Pharmaceutical Biology, 2009; 47(4): 285–288

o r i g i n a l a r t i c l e

Variation of total hypericin, phenolic and flavonoid compounds in Hypericum triquetrifolium during its phenological cycle

Zuhal Toker

Biology Department, Faculty of Science and Art, University of Dicle, Diyarbakır, Turkey

Address for Correspondence: Dr Zuhal Toker, University of Dicle, Faculty of Science and Art, Biology Department, 21280 Diyarbakır, Turkey. Tel.:+90-412-2488550 ext 3129. Fax: +90-412-2488039. E-mail: ztoker@dicle.edu.tr

(Received 1 May 2008; revised 18 August 2008; accepted 18 August 2008)

Introduction

Hypericum (Hypericaceae) species are herbaceous plants, naturally found in the temperate zones of Europe, Asia, and Africa (Campbell & Delfosse, 1984). The genus is well known for its anti-inflammatory, diuretic, and sedative properties and as a healing agent (Dias et al., 1998). Extracts of the crude drug are widely used in the treatment of depression and have also been identified as a possible anticancer agent (Hubner et al., 1994; Linde et al., 1996; Alecu et al., 1998; Kamuhabwa et al., 2000).

The genus Hypericum is traditional medicinal con-taining a broad spectrum of secondary metabolites (Patocka, 2003). Many pharmacological activities appear to be attributable to their hypericin derivatives and phenolic constituents (Agostinis et al., 2002; Kim et al., 2002).

Hypericin and pseudohypericin are photodynamic pigments, produced from dimerized emodin anthrone (Falk, 1999). Photodynamic hypericin activities dis-played under the influence of light are used for therapy in various diseases. Photoactivated hypericin generates

reactive oxygen species and its photocytotoxicity prop-erties have been proposed as photochemotherapeutic (Vandenbogaerde et al., 1997). These properties allow hypericin to act as an antiviral agent. Attention has been focused on its use against human immunodeficiency virus type 1 (Meruelo et al., 1988).

Recent studies have focused on the health functions of phenolics, including flavonoids and anthocyanins. Phenolic compounds are widely distributed in the plant kingdom. External stimuli such as microbial infections, ultraviolet radiation, and chemical stressors induce their synthesis. The phenolic compounds, flavonoids and furanocoumarins, have many ecologic functions and affect human health. Ecologic functions include defense against microbial pathogens and herbivorous animals. The synthesis of phenolic compounds in plants can be modulated by the application of herbicides and, to a lesser extent, insecticides and fungicides.

Phenolics possess a wide spectrum of biochemi-cal activities such as antioxidant, antimutagenic, and anticarcinogenic, as well as the ability to modify gene expression (Carbanaro et al., 2002; Floridi et al., 2003).

ISSN 1388-0209 print/ISSN 1744-5116 online © 2009 Informa UK LtdDOI: 10.1080/13880200802578983

abstractThe genus Hypericum (Hypericaceae) contains a variety of structurally diverse natural products which possess a wide array of biological properties. The present study examined quantitative variations in total hypericin, phenols, and flavonoids in Hypericum triquetrifolium Turra. in relation to phenological develop-ment. Plant material was harvested at different phenological stages (vegetative, full flowering, and mature fruiting stages). The amounts of bioactive compounds in whole shoots were assayed. The highest total hypericin, phenolic, and flavonoid contents were observed at the full flowering stage.

Key words: Flavonoids; Hypericaceae; Hypericum triquetrifolium; hypericin; phenolic compounds; phenological cycle

http://www.informapharmascience.com/phb

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

ueen

's U

nive

rsity

on

08/1

5/14

For

pers

onal

use

onl

y.

286 Z. Toker

Hypericum triquetrifolium, widespread in warm tem-perate areas throughout the world, is a perennial usu-ally growing on dry, stony, and sandy places (Robson, 1975). The aerial parts of H. triquetrifolium contain more hypericin than Hypericum perforatum L., the main and traditional source of hypericin (Kitanov, 2001; Alali et al., 2004). There are some literature reports on phytochemi-cal analyses of hypericin (Apaydın et al., 1999; Yeşilada et al., 1999) and on the antioxidant effects of ethanol and methanol extracts of H. triquetrifolium (Couladis et al., 2002; Conforti et al., 2007; Kızıl et al., 2008).

Based on the strong biological activity of hypericin and phenolic compounds, the aim of present study was focused on determination of the total hypericin, pheno-lics, and flavonoid contents of H. triquetrifolium during its phenological cycle.

Materials and methods

Plant material

H. triquetrifolium was collected in Diyarbakır in the area of south east of Turkey from May to September 2006 at the stages of vegetative, full flowering, and mature fruit-ing. Voucher specimens have been deposited at the her-barium of the Department of Biology, Faculty of Science and Art, Dicle University (voucher no. DUF-2512). They were identified by Dr A. Selçuk Ertekin from the same institution.

