Anal Bioanal Chem (2005) 383: 1098–1105
DOI 10.1007/s00216-005-0120-8
ORIGINA L PA PER
T. Ligor . A. Ludwiczuk . T. Wolski . B. Buszewski
Isolation and determination of ginsenosides in American ginseng
leaves and root extracts by LC-MS
Received: 13 May 2005 / Revised: 12 September 2005 / Accepted: 13 September 2005 / Published online: 9 November 2005
# Springer-Verlag 2005
Abstract Ginseng saponins (ginsenosides) were extracted
from the root and leaves of locally cultivated American
ginseng (Panax quinquefolium L.). For the isolation of
compounds from plant samples three different extraction
methods were utilized: accelerated solvent extraction, the
ultrasound-assisted solvent extraction and mechanical shak-
ing assisted solvent extraction. The separation of compounds
was achieved with a water–acetonitrile gradient system
using a C18 reversed-phase column. Target compounds
were identified in MS2 and MS3 experiments. The relative
distribution of these ginsenosides in each root and leaf
extract was established. The limit of detection of the method
was less than 30 ng/ml. Recovery of ginseng saponins in
spiked samples exceeded 80%, while the relative standard
deviation ranged from 7.1 to 9.1%. The total concentrations
of ginsenosides were 41 and 13 mg/g in root and leaves.
Keywords American ginseng . Leaves . Extraction .
Liquid chromatography/mass spectrometry
Introduction
The Panax genus consists of approximately 20 species
occurring in Asian countries and in North America. Gin-
seng has been used for thousands of years as a traditional
medicine in many oriental countries. Ginseng extracts have
wide pharmacological properties, such as antifatigue,
T. Ligor . B. Buszewski (*)
Department of Environmental Chemistry and Ecoanalytics,
Faculty of Chemistry, Nicolaus Copernicus University,
7 Gagarina Street,
87100 Toruń, Poland
e-mail: bbusz@chem.uni.torun.pl
A. Ludwiczuk . T. Wolski
Department of Pharmacognosy with Medicinal Plant
Laboratory, Skubiszewski Medical University,
1 Chodźki Street,
20093 Lublin, Poland
antidiabetes, vasodilation and antidepressant, and are
effective in stimulating the memory as well as in the
prevention of cancer and the ageing process. The most
abundant forms are Panax ginseng, P. japonicus and P.
quinquefolium, which grow in North America [1]. Both, P.
ginseng (Korean ginseng) and P. quinquefolium (American
ginseng) are the most used species in medicine. These
species differ imperceptibly in therapeutic properties [2].
Many scientists are of the opinion that the American
ginseng (P. quinquefolium) is less stimulating than the
Korean one. American ginseng is generally used as a mild
tonic; however its mechanism of action remains unclear
[3]. Recent publications indicated a weak central nervous
system (CNS) stimulant activity [4] and a hypoglycemic
activity and showed the immunomodulating properties [5].
Instead, P. quinquefolium is more effective as an adapto-
genic agent and a tonic against stress. In general, saponins
and polysaccharides are thought to be responsible for its
activity. American ginseng [2] is rich in the Rb1 group of
ginsenosides (Rb1, Rb2, Rc, Rd, mRb2, mRc), which can
explain the sedative and metabolic effects on the CNS.
Moreover, previous data suggested that administration of
American ginseng leaf extract reduced high blood glucose
levels [6]. Therefore, American ginseng leaves have
promising potential to be an additional source of ginseno-
sides, along with ginseng root, in the treatment of diabetes
mellitus. Popowich and Kitts [7] suggested that P.
quinquefolium leaves and roots are potentially a source of
rare ginsenosides Rh2 and Rg3. These ginsenosides are not
naturally present in American ginseng, but are products of
a thermal process (conversion of ginsenosides Rg1, Rc and
Rd). Rh2 can reduce the proliferation of a variety of
cultured cancer cells and can influence apoptosis [8]. Rg3
has also been shown to possess antitumor properties [9].
Ginsenosides can be described as a glycoside consisting
of an aglycone moiety and one or more covalently linked
sugars monomers. However, most ginsenosides contain
multiple oligosaccharide units at various positions. 20(S)-
Protopanaxadiol and 20(S)-protopanaxatriol are the core of
Rb1 group and Rg1 group (Re, Rf, Rg1, glc-Rc) ginseno-
sides, respectively (Fig. 1).
 1099
Fig. 1 Structure of 21
22 24
ginsenosides R3O 26
HO 20

