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Variation in the composition of the essential oils, phenolic compounds andmineral elements of Hypericum perforatum L. growing in EstoniaKati Helmjaa; Merike Vahera; Tõnu Püssab; Anne Orava; Anu Viitaka; Tuuli Levandia; Mihkel Kaljuranda

a Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn,Estonia b Department of Food Science and Hygiene, Estonian University of Life Science, Kreutzwaldi58A, 51014 Tartu, Estonia

Online publication date: 07 March 2011

To cite this Article Helmja, Kati , Vaher, Merike , Püssa, Tõnu , Orav, Anne , Viitak, Anu , Levandi, Tuuli and Kaljurand,Mihkel(2011) 'Variation in the composition of the essential oils, phenolic compounds and mineral elements of Hypericumperforatum L. growing in Estonia', Natural Product Research, 25: 5, 496 — 510To link to this Article: DOI: 10.1080/14786411003792165URL: http://dx.doi.org/10.1080/14786411003792165

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Natural Product ResearchVol. 25, No. 5, March 2011, 496–510

Variation in the composition of the essential oils, phenolic compounds

and mineral elements of Hypericum perforatum L. growing in Estonia

Kati Helmjaa*, Merike Vahera, Tonu Pussab, Anne Orava, Anu Viitaka,Tuuli Levandia and Mihkel Kaljuranda

aDepartment of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618Tallinn, Estonia; bDepartment of Food Science and Hygiene, Estonian University ofLife Science, Kreutzwaldi 58A, 51014 Tartu, Estonia

(Received 19 October 2009; final version received 19 March 2010)

A comprehensive investigation of the chemical composition of the aerialparts of Hypericum perforatum L. collected in three habitations in Estoniawas carried out. An analysis by gas chromatography–mass spectrometryand gas chromatography–flame ionisation detection established themain components of the essential oils. The phenolic compounds both inethanol and water extracts of the plant were analysed using liquidchromatography–mass spectrometry (LC–MS) and capillary zone electro-phoresis. In addition to the earlier published polyphenols, several newphenolic acids and flavonoids, such as quercetin hexoside malonates and anA-type catechin–epicatechin trimer were identified in this Hypericum forthe first time. The contents of the pharmaceutically important antidepres-sants hyperforin and hypericin were also estimated by LC–MS andcompared with the data in the literature. The composition of the mineralelements was determined by atomic absorption spectroscopy. The results ofthe study demonstrate a rather high variability in the content of differentsubstance groups in H. perforatum L. and, hence, the need for a survey ofthe raw material in the course of selection of raw materials for pharma-ceutical preparations.

Keywords: St. John’s wort; chromatography; capillary electrophoresis;essential oils; polyphenols

1. Introduction

Herbs belonging to the genus Hypericum, widely distributed in various parts of theworld such as Europe, Asia, North Africa and America (Peng, Yuan, & Ye, 2005;Silva, Ferreres, Malva, & Dias, 2005; Tatsis et al., 2007; Urbanek, Blechtova,Pospısilova, & Polasek, 2002), are very important in medicine. Among them,Hypericum perforatum L., known as St. John’s wort, is one of the most consumedmedicinal plants, and has gained a good reputation due to its wound-healing and

*Corresponding author. Email: helmja@staff.ttu.ee

ISSN 1478–6419 print/ISSN 1029–2349 online

� 2011 Taylor & Francis

DOI: 10.1080/14786411003792165

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anti-inflammatory properties (Huck, Abel, Popp, & Bonn, 2006; Peng et al., 2005;Tatsis et al., 2007; Urbanek et al., 2002). High antimicrobial and antiviral activitiesof the plant have also been reported (Peng et al., 2005; Urbanek et al., 2002).However, H. perforatum L. has received most of this attention due to its activityagainst mild-to-moderate depression (Gomez, Cerutti, Olsina, Silva, & Matrınez,2004; Hamoudova, Pospısilova, & Spilkova, 2006; Hansen et al., 1999; Huck et al.,2006; Peng et al., 2005; Tatsis et al., 2007). The curative value of the plant has beenattributed to a wide range of compounds – flavonol glycosides, biflavones, phenolicacids, procyanidins, phenylpropanes, phloroglucinols (hyperforin and adhyperforin),naphthodianthrones (hypericin, pseudohypericin, protohypericin and protopseudo-hypericin), xanthones, tannins, and also to a number of essential oils (Peng et al.,2005; Schmidt, Jaroszewski, Bro, Witt, & Stærk, 2008). Particularly, phloroglucinolsand naphthodianthrones are considered to be synergistically responsible for theantidepressive activities of H. perforatum L. (Bilia, Bergonzi, Mazzi, & Vincieri,2001; Charchoglyan et al., 2007; Hamoudova et al., 2006; Huck et al., 2006; Urbaneket al., 2002). Incidentally, there is an interesting hypothesis that hypericin is actuallyproduced by an endophytic fungus symbiotically living in the stems of H. perforatumL. (Kusari, Lamshoft, Zuhlke, & Spiteller, 2008). Several papers have been devotedto the liquid chromatographic analysis of phloroglucinols and naphthodianthronesinH. perforatum L. (Huck et al., 2006; Seger et al., 2004; Tatsis et al., 2007; Williams,Sander, Wise, & Girard, 2006) as well as in human plasma (Riedel et al., 2004). Muchis known also about the phenolic acids and flavonoids of H. perforatum L. (Broliset al., 1998; Skerget et al., 2005; Wei, Xie, Dong, & Ito, 2009). Phenolic acids arerepresented by chlorogenic acids, as well as by caffeic and coumaroylquinic acids, theflavonoids by proanthocyanidins, epigallocatechin, a number of various glycosidesof quercetin, a rhamnoside of diapigenin as well as by diapigenin. The flavonoidspresent in the extracts of H. perforatum L. presumably contribute to theantidepressive activity of phloroglucinols and naphthodianthrones (Hamoudovaet al., 2006; Schmidt et al., 2008; Urbanek et al., 2002).