Preparation of extracts

Aerial parts were dried for 10 days at room temperature. Each sample was extracted with 50 mL of 99% ethanol for 72 h at room temperature. The extracts were kept dark in a deep freezer until used for the determination of total phenolic and flavonoid contents.

Determination of total hypericin content

Dried plant material (200 mg) was placed in vials con-taining chloroform (10 mL) and extracted in a sonicator to remove chlorophyll. Next, chloroform was removed under vacuum. After removing chloroform, the samples were re-extracted with methanol (10 mL) in a sonica-tor. The methanol extraction process was repeated three times, and then the methanol was removed under vacuum. The final samples were dissolved in metha-nol and 1 mL of the supernatant was placed in a test tube. Absorbance was measured at 589 nm (CHEBIOS T80+UV/VIS spectrometer). Spectrophotometric analysis was performed by using an eight-point cali-bration curve generated with pure hypericin (Sigma, Italy) as standard (Figure 1). The hypericin content in

H. triquetrifolium was calculated and expressed as micrograms of hypericin per gram of dry material.

Determination of total phenolic content

The content of total phenolic compounds in the etha-nol extract of H. triquetrifolium was determined using Folin–Ciocalteus reagent according to the method of Singleton et al. (1999). Crude ethanol extract (40 L) of H. triquetrifolium (1 mg/mL) was mixed with 200 µL Folin–Ciocalteus reagent (Sigma Aldrich, Germany) and 1160 µL of distilled water, followed by 600 µL of 20% (v/v) Na

2CO

3 three minutes later. The mixture was shaken for

2 h at room temperature and absorbance was measured at 765 nm. Spectrophotometric analysis was performed by using a five-point calibration curve generated with pure gallic acid (Sigma Aldrich) as standard (Figure 2). The concentration of total phenolic compounds in H. triquetrifolium was determined as micrograms of gallic acid equivalents per milligram of extract using the fol-lowing equation obtained from the standard gallic acid graph (R2=0.9875):

Absorbance=0.0024 × gallic acid (µg).

y = 0.0331xR2 = 0.992

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 10 20 30 40 50Concentration (mg/mL)

Abs

orba

nce

Figure 1. Concentration–response curve of absorbance at 589 nm for the hypericin standard.

Figure 2. Concentration–response curve of absorbance at 765 nm for the gallic acid standard.

y = 0.0024xR2 = 0.9875

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 200 400 600

Abs

orba

nce

Concentration (mg/mL)

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

ueen

's U

nive

rsity

on

08/1

5/14

For

pers

onal

use

onl

y.

Bioactive compounds in Hypericum triquetrifolium 287

Determination of total flavonoid content

Measurement of flavonoid concentration of the extract was based on the method described by Park et al. (1997) with a slight modification. An aliquot of 1 mL of the solution (containing 1 mg of extract in metha-nol) was added to a test tube containing 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 M potassium acetate, and 3.8 mL of methanol. After 40 min at room tem-perature, the absorbance was determined at 415 nm. Spectrophotometric analysis was performed by using a five-point calibration curve generated with pure quercetin (Sigma) as standard (Figure 3). The flavonoid content in H. triquetrifolium was expressed in milli-grams of quercetin equivalents per gram fresh weight of plant material.

Statistical analyses

All analyses were performed in triplicate, and the results expressed as mean±standard deviation. Significant dif-ferences among the groups were determined by one-way ANOVA using the SPSS 12.0 software package. The results were considered significant if the value of p was less than 0.05.

Results

The sampled plant material included leaves and stems at the vegetative stage; reproductive parts, leaves, stems, and flowers at the full flower stage; and stems and brown capsules with seeds at the mature fruiting stage.

The total hypericin contents, expressed as µg of hyper-icin in a given dry material, of the H. triquetrifolium methanol extracts (1 mg each) from the vegetative, full flowering, and mature fruiting stage were 140.0, 242.3, and 125.0, respectively.

The total amounts of phenolic compounds, deter-mined as mg gallic acid equivalents/mg extract, of the H. triquetrifolium ethanol extracts (1 mg each) from the

vegetative, full flowering, and mature fruiting stage were 172.3, 212.6, and 133.3, respectively.

The total flavonoid contents, expressed as mg querce-tin equivalents/g fresh weight of plant material, from the vegetative, full flowering, and mature fruiting stage were 173.3, 217.3, and 168.3, respectively.

The highest levels of total hypericin, total flavonoids, and total phenolic compounds were observed at the full flowering stage. The concentration of these com-pounds decreased during fruit development. These differences were statistically significant (p<0.005) (Table 1).

