant tissue except for hyperforin which was not accumulated in Hypericum scabrum. The presence of hyperforin and the phenolic compounds examined in both species were reported by us for the first time.

Keywords::

• Hypericin
• pseudohypericin
• hyperforin
• phenolic constituents
• morphogenetic variation
• secondary chemistry

Introduction

Hypericum (Guttiferae) is a genus of about 400 species of flowering plants. These species have been used for medicinal purposes due to their various medicinal properties for hundreds of years (Demirci et al., 2005). In particular, extracts of Hypericum perforatum L. are now widely used in Europe as a drug for the treatment of depression (Patocka, 2003). Turkey is an important centre for Hypericum genus with 89 species, of which 43 are endemic (Davis, 1988).

Hypericum scabrum L. is an herbaceous perennial plant which is distributed in dry rocky slopes and open woodland of Turkey (Davis, 1988) and has been used by Turkish folk for its antispasmodic, sedative and anti-inflammatory properties (Tanker, 1971). In the folk medicine of Turkey, a skin ointment was prepared from aerial parts of the plant and used as remedy against psoriasis. H. scabrum has an important pharmacological potential, with its well documented antibacterial (Azırak & Erdoğrul, 2003) and antimicrobial (Erdoğrul et al., 2004) activities.

Hypericum bupleuroides Gris. is another herbaceous perennial species of Hypericum genus growing in damp forest places of high altitudes in north-east Turkey. In Turkish folk medicine, the plant has been used in the treatment of skin burns and intestinal disorders (Baytop, 1999).

Hyperforin, hypericins and phenolics are thought to be the main ingredients of Hypericum extract, which is available over the counter for treatment of several diseases (Chu et al., 2000; Bauer et al., 2001). Results from recent studies have indicated hyperforin as the main compound, responsible for antidepressant effects of Hypericum extracts (Roz & Rehavi, 2004). It also exhibits anti-inflammatory (Feisst & Werz, 2004), antitumoral (Schwarz et al., 2003) and antiangiogenic (Dona et al., 2004) effects. The naturally occurring red pigments hypericin and pseudoyhpericin have been reported to exhibit important biological activities, namely photodynamic, antiviral, antiretroviral, antibacterial, antipsoriatic, antidepressant and antitumoral activities (Guedes & Eriksson, 2005). Hypericins have been found only in Hypericum species, thus, are also important chemotaxonomically for the infrageneric classification of Hypericum genus (Kitanov, 2001). Flavonoids are a group of bioactive phenolics of Hypericum plants. Results from clinical studies have indicated the possible role of flavonoids in prevention of cardiovascular diseases and some kinds of cancer (Chu et al., 2000). Although hyperforin and hypericins have been reported to mainly contribute to the pharmacological effects of Hypericum extracts, flavonoids have also made an important contribution to the antidepressant activity (Noldner & Schotz, 2002; Gastpar & Zeller, 2005).

Increased market demand for Hyperici herba has led to intensive studies on the chemistry and biological activities of Hypericum species. This is especially true for H. perforatum, which is the most common and well known plant. 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. Several previous investigations were carried on the concentrations of hypericins in H. scabrum and H. bupleuroides (Zevakova et al., 1991; Kitanov, 2001; Ayan et al., 2004, 2008). However, corresponding species have not yet been studied for the presence of hyperforin and phenolics to date. Thus, the aim of the present study was to determine the accumulation of the phloroglucinol derivative hyperforin, the naphthodianthrones hypericin and pseudohypericin, the phenylpropane chlorogenic acid and the flavonoids such as rutin, hyperoside, apigenin-7-O-glucoside, kaempferol, quercitrin, quercetin, and amentoflavone in H. scabrum and H. bupleuroides.

