C. ÇIRAK, J. RADUŠIENĖ, V. JANULIS, L. IVANAUSKAS, N. ÇAMAŞ, A. K. AYAN

Turk J Biol 35 (2011) 449-456 © TÜBİTAK doi:10.3906/biy-1002-36

Phenolic constituents of Hypericum triquetrifolium Turra (Guttiferae) growing in Turkey: variation among populations and plant parts

Cüneyt ÇIRAK1,*, Jolita RADUŠIENĖ2, Valdimaras JANULIS3, Liudas IVANAUSKAS3, Necdet ÇAMAŞ1, Ali Kemal AYAN1 1Ondokuz Mayıs University, Bafra Vocational High School, 55040 Samsun - TURKEY 2Institute of Botany, Zaliuju ezeru 49, Vilnius LT-08406 - LITHUANIA 3Kaunas University of Medicine, Mickeviciaus 9, LT-44307 - LITHUANIA

Received: 19.02.2010

Abstract: Hypericum triquetrifolium Turra (Guttiferae) has received considerable scientifi c interest in recent years, as it is a source of a variety of biologically active compounds including phenolics. Th e present study was conducted to determine the variation in the content of several phenolics, namely phenylpropane chlorogenic acid and fl avonoids such as , hyperoside, -7-O-glucoside, , , and amentofl avone among four H. triquetrifolium populations from the Central Black Sea Region of Turkey. Th e aerial parts representing a total of 30 shoots were collected at full fl owering in each population. Aft er drying at room temperature, they were dissected into fl oral, leaf and stem tissues and subsequently assayed for phenolic compounds by HPLC. Th e populations varied signifi cantly in phenolic content. Among diff erent plant parts, the fl owers were found to be the principle organ for chlorogenic acid, hyperoside, apigenin-7-O-glucoside, kaempferol, quercetin and amentofl avone accumulations, while rutin and quercitrin were accumulated mainly in the leaves. Th e presence of apigenin-7-O-glucoside and amentofl avone in H. triquetrifolium is reported by us for the fi rst time. Th e chemical variation among the populations and plant parts is discussed as being possibly the result of diff erent genetic, environmental and morphological factors.

Key words: Chlorogenic acid, fl avonoids, HPLC, Hypericum triquetrifolium, population variability

Türkiye’de yetişen Hypericum triquetrifolium Turra (Guttiferae) un fenolik bileşenleri: popülasyon ve bitki kısımları arasındaki varyasyon

Özet: Hypericum triquetrifolium Turra (Guttiferae) son yıllarda bilimsel dikkatleri üzerine çeken bir bitki olup, fenoliklerin de dahil olduğu çok sayıda biyoaktif bileşen içermektedir. Bu çalışma, ülkemizin Orta Karadeniz bölgesinde bulunan dört H. triquetrifolium populasyonunda bazı fenolik bileşenlerin (sırasıyla fenilpropan bileşiği olan klorojenik asit, ve fl avonoid bileşikleri rutin, hiperozit, apigenin-7-O-glukozit, kemferol, kersitrin, kersetin ve amentofl avon) varyasyonunu belirlemek amacıyla yürütülmüştür. Tam çiçeklenme döneminde her popülasyon için toplam 30 bitki toplanmıştır. Materyal oda sıcaklığında kurutulduktan sonra çiçek, yaprak ve sap kısımlarına ayrılmış; fenolik içerikleri belirlenmek üzere HPLC analizlerine tabi tutulmuşlardır. Popülasyonların fenolik içerikleri önemli seviyede farklı bulunmuştur. Farklı bitki kısımları içerisinde klorojenik asit, hiperozit, apigenin-7-O-glukozit, kemferol, kersetin ve

449 Phenolic constituents of Hypericum triquetrifolium Turra (Guttiferae) growing in Turkey: variation among populations and plant parts

amentofl avon’un esas olarak çiçeklerde, rutin ve kersitrin’in ise yapraklarda biriktiği belirlenmiştir. H. triquetrifolium’un apigenin-7-O-glukozit ve amentofl avon içerdiği ilk olarak bu çalışmada rapor edilmiştir. Popülasyonlar ve bitki kısımları arasında gözlenen kimyasal varyasyon farklı genetik, çevresel ve morfolojik faktörlerin muhtemel bir sonucu olduğu bağlamında tartışılmıştır.