12 17 27

19 18 14 15
1 9
3 5 30
6
R1O
29 28
R2

Ginsenoside R1 R2 R3
20 (S)-protopanaxadiol
Rb1 glc-glc H glc-glc
Rb2 glc-glc H glc-ara (p)
Rc glc-glc H glc-ara (f)
Rd glc-glc H glc
20 (S)-protopanaxatriol
Re H -O-glc-rha glc
Rf H -O-glc-rha H
Rg1 H -O-glc glc
Rg2 H -O-glc-rha H
Rh1 H -O-glc H

An important part of the recovery and purification
process of active compounds from plant material is ex-
traction. Ginseng saponins from different species of gin-
seng were extracted with water–methanol mixtures using
ultrasonic extraction [10] and a mechanical shaker at room
temperature for 12 h [11]. Others workers applied methanol
with heating under reflux [12] and Soxhlet apparatus for
extraction of ginsenosides [13]. These methods are
commonly used because of their simplicity and low cost.
However, the conventional methods require large volumes
of solvents, which are often expensive or hazardous. The
other disadvantages include long extraction time, labor-
intensive procedures, unsatisfactory extraction efficiency
or poor reproducibility. The desire to reduce these disad-
vantages has led to the development of newer techniques
for ginsenosides extraction, such as micelle-mediated
extraction [14] and supercritical fluid extraction [15].
A relatively new technique, known as pressurized liquid
extraction or accelerated solvent extraction (ASE), has
shown good potential in overcoming major drawbacks of
classical extraction methods. ASE was used for extraction
of active ingredients from medicinal plants and herbal
materials [16, 17] such as berberine [18] and ginsenosides
[18, 19]. Recently, some investigators have focused on
development of microwave-assisted extraction (MAE) as
an alternative method to the time-consuming conventional
method [20]. The results of 15-min MAE were better than
that from 10-ho conventional solvent extraction [21].
There are many methods for analyzing ginsenosides,
e.g., thin layer chromatography [1], gas chromatography
(GC) [22] and high-performance liquid chromatography
(HPLC) [23]. Among these, GC methods will give good
sensitivity and resolution but needs additional sample pre-
paration such as hydrolysis followed by trimethylsilyla-
tion; therefore, only protopanoxadiol and protopanoxatriol
can be analyzed [22]. The HPLC methods are widely used
for analyzing ginsenosides. Owing to ginsenosides being
poor chromophores, the detection is limited to short wave-
lengths (e.g., 205 nm) and many compounds may interfere
with target compounds. Recently, a method based on
evaporative light scattering detection was applied [24]. The
limits of detection for Rg1, Re, Rb2 and Rd are 50, 65, 85
and 40 ng, respectively. Ion chromatography with pulsed
amperometric detection has good sensitivity, and the limits
of detection for Rg1 and Re are 0.8 and 1.0 ng, respectively
[25]. Liquid chromatography (LC) coupled with tandem
mass spectrometry (MS/MS) was developed during the last
decade [26–28]. This technique is very useful for charac-
terization and quantification of ginseng extracts. The limit
of detection for ginsenosides can be at the level of 2 pg,
showing that it is the most sensitive method [29].
In this investigation LC and MS with multiple frag-
mentation for analysis of root and leaves of locally culti-
vated P. quinquefolium were applied. Different extraction
methods such as ASE, ultrasound-assisted solvent extrac-
tion and shaking solvent extraction followed by solid-
phase extraction (SPE) for sample preparation and cleanup
were tested. The characterization of target molecules was
carried out using the collision-induced dissociation (CID)
spectra from the sodium adduct spectra. The extraction
procedures were compared and the relative distribution of
ginsenosides in leaves and root of American ginseng ex-
tract was established.
1100
Experimental
Apparatus
The apparatus consisted of a HPLC 1100 (Agilent,
Waldbronn, Germany) system with a quaternary pump, a
UV–vis detector, an automatic sample injector and an
Agilent 1100 series LC/MSD Trap VL mass spectrometer
(Agilent, Waldbronn, Germany) with an electrospray
ionization interface. For the MS, the following conditions
were used: capillary voltage 4.0 kV; skimmer 1 65.8;
capillary exit offset 91.6 V; trap drive 43.9. The drying
temperature was set at 325°C, the drying flow at 7.5 l/min
and the nebulizer pressure at 8 psi. The mass spectrometer
was operated in the positive ion mode and with a scan
range m/z 200–1,300 amu. The ASE system (ASE 100)
was purchased from Dionex Corporation (Sunnyvale, CA
USA). The HPLC separation was performed on the on a
150×4.6-mm inner diameter, 5-μm octadecyl L-column
(Waters). The flow rate through the HPLC column was
0.9 ml/min and the entire effluent was directed to the mass
spectrometer. The binary gradient consisting of water and
acetonitrile was used as follows: 0–30 min, isocratic 18%
acetonitrile; 35 min, 30% acetonitrile; 35–40 min, isocratic
30% acetonitrile; 45 min, 35% acetonitrile; 52 min, 40%
