Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 64 No. 4 pp.

327ñ333, 2007 ISSN 0001-6837

Polish Pharmaceutical Society

THE QUALITATIVE ANALYSIS OF ETHANOL EXTRACTS OF HERBAL RAW

MATERIALS BY METHOD OF HIGH PRESSURE LIQUID
CHROMATOGRAPHY
ZENONA KALVËNIENË, SAULË VELÞIENË, KRISTINA RAMANAUSKIENË*, ARÛNAS
SAVICKAS, LIUDAS IVANAUSKAS1 and VALDAS BRUSOKAS2.

Department of Pharmaceutical Technology and Social Pharmacy, 1Department of Analytic and

Toxicological Chemistry, 2Department of Pharmaceutical Chemistry and Pharmacognosy, Kaunas
University of Medicine, A. MisckeviËiaus g. 9 LT 44307 Kaunas, Lithuania
Abstract: The article describes the qualitative analysis of ethanol extracts from St. Johnís wort, cinnamon bark,
rosemary leaves, lavender flowers, and ginger rhizomes, and the extract of their mixture using the method of
high pressure liquid chromatography in order to identify flavonoids. The aim of the study was to identify the
amounts of rutin, hyperoside, quercetin, and other flavonoids in the multi-component extract as well as in the
control extracts of its compositional parts using the method of high pressure liquid chromatography, by evalu-
ation of the correlation between the amounts of flavonoids in the extracts. The evaluation of the results of the
study showed that flavonoid amount in the control extracts of the herbal material and flavonoid amount in the
ethanol extracts of the mixture of raw material did not differ significantly. It was found that the largest amount
of flavonoids was extracted from St. Johnís wort. The results of the analysis confirmed the assumption that the
yield of flavonoids from St. Johnís wort has no influence on the active substance of other medicinal herbal raw
materials.
Keywords: St. Johnís wort, ginger, cinnamon, lavender, rosemary, flavonoids

At present, the use of complex liquid herbal
preparations has been increasing. Taking into con-
sideration the needs of Lithuanian consumers, we
chose to produce a new sedative, indigestion-reliev-
ing complex phytopreparation. For the preparation
of the multi-componential herbal extract, we select-
ed herbal raw material (ginger rhizomes, cinnamon
bark, rosemary leaves, and lavender flowers) with
sedative and indigestion-relieving effects of its bio-
logically active substances. During the selection of
the raw material, attempts were made to achieve that
the biologically active substances in the composition
of herbal raw materials, i.e. essential oils (borneol,
bisabolene, cineol, camphor, linalool, terpineol,
eugenol, cinnamaldehyde, gingerol, shogaol, geran-
iol) and flavonoids (hyperforin, quercitrin, iso-
quercitrin, rutin), would be targeted at particular
symptoms and would complement each otherís
effect. Four herbal raw materials with positive
effects of their active substances on digestive tract
disorders were selected. According to literature, the
main active substances of the component raw mate-
rials, i.e. ginger rhizomes, rosemary leaves, cinna-
mon bark, and lavender flowers are essential oils (1-
8). All these raw materials can be used for the treat-
ment and prophylaxis of digestion disorders.
The herbal raw material of St. Johnís wort
(Hyperici herba) with sedative and anti-depressive
action of its biologically active substances was
selected. It is one of the most popular herbs in
Lithuania, which is attributed to the traditional
group of medicinal plants. St. Johnís wort accumu-
lates up to 13 per cent of enzymes, 0.1 ñ 1.25 per
cent of essential oils, up to 8 per cent of flavonoids
(rutin, quercetin, quercitrin, hyperoside) as well as
antrachinones (hypericin and its derivatives), hyper-
phorin, carotinoids, and resins (2, 9, 10).
Flavonoids, i.e. quercetin and quercitrin are distin-
guished by their sedative and antidepressive effects
of monoamine oxydase A type. Xantones also act as
monoamine oxydase A inhibitors: they inhibit anxi-
ety. Biflavonoids possess great affinity to benzodi-
azepine receptors. They relieve anxiety and stimu-
late sleep (11-15). R. F. Weiss recommended St.
Johnís wort ëfor the treatment of somatogenic and
psychogenic forms of depressioní (16). In the
Republic of Lithuania, the following indications of
St. Johnís wort are approved: the complementary

* Correspondence: Kristina RamanauskienÎ , tel +370 37 327255, +370 601 02567, e-mail povvis@gmail.com;

327
328 ZENONA KALVËNIENË et al.