Anahtar sözcükler: Klorojenik asit, fl avonoidler, HPLC, Hypericum triquetrifolium, popülasyon değişkenlikleri

Introduction study has been done on the variation in the phenolic Hypericum triquetrifolium Turra (Guttiferae), constituents in H. triquetrifolium. Th us, the present native to Eastern Europe and the Mediterranean study was conducted to determine the variation in the area, is a herbaceous perennial plant that grows in content of the phenylpropane chlorogenic acid and open dry stony/sandy ground and cultivated fi elds fl avonoids, namely rutin, hyperoside, apigenin-7- in Turkey (1). It has traditionally been used in the O-glucoside, kaempferol, quercitrin, quercetin, and treatment of burns and gastrointestinal diseases in amentofl avone among H. triquetrifolium populations Turkish folk medicine (2). Results from recent studies from Turkey. So far, apigenin-7-O-glucoside and reporting the antinociceptive (3), anti-infl ammatory amentofl avone have not yet been detected in H. (4), antioxidant (5), antibacterial (6), antifungal triquetrifolium. Th is article also describes the fi rst (7), and cytotoxic (8) activities of H. triquetrifolium occurrence of these compounds in this species. demonstrate the great potential of this species for use as a medicinal plant. Materials and methods Th e methanolic extract of the aerial parts Brief description of plant materials of Hypericum species has been reported to contain many bioactive compounds, namely the Th e plant materials have been described in our naphthodianthrones hypericin and pseudohypericin previous studies (33). Th e species were identifi ed (9), the phloroglucinol derivatives hyperforin and by Dr. Hasan Korkmaz, Faculty of Science and Art, adhyperforin, fl avonoids (10), essential oils (11), Department of Biology, 19 Mayıs University, Samsun, xanthones (12), tannins (13), procyanidins, and Turkey. other water-soluble components (14) that possess a Experimental procedures wide array of biological properties. Th e aerial parts of H. triquetrifolium plants Phenolic compounds are well-known antioxidant representing a total of 30 shoots were collected at agents, some of which exert therapeutic antiviral, full fl owering from 4 sites in northern Turkey (Table anti-allergic, anti-platelet, and anti-infl ammatory 1). Th e sampling sites were not grazed or mown activities (15-18). Flavonoids have attracted during the plant-gathering period. Th e top 2/3 of the considerable interest as dietary constituents and the plants were harvested between 1100 and 1300 hours. results of clinical studies have indicated their possible Conditions on the day of collection were clear and role in preventing cardiovascular diseases and several sunny at all the sites. Temperatures ranged from 24 kinds of cancer (19). Although hypericins and to 35 °C. Aft er collection, 10 individuals were kept as hyperforin have been reported to mainly contribute whole plants and the rest were dissected into fl oral, to the pharmacological eff ects of Hypericum extracts, leaf and stem tissues. Th e plant materials were dried fl avonoids have also made an important contribution at room temperature (20 ± 2 °C), and subsequently to antidepressant activity (20,21). For these reasons, assayed for their chemical contents by HPLC. many individual or groups of Hypericum species have been investigated for the presence of phenolics Preparation of plant extracts (22-31). H. triquetrifolium was reported previously Samples of 0.5-1.0 g of air-dried plant material to contain chlorogenic acid, rutin, hyperoside, with a moisture content of 10.0% were mechanically quercitrin, quercetin (6), kaempferol (5), and ground with a laboratory mill to obtain a homogeneous phenolic and fl avonoid compounds (32). However, no drug powder and extracted with 96% EtOH (50 mL)

450 C. ÇIRAK, J. RADUŠIENĖ, V. JANULIS, L. IVANAUSKAS, N. ÇAMAŞ, A. K. AYAN

Table 1. Geographical data and seasonal climatic conditions of Hypericum triquetrifolium growing localities from northern Turkey.

