Extraction and Hydrolysis Parameters for Determination of Quercetin in Hypericum perforatum

Extraction and Hydrolysis Parametersfor Determination of Quercetin in Hypericumperforatum

M. Biesaga, A. Stafiej, K. Pyrzynska&

Department of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland;E-Mail: [email protected]

Received: 12 December 2006 / Revised: 6 February 2007 / Accepted: 12 February 2007Online publication: 4 May 2007

Abstract

The optimum conditions for extraction of rutin and quercetin from Hypericum perforatum wereinvestigated. The best efficiency of extraction was achieved with aqueous methanol 40–80% (v/v).For quercetin analysis as aglycone the effect of acid concentration and hydrolysis time on theextraction recovery were also studied. Hydrolysis for 5 min in the presence of 2.8 mol L)1 HCl aswell as for 10 min with 1.1 mol L)1 HCl efficiently released quercetin from rutin. The content ofquercetin)3-O-glycosides (rutin, hyperoside and quercetrin) and quercetin aglycone as well aschlorogenic and caffeic acids in H. perforatum leaves and flowers were determined by HPLC withphotodiode-array detection and confirmed by electrospray mass spectrometry.

Keywords

Column liquid chromatographyExtraction and hydrolysis parametersFlavonoidsHypericum perforatum

Introduction

Hypericum perforatum L. is a herbaceous

perennial plant widely distributed in the

world. It is well known as ‘‘St. John’s

wort’’ in Anglo-Saxon folk medicine and

‘‘Erba di S. Giovanni’’ in Italy. The

extract of H. perforatum aerial parts,

usually gathered during the flowering

period or shortly before, has been used

throughout history as an antiinflamma-

tory, antidepressive and healing agent [1].

The part of this activity is attributed also

to the drug antimicrobial activity [2].

Hypericum extracts are marketed not

only as herbal medicinal products but

also in the form of dietary supplements

[3]. In recent years, the consumption of

H. perforatum-derived products has

increased dramatically and presently it is

one of the most consumed medicinal

plant over the world [4].

Hypericum perforatum contains a

number of constituents with documented

biological activity including naphtho-

dianthrones (hypericin, on whose con-

tent most of the available preparations

are standardized), phloroglucinol deriv-

atives (such as hyperforin), a broad

range of flavonoids and biflavonoids

as well as chlorogenic acid. The flavo-

noids comprising the aglycone quercetin

and several glycosides, namely hypero-

side (quercetin-3-galactopyranoside), ru-

tin (quercetin-3-rhamnosylglucoside) and

quercitrin (quercetin-3-rhamonopyrano-

side). These compounds have received

special attention as dietary constituents

during the last few years. The epidemi-

ological studies point out to their pos-

sible role in preventing cardiovascular

diseases and cancer [5]. This health-

promoting activity seems to be related to

the antioxidant (free-radical scavenging)

activity to flavonoids [6]. Quercetin,

3,3¢,4¢,5,7-pentahydroxylflavone, is one

of the most abundant flavonoids present

as aglycones and glycosides, in which

one or more sugar groups is bound to

phenolic groups by glycosidic bond at

the 3-position. Almost 180 different

glycosides of quercetin have been de-

scribed in nature, with rutin being one of

the most common [7].

Generally, HPLC methods of H. per-

foratum preparations are concerned with

the determination of hypericin and

pseudohypericin content [8]. As the bio-

logical effect of these preparations are

considerably arising, the analysis of mix-

ture of glycosides as well as aglycones is

desirable. Silva et al. [9] demonstrated

that compounds other than hypericins

are highly relevant for both radical-

scavenging and inhibition of lipid perox-

idation activities. The antioxidant activity

of the extracts could be attributed to

flavonoid glycosides and chlorogenic acid

[10].

2007, 65, 701–706

DOI: 10.1365/s10337-007-0204-z0009-5893/07/06 � 2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

Original Chromatographia 2007, 65, June (No. 11/12) 701

Determination of compounds in plant

materials requires extraction from the

sample matrix prior to injection into the

HPLC system. Flavonoids are commonly

extracted from herbal samples with pure

methanol, ethanol or their combination

with water [9–11], but in some cases

ethyl acetate [2] or acetone [12] have

been used. The optimum extraction

conditions varied depending on the

active compounds as well as on the kind

of plant. Determination of individual

flavonoid glycosides in plant materials is

difficult, due to their large number. In

many cases the knowledge of the total

aglycone content for each flavonoid is

required. Walle et al. [13] reported, that

the dietary form of flavonoids, the fla-

vonoid glycosides, may be hydrolyzed in

the oral cavity by bacteria to deliver the

biologically active aglycones at the sur-

face of the epithelial cells. Hydrolysis of

all glycosides to aglycones allows to ob-

tain more accurate data on flavonol

concentrations in food samples. This

process is dependent on the concentra-

tion of HCl, hydrolysis time and tem-

perature and the composition of the

extraction solvent. For example, higher

temperatures may provoke decomposi-

tion of some active components [14].