Table 1. The composition of herbal extracts.

Composition of a multi-component Ratio of crude drug and ethanol in Ratio of crude drug and ethanol in
extract a multi-component extract control extract

St. Johnís wort herb (4 parts),
Ginger rhizome (3 parts),
Cinnamon bark (1 part),
1. St. Johnís wort herb 1 part, 1. St. Johnís wort herb extract, ratio
ethanol 12.5 parts 1:12.5
2. Ginger rhizome 1 part, ethanol 2. Ginger rhizome extract, ratio
16.7 1:16.7

Rosemary leaves (1 part), 3. Cinnamon bark 1 part, ethanol 3. Cinnamon bark extract, ratio
50 parts 1:50
Lavender flowers (1 part)
4. Rosemary leaves 1 part, ethanol 4. Rosemary leaves extract, ratio
1 part of crude drug mixture 50 parts 1:50
requires 5 parts of ethanol
5. Lavender flowers 1 part, ethanol 5. Lavender flowers extract, ratio
50 parts 1:50

Table 2. The quantitative identification of active compounds in herbal extracts.

Active Extract of Extract of Extract of Extract of Extract of Extract of

compounds, St. Johnës ginger cinnamon lavender rosemary the mixture of
µg/mL wort rhizomes bark flowers leaves herbal raw materials
Quercetin 700.04 1.11 568.1
Hyperoside 477.82 39.9 11.42 664.78
Rutin 531.49 11.68 658.22
Chlorogenic
acid 169.58 1.96 171.86
Caffeic acid 0.83
Astragalin
Keampferol 0.62 84.0
Quercitrin 1.90
Apigenin-
7-glycosyde 0.49
Vitexin
rhamnoside 19.5
Vicenin 20.68
Orientin 0.43

remedy for the treatment of neurotic depressions,
sleep disorders, and anxiety; the anti-inflammatory
and wound-healing remedy (17). For the study of
herbal extracts, the same methods of analysis as
those for chemical drugs are applied, and their
results are evaluated according to analogous criteria.
High pressure liquid chromatography (HPLC) was
selected as a reliable method of analysis. This
method allows for a qualitative and quantitative
analysis of components of herbal extracts (18-20). It
seems relevant to analyze the chemical composition
of the prepared extracts and to prove that St. Johnís
wort is a major source of flavonoids.
The aim of the study was to identify the
amounts of rutin, hyperoside, quercetin and other
flavonoids in a multi-component extract using the
method of high pressure liquid chromatography, by
estimating the correlation among the amounts of
flavonoids in the extract.
MATERIALS AND METHODS
The object of the analysis is a multi-component
herbal extract containing: herbs of St. Johnís wort,
ginger rhizomes, cinnamon bark, rosemary leaves,
lavender flowers, and 70 % ethanol (Table 1).
For the qualitative and quantitative identifi-
cation of flavonoids in the extracts of the crude
drug, control extracts were prepared, with the ratio
of crude drug and the vehicle matching the ratio of
 The qualitative analysis of ethanol extracts of herbal raw... 329