Mean Sites Collection date Voucher no. Latitude (N) Longitude (E) Elevation (m) Precipitation (mm) Habitat temperature (°C) Şahinler 20 July 2007 BMYO # 1/1 40°41ʹ 36°23ʹ 640 11.3 653 Rocky and open slopes

Taşova 18 July 2007 BMYO # 1/2 40°43ʹ 36°24ʹ 272 10.6 578 Arid pasturelands

Niksar 19 July 2007 BMYO # 1/3 40°27ʹ 36°44ʹ 713 11.7 450 Arid pasturelands

Erbaa 14 July 2007 BMYO # 1/4 40°39ʹ 36°37ʹ 264 13.2 350 Sides of cultivated area for 72 h, at room temperature. Th e prepared extracts min 55% A, 45% B; 50-55 min 55%-95% A, 45%- were kept in the dark in a refrigerator until used (34). 5% B. Flow rate: 0.4 mL/min. Injection volume: 10 Before HPLC separation, the extracts were fi ltered μL. Th e column temperature was 20 °C. Th e elution through a membrane fi lter with a pore size of 0.22 was monitored at 360 nm and the data obtained were μm (Carl Roth GmbH, Karlsruhe, Germany). compared with authentic samples of the respective HPLC analysis compounds (35). HPLC analysis of phenolic acid and fl avonoids Th e quantity of compounds was calculated from was performed using a Waters model 2690 gradient an external standard calibration in the concentration pump with Waters 2487 UV-detector and XTerra range of 0.5-100.0 μg/mL (r2 = 0.9999). Each sample RP18 column (150 × 3.9 mm, 3.5 μm). Compounds was analyzed twice and the mean value was used on the column were separated with 5% water for calculation. A typical HPLC chromatogram of including 0.1% trifl uoracetic acid (solvent A) and a fl ower extract is shown in Figure 1. All solvents 95% acetonitrile containing 0.1% trifl uoracetic acid and standards of reference substances were of HPLC (solvent B), using the following gradient elution grade and purchased from Roth Chemical Company program: 0-45 min 95%-55% A, 5-45% B; 45-50 (Karlsruhe, Germany).

3 0.140 0.130 0.120 0.110 0.100 0.090 0.080 6

AU 0.070 0.060 0.050 5 0.040 0.030 0.020 0.010 1 4 2 7 8 0.000 200 400 600800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 32003400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 Minutes Figure 1. Typical chromatograms of Hypericum triquetrifolium fl ower extracts obtained by HPLC separation. Peak identifi ed: 1-chlorogenic acid, 2-rutin, 3-hyperoside, 4-apigenin-7-O- glucoside, 5-quercitrin, 6-quercetin, 7-kaempferol, and 8-amentofl avone.

451 Phenolic constituents of Hypericum triquetrifolium Turra (Guttiferae) growing in Turkey: variation among populations and plant parts

Data analysis of which was produced by plants from the Niksar Data for the chlorogenic acid, rutin, hyperoside, population (0.02 mg/g DW) (Table 2). apigenin-7-O-glucoside, kaempferol, quercitrin, Th e stems, leaves and reproductive tissues quercetin, and amentofl avone contents of the plant diff ered signifi cantly with regard to the accumulation material including whole plant, stem, leaf, and fl ower of phenolics (Figures 2 and 3). Th e highest levels of were subjected to ANOVA and signifi cant diff erences chlorogenic acid, hyperoside, quercetin, apigenin- among mean values were tested with the Duncan’s 7-O-glucoside, and amentofl avone were detected in multiple range test (P < 0.01) using MSTAT statistical the fl owers of all populations. Similarly, kaempferol soft ware. Th e mean values of the chemical contents was accumulated mainly in the fl owers of plants were normalized using x’ = √ x + 1 transformation from the Taşova, Niksar, and Erbaa populations, before conducting ANOVA, when necessary, because while the highest level of this compound was found some chemicals were not detected in several cases. in the leaves of plants from the Şahinler population. Of note was that the leaves were found to be superior Results and discussion to the fl owers in terms of rutin and quercitrin accumulations. Leaves produced the highest level Th e content levels of chlorogenic acid, rutin, of these compounds at all sites. As for the stems, hyperoside, quercitrin, quercetin, kaempferol, signifi cantly lower accumulation levels of the apigenin-7-O-glucoside, and amentofl avone in the secondary metabolites were observed in the organs plant materials including whole shoots, stem, leaf, and they did not produce amentofl avone. and fl ower tissues varied signifi cantly among the populations (P < 0.01). Th e chlorogenic acid, rutin, Variations in the levels of bioactive secondary quercitrin, and apigenin-7-O-glucoside contents of metabolites in populations of Hypericum plants have the whole shoots were similar among the populations an important impact on the pharmacological activity and plants, while plants from the Taşova population of the tested extracts (36). Hence, investigations of produced the highest amount of corresponding population variability in the content of secondary compounds (4.04, 5.22, 2.87, and 0.27 mg/g DW for metabolites from diff erent species of Hypericum chlorogenic acid, rutin, quercitrin, and apigenin-7-O- have been performed over several decades. In the glucoside, respectively). Th e hyperoside, quercetin, present study, signifi cant diff erences were detected and kaempferol contents of the whole shoots reached in phenolic accumulation among wild populations the highest level in the Şahinler population (15.97, of H. triquetrifolium. Th e populations were located in 2.08, and 0.02 mg/g DW for hyperoside, quercetin, diff erent parts of northern Turkey and their growing and kaempferol, respectively). Generally, lower sites diff ered from each other in terms of climatic accumulation levels were observed in the Niksar and and geographic factors (Table 1). As a result of these Erbaa populations for all examined compounds with environmental diff erences, each population’s growing the exception of amentofl avone, the highest level site has specifi c ecological conditions and habitat.