There are also a number of literature reports on the analyses of the plant’s essentialoil, which is a complex mixture of volatiles derived from plant secondary metabolites(Bruni et al., 2005; Cakir et al., 1997; Couladis, Baziou, Petrakis, & Harvala, 2001;Gudzic, Nedeljkovic, Dordevic, & Comor, 1997;Mockute, Bernotiene, & Judzentiene,2003; Pintore et al., 2005; Saroglou, Marin, Rancic, Veljis, & Skaltsa, 2007; Schwob,Bessiere, Masotti, & Viano, 2004; Smelcerovic, Lepojevic, & Djordjevic, 2004;Tognolini et al., 2006; Weyerstahl, Splittgerber, Marschall, & Kaul, 1991). The maincomponents of these oils are isoalkanes (2-methyl octane, 2-methyl nonane, 2-methyldecane), monoterpenes (�-pinene), sesquiterpenes (�-caryophyllene, germacrene D),oxygenated sesquiterpenes (spathulenol and caryophyllene oxide) and aliphaticalcohols (n-dodecanol and n-tetradecanol).

In this work, H. perforatum L. collected from three habitations in Estonia wasput under complex investigation. The analyses of essential oils of the aerial parts ofthe plant by gas chromatography–mass spectrometry (GC–MS) and gas chroma-tography–flame ionisation detector (GC–FID) were carried out to reveal the mainconstituents of the essential oils. The composition of the groups of phenolic acidsand flavonoids both of ethanol and water extracts of H. perforatum L. werecompared by high-performance liquid chromatography–UV diode array detection–electrospray ionisation–tandem mass spectrometry (LC-DAD-ESI-MS/MS) and

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capillary zone electrophoresis (CZE). The main phloroglucinols and naphthodian-

thrones were also identified and quantified by LC-DAD-ESI-MS/MS. Further, thecomposition of mineral elements as essential or toxic agents in metabolism was

determined by atomic absorption spectroscopy (AAS) and their content in different

plants and in the water and ethanol extracts was compared.

2. Results and discussion

2.1. Identification of the oil components

The qualitative analysis of the constituents of essential oils in three different

H. perforatum L. plants by GC–FID and GC–MS was carried out and the identified

components are reported in Table 1. The qualitative analysis was based on acomparison of the retention indices (RI) on the columns of different polarity and

mass spectra with the corresponding data in the literature (Davies, 1990; Zenkevich,1996), as well as with the RI data of authentic samples and computer mass spectra

libraries (NIST). The percentage composition of the essential oils was determined

from GC peak areas without correction factors.The SDE method of the dried aerial parts of H. perforatum L. gave the oil with a

yield of 0.7–1.9mg g�1; amounting in Sample 1 about 95%, in Sample 2 over 94%

and in Sample 3 over 95% of the total oil content. The main components (Table 1) of

the studied oils were 2-methyloctane (0.8–11.3%), �-pinene (3.1–14.3%), �-pinene(1.2–6.8%), (E)-�-caryophyllene (1.8–19.2%), �-muurolene (0.3–8.7%), germacrene

D (2.0–13.7%), �-cadinene (1.6–5.4%), spathulenol (2.9–4.7%), caryophyllene oxide(2.5–4.1%), globulol (0.5–5.5%), �-cadinol (0.9–5.0%) and sesquiterpene alcohol

(0.3–4.1%). However, the composition of the oils in the three analysed samples

varied greatly.Two of our H. perforatum L. samples (Samples 1 and 3) were rich in

monoterpenes and isoalkanes, particularly in �-pinene and 2-methyloctane

(13.6–25.6%). These compounds were found in high quantities in the H.perforatum L. essential oils from Italy (27.0–61.3%) (Pintore et al., 2005; Skerget

et al., 2005; Smelcerovic et al., 2004), Greece (33.6%) (Petrakis, Couladis, & Roussis,

2005) and Serbia (10.0–34.2%) (Gudzic, Dordevic, Palic, & Stojanovic, 2001;Saroglou et al., 2007).

