Chinese Medicine, 2010, 1, 23-27
doi:10.4236/cm.2010.11004 Published Online June 2010 (http://www.SciRP.org/journal/cm)
Determination of Cichoric Acid as a Biomarker in
Echinacea Purpurea Cultivated in Iran Using
High Performance Liquid Chromatography
Javad Zolgharnein1*, Ali Niazi2, Somieh Afiuni-Zadeh1, Khosrow Zamani1
1Department of Chemistry, Faculty of Science, University of Arak, Arak, I.R. Iran
2Department of Chemistry, Faculty of Science, Azad University of Arak, Arak, I.R. Iran
Received March 4, 2010; revised April 29, 2010; accepted May 10, 2010
Abstract
Echinacea purpurea (Purple coneflower) is an immunostimulating drug, containing multiple substances. The
most important substance in activity is polysaccharide, caffeic acid derivatives (cichoric acid), alkamides and
glycoproteins. It is not clear yet, which substances are responsible for activity. Cichoric acid is an appropri-
ate marker of the quality of Echinacea purpurea containing product, because it has immune stimulatory ef-
fects and it is susceptible to degradation. In this study a TLC scanner system and HPLC method has been
used for identification and determination of cichoric acid in aerial parts of Echinacea purpurea. The results
showed that the cichoric acid content of Echinacea purpurea cultivated in Iran is about 1.50 ± 0.65% (w/w)
which is comparable with cichoric acid content in native plants. The local conditions have no significant ef-
fect on cichoric acid content as a biomarker of Echinacea purpurea quality.
Keywords: Echinacea Purpurea, Cichoric Acid, TLC, HPLC, Iran
1. Introduction
Herbal medicine has lower side effects in comparison
with chemical drugs. Attention and stimulation for using
herbal drugs are increasing in recent years. Systematic
study about characteristics properties of herbal drug has
been increased. Echinacea purpurea Monech (purple
coneflower), originated in the United States of America
and was brought to Europe in the late 19th century [1].
Recently, Echinacea purpurea has been cultivated in
some parts of Iran (e.g. Isfahan) and used in some herbal
drugs [2]. It exhibits antioxidative, antibacterial, antiviral
and antifungal activities, but most of all it affects on
various immune parameters [3-21]. Because of its im-
muno-enhancing activity, it has been recently used in
AIDS therapy [22]. It is used for treating flu and cold,
against which symptomatic treatment is an established
procedure
The most important potential active compounds in
purple coneflower are polysaccharides, caffeic acid de-
rivatives (especially cichoric acid), alkamides and gly-
coproteins [6,7]. Cichoric acid is an appropriate marker
because it has immune stimulatory effects and is proba-
bly the most effective substance in Echinacea purpurea.
Climate and cultivation conditions could affect cichoric
acid content in Echinacea purpurea. Recently, neverthe-
less, the lack of studies on the adaptability of the culture,
the productivity and the quality of the product was ap-
parent. Various methods for determination of cichoric
acid such as HPLC, and capillary electrophoresis have
been used [22-31].
In this study, a TLC scanner system and an HPLC
method was used for identification and quantification of
cichoric acid content in aerial parts of Echinacea pur-
purea which is cultivated in Iran. The result was com-
pared with native plants in Europe and America.
2. Experimental
2.1. Chemicals
2-aminoethyl diphenyl borinate (Sigma), caffeic acid,
chlorogenic acid and all solvents; methanol, ethyl acetate,
formic acid was analytical grade and purchased from
Merck. All solvents used for running HPLC, were HPLC
grade. Dionized, double distilled water was used.