Table 2. Chlorogenic acid, rutin, hyperoside, quercitrin, quercetin, kaempferol, apigenin-7-O-glucoside, and amentofl avone contents of Hypericum triquetrifolium whole shoots from wild populations located in northern Turkey (mg/g DW).

Populations Chlorogenic acid Rutin Hyperoside Quercitrin Quercetin Kaempferol Apigenin-7-O-glucoside Amentofl avone

Şahinler 1.77 ba 2.39 b 15.97 a 1.32 b 2.08 a 0.02 a 0.12 b 0.007 b

Taşova 4.04 a 5.22 a 5.46 c 2.87 a 1.76 b 0.005 b 0.27 a 0.006 b

Niksar 2.11 b 2.53 b 3.88 d 2.74 a 0.20 c 0.01 a 0.10 b 0.04 a

Erbaa 0.54 c 2.01 b 8.68 b 1.22 b 1.13 b 0.01 a 0.10 b 0.02 a aValues followed by diff erent small letters in each column are signifi cantly diff erent (P < 0.01) according to Duncan’s multiple range test.

452 C. ÇIRAK, J. RADUŠIENĖ, V. JANULIS, L. IVANAUSKAS, N. ÇAMAŞ, A. K. AYAN

a 7 9 (A) a a (B) 6 7.5 5 b a 6 a 4 b 4.5 3 a 3 a 2 b b b b c a a b b Rutin (mg/g DW) 1.5 b 1 c c b c c 0 0

Chlorogenic acid (mg/g DW) Şahinle Taşova Niksar Erbaa Şahinle Taşova Niksar Erbaa Sites Sites a 6 21 (C) (D) a 18 5 15 a 4 a b a 12 3 b 9 a 2 a b a a 6 b b b b 1 b c 3 c b c c c b Qercitrin (mg/g DW) Qercitrin Hyperoside (mg/g DW) Hyperoside 0 0 Şahinle Taşova Niksar Erbaa Şahinle Taşova Niksar Erbaa Sites Sites Stem Leaf Flower

Figure 2. Chlorogenic acid (A), rutin (B), hyperoside (C), and apigenin-7-O-glucoside (D) contents in stem, leaf, and fl ower of Hypericum triquetrifolium plants from wild populations located in northern Turkey (values with diff erent small letters—a, b, c—within columns for each site diff er signifi cantly at the level of P < 0.01; Bars are ± s.e.).

a 4 (A) 0.04 (B) a a a 3 0.03 b a a 2 b a 0.02 b b 1 b a a b 0.01 b c Qercetin (mg/g DW) Qercetin c c b c c c c 0 Kaempferol (mg/g DW) 0 Şahinle Taşova Niksar Erbaa Şahinle Taşova Niksar Erbaa Sites Sites

0.4 (C) a a 0.06 (D) a 0.05 0.3 a 0.04 0.2 a 0.03 a a a b b b 0.02 0.1 b b b b c 0.01 b c c b

0 (mg/gDW) 0 Şahinle Taşova Niksar Erbaa Şahinle Taşova Niksar Erbaa

Apigenin-7-O-glucoside (mg/g DW) Sites Sites

Stem Leaf Flower

Figure 3. Kaempferol (A), quercitrin (B), quercetin (C), and amentofl avone (D) contents in stem, leaf and fl ower of H. triquetrifolium plants from wild populations located in northern Turkey (values with diff erent small letters—a, b, c—within columns for each site diff er signifi cantly at the level of P < 0.01; Bars are ± s.e.).