acetonitrile; and 100% acetonitrile from 52 to 55 min at a
flow of 1.5 ml. Both solvents contained 0.01 % acetic acid.
Chemicals and reagents
Ginsenosides Rb1, Rb2, Rg1, Rc and Rd were purchased
from Roth (Karlsruhe, Germany). Deionized water was
from a Milli-Q system (Millipore, USA); acetonitrile and
methanol (both LC–MS grade) and octadecyl C18 Baker
Bond were from Baker(Deventer, The Netherlands). Acetic
acid 99.99+% was purchased from Sigma-Aldrich (Stein-
heim, Germany).
Plant material and extraction
A four-year-old American ginseng plant (P. quinquefolium)
cultivated in the Department of Industrial and Medical
Plants, University of Agriculture (Lublin, Poland) and
collected in September 2001 was used in the investigations.
The plant was authenticated by the taxonomist of the Marie
Curie-Skłodowska University (Lublin, Poland).
Three different extraction methods were applied: me-
chanical shaking, ultrasound-assisted extraction and ASE.
In all cases dried and pulverized plant material (2.0 g) and
50% (v/v) aqueous methanol as a solvent were used.
1 Shaking: The plant sample was extracted three times
(20 min for each extraction) with 50 ml of fresh sol-
vent in a mechanical shaker and the extracts were
combined.
2 Ultrasound-assisted extraction: The mixture of sample
and solvents was transferred to an Erlenmeyer flask
and placed in ultrasonic bath. Ultrasonification was
carried out for 20 min. The extraction was repeated
three times with 50 ml of solvent and then the extracts
were combined.
3 ASE: The sample was placed in a stainless steel cell and
extracted at 120°C. Subsequently, the cell was heated at
the preset extraction temperature and the solvent was
continuously pumped through the sample. When the
cell was full of the extraction solvent, the cell was
heated and pressurized (10 MPa) for a fixed time to
ensure that the sample reached thermal equilibrium.
Afterward, a static period occurred during which the
sample was extracted for 5 min with three static periods
at the preset extraction temperature and pressure. The
total extraction time was approximately 25 min (heat-
ing plus three static periods).
Each extract was filtered and evaporated to dryness by a
rotary vacuum evaporator at 60°C. The residue was redis-
solved in 25 ml of 50% (v/v) aqueous methanol. A 10-ml
aliquot of this solution was applied to a C18 SPE column
(500 mg of bed) that was conditioned with 10 ml methanol
followed by 10 ml water. After washing with 10 ml water
followed by 10 ml of 30% aqueous methanol, in order to
remove excessive interferences, the ginsenosides were
eluted with 10 ml methanol.
Calibration curve
The standard curves for five concentrations ginsenosides
were prepared for determination of saponins in plant
extracts. Standard solutions of Rb1, Rb2, Rg1, Rc and Rd
were prepared in 90% acetonitrile by sequential dilutions of
the stock solution to a concentration of 0.5 mg/ml.
The calibration curve was established for each com-
pound at a concentration level of 0.08–1.20 μg/ml. Stan-
dards were analyzed before the analysis of each batch of
samples.
Results and discussion
Several methods for the analysis of Panax species have
been published to date. A number of works reported that
the separation of ginseng extract constituents was accom-
plished using a long gradient of up to 60 min in with a
composition of water and acetonitrile [10, 12, 14]. To
obtain an acceptable separation of major saponins and
other interference compounds (saccharides, chlorophylls,
etc.) a 52-min chromatographic run was used. From the
analytical point of view, the baseline resolution between
Rg1 and Re is rather difficult, especially in the presence of
higher concentrations of one them.
SPE is a widely used pretreatment method which has
the property of removing interferences from a sample ma-
trix. Therefore, this method can be used for cleanup og
ginseng extracts. Recoveries were determined by addition
 1101
Table 1 Recoveries of ginsenosides (n =4) obtained for SPE shows that the recovery for all ginseno-
Compound Recovery (%) sides was between 83.8 and 89.6% for all saponins. Lower
values were reported for Rb2 and Rd—83.8 and 89.6%,
Rb1 91.2±8.8 respectively. The relative standard deviation (RSD) did not
Rb2 83.8±7.9 exceed 9.5% for any of the compounds. For Rg1, the lowest
Rc 92.1±8.3 value of the RSD (7.1%) was observed.
Rd 89.6±9.1 The ginseng saponins extracted from root and leaves
Rg1 85.3±7.1 were analyzed using HPLC with MS detection. The [M
+Na]+ ions were observed in positive MS mode. The
abundances of protonated species [M+H]+ were very weak
of 12.5 ml of standard solution to 12.5 ml of 50% metha- or not observed at all. Figure 2 shows the mass spectra
nol–water extract. The average recovery and standard obtained from ginsenoside; it does not contain traces of
deviation are shown in Table 1. The analysis of the results protonated cations. Probably, the labile nature of proton-