Sesquiterpenes were the main component group of our Sample 2 (63.3%).

Similar results were reported by Mockute et al. (2003) (35.6–51.9%), Schwob et al.

(2004) (38.8–84.4%), Gudzic et al. (1997) (68.3%) and by Bruni et al. (2005) (63.5%).Germacrene D was identified as the dominant constituent in the oils of

H. perforatum L. from two localities in Lithuania by Mockute et al. (2003)(16.1–31.5%) and from three localities in southern France by Schwob, Bessiere and

Viano (2002) (17.8–37.3%). Germacrene D was the second major constituent in

Sample 3 (13.7%).The high content of oxygenated sesquiterpenes (44.1%) in our Sample 1 is

comparable with the content of this component group in the H. perforatum L. oils

from Lithuania (39.2–63.3%) (Mockute et al., 2003; Singleton, Orthofer, &

Lamuela-Raventos, 1999), Greece (35.2%) (Pavlovic, Tzakou, Petrakis, &Couladis, 2006), and from two localities of France (44–44.1%) (Schwob et al., 2002).

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Table 1. Composition of the essential oil ofH. perforatum L. from three different habitations.

RI Concentration (%)

CompoundIdentification

method SPB-5 SW-10 Sample 1 Sample 2 Sample 3

n-Hexanal 1, 2, 3 800 1092 0.4 0.1 0.3(E)-2-Hexenal 1, 2, 3 845 1226 0.1 0.2 0.22-Methyloctane 1, 2, 3 856 850 5.5 0.8 11.3n-Nonane 1, 2, 3 900 900 7.0 0.3 2.0�-Thujene 1, 2, 3 922 1027 0.3 0.2 1.4�-Pinene 1, 2, 3 929 1023 8.1 3.1 14.3Sabinene 1, 2, 3 966 1123 0.7 0.1 2.83-Methylnonane 2, 3 968 960 1.2 0.1 2.1�-Pinene 1, 2, 3 969 1110 1.7 1.2 6.86-Methyl-5-hepten-2-one 1, 2, 3 984 1344 0.2 0.2 tr�-Myrcene 1, 2, 3 988 1167 0.4 0.2 1.6�-Phellandrene 1, 2, 3 1000 1168 tr tr 0.1�-Terpinene 1, 2, 3 1012 1182 0.4 0.2 0.9p-Cymene 1, 2, 3 1019 1273 0.6 0.2 0.5Limonene 1, 2, 3 1024 1202 0.2 0.1 0.5�-Phellandrene 2, 3 1026 1210 0.2 0.1 0.7(Z)-�-Ocimene 1, 2, 3 1034 1240 0.1 0.1 0.6(E)-�-Ocimene 1, 2, 3 1045 1256 0.2 0.2 2.42,6-Dimethyl-5-heptenal 1, 2, 3 1049 1439 tr tr tr�-Terpinene 1, 2, 3 1053 1246 0.8 0.3 1.32-Methyldecane 1, 2, 3 1061 1050 0.2 0.1 0.7Terpinolene 1, 2, 3 1083 1284 0.2 0.1 0.5n-Undecane 1, 2, 3 1100 1100 0.7 0.1 1.0n-Nonanal 1, 2, 3 1102 1358 0.1 0.1 trIsoamyl isovalerate 1, 2, 3 1104 1300 tr tr trMenthone 1, 2, 3 1145 1456 tr 0.1 –(E)-2-Nonenal 1, 2, 3 1160 1548 – 0.1 trTerpinen-4-ol 1, 2, 3 1171 1602 1.4 0.3 1.4�-Terpineol 1, 2, 3 1186 1702 0.4 0.1 0.6Estragol 1, 2, 3 1193 1660 tr 0.1 trDecanal 1, 2, 3 1200 1485 0.1 0.1 trCarvone 1, 2, 3 1237 1721 tr 0.3 –2-Methyldodecane 1, 2 1267 1256 – – 0.4n-Undecanal 1, 2, 3 1300 1570 – 0.2 tr�-Longipinene 1, 2, 3 1338 1456 tr 0.3 –�-Cubebene 1, 2, 3 1341 1452 0.3 1.8 tr�-Ylangene 1, 2, 3 1361 1473 tr 0.6 tr�-Copaene 1, 2, 3 1366 1482 0.4 1.3 trLongicyclene 1, 2 1372 1500 tr 0.2 –�-Ylangene 1, 2, 3 1376 1508 – 0.5 tr�-Bourbonene 1, 2, 3 1379 1519 0.1 0.1 tr�-Cubebene 1, 2, 3 1384 1530 0.1 0.7 0.6Isolongifolene 1, 2 1395 1520 0.1 0.1 0.2�-Cedrene 1, 2, 3 1398 1562 0.4 1.0 0.3(E)-�-Caryophyllene 1, 2, 3 1409 1589 1.8 19.2 3.8�-Funebrene 1, 2 1417 1581 0.5 1.7 tr�-Gurjunene 1, 2, 3 1423 1630 tr 1.0 trAromadendrene 1, 2, 3 1434 1594 – 0.9 tr�-Humulene 1, 2, 3 1440 1655 0.3 1.0 0.4

(Continued )

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Table 1. Continued.