the multi-component extract (Table 1). The
method of the preparation was percolation (21-23).
The solvent of the crude herbal materials is 70 %
ethanol. Identification of flavonoids was per-
formed using high pressure liquid chromatogra-
phy; the equipment used was a chromatograph
Waters 2690 with the spectrophotometric detector
Waters 2487; chromatographic column ÑX-Terraì
RP18 3,5 µm, column dimensions 3.0 × 150 mm;
the volume of injected sample was 10 µl. Elution
is gradient, i.e. the composition of the gradient
changes with time. Eluent A: acetonitrile and tri-
fluoroacetic acid (99.9:0.1, v/v). Eluent B: dis-
tilled water and trifluoroacetic acid (99.9:0.1, v/v).
Gradient shift: from 0 min ñ 5% of eluent A and
95% of eluent B; from 45.0 min ñ 45% of eluent A
and 55% of eluent B; from 60.0 min ñ 5% of elu-
ent A and 95% of eluent B. The eluent flow rate
was 0.4 mL/min. Preparation of the tested solu-
tion: the extract was filtered through a membrane
filter with pore diameter of 5 µm and diluted with
70% vol. ethanol solution, ratio 1 : 1. Flavonoids
were identified at 360 nm wavelength. The com-
pounds were identified and their amounts in
extract calculated according to the length of reten-
tion of the extracted components, peak areas, and
ratios between individual peak areas and those of
standard peaks.

Figure 1. The chromatogram of flavonoid identification by HPLC in the extract of the multi-component herbal mixture.
1 ñ chlorogenic acid; 2 ñ caffeic acid; 3 ñ vicenin; 4 ñ orientin; 5 ñ vitexin-4-rhamnoside; 6 ñ vitexin; 7 ñ isovitexin; 8 ñ rutin; 9 ñ hyper-
oside; 10 ñ apigenin-7-glucoside; 11 ñastragalin; 12 ñ quercitrin; 13 ñ luteolin; 14 ñ quercetin; 15 ñ apigenin; 16 ñ kaempferol.

Figure 2. The chromatogram of flavonoid identification by HPLC in the control extraction of St. Johnís wort.
1 ñ chlorogenic acid; 2 ñ caffeic acid; 3 ñ vicenin; 4 ñ orientin; 5 ñ vitexin-4-rhamnoside; 6 ñ vitexin; 7 ñ isovitexin; 8 ñ rutin; 9 ñ hyper-
oside; 10 ñ apigenin-7-glucoside; 11 ñastragalin; 12 ñ quercitrin; 13 ñ luteolin; 14 ñ quercetin; 15 ñ apigenin; 16 ñ kaempferol.
330 ZENONA KALVËNIENË et al.

Figure 3. The chromatogram of flavonoid identification by HPLC in the control extraction of ginger rhizomes.
1 ñ chlorogenic acid; 2 ñ caffeic acid; 3 ñ vicenin; 4 ñ orientin; 7 ñ isovitexin; 8 ñ rutin; 10 ñ apigenin-7-glucoside; 11 ñastragalin; 12 ñ
quercitrin; 13 ñ luteolin; 14 ñ quercetin; 16 ñ kaempferol.

Figure 4. The chromatogram of flavonoid identification by HPLC in the control extract of cinnamon bark.
1 ñ chlorogenic acid; 2 ñ caffeic acid; 3 ñ vicenin; 4 ñ orientin; 5 ñ vitexin-4-rhamnoside; 6 ñ vitexin; 7 ñ isovitexin; 8 ñ rutin; 9 ñ hyper-
oside; 10 ñ apigenin-7-glucoside; 11 ñastragalin; 12 ñ quercitrin; 13 ñ luteolin; 14 ñ quercetin; 15 ñ apigenin; 16 ñ kaempferol.

RESULTS Furthermore, not only flavonoids but also two phe-

In the prepared multi-component extract
(MCE), the largest amounts of the following
flavonoids were identified: hyperoside, quercetin,
rutin; chlorogenic acid was also identified (Table 2).
Figure 1 shows a chromatogram in which other
active substances in addition to the above-men-
tioned flavonoids in the multi-component herbal
extract were found. In the control extract of St.
Johnís wort (SJWCE), similar flavonoids were iden-
tified and their amounts measured (Table 2).
nolic acids ñ caffeic acid and chlorogenic acid ñ
were identified in SJWCE (Fig. 2). The amount of
chlorogenic acid was measured (Table 2). In the
control extract of ginger (GCE), phenolic acids, i.e.
caffeic acid and chlorogenic acid were identified
and their amounts measured (Table 2). The results of
the analysis have shown a small amount of
flavonoids in ginger rhizome extract, which is con-
gruent with the data in the literature (1-3, 5, 11).
Flavonoids were not detected in this extract (Fig. 3).
In the control extract of cinnamon bark (CCE), very
 The qualitative analysis of ethanol extracts of herbal raw... 331