Discussion and conclusions

Concentrations of secondary metabolites in plants may vary substantially with developmental stage. Therefore, investigations on the ontogenic variation of secondary metabolites are worthwhile. In the present study, we investigated the variation in total hypericin, flavonoid, and phenolic contents of H. triquetrifolium during its phenological cycle. The highest hypericin content was obtained from the full flowering stage. This finding is in agreement with previous published results (Alali et al., 2004; Ayan & Çırak, 2008). Similarly, the highest content of hypericin in H. perforatum L. (Kazlauskas & Bagdonaite, 2004; Couceiro et al., 2006), Hypericum prunatum, Hypericum avicularifolium (Çırak et al., 2006), Hypericum perfoliatum (Çırak et al., 2007b) and Hypericum origanifolium Willd. (Çırak et al., 2007b) was determined during flower ontogenesis. The antioxidant activity of herbs results mainly from phenolic com-pounds (Malenčić et al., 2007; Radulovic et al., 2007). In the present work, the total phenolic and flavonoid contents of H. triquetrifolium were found to change dur-ing the phenological cycle. The highest total phenolic and flavonoid contents were also obtained from the full flowering stage.

The present results therefore suggest that, for medici-nal purposes, plant material of H. triquetrifolium should be collected during the full flowering stage, in which bioactive compounds reach their highest level.

y = 0.0154xR2 = 0.9994

0.0

0.2

0.4

0.60.8

1.0

1.2

1.4

0 20 40 60 80

Abs

orba

nce

Concentration (mg/mL)

Figure 3. Concentration–response curve of absorbance at 415 nm for the quercetin standard.

Table 1. Variation in total hypericin, total flavonoid and total phenolic contents (g/g dry weight) of Hypericum triquetrifolium during its phenological cycle.

Phenological stage Hypericin Flavonoids Phenolics

Vegetative 140.0±2.3b 173.3±3.4b 172.3±3.7b

Full flowering 242.3±2.4a 217.3±1.2a 212.6±6.0a

Mature fruiting 125.0±1.1b 168.3±3.5b 133.3±2.7c

Data expressed as mean±standard deviation. a,b,cMean values within a column with unlike superscript letters are significantly different according to Duncan’s multiple range test (p<0.005).

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

ueen

's U

nive

rsity

on

08/1

5/14

For

pers

onal

use

onl

y.

288 Z. Toker

Acknowledgements

The work was supported by research grants from the Dicle University Research Council (DUAPK; project numbers DUAP-2000-FF-410).

Declaration of interest: The authors report no conflicts of interest.

References

Agostinis P, Vantieghem A, Merlevede W, De Witte D (2002): Hypericin in cancer treatment: more light on the way. Int. J Biochem Cell Biol 34: 221–241.

Alali F, Tawaha K, Al-Eleimat T (2004): Determination of hypericin content in Hypericum triquetrifolium Turra (Hypericaceae) growing wild in Jordan. Nat Prod Res 18: 147–151.

Alecu M, Ursacıuc C, Halalau F, Coman G, Merlevede W, Waelkens E, De Witte P (1998): Photodynamic treatment of basal cell carci-noma and squamous cell carcinoma with hypericin. Anticancer Res 18: 4651–4654.

Apaydın Ş, Zeybek U, İnce İ, Elgin G, Karamenderes C, Öztürk B, Tuğlular I (1999): Hypericum triquetrifolium Turra. extract exhibits anticiceptive activity in the mouse. J Ethnopharmacol 67: 307–312.

Ayan AK, Çirak C (2008): Hypericin and pseudohypericin contents in some Hypericum species growing in Turkey. Pharm Biol 46: 288–291.

Campbell MH, Delfosse ES (1984): The biology of Australian weeds. 13. Hypericum perforatum L. J Aust Inst Agric Sci 50: 63–73.

Carbanaro M, Mattera M, Nicoli S, Bergamo P, Cappelloni M (2002): Modulation of antioxidant compounds in organic vs conven-tional fruit (peach, Prunus persica L., and pear, Pyrus communis L.). J Agric Food Chem 50: 5458–5462.

Çırak C, Sağlam B, Ayan AK, Kevseroğlu K (2006): Morphogenetic and diurnal variation of hypericin in some Hypericum species from Turkey during the course of ontogenesis. Biochem Syst Ecol 34: 1–13.

Çırak C, Radusiene J, Janulis V, Ivanauskas L (2007a): Secondary metabolites in Hypericum perfoliatum variation among plant parts and phenological stages. Botanica Helvatica 117: 29–36.

Çırak C, Radusiene J, Ivanauskas L, Janulis V (2007b): Variation of bioactive secondary metabolites in Hypericum origanifolium during its phenological cycle. Acta Physiol Plant 29: 197–203.

Conforti F, Loızzo MR, Statti AG, Menichini F (2007): Cytotoxic activ-ity of antioxidant constituents from Hypericum triquetrifolium Turra. Nat Prod Res 21: 42–46.