453 Phenolic constituents of Hypericum triquetrifolium Turra (Guttiferae) growing in Turkey: variation among populations and plant parts

Th e chemical variability in the content of phenolics Th e diff erences in chemical composition between evaluated among the populations may be attributed the leaves and fl owers for H. triquetrifolium did not to the diff erent environmental conditions of the correspond to those described for other Hypericum sampling sites. However, the present fi ndings also species. Th e fl oral parts had the highest level of indicate a signifi cant genetic diff erence/similarity quercitrin, quercetin, and apigenin-7-O-glucoside, among the populations. For example, the Şahinler and whereas the leaves were superior to generative tissues Niksar populations are separated by a distance of 170 with respect to chlorogenic acid and hyperoside km and have a substantially diff erent environment accumulation in H. origanifolium and H. perfoliatum (mean temperature, precipitation, elevation etc.). (29,30). Th e fl owers of H. montbretii accumulated Hence, they should represent distinct populations. the highest level of apigenin-7-O-glucoside and the However, both populations produced similar highest content of quercitrin was found in stems amounts of chlorogenic acid, rutin, kaempferol, of fl owering plants, while leaves were superior to and apigenin-7-O-glucoside. In contrast, although the other tissues with respect to chlorogenic acid the Şahinler and Taşova populations are separated and hyperoside accumulation (31). In the case of by only 8 km and have very similar environmental H. perforatum, the fl owers accumulated higher conditions, 3- and 4-fold diff erences were detected amounts of hypericin, quercetin, and quercitrin, between the populations in terms of hyperoside and while the leaves had the highest levels of hyperoside kaempferol contents, respectively. and rutin (10,24). Th e diff erences in the phenolic Hypericum plants are characterized by the content of plant tissues among Hypericum plants presence of secretory structures, including light may be attributed to the genotypic diff erences of the glands, dark glands, and secretory canals, in which corresponding species. biologically active substances are synthesized and/ Results from the present study indicate that H. or accumulated (37). Th e localization of the various triquetrifolium accumulates lower concentrations types of secretory structures varies among the plant of rutin and quercetin; comparable concentrations tissues, and the levels of phytomedicinal compounds of chlorogenic acid, apigenin-7-O-glucoside, present in a particular Hypericum tissue depend on and quercitrin; and higher concentrations of the relative abundance of these secretory structures hyperoside and amentofl avone when compared to H. on the harvested material. In the present study, the perforatum, a well known and commercial source of fl owers were found to be the main organs for the the compounds examined (Table 3). Th e results also accumulation of the phenolics tested, except for rutin indicate that both species contain a similar array of and quercitrin. phenolic constituents.

Table 3. Comparison of the content (mg/g DW) of chlorogenic acid, rutin, hyperoside, quercitrin, quercetin, kaempferol, apigenin-7- O-glucoside, and amentofl avone in Hypericum triquetrifolium and H. perforatum.

Compounds H. triquetrifolium H. perforatum References Chlorogenic acid 0.54-4.04a 1.11-2.19 Maggi et al. (38) Radusiene et al. (10); Rutin 2.01-5.22 0.19-34.71 Martonfi and Repçak (22) Hyperoside 3.88-15.97 2.07-7.69 Maggi et al. (38) Apigenin-7-O-glucoside 0.10-0.27 0.05-0.2 Çırak et al. (30) Radusiene et al. (10) Quercitrin 1.22-2.87 0.05-4.77 Martonfi and Repçak (22) Radusiene et al. (10) Quercetin 0.2-2.08 0.05-24.12 Martonfi and Repçak (22) Kaempferol 0.005-0.02 - No previous report Amentofl avone 0.006-0.02 0.001-0.005 Greeson et al. (14) aTh e values are the lowest and highest level of the corresponding phenolics in the whole shoots of plants from diff erent populations.