RI Concentration (%)

CompoundIdentification

method SPB-5 SW-10 Sample 1 Sample 2 Sample 3

�-Copaene 1, 2, 3 1447 1631 0.8 0.3 0.1(E)-�-Farnesene 1, 2, 3 1450 1658 0.1 0.8 tr�-Acoradiene 1, 2, 3 1453 1669 1.5 0.8 0.4Alloaromadendrene 1, 2, 3 1455 1627 0.1 0.8 0.4�-Muurolene 1, 2, 3 1467 1678 1.3 8.7 0.3Germacrene D 1, 2, 3 1470 1696 2.3 2.0 13.7�-Amorphene 1, 2, 3 1485 1711 0.5 6.5 1.6�-Muurolene 1, 2, 3 1485 1716 0.5 0.6 0.2(Z,E)-�-Farnesene 1, 2, 3 1489 1729 – 0.6 0.3Viridiflorene 1, 2 1492 1665 0.2 0.4 0.2n-Tridecanal 1, 2 1495 1783 0.1 0.7 0.2�-Guaiene 1, 2, 3 1500 1721 0.1 0.3 1.6�-Cadinene 1, 2, 3 1505 1750 1.6 4.0 0.9(Z)-Calamenene 1, 2, 3 1514 1822 0.1 0.9 –�-Cadinene 1, 2, 3 1513 1752 2.7 5.4 1.6Cadina-1,4-diene 1, 2, 3 1523 1820 0.1 0.2 tr�-Sesquiphellandrene 1, 2, 3 1526 1765 0.2 tr –(Z)-Nerolidol 1, 2 1528 2000 0.1 0.6 0.1�-Cadinene 1, 2, 3 1531 1771 0.3 0.5 0.2�-Calacorene 1, 2, 3 1540 1904 0.3 0.1 0.2Caryophyllene epoxide 1, 2, 3 1550 1976 0.3 0.1 0.1(Z)-3-Hexenyl benzoate 1, 2, 3 1558 2127 0.5 0.3 tr(E)-Nerolidol 1, 2, 3 1563 2050 0.9 1.7 0.9Spathulenol 1, 2, 3 1565 2120 4.7 3.5 2.9Caryophyllene oxide 1, 2, 3 1571 1968 4.1 3.4 2.5Sesquiterpene compound I 1, 2, 3 1582 2120 2.0 0.9 –Globulol 1, 2, 3 1592 2020 5.5 0.7 0.9Viridiflorol 1, 2, 3 1597 2053 1.0 0.2 0.2Humulene epoxide 1, 2, 3 1600 2018 0.5 0.3 trSesquiterpene compound II 1, 3 1610 1992 2.5 0.1 0.1Ledol 1, 2, 3 1619 2100 0.8 0.4 trMethyltridecanol 1, 2 1621 2140 – 0.3 –Cubenol 1, 2, 3 1629 2120 0.1 0.8 tr�-Cadinol 1, 2, 3 1632 2182 1.4 0.7 0.9�-Bisabolol 1, 2 1632 2165 1.2 0.4 0.5�-Muurolol 1, 2, 3 1635 2170 0.1 0.3 trT-Muurolol 1, 2 1638 2192 0.4 0.3 0.7(E)-�-Cadinol 1, 2, 3 1647 2230 5.0 0.9 0.9Cadina-1,4-dien-3-ol 1, 2 1648 2354 1.5 1.3 0.1(Z)-�-Cadinol 1, 2 1650 2227 0.3 0.4 trCaryophylla-3(15),7-dienol 1, 2 1667 2309 0.8 0.7 tr�-Bisabolol 1, 2, 3 1680 2220 0.2 0.3 0.1Sesquiterpenic compound III 1, 2, 3 1682 2287 4.1 1.6 0.3(Z,Z)-�-Farnesol 1, 2 1685 2350 4.0 0.3 0.1(Z,E)-�-Farnesol 1, 2 1685 2332 1.5 0.3 trBenzyl benzoate 1, 2, 3 1750 2619 0.6 0.3 0.16,10-Dimethylpentadecanone 1, 2, 3 1800 2221 0.4 0.2 0.2n-Nonadecane 1, 2 1900 1900 0.1 tr 0.4n-Heneicosane 1, 2, 3 2100 2100 – 0.1 0.2