Intens.
x106 Intens.
x10 4
+M S, 36 .0m in (#2394) Rg1 +M S, 36 .0m in (#2394)
5 4 [M+H]+ 801 m/z 823. 7
3 773.5
4
2 778. 8 780. 4 801.6
776.9
793.5 805.0
772.4 79 9.8 802.5
3 1 785.2
788.5
795.0 796.3 807.1 809.7
0
770 775 780 785 790 795 800 805 m/z
2

1
643.6 719.6
0
200 400 600 800 1000 m/z

Intens. +M S, 46.0m in (#3085)

x106 Intens.
x10 4
1071. 6
+
+M S, 46.0m in (#3085) Rb1
6 1. 5
1083. 4 [M+H] 1109 m/z 1119.7

1. 0 1093. 2
1099. 6 1113. 6 1131 .8
1097. 5 1105.2
1064. 9 1117. 7
1075.7
4 0. 5 1090.9
1106.8
109 5.1
0. 0
1060 1070 1080 1090 1100 1110 m/z
2

789. 7
0
200 400 600 800 1000 m/z

Intens. +M S, 47.1m in (#3164)

x106 In tens.
x104
+
+M S, 47 .1m in (#3164) Rc
5
5

4 1027.8
[M+H] 1079 m/z 1101.7
3
4 1019.1
2 1039.6
10 44.5 1054.6
3 1 1012.6
1033.5
1048.8
1064. 4 1072.6
1075.7
1079.4 1083.6
1001. 3 1009.5 1024.2 1060.4
0
2 1000 1010 1020 1030 10 40 1050 1060 1070 1080 m/z

1
789. 6
0
200 400 600 800 1000 m/z

Intens. +M S, 4 8.2m in (#3927 )

x10 7 Intens.
x105
+MS, 48.2m in (#3927) Rb2
6
5 [M+H]+ 1079 m/z
4 1101.7
3
4 1080.8
2
9 97.6 1045. 5 1062.0 1078.2 1090. 7
1
1014. 7 1027. 5
0
2 1000 1010 1020 1 030 1040 105 0 1060 1070 1080 m/z