Figure 5. The chromatogram of flavonoid identification by HPLC in the control extract of lavender flowers.
1 ñ chlorogenic acid; 2 ñ caffeic acid; 3 ñ vicenin; 4 ñ orientin; 5 ñ vitexin-4-rhamnoside; 6 ñ vitexin; 7 ñ isovitexin; 8 ñ rutin; 9 ñ hyper-
oside; 10 ñ apigenin-7-glucoside; 11 ñastragalin; 12 ñ quercitrin; 13 ñ luteolin; 14 ñ quercetin; 15 ñ apigenin; 16 ñ kaempferol

Figure 6. The chromatogram of flavonoid identification by HPLC in the control extraction of rosemary leaves.
2 ñ caffeic acid; 5 ñ vitexin-4-rhamnoside; 6 ñ vitexin; 7 ñ isovitexin; 8 ñ rutin; 9 ñ hyperoside; 10 ñ apigenin-7-glucoside; 11 ñastragalin;
12 ñ quercitrin; 13 ñ luteolin; 14 ñ quercetin; 16 ñ kaempferol

small amounts of the following substances were
identified: quercetin, apigenin-7-glycoside,
quercitrin, and kaempferol (Table 2). In the chro-
matogram (Fig. 4), hyperoside, rutin, luteolin,
chlorogenic and caffeic acids were identified; how-
ever, their amounts were not significant. The largest
amount of biologically active substances in crude
cinnamon belongs to essential oils (1-3, 11), and the
amount of the identified flavonoids is not pharma-
cologically relevant (Table 2). In the control extract
of lavender flowers (LCE), hyperoside, vitexin,
rhamnoside, vicenin, and rutin were identified
(Table 2). In addition to these, luteolin, apigenin,
and apigenin-7-glycoside were also found in LCE
(Fig. 5). In the control extract of rosemary leaves
(RCE), the following compounds and their amounts
were identified: caffeic acid, isovitexin, luteolin
(Fig. 6). A big part of the area under peaks belongs
to some unidentified compounds released during 31
and 34-36 min., and the length of their release sig-
nificantly differs from the release of the standards
from the column (Fig. 6).
The study showed that the highest amount of
flavonoids (hyperoside, rutin, quercetin) and chloro-
genic acid was released from the control extract of
St. Johnís wort (Fig. 7). Figure 7 shows that the con-
trol extract of St. Johnís wort yields the largest
amount of quercetin and the smallest amount of
chlorogenic acid. The identified amounts of
flavonoids in the control extract of lavender, cinna-
332 ZENONA KALVËNIENË et al.
Figure 7. Correlation of flavonoid amounts in extracts.
mon and rosemary are relatively small if compared
to the control extract of St. Johnís wort (Fig. 7).
Therefore, it can be stated that the largest amount of
flavonoids in the multi-component extract is extract-
ed from crude herbs of St. Johnís wort. This coin-
cides with the data in the literature, in particular that
the main flavonoids in the herbs of St. Johnís wort
are hyperoside, rutin and quercetin (2, 9, 10). The
comparison of the findings in control extract and in
the extract of the mixture of the herbal raw materi-
als showed that the amount of flavonoids differed
insignificantly, which supports our assumption that
other components of the herbal mixture have no
influence on the yield of flavonoids. The method of
high pressure liquid chromatography has confirmed
the compatibility of biologically active substances
that were extracted from the mixtures of herbal raw
materials.
CONCLUSION
Using the method of high pressure liquid chro-
matography it was found that the largest amount of
flavonoids is extracted from crude herbs of St.
Johnís wort; therefore, the amount of flavonoids in
a multi-component extract differs insignificantly
from the flavonoid amount in the extract of St.
Johnís wort.
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Received: 5.01.2007