(Continued )

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2.2. Analysis of phenolic acids and flavonoids

By a conventional spectrophotometric method (Singleton et al., 1999), the total

phenolic content in water and ethanol extracts was estimated to be in the range of

1149–1162mg and around 2206mg of gallic acid equivalent in 100 g of dry weight,

respectively. Besides, the total flavonoid content of the different H. perforatum L.

samples wasmeasured by the protocol proposed in (Lenucci, Cadinu, Taurino, Piro, &

Dalessandro, 2006). The total flavonoid content value in water and ethanol extracts

varied between 598–629mg and 635–690mg, respectively, in 100 g of dry weight. Thus,

the ethanol extracts are richer in phenolic compounds compared to the water extracts.Two different extracts of H. perforatum L. samples were compared and the

polyphenols were determined by LC-ESI-MS/MS. Figure 1 illustrates the analysis

results of two samples (Samples 2 and 3), the chromatogram of the Sample 1 looks

intermediate.The comparison of the chromatograms (Figure 1(a) and (b)) reveals that

substantially more polyphenolic compounds were extracted by ethanol. The ethanol

extracts of three different samples showed no significant differences in the qualitative

but a rather big variability in the quantitative composition of the solutions. The

qualitative analyses ofH. perforatum L. indicated that most of the polyphenols are in

a glycoside form and the main flavonoid aglycone is quercetin. The other aglycones

are quinic, protocatechuic, vanillic, caffeic, coumaroylquinic and OH-phenylpro-

pionic acids, catechin and epicatechin monomers, and oligomers. H. perforatum L.

contains three different chlorogenic acids – chlorogenic acid (5-O-caffeoylquinic

acid), neochlorogenic acid (3-O-caffeoylquinic acid) and cryptochlorogenic acid (4-

O-caffeoylquinic acid), identified by their negative MS/MS fragmentation spectra

(comparison with commercial chlorogenic acid and literature data) as well as by

chromatographic retention times (Weisz, Kammerer, & Carle, 2009). Of special

interest is the quite rare A-type catechin–epicatechin trimer with a molecular weight

of 864 that was found in small amounts in the ethanolic extracts and was identified

by comparing with its positive MS/MS fragmentation spectrum from the literature

(Anderson et al., 2004). A-type procyanidin oligomers are considered to have a

Table 1. Continued.

RI Concentration (%)

CompoundIdentification

method SPB-5 SW-10 Sample 1 Sample 2 Sample 3

(Z)-Phytol 1, 2, 3 1952 2619 1.8 0.3 1.4Tricosane 1, 2, 3 2300 2300 tr 0.1 –Total (%) 94.4 95.0 95.2Monoterpenes 16.1 6.1 19.0Oxygenated monoterpenes 1.8 0.9 2.0Sesquiterpenes 16.7 63.3 27.0Oxygenated sesquiterpenses 44.1 20.9 12.8Aliphatic compounds 13.9 3.8 34.4Oil yield (mg g�1) 0.68 1.88 0.96

Note: tr, trace level (50.05%); RI, retention indices. Identification method: 1 – RISPB-5;2 – RISW-10; and 3 – GC–MS.

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strong antidiabetic effect. In addition, flavonoid hexoside malonates were, for thefirst time, identified in H. perforatum L. by their MS/MS fragmentation spectra(Figure 1) (Maatta, Kamal-Eldin, & Torronen, 2003).

The complex with the most intensive peaks 17–19 (Figure 1(b)) representsquercetin glucoside, glucuronide, rutinoside and galactoside. According to thepeak areas of the UV-chromatograms (AUC) between 15 and 41min of a

Figure 1. HPLC UV-chromatograms at �¼ 280 nm of water (a) and ethanol (b) extracts ofH. perforatum L. from two habitations in Estonia: Sample 2 (black line); Sample 3 (light line).Notes: Identified compounds: 1 – quinic acid; 2 – protocatechuic acid glucoside; 3 – vanillic acidglucoside; 4 – neochlorogenic acid; 5 – caffeic acid glucoside; 6 – coumaroylquinic acid andprocyanidin A 1; 7 – 3-OH-phenylpropionic acid glucoside; 8 – catechin; 9 – chlorogenic acid; 10– procyanidin A 2; 11 – cryptochlorogenic acid; 12 – epicatechin; 13–15 – quercetin dihexosides;16 – quercetin glucoside rhamnoside; 17 – quercetin galactoside; 18 – quercetin glucuronide andquercetin rutinoside; 19 – quercetin glucoside; 20 – quercetin pentoside; 21 – quercetinhexoside malonate 1; 22 – quercetin rutinoside malonate; 23 – quercetin hexoside malonate 2;and 24 – quercetin.