Hydrolysis of flavonoid glycosides from

food samples was mainly carried out in

1.2 M HCl at 90 �C for 2 h according to

a procedure presented by Hertog et al.

[15]. Quantitative data reported in the

literature are often obtained by different

analytical methods leading to some dif-

ficulties in the phytochemical compari-

son of the various plant samples.

Moreover, different extraction proce-

dures are indicated in the various Phar-

macopeias.

The aim of this work was to investi-

gate the optimum conditions for efficient

extraction of rutin and quercetin from H.

perforatum. For quercetin analysis as

aglycone the effects of acid concentration

and hydrolysis time on the extraction

recovery were also investigated.

Experimental

Reagents

The standards of flavonoids as well as

chlorogenic and caffeic acids, of which

chemical structures are presented in

Fig. 1, were purchased from Sigma-

Aldrich (Steinheim, Germany) and

Merck (Darmstadt, Germany). Solvents

and hydrochloric acid were obtained

from Merck (Darmstadt, Germany).

Hypericum perforatum were collected

from plants grown in the Botanic Garden

of the Warsaw University in the middle

of August 2004. The extracts were pre-

pared from dried leaves and dried flow-

ering tops.

HPLC Analysis

The chromatographic analysis was car-

ried out with an HPLC system from

Merck, which consisted of a gradient

pump, an autosampler with a 20 lL loop,

and diode array detector. The data was

collected and evaluated by the Merck

chromatographic software D-7000. The

analytical column was a Luna C-18(2)

25 cm · 4.6 mm i.d. (Phenomenex). A

gradient elution method was developed

based on modification of our earlier

procedure [16]. The mobile phase con-

sisted of 25 mM phosphate buffer at pH

2.5 (phase A) and methanol (phase B).

Gradient elution was used with the fol-

lowing eluents: time 0. 5% B; time

35 min, 10% B; time 50 min, 80% B;

time 52 min, 100% B. Eluent was deliv-

ered at a flow rate of 1 mL min)1 at

ambient temperature. Measurements

were performed at 350 nm for flavonoids,

whereas phenolic acids were detected at

280 nm. Major components were identi-

fied by comparison of retention times and

UV–vis spectra with reference standards.

The peak area of each selective com-

pound was measured for quantitative

analysis.

Sample extracts were also analyzed

using a Shimadzu LC system consisting

of binary pumps LC20-AD, degasser

DGU-20A5, column oven CTO-20AC,

autosampler SIL-20AC, and a detector

UV SPD 20A connected to a 3200

QTRAP mass spectrometer (Applied

Biosystem/MDS SCIEX). Compounds

for HPLC–MS were separated on a

150 · 2 mm, 3 mm particle size, column

Luna C-18 (2) from Phenomenex. As

eluent A 0.1% formic acid and eluent B

methanol were used. The mobile phase

was delivered at 0.2 ml min)1 in gradi-

ent mode: 0–5 min 20% B, 30 min 60%

B, 50 min 100% B, 55–60 min 20% B.

A MS system equipped with electro-

spray ionization (ESI) source opera-

ted in the negative-ion mode and a

quadrupole mass analyzer in a scan

mode from 50 to 1,000 m/z. ESI ioni-

Quercetin R = H

Hyperoside R = Gal

Quercitrin R = Rha

Rutin R = Glu-Rha

Chlorogenic acid Caffeic acid

- R

Fig. 1. Main constituents of Hypericum perforatum

Table 1. Retention data and ESI mass spectra data in the negative mode

Compound Molecularmass (Da)

Retentiontime (min)

Negative ion(M–H))(m/z)

Otherion (m/z)

Chlorogenic acid 354 7.4 353 191, 163, 137Caffeic acid 180 9.6 179 135Hyperoside 464 18.9 463 No*

Rutin 610 19.6 609 463Quercetrin 448 22.6 447 No*

Quercetin 302 26.4 301 No*

No* Not observed

702 Chromatographia 2007, 65, June (No. 11/12) Original

zation were as follows: curtain gas at

10 units declustering potential )50 V.