J. ZOLGHARNEIN ET AL. 24
2.2. Samples
Several different batches of aerial parts of aerated and
dried Echinacea purpurea were collected and ground
with a 40 mm mesh to a fine powder. Samples were pre-
pared by solving the powder in methanol/water (70:30)
with the ratio of 1 g in 10 ml using an ultrasonic bath for
approximately 5 min and then filtered. Chlorogenic acid
and caffeic acid were dissolved in alcohol/water solu-
tions which are stable at least for 30 hours if light pro-
tected.
2.3. TLC and HPLC
The CAMAG TLC system with auto sampler and scan-
ner which is controlled by Wincats software was used for
identification. The plates were prepared by TLC silica
gel HF254 (5 µm). The mobile phase composed of an-
hydrous formic acid, ethyl acetate and water (2:1:17).
Sample solution and standard solution were prepared
respectively by dissolving 0.5 g powder in 5 ml solvent
and 0.05 mg of caffeic acid and 0.05 mg of chlorogenic
acid in 10 ml solvent 25 µl of sample solution and 10 µl
of standard solutions were applied as TLC bands [31,32].
For detection of stains under UV366 lamp, 2-amino di-
phenyl borinate in methanol (10 mg/ml) was used as re-
agent which is famous as natural product reagent.
2.4. High Performance Liquid Chromatography
(HPLC)
The Cecil 1100 HPLC system was used for quantitative
assay. This system equipped with a 20 µl injection loop,
a C18 Lichrosorb column with 0.25 m length, and 4.6 mm
diameter and UV-Vis detector which was set at 330 nm.
Acetonitrile and phosphate buffer (phosphoric acid/water
(1:99 V/V) were used as mobile phases in a gradient elu-
tion of solvents with 1.5 ml/min flow rate according to
set programs in EP and USP [31,32]. Sample solution
and standard solution were prepared by dissolving 0.25
mg of powder of sample and 0.47 mg chlorogenic acid in
10 ml solvent respectively. Chlorogenic acid was used as
external standard which was cheaper and available in
Iran [22].
The percentage of cichoric acid content was calculated
by the following equation [22,31,32]:
Cu% = 100 (Au × Cs × 0.695)/(As × Ct) (1)
where Cu is the concentration of cichoric acid in sample
peak; Au, the peak area for cichoric acid; Cs, the concen-
tration of standard chlorogenic acid solution (mg/ml); As,
the peak area for standard chlorogenic acid; Ct, amount
of powder in sample solution (mg/ml); 0.0695, the re-
sponse factor of cichoric acid relative to that of chloro-
genic acid. The detector response factor was planned as
the ratio of slope of the two calibration curves prepared
with standard solutions [22,31,32].
3. Results and Discussion
The chemical structures of Cichoric acid (a), Chloro-
genic acid (b) and Caffeic acid (c) are shown in Figure 1.
Figure 2 shows the image of TLC plate of sample and
standard solutions. In the left track, the yellow florescent
zones are related to chlorogenic acid (Rf = 0.45) and caf-
feic acid (Rf = 0.84) contained in the standard solutions.
In the right track of TLC plate, the fluorescent zones are
related to caftaric acid (Rf = 0.52) and cichoric acid (Rf =
0.86) contained in sample solution. Figure 3, clearly
confirmed the presence of cichoric acid and absence of
chlorogenic acid in sample solution [31,32].
The chromatograms related to standard and sample are
shown in Figures 4 and 5 respectively. According to
literatures the relative retention time for cichoric acid
respect to chlorogenic acid is about 2.3 [31]. While the
chlorogenic acid peak appears at 8 minute, the cichoric
(a)
(b)
(c)
Figure 1. (a) The structure of Cichoric acid, (b) Chlorogenic
acid and (c) Caffeic acid.

J. ZOLGHARNEIN ET AL.25
Figure 2. TLC image of sample solution containing caftaric
acid and cichoric acid (right track) and standard solution
containing chlorogenic acid and caffeic acid (left. track).
Figure 3. TLC schematic figure of sample containing caftaric
acid and cichoric acid (right track) and standard solution
containing chlorogenic acid and caffeic acid (left. track).
Figure 4. HPLC chromatogram of 0.25 mg/5 ml standard
solution (chlorogenic acid).
Figure 5. HPLC chromatogram of 2.5 mg/10 ml sample
solution.
acid peak should appear at about 18 minute. Then the
cichoric acid concentration is calculated, based on equa-
tion which was mentioned in Equation (1). The HPLC
experimental data obtained is shown in Table 1.
Calculation results shows that cichoric acid concentra-
tion is about (1.5 ± 0.65)% (w/w) in the aerial parts of
Echinacea purpurea cultivated in Iran. This amount show
good agreement with native plant (1.2-3.1)%. This means
that the local conditions of cultivation of this herbal drug
have no significant effects on its medicine marker.
Table 1. The HPLC experimental data.
Cs 0.047 mg/mL
Ct 2.5 mg/mL
As 4934823
Au 5977445
Cu 1.58%
SD ±0.65%
Cs: concentration of standard solution (chlorogenic acid).
As: the peak area for chlorogenic acid.
Ct: concentration of sample solution containing cichoric acid.
Au: the peak area for cichoric acid.
Cu: concentration of cichoric acid in sample solution.
SD: Standard deviation (n = 3).

J. ZOLGHARNEIN ET AL. 26
.
4. Conclusions
The qualitative and quantitative analysis of Cichoric acid
content in Echinacea purpurea which is cultivated in Iran
has been studied. Cichoric acid is probably one of the
most important markers for controlling the quality of
drugs containing Echinacea purpurea. The results showed
that the cichoric acid content of Echinacea purpurea is
appropriate for drugs in comparison with native plants.
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