454 C. ÇIRAK, J. RADUŠIENĖ, V. JANULIS, L. IVANAUSKAS, N. ÇAMAŞ, A. K. AYAN

Th e main issue in medicinal plant standardization signifi cance of the corresponding compounds and is the huge variability in the secondary metabolite the phytochemical evaluation of H. triquetrifolium. profi le of a given plant material. Screening the wild plant populations to obtain the improved phytochemical profi les seems to be fi rst step in Acknowledgment identifying superior germplasm. Th e results of the Th e chemical evaluation was supported by present study indicate a signifi cant variation in the the Scientifi c Foundation of Kaunas University of phenolic constituent content of H. triquetrifolium Medicine. among Turkish populations. Th e regional distribution of this medicinal plant may be an important source Corresponding author: of chemical variability and should be considered while optimizing the processing methodology Cüney ÇIRAK of wild-harvested plant material. In the present Ondokuz Mayıs University, study, the presence of apigenin-7-O-glucoside and Bafra Vocational High School, amentofl avone in H. triquetrifolium was reported by us for the fi rst time. Such data could also be 55040 Samsun - TURKEY useful for the elucidation of the chemotaxonomic E-mail: [email protected]

References

1. Davis PH. Flora of Turkey and the East Aegean Islands. 11. Bertoli A, Menichini F, Mazzetti M et al. Volatile constituents Edinburgh University Press. Edinburgh; 1988. of the leaves and fl owers of Hypericum triquetrifolium Tura. 2. Baytop T. Th erapy with Medicinal Plants in Turkey. İstanbul Flavour Fragr J 18: 91-94, 2003. University Press, İstanbul; 1999. 12. Tanaka N, Takaishi Y. Xanthones from Hypericum chinense. Phytochemistry 67: 2146-2151, 2006. 3. Apaydın S, Zeybek U, Ince I et al. Hypericum triquetrifolium Turra. extract exhibits antinociceptive activity in the mouse. J 13. Dall’Agnol R, Ferraz A, Bernardi AP et al. Antimicrobial Ethnopharmacol 3: 307-312, 1999. activity of some Hypericum species. Phytomedicine 10: 511- 516, 2003. 4. Ozturk B, Apaydin S, Goldeli E et al. Hypericum triquetrifolium Turra. extract exhibits antiinfl ammatory activity in the rat. J 14. Greeson J, Sanford B, Monti DA. St. John’s wort (Hypericum Ethnopharmacol 80: 207-209, 2002. perforatum L.): A review of the current pharmacological, toxicological and clinical literature. Psychopharmacol 153: 5. Conforti F, Statti GA, Tundis R et al. Antioxidant activity of 402-414, 2001. methanolic extract of Hypericum triquetrifolium Turra aerial part. Fitoterapia 6: 479-483, 2002. 15. Russo A, Acquaviva R, Campisi A et al. Biofl avonoids as antiradicals, antioxidants and DNA cleavage protectors. Cell 6. Pistelli L, Bertoli A, Morelli I et al. Chemical and antibacterial Biol Toxicol 16: 91-98, 2000. evaluation of Hypericum triquetrifolium Turra. Phytother Res 19: 787-791, 2005. 16. Gülçin İ, Elias R, Gepdiremen A et al. Antioxidant secoiridoids from fringe tree (Chionanthus virginicus L.). Wood Sci Technol 7. Fraternalea D, Bertoli A, Giamperi L et al. Antifungal 43: 195-212, 2009. evaluation of Hypericum triquetrifolium polar extracts against Fusarium spp. Nat Prod Commun 1: 1117-1122, 2006. 17. Gülçin İ. Comparison of in vitro antioxidant and antiradical activities of L-tyrosine and L-Dopa. Amino Acids 32: 431-438, 8. Conforti F, Loizzo MR, Statti AG et al. Cytotoxic activity of 2007. antioxidant constituents from Hypericum triquetrifolium Tura. Nat Prod Res 21: 42-46, 2007. 18. Isfahlan AJ, Mahmoodzadeh A, Hasanzadeh A et al. Antioxidant and antiradical activities of phenolic extracts from 9. Kitanov GM. Hypericin and pseudohypericin in some Iranian almond (Prunus amygdalus L.) hulls and shells. Turk J Hypericum species. Biochem Syst Ecol 29: 171-178, 2001. Biol 34: 165-173, 2010. 10. Radušienė J, Bagdonaite E, Kazlauskas S. Morphological 19. Chu YH, Chang CL, Hsu HF. Flavonoid content of several and chemical evaluation on and H. vegetables and their antioxidant activity. J Sci Food Agric 80: maculatum in Lithuania. Acta Hort (ISHS) 629: 55-62, 2004. 561-566, 2000.