Negative ionization mode: source volt-

age )4.5 kV. Retention times and elec-

trospray ionization mass spectra for

investigated compounds in negative

mode are presented in Table 1.

Extraction and Hydrolysisof Plant Material

One gram of air-dried plant was ex-

tracted with 50 mL of appropriate sol-

vent and heated in a water bath for

30 min. Two 200 lL samples of plant

extract were dried in a desiccator under

vacuum. To the first sample 200 lL of

water/methanol (60:40 v/v) were added,

filtered and it was immediately injected

into the HPLC system. The second

sample was dissolved in 1.1 M HCl–

methanol (60:40 v/v) mixture and heated

in a water bath for the next 30 min,

dried in a desiccator, dissolved in water/

methanol (60:40, v/v), filtered and injec-

ted into the HPLC. Quantification was

obtained against calibration curves

based on the analysis of standard com-

pounds. Values represent the mean of

analysis of three independently extracted

and hydrolysed samples with the stan-

dard deviation.

Results and Discussion

Flavonoids, particularly quercetin and

glycolysated derivatives, are a major

class of compounds present in the H.

perforatum extracts [9, 17]. Silva et al. [9]

found that they represent almost 57% of

the total phenols, whereas hydroxycin-

namates only up to 10%. These com-

pounds are well known as antioxidants

and the observed properties of H. per-

foratum extract could be responsible for

some of the medicinal claims that are

attributed to this plant, namely those

related to free radical generation, such

as antiinflammatory properties. The

substitution of hydroxyl groups by

glycolysation decreases the antioxidant

activity of the aglycone, probably due to

steric hindrance [18]. The consumption

of H. perforatum extracts, either as

pharmaceutical preparations or included

in food products, has the potential to

confer antioxidant properties with bene-

fit to health.

Extraction

Several solvents were investigated,

including water, methanol, ethanol, ethyl

acetate and dimethylformamide (DMF)

to check their extraction efficiency from

H. perforatum. Rutin and quercetin, a

flavone glycoside and its aglycone, were

chosen as a model flavonoid compound

because they are the most widely and

abundantly present in herbs and plant

foods. The content of these compounds

Water

MeOH

EtOH

DMF

EtOAc

0 3 6 9 12 15 18

Concentration (mg g-1)

RutinQuercetin

Fig. 2. The efficiency of extraction of rutin and quercetin from Hypericum perforatum leaves usingdifferent solutions (1 g of herb with 50 mL of appropriate solvent)

0

40

60

80

100

0 5 10 15 20 25

)v/v(%,

HOe

MfotnetnoC Rutin

Quercetin

Concentration (mg g-1)

Fig. 3. Effect of methanol concentration on the extraction yield of rutin and quercetin fromHypericum perforatum leaves

Table 2. Analytical characteristics of the calibration graphs for phenolic compounds

Compound Linear range(mg L)1)

y = ax + b* Regressioncoefficient

LOD(lg L)1)

Recovery (%)(n = 4)

slope(x-10)3)

intercept

Chlorogenic acid 0.5–4.0 3.31 1.43 0.991 131 95.2 ± 3Caffeic acid 0.5–4.0 1.19 1.32 0.998 41 102.5 ± 4Rutin 0.4–32 3.74 2.76 0.992 62 99.6 ± 2Hyperoside 0.4–32 3.27 2.41 0.991 43 87.0 ± 4Quercitrin 0.4–32 1.68 2.85 0.990 56 89.0 ± 2Quercetin 0.4–32 1.22 2.15 0.990 59 92.9 ± 2

* y Concentration (mg L)1), x-peak area


(expressed in mg per gram of the dry

plant material) in the appropriate

extracts is presented in Fig. 2.

The extract composition was the same

in each case, but the flavonoids yields

were different. Extraction of quercetin as

well as rutin from H. perforatum with

methanol gave the highest efficiency, that

confirmed earlier results [11, 19]. With

dried plant materials, low polarity sol-

vents such as ethyl acetate, simply leach

the sample whereas alcoholic solvents or

water presumably rupture cell mem-

branes and enhance the extraction of

endocellular materials [20]. Because

methanol is more convenient as a com-

ponent of mobile phase in HPLC analy-

sis, we continued our experiments with

methanol as an extraction solvent

checking its concentration.