Materials and methods

The plant materials of H. scabrum and H. bupleuroides were collected from rocky slopes within the Maçka district of Trabzon province, Turkey (40° 49′ N; 39° 37′ E; 970 m above sea level) in July, 2007. The species (identified by Prof. Güray Kutbay, Ondokuz Mayis University, Faculty of Science and Art, Department of Biology, Turkey) were determined according to Davis (1988), and voucher specimens were deposited in the herbarium of Ondokuz Mayis University Vocational School of Bafra (BMYO No. 2 for H. scabrum and BMYO No. 3 for H. bupleuroides). For each species, 30 plants were selected randomly at full flowering and the top two thirds of the shoots were harvested between 12:00 p.m and 13:00 p.m. In the laboratory they were dissected into floral, leaf and stem tissues and pooled. The samples were dried at room temperature (20° ± 2°C) to a constant moisture content of 10%.

Air-dried plant material was ground in a laboratory mill (6000 rpm) to obtain a homogenous drug powder. Three sub-samples (0.5-1 g) of each sample were extracted with 50 mL of 96% EtOH for 72 h at room temperature. The extracts were kept dark in a refrigerator (+4°C) until used. Before the HPLC analysis extracts were exposed to light for 30 min to allow the conversion of protohypericins into hypericins (Kurth & Spreemann, 1998; Michelitsch et al., 2000). Aliquots of 1 mL of the extracts were analyzed undiluted for hypericins and hyperforin, and diluted with 19 mL of EtOH for phenolics. All solvents and standards were of HPLC grade and purchased from Roth (Karlsruhe, Germany).

HPLC analysis with UV/PDA was performed using a Waters model 2690 chromatography system (Waters, Milford, USA), equipped with Waters 2487 UV/Vis and Waters 996 PDA detectors. Separation of hyperforin and hypericins was carried out using a Hichrom column Hypersil H5ODS-150A (150 × 4.6 mm, 5 μm) (Hichrom, Berkshire, UK) and an H5ODS-10C guard-cartridge. The hyperforin elution program was isocratic. The solvent system of the mobile phase consisted of 25% water containing 0.1% trifluoroacetic acid (TFA) and 75% acetonitrile containing 0.1% TFA. The flow rate was kept at 1.5 mL/min. The detector monitored the eluted components at 270 nm, depending on λ_max of PDA spectrum of hyperforin. The samples (10 μL) were injected. The eluted hyperforin was identified on the basis of the retention time by comparison with the retention time of the reference standard. The identity of the constituent was also confirmed with PDA detector by comparison with UV spectra of the reference standard at the wavelength range 190–400 nm.

Hypericins were analyzed according to a modified HPLC method (EDQM, 2004). The elution program was isocratic. The mobile phase consisted of ethyl acetate/15.6 g/L solution of sodium dihydrogen phosphate NaH₂PO₄ and methanol (39:41:160). The flow rate was 1 mL/min, injection volume was 10 μL and the column temperature was 20°C. The elution was monitored at 590 nm and obtained data were compared with those of hypericin and pseudohypericin standard samples.

Separation of phenolics was carried out using an XTerra RP18 column (150 × 3.9 mm, 3.5 μm) and Supelguard^™Ascentis^™ RP-Amide 20 × 4 mm guard- precolumn. A binary solvent system of the mobile phase was performed consisting of solvent A (5% water containing 0.1% TFA) and solvent B (95% acetonitrile containing 0.1% TFA). The following gradient elution program was used: 0-45 min 95-55% A, 5-45% B; 45-50 min 55% A, 45% B; 50-55 min 55-95% A, 45-5% B. Flow rate was 0.4 mL/min; injection volume: 10 μL. The column temperature was 20°C. The elution was monitored at 360 nm. The eluted components were identified on the basis of the retention time by comparison with the retention time of the reference standard. Identity of constituents was also confirmed with PDA detector by comparison with UV spectra of reference standards in the wavelength range from 220 to 380 nm.

Each extract was analyzed twice, and the mean value was used for the calculation of concentrations in plant material.

Results and discussion

The concentration of compounds in the extracts was calculated from an external standard calibration in the concentration range of 0.5–100 μg/mL (r² = 0.99994 for hyperforin, r² = 0.99461 for hypericins, r² = 0.99985 for amentoflavone, r² = 0.99909 for chlorogenic acid, r² = 0.99921 for rutin, r² = 0.99984 for hyperoside, r² = 0.99968 for quercitrin, r² = 0.99974 for quercetin, r² = 0.99970 for apigenin-7-O-glucoside and r² = 0.999754 for kaempferol). Typical HPLC chromatograms of hyperforin, hypericins and phenolics of flower extracts are shown in Figures 1, 2 and 3.