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chromatographic run, flavonol quercetin and its derivatives, the largest family ofpolyphenols in H. perforatum L., constitute roughly 50–60% (250 nm) or 30–50%(280 nm) of all the polyphenols.

The total content of quercetin and its main glycosides can be estimated to be 4.3(Sample 1), 5.3 (Sample 2) and 4.8mg g�1 (Sample 3), by the AUC values of thechromatograms of the ethanol extracts at 370 nm and characteristic wavelength forflavonols, between tR¼ 15–45min, using the mean value of the absorbances of themethanol solution of quercetin and rutin at the same wavelength as a basis for thecalculation.

The concentration of five aglyconic polyphenols in different samples wasestimated in the ethanolic extracts of H. perforatum L. by the calibration curvesconstructed for the same or a closely related compound (catechin for epicatechin andchlorogenic acid 1 for chlorogenic acid 2). The results are given in Table 2.

In addition, CZE was applied to the analysis of different extracts ofH. perforatum L. By comparing the water and ethanol extracts of the plant fromdifferent habitations, it is observed that the samples have similar qualitativefingerprints. However, it was revealed that more components were extracted withethanol as it was observed with the analysis spectrophotometrically. Although thecontent of phenolic acids was higher in water extracts, the ethanol extracts appearedto be richer in flavonoid glycosides.

2.3. Analysis of phloroglucinols and naphthodianthrones

Phloroglucinols and naphthodianthrones, compounds of a high pharmaceuticalimportance, were quantified in the ethanolic extracts of H. perforatum L. by themethod of external standards. These compounds were identified by comparing theMS/MS fragmentation spectra obtained with the respective spectra of the commercialstandards as well as with corresponding spectra from the literature (Riedel et al., 2004;Sleno, Daneshfar, Eckert, Muller, & Volmer, 2006; Tatsis et al., 2007). It was revealedthat all the three H. perforatum L. samples contained remarkable but differentamounts of the phloroglucinol hyperforin as well as the naphthodianthroneshypericin and pseudohypericin. The content of these compounds in H. perforatumL. was estimated by using the calibration curves of commercial hyperforin andhypericin (the last used also for the quantitation of pseudohypericin) constructed atUV-vis wavelengths of 280 and 591 nm, respectively (Table 2).

Table 2. Content of a number of aglyconic compounds (mg g�1) in threesamples of H. perforatum L.

Sample 1 Sample 2 Sample 3

Catechin 0.12 0.07 0.18Epicatechin 0.23 0.36 0.67Chlorogenic acid 0.04 0.06 0.36Neochlorogenic acid 0.12 0.17 0.28Quercetin 0.04 0.06 0.06Hyperforin 1.10 0.71 0.52Hypericin 0.012 0.025 0.036Pseudohypericin 0.036 0.036 0.126

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Various authors have reported rather different values of concentration ofhypericins and hyperforins in H. perforatum L. The content of hyperforin in oursamples is comparable with the results of Smelcerovic, Spiteller and Zuehlke (2006),using a similar extraction method with methanol, but the content of hypericin isabout five times lower. Sufficiently higher, but variable contents of hyperforinhave been obtained by supercritical fluid extraction with carbon dioxide (Glisic,Smelcerovic, Zuehlke, Spiteller, & Skala, 2008).

2.4. Analysis of the composition of mineral elements

The comparison of the composition of mineral elements of different extractsof H. perforatum L. was carried out by AAS and the results of the distribution ofmineral elements in H. perforatum L. and extracts are given in Table 3. As thesamples were obtained from different habitations, which differ in soil physico-chemical parameters, the constituents of the mineral elements, thus, enable us tounderstand the effect of soil on its constituents.

In this study, measurable amounts of K, Mg, Mn, Pb, Zn, Co and Cr weredetected in the plant and in the extracts. From the results obtained (Table 3),it is seen that K, Mg, Pb and Cr were found at higher concentrations inSample 2, whereas Sample 1 contained more Mn and Co. Sample 3 showeda higher content of Zn.

The comparison of the extracts showed that there exist differences between thevalues of the extracted metals. A higher content of metals was detected in waterextracts (K and Pb about 70%; Zn, Mn, Mg, Co and Cr between 10 and 25%), but inthe case of ethanol extracts, the values of the extracted metals were approximately10–20 times lower.

Besides the mineral elements found in the current work, metals like Li, Na, Feand Ni have been determined in H. perforatum L. plant (Gomez et al., 2004; Razic,Onjia, Dogo, Slavkovic, & Popovic, 2005). According to the data reported in theliterature, dried powdered leaves and flowers of H. perforatum L. plant containedmore Cu, Co and Pb than commercial tinctures of the plant (Gomez, Cerutti,Sombra, Silva, & Martınez, 2007).