The best efficiency of extraction of

quercetin and rutin was achieved with

aqueous methanol in the range of 40–

80% (v/v) (Fig. 3). Similar results were

obtained when ethanol was used for

extraction of tested flavonoids from H.

perforatum. However, the concentration

of rutin in the final extract was much

lower (15.1 mg g)1 dry weight). The

increase in extraction time above 30 min

did not result in the increase of the

amount of quercetin or rutin. In the case

of the extraction with aqueous mixtures,

the required proportion of water in the

used solution is lower with fresh samples

than with dried samples [20]. Avato and

Guglielmi [12] reported that the applica-

tion of sonication improves extraction of

flavonoids. In our studies, however, when

the extraction from H. perforatum with

methanol/water (40:60 v/v) solution was

carried out on an ultrasonic bath at room

temperature, the efficiency of this process

was about 50% lower in comparison to

the heating on a water bath.

Hydrolysis

The effect of hydrolysis conditions was

examined for pure rutin standard. After

addition of different concentration of

HCl in 40% v/v methanol, the samples

were refluxed at 90 �C for an appropriate

time (5–20 min). The efficiency of rutin

hydrolysis was increasing with time due

to breaking the glycosidic bonds.

Hydrolysis for 5 min in the presence of

2.8 mol L)1 HCl as well as for 10 min

with 1.1 mol L)1 HCl efficiently released

quercetin from rutin. For most flavonoid

glycosides, the 2 h refluxing at 80 �C with

1.2 mol L)1 HCl was proposed [15].

However, the extended exposure time to

HCl could cause degradation of quercetin

[21]. Generally for the hydrolysis process,

optimum compromise is to achieve com-

plete release of aglycones and to minimize

degradation reactions of compounds

involved. The study presented by Lom-

bard [14] indicated that the main quer-

cetin glycosides are relatively heat stable,

thus, higher temperature and shorter time

for their hydrolysis could be employed.

Figure 4 presents the chromatograms

obtained with and without acidic hydro-

lysis of the standard mixture of the main

constituents of H. perforatum. The ex-

tract after hydrolysis (1.1 mol L)1 HCl

for 30 min) contained only traces of rutin

and all the glycosidic bonds in hyperoside

as well as in isoquercitrin were broken,

resulted in an extreme increase of

Fig. 4. The chromatograms of the standard mixture a without and b after acidic hydrolysis.Peaks 1 = chlorogenic acid, 2 = caffeic acid, 3 = hyperoside, 4 = rutin, 5 = quercitrin,6 = quercetin

Fig. 5. HPLC chromatograms of Hypericum perforatum: a methanolic extract; b extract afterhydrolysis. A Hypericum leaves; B Hypericum flowers. Peak identification as in Fig. 4


quercetin (Fig. 5b). Increasing of hydro-

lysis time in the range of 30–60 min did

not improve decomposition of rutin. In

addition, lower content of free quercetin

was observed, probably due to the deg-

radation process. Thus, in the further

experiments Hypericum extracts were

hydrolysed for 30 min.

Validation Procedure

To check the linearity of the response of

detector, a linear regression analysis of

peak area versus concentration of the

studied compounds was used. The line-

arity was determined by the square cor-

relation coefficients of the calibration

curves generated by three repeated injec-

tions of standard solutions at five con-

centration levels. The limit of detection

was estimated from the residuals of cali-

bration graphs. Calibration parameters

are shown in Table 2. All the compounds

showed a good linearity with regression

coefficients ‡0.990 and the limit of

detection (LOD) for S/N = 3 ranging

from 43 lg L)1 for hyperoside to

131 lg L)1 for chlorogenic acid.

The repeatability of peak area and

retention times were calculated by the

RSD of five injections carried out on the

same day. The RSD for the retention

times of all peaks was <1% and the

coefficient of variation for the peak areas

was <2%. The precision was established

by assaying six different extracts of the

same herb sample with the proposed

chromatographic analysis. The RSDs

were in the range of 1.2–2.4% for the

studied compounds.

Recovery experiments were performed

in order to study the accuracy of the

method. Known amounts of pure stan-

dards were added to a herb sample at

6 mg L)1 concentrations (Table 2).

Recoveries ranged between 87 and

102.5% and these values testify the

accuracy of the proposed method. The

reproducibility of the method was evalu-

ated during recovery experiments and the

RSD were always <7%.