Couceiro MA, Afreen F, Zobayed SMA, Kozai T (2006): Variation in concentrations of major bioactive compounds of St. John’s wort: effects of harvesting time, temperature and germplasm. Plant Sci 170: 128–134.

Couladis M, Bazıou P, Verykokıdeu E, Loukıs A (2002): Antioxidant activity of polyphenols from Hypericum triquetrifolium Turra. Phytother Res 16: 769–770.

Dias ACP, Francisco A, Barberan T, Ferreria F, Ferreres F (1998): Unusual flavonoids produced by callus of Hypericum perfora-tum. Phytochemistry 48: 1165–1168.

Falk H (1999): From the photosensitizer hypericin to the photorecep-tor stentorian: the chemistry of phenanthroperylene quinines. Angew Chem Int Ed Engl 38: 3116–3136.

Floridi S, Montanari L, Ombretta M, Fantozzi P (2003): Determination of free phenolic acids in wort and beer by coulometric array detection. J Agric Food Chem 51: 1548–1554.

Hubner WD, Lande S, Podzuweit H (1994): Hypericum treatment of mild depression with somatic symptoms. J Geriatr Psychiatry Neurol 7: 12–14.

Kamuhabwa AR, Agostinis P, Kasran A, De Witte PA (2000): Photodynamic activity of hypericin in human urinary bladder carcinoma cell. Anticancer Res 20: 2579–2584.

Kazlauskas S, Bagdonaite E (2004): Quantitative analysis of active substances in St. John’s wort (Hypericum perforatum L.) by the high performance liquid chromatography method. Medicina (Kaunas) 40: 975–981.

Kim DO, Lee KW, Lee HJ, Lee CY (2002): Vitamin C equivalent anti-oxidant capacity (VCEAC) of phenolic phytochemicals. J Agric Food Chem 50: 3713–3717.

Kitanov GM (2001): Hypericin and pseudohypericin in some Hypericum species. Biochem Syst Ecol 29: 171–178.

Kızıl G, Kızıl M, Yavuz M, Emen S, Hakimoğlu F (2008): Antioxidant activities of ethanol extracts of Hypericum triquetrifolium and Hypericum scabroides. Pharm Biol 46: 231–242.

Linde K, Ramirez G, Mulrow CD, Pauls A, Weidenhammer W, Melchart D (1996): St John’s wort for depression: an overview and meta-analysis of randomised clinical trials. BMJ (Clin Res Ed) 313: 253–258.

Malenčić D, Popović M, Miladinović J (2007): Phenolic content and antioxidant properties of soybean (Glycine max (L.) Merr.) seeds. Molecules 12: 576–581.

Meruelo D, Lavie G, Lavie D (1988): Therapeutic agents with dramatic antiretroviral activity and little toxicity and effective doses, aro-matic polycyclic diones hypericin and pseudohypericin. Proc Natl Acad Sci U S A 85: 5230–5234.

Moreno MIN, Isla MI, Sampietro AR, Vattuone MA (2000): Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. J Ethnopharmacol 71: 109–114.

Park YK, Koo MH, Ikegaki M, Contado JL (1997): Comparison of the flavonoid aglycone contents of Apis melliferapropolis from vari-ous regions of Brazil. Arq Biol Technol 40: 97–106.

Patocka J (2003): The chemistry, pharmacology, and toxicology of the biologically active constituents of the herb Hypericum perfora-tum L. J Appl Biomed 1: 61–73.

Radulovic N, Stankov-Jovanovic V, Stojanovic G, Melcerovic AS, Spiteller M, Asakawa Y (2007): Screening of in vitro antimicro-bial and antioxidant activity of nine Hypericum species from the Balkans. Food Chem 103: 15–21.

Robson NKB (1975): Hypericum L. In: Davis PH, editor. Flora of Turkey and the East Aegean Islands. Edinburgh: Edinburgh University Press. pp 355–401.

Singleton VL, Orthofer R, Lamuela-Raventos RM (1999): Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent. In: Packer L, editor. Methods in Enzymology. Vol. 299: Oxidants and antioxidants (Part A). San Diego, CA: Academic Press. pp 152–178.

Yeşilada E, Gürbüz I., Shitabo H (1999): Screening of Turkish anti-

ulcergenic folk remedies for anti-Helicobacter pylori activity. J Ethnopharmacol 66: 289–293.

Vandenbogaerde AL, Cuveele JF, Proot P, Himpens BE, Merlevede W, and de Witte PA (1997): Differential cytotoxic effects induced after photosensitization by hypericin. J Photochem Photobiol B 38: 136–142.

Phar

mac

eutic

al B

iolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Q

ueen

's U

nive

rsity

on

08/1

5/14

For

pers

onal

use

onl

y.