3. Experimental

3.1. Chemicals

All the chemicals were of analytical grade and were used as received. Sodiumtetraborate and hydroxide, as well as quercetin, rutin, chlorogenic acid, hypericin,hyperforin, ethanol, n-hexane and n-tetradecane were obtained from Sigma–Aldrich

Table 3. Mineral element content in the samples of H. perforatum L.

PlantZn

(mg g�1)Mn

(mg g�1)K

(mg g�1)Mg

(mg g�1)Pb

(ng g�1)Co

(ng g�1)Cr

(ng g�1)

Sample 1 29.4� 0.4 58.4� 1.4 9471� 59 1875� 52 167� 7.9 175� 4.0 240� 11.1Sample 2 32.6� 0.6 51.1� 1.3 12,429� 144 2234� 50 220� 8.1 150� 5.6 243� 10.0Sample 3 35.5� 0.6 31.8� 1.2 7612� 40 1889� 30 120� 6.4 102� 5.1 122� 6.8

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(St. Louis, MO, USA). Catechin, polyvinylpyrrolidone K-90 (Mr� 360,000) andpalladium (II) chloride anhydrous were purchased from Fluka (Buchs, Switzerland).Boric acid, formic acid and hydrogen peroxide (35%) were from Riedel-de Haen(Seelze, Germany). Nitric acid 66% ‘suprapure’ grade was from Merck (Darmstadt,Germany) and hydrochloric acid (36%) ‘for trace metal analysis’ was from Baker(Phillipsburg, USA). Methanol was purchased from Rathburn Chemicals(Walkerburn, Scotland) and acetonitrile from Romil (Cambridge, UK). Argon(99.998% of purity), acetylene (99.998% of purity) and helium were purchased fromEesti AGA (Tallinn, Estonia).

The stock atomic spectroscopy standard solutions (1000mgL�1) of Pb, Cr, Co,Mn, Mg, K and Zn were purchased from Fluka (Switzerland). Multielement QualityControl Standard 26 (High-purity standards, Charleston, USA) was graduallydiluted with 4% nitric acid solution before use. The deionised water (Milli-Q,Millipore S.A., Molsheim, France) was used for the preparation of all solutions.

3.2. Sample preparation

Plant samples of H. perforatum L. were collected in three different locationsin Estonia during the year of 2007: Sample 1 – cultivated in the southern part,Sample 2 – in the central part and Sample 3 – in the northern part of Estonia. Ingeneral, there are no variations in biotic and abiotic factors. The voucher specimensare deposited in the herbarium of the Department of Pharmacy (University of Tartu,Estonia). For the analyses, 20 g of each sample (mix of young and old plants) werecollected and the aerial part of the plant at the flowering stage was used. The sampleswere air-dried at ambient temperature and the ground material was sieved througha 0.5mm sieve.

The ultrasonic extraction was performed using both ethanol and water. For theextraction, 0.5 g of the ground and sieved material was weighed and 5mL ofthe respective extraction solvent was added. The sample was kept at roomtemperature for 60min and in an ultrasonic bath at room temperature for 20min.The extract was filtered through a 0.45 mm filter and stored at �20�C. The extractionwas performed in triplicate and the extracts were concentrated by a vacuum rotator.Before the analyses by HPLC and capillary electrophoresis (CE), the concentrateswere diluted with the respective solvents to 0.5mL of ethanol or water solution.

For the analysis by AAS, 0.2 g of an air-dried sample was mineralised by 5mL ofconcentrated nitric acid in Teflon bombs in a microwave oven (Anton PaarMultiwave 3000, Graz, Austria) at temperatures of up to 180�C for 30min. Aftercooling down, the solution in the bombs was transferred to volumetric flasks (15mL)with Milli-Q water for the determination of the total mineral element constituent.But in the analysis of ethanol and water extracts, the sample was heated to drynessand mineralised with nitric acid at 80�C for 2 h on the water bath. All theexperiments were conducted in triplicate.

3.3. Isolation of the essential oil

Approximately 12–15 g of air-dried plant was hydrodistilled in the Marcusson typemicro simultaneous distillation extraction (SDE) apparatus (Bicchi, D’Amato,

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Nano, & Frattini, 1990) with n-hexane as the solvent (0.5mL) for 2 h. Puren-tetradecane (2 mL) was used as the internal standard for the oil yield determination.

3.4. Gas chromatography

GC–FID analyses were carried out using Chrom-5 systems equipped with SPB-5(poly(5%-diphenyl-95%-dimethyl)siloxane) and SW-10 (polyethylene glycol) capil-lary columns (30m� 0.25mm, film thickness 0.25mm) from Supelco (USA). Thecarrier gas was helium, at a flow rate of 1.4mLmin�1, split 1 : 150. The thermalprogramme was 50–250�C at the rate of 2�Cmin�1. The injector temperature was250�C. A Spectra-Physics SP4100 computing integrator was used for data processing.