Figure 1.  Typical chromatogram of H. bupleuroides flower extract obtained by HPLC separation at 270 nm. Peak identified: 1 –hyperforin.

Figure 2.  Typical chromatograms of Hypericum scabrum (A) and H. bupleuroides (B) flower extracts obtained by HPLC separation at 590 nm. Peak identified: 1–pseudohypericin, 2–hypericin.

Figure 3.  Typical chromatograms of Hypericum scabrum (A) and H. bupleuroides (B) flower extracts obtained by HPLC separation at 360 nm. Peak identified: 1–chlorogenic acid, 2–rutin, 3–hyperoside, 4–apigenin-7-O-glucoside, 5–quercitrin, 6–quercetin, 7–kaempferol, 8–amentoflavone.

All of the secondary metabolites examined were detected in both species at various levels depending on plant tissue except for hyperforin which was not accumulated in stems, leaves, and flowers of H. scabrum. In H. scabrum, hypericin and pseudohypericin were detected only in flowers. Stems and leaves did not produce those secondary metabolites. Similarly, amentoflavone did not accumulate in stems. Among different plant parts, leaves were found to be superior over flowers and stems in terms of chlorogenic acid, rutin, hyperoside, apigenin-7-O-glucoside, and quercetin accumulations, while flowers produced higher levels of kaempferol, quercitrin, and amentoflavone. In H. bupleuroides, hypericin, pseudohypericin, and hyperforin did not accumulate in stems and leaves whereas flowers produced those secondary metabolites. The highest accumulation levels of the secondary metabolites examined were established in flowers with the exception of the flavonoid apigenin-7-O-glucoside which was produced mainly by leaves (Table 1).

Table 1.  Secondary metabolite concentrations in stem, leaf and flower of Hypericum scabrum and H. bupleuroides (mg/g DW). Data are means of three extracts prepared from a pooled sample of 30 plants for each species.

	Hypericum scabrum			Hypericum bupleuroides
Compounds	Stem	Leaf	Flower	Stem	Leaf	Flower
Hyperforin	0	0	0	0	0	0.451
Hypericin	0	0	0.071	0	0	0.352
Pseudohypericin	0	0	0.191	0	0	0.073
Chlorogenic Acid	0.038	1.01	0.170	0.306	1.300	5.608
Rutin	0.007	1.880	0.749	0.058	0.028	0.141
Hyperoside	0.408	1.480	1.123	2.827	4.519	7.386
Apigenin-7-O-glucoside	0.104	1.347	0.073	0.099	0.285	1.948
Kaempferol	0.048	0.179	0.649	0.134	0.268	0.693
Quercitrin	0.002	0.003	0.014	0.006	0.005	0.014
Quercetin	0.233	1.202	0.672	0.102	1.362	1.799
Amentoflavone	0	0.003	0.044	0	0.003	0.054

The occurrence of hypericins in H. scabrum and H. bupleuroides has long been a disputed subject. Hypericin and pseudohypericin were not detected in both species by Kitanov (2001) and Ayan et al. (2004). Ayan et al. (2008) reported that H. scabrum contained both hypericin forms and Zevakova et al. (1991) determined only hypericin, not pseudohypericin in this species. The different results of the present and previous works can potentially be attributed to the analytical methods used for hypericin and pseudohypericin determinations. Kitanov (2001) and Ayan et al. (2004) used DAC (1986) method concerning spectrophotometric assay for total hypericins. In our case, we used HPLC method for hypericin and pseudohypericin determination as adopted by Zevakova et al. (1991) and Ayan et al. (2008).