Analysis of Plant Materials

On this basis, the analysis of phenolic

acids, three quercetin-3-O-glycosides and

their aglycone quercetin was performed

in the methanolic extracts of H. perfora-

tum leaves and flowers. The presence of

constituents was confirmed by checking

their MS spectra. The MS product ion

spectra for [M–H]) ions of determined

compounds are presented in Fig. 6. A

typical fragment of caffeic acid was ion at

m/z 135 due to the loss of carboxylic

group [M-45]). Chlorogenic acid is ester

of caffeic acid and fragments at m/z 163 is

caffeic acid without OH group [180–17]

and 191 represents 1,3,4,5-tetrahydroxy-

cyclohexanecarboxylic acid [192-H]).

According to La Torre [22] fragmenta-

tion of flavonoids occurred only at )80 V

deflector voltage, whereas the sugar could

be lost. The loss of sugar rhamonose in

rutin was observed under our conditions

()50 V) peak at and 463 m/z ion

occurred. The content of these com-

pounds (expressed as percentage in the

dry mass of plant material analysed) were

also determined after the acidic hydroly-

sis process (Table 3).

The hydrolysis process significantly

reduced the content of glycosides as

well as chlorogenic acid, which is

decomposed under acidic medium at

Fig. 6. ESI–MS spectra of compounds in the extract of Hypericum perforatum in negative mode


elevated temperature to caffeic acid. The

data showed that the levels of chlorogenic

acid are reduced by a factor of 9.5 and

the levels of caffeic acid increase by 3.7

after hydrolysis. The hydrolysis process

resulted in an extreme increase of quer-

cetin; its content was found as 0.7% in

leaves and 1.2% in flowers (calculated on

the dry mass of the herb).

The comparison of the flowers and

leaves of Hypericum herb shows a sig-

nificant higher content of quercetin in the

flowers. The amount of caffeic acid is

similar in these two parts and higher rutin

concentration was found in the leaves.

The data showed that leaves of Hyperi-

cum contain the highest amount of rutin,

while quercetin dominates in the flowers

of this herb. Umek et al. [17] found that

the content of rutin in Hypericum was in

a strong positive correlation with the

altitude of the growing site, opposite to

the quercitrin content. Recently, in Italy,

the existence of a specific chemotype of

H. perforatum, which does not produce

rutin, was reported [12]. However, it is

impossible to compare our results found

for the individual flavonoids with the

corresponding HPLC results published

previously because the abundance and

ratio of these analytes in the extracted

samples are variable depending on the

origin of the parent plant, on the period

of herb harvest as well as on the flower

development stage. Moreover, quantita-

tive data reported in the literature are

often obtained by different extraction

procedures.

Conclusion

The accurate quantification of rutin and

quercetin in H. perforatum samples is

dependent on the selection of appropriate

extraction conditions. The best efficiency

was achieved with aqueous methanol in

the range of 40–80% (v/v). Hydrolysis for

5 min in the presence of 2.8 mol L)1 HCl

as well as for 10 min with 1.1 mol L)1

HCl efficiently released quercetin from

rutin.

The proposed HPLC method for the

analysis of H. perforatum extracts

allowed the identification and quantifi-

cation of two phenolic acids, quercetin

and three glycosides of quercetin.

Chlorogenic acid was the predominant

phenolic acid in leaves and flowers of

this herb. After hydrolysis of the extract

significant decomposition of chlorogenic

acid to caffeic acid was observed.

The quercetin-3-O-glycosides were also

decomposed in applied hydrolysis con-

ditions, although the small amount of

rutin still remained.

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Table 3. Content of main compounds ofHypericum perforatum leaves and flowers after extractionand acidic hydrolysis (in mg g)1 calculated on the dry mass)

Analyte Leaves Flowers

Extraction Hydrolysis Extraction Hydrolysis

Chlorogenic acid 1.19 ± 0.06 0.13 ± 0.006 3.28 ± 0.15 0.31 ± 0.015Caffeic acid 0.15 ± 0.007 0.57 ± 0.02 0.17 ± 0.009 0.59 ± 0.03Rutin 3.45 ± 0.16 0.54 ± 0.03 1.49 ± 0.06 0.61 ± 0.03Hyperoside 1.78 ± 0.09 nd 4.70 ± 0.23 0.52 ± 0.02Quercitrin 0.38 ± 0.018 nd 1.84 ± 0.09 0.71 ± 0.03Quercetin 0.31 ± 0.016 6.94 ± 0.34 7.52 ± 0.36 12.4 ± 0.62

nd Not detected (limit of detection-0.04 mg g)1)Values represent mean ± SD, n = 3


Extraction and Hydrolysis Parameters for Determination of Quercetin in Hypericum perforatum

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Transcript of Extraction and Hydrolysis Parameters for Determination of Quercetin in Hypericum perforatum