0
200 400 600 800 1000 m/z

Intens. +M S, 50.6m in (#3432)

x10 6
8
Intens.
x104
9 30.6 +MS, 50.6m in (#3432) Rd
3
[M+H]+ 947 m/z 952.5 9 54.6
923.6
6 96 9.7
2 960.1
929. 8 958.8
934. 9 949. 5 951. 3
937.6
1 925. 6
4 927. 4
932.4 944. 7 945.9
947.8
928.6 955. 5
0
920 925 930 93 5 940 945 95 0 955 m/z
2
789. 7
0
200 400 600 800 1000 m/z

Fig. 2 Positive ion mass spectra of ginsenosides: Rg1, Rb1, Rc, RB2, RD. IN enlarged views protonated cation regions
1102

A
Intens. +MS2(1131.8), 9.4min (#246)
x106
[M+Na]+
[M-Ogluglu+Na]+
1131.7

2.0

789.6
1.5

[Ogluglu+Na]+
1.0

0.5
365.3

0.0
400 600 800 1000 1200 1400 m/z

B
Intens.
[Ogluglu+Na]+
+MS3(1131.8->789.6), 9.5min (#197)
x10 4

365.2
2.0

1.5

[M-Ogluglu-glu+Na]+
1.0

0.5 789.6
627.5 1178.2
705.6
0.0
400 600 800 1000 1200 1400 m/z

n
Fig. 3 The MS spectra of Rb1. a 1,131.7→fragment scan; b 1,131.7→789.6→fragment scan

ated molecules might have resulted in decomposition of ized by R2 with a value higher than 0.990. The highest
these species in the ion source region. value was noted for Rb1 (0.999). A lower value of R2 was
However, the sodium adducts for ginsenosides were reported for Rg1 (0.992). These data show that the varia-
observed by other researchers [3, 30–32]. The LC–MS data bles are characterized by high correlation in the concen-
collected, which yielded [M+Na]+ parent ions and a very tration range examined.
little fragmentation, provided evidence for ginsenosides. The method validation (repeatability, intermediate pre-
Besides, these ion sodium adducts can be analyzed in MS2 cision and linearity) showed that this method is suitable for
and MS3 mode and yielded characteristic fragmentation the analysis of ginseng saponins. The recovery was in the
patterns including evidence of sugar loss. On the basis of range 83.8–92.1%. Repeatability was evaluated on three
these data ginsenosides were easily identified in purified samples; each sample was injected six times. The RSD of
ginseng fractions after SPE cleanup. the results did not exceed 10%. For intermediate precision
The cleavage reaction in MSn occurs predominantly at (two workers, each preparing three samples) the RSD was
the glycosidic linkage [25]. The glycosidic linkages and below 14.2%.
attached sugars can be determined from the CID spectra of This method was applied for the identification and the
sodiated ions, both molecular and fragment. Figure 3 quantification of target compounds in ginseng extracts. The
shows CID mass spectra obtained of ginsenoside Rb1. The identification of the compounds was established by compar-
ion observed at m/z 789 corresponds to a fragment ion ison with the authentic reference compounds in terms of
arising from loss of sugars (−R3) from Rb1. However, the retention times and mass spectra.
ion at m/z 365 is common to fragment [Ogluglu+Na]+. The
ion at m/z 627 corresponds to loss of glucose (at −R1) from
sodiated species [M−Ogluglu+Na]+.
Sodiated molecular ions [M+Na]+ of ginsenosides were Table 2 Sodiated molecular ions of ginsenosides
integrated and the peak areas were used for quantification
(Table 2). Compound Molecular Molecular ion sodium
The limits of quantification and data on precision are weight adduct [M+Na]+m/z
shown in Table 3. The limit of detection of the described RB1 1,108 1,131
method was below 30 ng/ml. The repeatability of the
RB2 1,078 1,101
method expressed as a RSD for peak areas at the lowest
RG1 800 823
concentration was generally below 4.5%. Only one com-
RC 1,078 1,101
pound (Rg1) was characterized by a RSD value higher than
RD 946 969
5%. The linearity of the calibration curves was character-
 1103
Table 3 Regression parameters of calibration curves after sonically assisted extraction the concentration of
Compound Calibration range R 2
RSD (%) a
LOQ ginsenosides was similar—26.02 mg/g.
(μg/ml) (μg/ml) The highest extraction efficiency was observed during
shaking extraction (total concentration 41.02 mg/g). The
Rg1 0.08–0.120 0.997 5.6 80 analysis of the results obtained for shaking extraction
Rb1 0.08–0.120 0.999 4.4 80 shows that the concentration of ginsenosides was between
Rc 0.08–0.120 0.995 3.6 80 0.32 mg/g for Rg1 and 30.52 mg/g for Rb1. A lower ef-
Rd 0.08–0.120 0.998 4.2 80 ficiency of extraction was reported for ultrasound-assisted
Rb2 0.08–0.120 0.992 3.9 80 extraction. The lowest concentration was observed for Rb2
and Rg1 (0.82 and 0.87 mg/g, respectively) but the highest
RSD relative standard deviation, LOQ limit ofquantification
a
Calculated for n=6, at a concentration of 0.08 mg/ml of each concentration was detected for Rb1. The amount of com-
compound pounds in the extracts obtained after ASE was similar to that
mentioned before. The highest concentration was noted for
Rb1 (17.26 mg/g) and the lowest for Rg1 (0.55 mg/g). The
amount of Rb1 was always significantly higher than that of
Target ginsenosides were detected in all extracts. other ginsenosides; however, the concentration of Rg1 did
Figure 4 shows typical chromatograms obtained from not exceed 0.87 mg/g.
root and leaf extracts. The method precision calculated as a RSD was found to
The results in Table 4 show that the extraction efficiency range from 3.9 to 10.4% for saponins. The RSDs were
of saponins in American ginseng by ASE is comparable to quite similar for all the methods tested, but higher values
that obtained by sonication. The total amount of saponins were observed for lower concentration of ginsenosides
after ASE extraction from ginseng root was 26.03 mg/g but in plant extracts. The lower method precision can be ex-