GC–MS analyses were recorded on a GCMS-QP2010 (Shimadzu, Japan) on afused silica capillary column (30m� 0.32mm; film thickness of 0.25mm) with abonded stationary phase poly(5%-diphenyl-95%-dimethyl)siloxane (ZB-5, Zebron,USA). Helium as the carrier gas with a split ratio of 1 : 17, and a flow rate of1.8mLmin�1 was applied. The temperature programme was 2 min at 60�C, followedby 60�–280�C at 12�Cmin�1. The injector temperature was 280�C and MSconditions were as follows: ionisation voltage 70 eV, scan rate 1 scan s�1, massrange 35–300Da and ion source temperature 280�C.

The analyses of the plant by GC–MS revealed three unknown sesquiterpenecomponents and their mass fragmentation patterns are given in Table 4.

3.5. Liquid chromatography

The liquid chromatographic 1100 Series system of Agilent Technologies(Palo Alto, USA) was used. For the separation of compounds, a reversed phaseHPLC Zorbax 300SB-C18 column (2.1� 150mm; 5 mm; Agilent Technologies) wasused in a stepwise mobile phase gradient of 0.1% formic acid (solvent A)and acetonitrile (solvent B) at a flow rate of 0.3mLmin�1 at 35�C. The sampleinjection volume was 10 mL. For the detection and identification of substances, theAgilent 1100 Series UV-vis DAD and 1100 Series LC/MSD Trap-XCT with an

Table 4. Mass fragmentation patterns for unknown compounds in H. perforatum L.by GC–MS.

Compound [Mþ]MS fragmentation iona

(in the descending order of intensity)

Sesquiterpene compound I – 123 (100), 81 (65), 41 (32), 93 (16), 107 (16), 79 (14)Sesquiterpene compound II 220 159 (100), 41 (90), 105 (75), 91 (70), 119 (65), 79 (58),

43 (50), 131 (50), 202 (42)Sesquiterpene compound III 220 (18) 41 (100), 43 (90), 91 (85), 159 (65), 93 (58), 55 (55),

105 (55), 79 (53)

Note: aThe relative intensity is given in parentheses, calculated by the most abundant iontaken as 100.

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electrospray ionisation interface (ESI) were connected to an Agilent 1100 Series

instrument consisting of an autosampler, a solvent membrane degasser, a binarypump and a column thermostat. The HPLC 2D ChemStation software with a

ChemStation Spectral SW module was used for the process guidance. The conditionsof the negative ion MS detection: m/z interval 50–1000; target mass 400;

number of precursor ions 2; maximum accumulation time 100ms; compoundstability 100%; flow rate of the drying gas (N2 from the generator) 10Lmin�1, gas

temperature 350�C and nebuliser pressure 30 psi and collision gas He pressure

6� 10�6mbar.DAD was working at an interval of 200–600 nm and the absorbance of the eluate

was continuously monitored at wavelengths of 250, 280, 370, 450 and 591 nm.

3.6. Capillary electrophoresis

All experiments were performed using an Agilent CE System (Agilent Technologies,Waldbronn, Germany) with DAD. This apparatus automatically performed all steps

of measurement protocols, including capillary conditioning, sample introduction,voltage application and detection. A CE ChemStation (Agilent Technologies) was

used for instrument control, data acquisition and data handling. The separation of

polyphenols was performed in a fused silica capillary (60� 50� 10�6 cm; PolymicroTechnology, Phoenix, AZ, USA) with an effective length of 52 cm. Prior to use, the

capillary was rinsed with a 0.1mol NaOH solution for 5min and with a separationbuffer for 5min. As a separation buffer, 50mmol sodium tetraborate (pH 9.3) was

used. The applied voltage for the separation of polyphenols was þ20 kV.The analyses of the fingerprints of phenolic compounds found in plant extracts

were based on the protocol developed by Helmja et al. (2007) and Orav, Viitak and

Vaher (2010).

3.7. Atomic absorption spectroscopy

Spectra AA 220Z and 220F (Varian, Mulgrave, Australia) atomic absorption

spectrometers (AAS) equipped with a side-heated GTA-110Z graphite atomizer,

Zeeman-effect background correction and integrated autosampler and graphitetubes with a coating and platforms made of pyrolytic graphite were in use. As the

purge gas, argon of 99.998% purity was used and in flame atomic absorptionspectrometry (FAAS), acetylene of 99.998% purity was used. The total volume of

the sample for the analysis of Pb injected into the atomizer was 30 mL, containing10 mL of a colloidal modifier, which was synthesised according to the procedure

described in Viitak and Volynsky (2006). Cr, Zn and Cu were injected without amodifier. The concentration of the modifier solution was 1mgmL�1.

Acknowledgements

The authors greatly acknowledge Tiiu Kailas and Dr Mati Muurisepp for their help in theanalysis of GC–MS.

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