Hypericum plants are characterized morphologically by three kinds of secretory structures, including light glands, dark glands and secretory canals, in which biologically active substances are synthesized and/or accumulated (Ciccarelli et al., 2001). Generally, the localization of the various types of secretory structures 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. Hence, organ-dependence of a given chemical is common among Hypericum species. The differences in chemical composition between leaves and flowers for H. scabrum and H. bupleuroides did not correspond in exactly the same way to each other as well as those described for other Hypericum species. Floral parts had the highest level of quercitrin, quercetin, and apigenin-7-O-glucoside, whereas leaves were superior to generative tissues with respect to chlorogenic acid and hyperoside accumulations in Hypericum origanifolium Willd (Çırak et al., 2007a). Flowers of Hypericum montbretii Spach accumulated the highest level of hyperforin and apigenin-7-O-glucoside and the highest concentration of quercitrin was found in stems of flowering plants while leaves were superior to the other tissues with respect to chlorogenic acid and hyperoside accumulations (Çırak & Radusiene, 2007). In the case of H. perforatum, flowers accumulated larger amounts of hypericin, quercetin, and quercitrin, and leaves had the highest levels of hyperoside and rutin (Kazlauskas & Bagdonaite, 2004; Radusiene et al., 2004; Çırak et al., 2007b). The difference in localizations of secondary metabolites among Hypericum plants may be attributed to the genotypic differences of corresponding species.

Results from the present study indicate that H. scabrum accumulates lower concentrations of hypericin, chlorogenic acid, rutin, hyperoside, quercitrin, and quercetin, comparable amounts of pseudohypericin and higher quantities of apigenin-7-O-glucoside and amentoflavone, while H. bupleuroides accumulates lower concentrations of hyperforin, hypericin, pseudohypericin, rutin, quercitrin, and quercetin, comparable concentrations of hyperoside and higher concentrations of chlorogenic acid, apigenin-7-O-glucoside and amentoflavone when compared to H. perforatum, a well known and commercial source of the secondary metabolites examined (Table 2). The presence of kaempferol has not been reported for H. perforatum and might possibly be specific for H. scabrum and H. bupleuroides.

Table 2.  Comparison of the contents of secondary metabolites (mg/g DW) in H. scabrum, H. bupleuroides (this study) and H. perforatum (compiled from various sources).

Compounds	H. scabrum	H. bupleuroides	H. perforatum	References*
Hyperforin	0**	0-0.451	8.35-150	1-3
Hypericin	0-0.071	0-0.352	0.01-2.37	4-7
Pseudo-hypericin	0-0.191	0-0.073	0.05-1.24	8
Chlorogenic acid	0.038-1.01	0.306-5.608	1.11-2.19	2
Rutin	0.007-1.880	0.028-0.141	0.19-34.71	9, 10
Hyperoside	0.408-1.480	2.827-7.386	2.07-7.69	2
Apigenin-7-O-glucoside	0.073-1.347	0.099-1.948	0.05-0.2	11
Quercitrin	0.002-0.014	0.005-0.014	0.05-4.77	9, 10
Quercetin	0.233-1.202	0.102-1.799	0.05-24.12	9, 10
Kaempferol	0.048-0.649	0.134-0.693	-	No previous report
Amentoflavone	0-0.044	0-0.054	0.001-0.005	3

*References: ¹Kirakosyan et al., (2003); ²Maggi et al., (2004); ³Greeson et al., (2001); ⁴Walker et al., (2001); ⁵Sirvent et al., (2002); ⁶Southwell and Bourke (2001); ⁷Çırak et al., (2006); ⁸Ayan and Çırak (2008); ⁹Radusiene et al., (2004); ¹⁰Martonfi and Repçak (1994); ¹¹Çırak et al., (2007b)

**Data for H. scabrum, H. bupleuroides are the lowest and highest levels of the corresponding compounds observed in the present study.

Conclusions

Considering the pharmacological significance of hyperforin, hypericins, and phenolics, their possible use in therapeutics and the growing interest in analytical data on plant secondary metabolites, it is important to find new sources of these compounds. The results encourage further studies on documenting the agricultural and pharmacological potentials of H. scabrum and H. bupleuroides. In the present study, the presence of hyperforin and the phenolic compounds examined in both species was reported by us for the first time. Such data could also be useful for elucidation of the chemotaxonomic significance of the corresponding compounds.

Declaration of interest: The chemical evaluation was supported by the Scientific Foundation of Kaunas University of Medicine. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.