A
Intens.
x10 6
6
3

3
2
4
2
1

5
1

0
35 40 45 50 55 60 Time [min]

B
Intens.
x10 6
6
2.5

2.0

2 5
1.5

1.0 1
4
3
0.5

0.0
35 40 45 50 55 Time [min]

Fig. 4 The total ion chromatogram of root (a) and leaf (b) extracts. Ginsenosides are eluted in the following order: 1-Rg1, 2-Re; 3-Rb1,
4-Rc, 5-Rb2, 6-Rd
1104
Table 4 Comparison of extraction methods
Compound ASE (root) ASE (leaves) Ultrasound assisted Mechanically shakena Fraction of ginsenosides
Concentration RSD Concentration RSD Concentration RSD Concentration RSD Root Leaves
(mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) (%)

Rg1 0.55 3.9 0.22 4.2 0.87 8.5 0.32 10.4 0.8 1.6
Rb1 17.26 9.8 0.58 4.2 16.81 3.9 30.52 3.9 73.3 4.2
Rc 2.67 5.8 1.32 6.1 2.65 4.1 2.98 4.8 7.2 9.5
Rb2 0.62 10.2 3.78 5.2 0.82 6.3 0.65 7.2 1.6 27.4
Rd 4.93 4.3 7.91 7.3 4.91 5.1 7.15 4.3 17.2 57.2
Total 26.03 13.81 26.06 41.02