455 Phenolic constituents of Hypericum triquetrifolium Turra (Guttiferae) growing in Turkey: variation among populations and plant parts

20. Noldner M, Schotz K. Rutin is essential for the antidepressant 30. Çırak C, Radušienė J, Janulis V et al. Chemical constituents activity of Hypericum perforatum extracts in the forced of some Hypericum species growing in Turkey. J Plant Biol 50: swimming test. Planta Med 68: 577-80, 2002. 632-635, 2007. 21. Gastpar M, Zeller K. Hypericum-extrakt STW3 und 31. Çırak C, Radušienė J, Sağlam B. Variation of bioactive sertralin zur behandlung der mittelschweren depression. substances in Hypericum montbretii during plant growth. Nat Psychopharmakotherapie 12: 146-153, 2005. Prod Res 22: 246-252, 2008. 22. Marfonti P, Repcak M. Secondary metabolites during fl ower 32. Toker Z. Variation of total hypericin, phenolic and fl avonoid ontogenesis of Hypericum perforatum L. Zahradnictvi 21: 37- compounds in Hypericum triquetrifolium during its 44, 1994. phenological cycle. Pharm Biol 47: 285-288, 2009. 23. Tekel’ova D, Repcak M, Zemkova E et al. Quantitative changes 33. Ayan AK, Çırak C. Hypericin and pseudohypericin contents of dianthrones, hyperforin and fl avonoids content in the fl ower in some Hypericum species growing in Turkey. Pharm Biol 46: ontogenesis of Hypericum perforatum. Planta Med 66: 778-780, 288-291, 2008. 2000. 34. Gülçin İ, Kireçci E, Akkemik E et al. Antioxidant and 24. Kazlauskas S, Bagdonaite E. Quantitative analysis of active antimicrobial activities of an aquatic plant: Duckweed (Lemna substances in St. John’s wort (Hypericum perforatum L.) by the minor L.). Turk J Biol 34: 175-188, 2010. high performance liquid chromatography method. Medicina 35. Kovacs G, Kuzovkina IN, Szoke E et al. HPLC determination (Kaunas) 40: 975-981, 2004. of fl avonoids in hairy-root cultures of Scutellaria baicalensis 25. Abreu IN, Porto ALM, Marsaioli AJ et al. Distrubution of Georgi. Chromatographia 60: 81-85, 2004. bioactive substances from Hypericum brasiliense during plant 36. Bergonzi MC, Bilia AR, Gallori S et al. Variability in the growth. Plant Sci 167: 949-954, 2004. content of the constituents of Hypericum perforatum L. and 26. Ozen T, Ayan AK, Çırak C et al. Total phenol content of some some commercial extracts. Drug Dev Ind Pharm 27: 491-497, Hypericum species growing in Turkey. Chem Nat Compd 41: 2001. 232-233, 2005. 37. Ciccarelli D, Andreucci AC, Pagni AM. Translucent glands 27. Martonfi P, Repcak M, Zanvit P. Secondary metabolites and secretory canals in Hypericum perforatum, Morphological, variation in Hypericum maculatum and its relatives. Biochem anatomical and histochemical studies during the course of Syst Ecol 34: 56-59, 2006. onthogenesis. Ann Bot-London 88: 637-644, 2001. 28. Ayan AK, Çırak C, Yanar O. Variations in total phenolics 38. Maggi F, Ferretti G, Pocceschi N et al. Morphological, during ontogenetic, morphogenetic, and diurnal cycles in histochemical and phytochemical investigation of the genus Hypericum species from Turkey. J Plant Biol 49: 432-440, 2006. Hypericum of the Central Italy. Fitoterapia 75: 702-711, 2004. 29. Çırak C, Radušienė J, Janulis V et al. Variation of bioactive secondary metabolites in Hypericum origanifolium during its phenological cycle. Acta Physiol Plant 29: 197-203, 2007.

456