plained by both the heterogeneity of the sample and a
matrix effect; however, this problem is frequently con-
nected with biological samples.
The contribution of both Rb1 and Rd to the total content
of ginsenosides was 60 and 25%, respectively. Ginsenoside
Rc was detected at a lower concentration than Rb1 and Rd
and its contribution to the total content was about 7.20%.
However, the contribution of Rb2 and Rg1 was 1%. The
contributions of ginsenosides in leaf extract are different
from those mentioned before. Ginsenoside Rd was detected
at a concentration of 7.91 mg/mg. The contribution of this
compound was about 57.20% to the total content. Com-
paring the contribution of Rd, we found that its level was
significantly higher in leaves and than in the root (17.20%).
Other ginsenoside contributions to the total concentration
are as follows: Rb2 27.4%; Rc 9.5%; Rb1 4.2%; Rg1 0.8.
Conclusions
The results show the performance of the method for the
analysis of P. quinqefolium extracts present in concentra-
tions usually ranging from 0.22 to 30.52 mg/g. The limit of
detection was 1.6–2.3 mg/ml. The selectivity and the
sensitivity can be improved by the selection of multiple
reaction monitoring mode during quantification However,
the concentration of ginsenosides present in American
ginseng extracts does not require the limit of detection to be
enhanced from that established for full-scan mode. In this
study different extraction methods were used for extraction
of ginsenosides from plant material. Shaking extraction
with a 50% methanol–water mixture (1:1) allows markedly
higher extraction yields than ultrasonic and ASE methods.
ASE and ultrasonic extraction give similar results–the total
concentration of five ginsenosides was 26 mg/g. Quanti-
tative determination of ginsenosides in root and leaves
from American ginseng cultivated in Poland was made.
The total concentrations of saponins in leaf extract are
close to 2 times lower than in root extract—13.81 and
26.03 mg/g, respectively. Additionally, leaves are rich in
Rd (7.91 mg/g) and its concentration is similar to the
concentration reported in root extract.
Therefore, on the basis of the concentration of major
saponins, leaf extract can be an alternative to root extract as
a source of ginsenosides used in herbal preparations.
Acknowledgements The authors wish to thank to S. Witkowski (S.
Witko, Łódż, Poland) for donation of LC–MS grade solvents and E.
Cichaczewska (Nicolaus Copernicus University, Toruń) for technical
assistance during the experiments. This work was supported by
UMK grant No. 328-Ch.
References
1. Ludwiczuk A, Wolski T, Berbeć S (2002) J Planar Chromatogr
15:147–150
2. Corthout J, Naessens T, Apers S, Vlietinck AJ (1999) J Pharm
Biomed Anal 21:187–192
3. Gafner S, Bergson C, McCollom MM, Cooper LM, McPhail
KL, Gerwick WH, Angerhofer CK (2004) J Agric Food Chem
52:1546–1550
4. Li Z, Guo YY (1999) J Pharm Pharmacol 51:435–440
5. Assinewe VE, Arnason JT (2002) Phytomedicine 9:398–404
6. Xie JT, Mehendale SR, Wang A, Han AH, Wu JA, Osinski J,
Yuan CS (2004) Pharmacol Res 49:113–117
7. Popovich DG, Kitts DD (2004) Phytochemistry 65:337–344
8. Fei XF, Wang BX, Tashiro S, Li TJ, Ma JS, Ikejima T (2002)
Acta Pharmacol Sin 23:315–322
9. Keum YS, Han SS, Chun KS, Park KK, Park JH, Lee SK, Surh
YJ (2003) Mutat Res 75–85:523–524
10. Li W, Gu C, Zhang H, Awang DVC, Fitzloff JF, Fong HHS,
Bremen RB (2000) Anal Chem 72:5417–5422
11. Fuzzati N, Bagetta B, Jayakar K, Pace R, Peterlongo F (1999) J
Chromatogr A 857:69–79
12. Chan TWD, But PP, Chang SW, Kwok IMY, Lau FW, Xu HX
(2000) Anal Chem 72:1281–1287
13. Vongsangnak W, Gua J, Chauvatcharin S, Zhong JJ (2004)
Biochem Eng J 18:115–120
14. Fang Q, Yeung HW, Leung HW, Huie CW (2000) J Chro-
matogr A 904:47–55
15. Wang HC, Chen CR, Chang CJ (2001) Food Chem 72:505–509
16. Benthin B, Danz H, Hamburger H (1999) J Chromatogr A
837:211–219
17. Suomi J, Siren H, Hartonen K, Riekkola ML (2000) J Chro-
matogr A 868:73–78
18. Schieffer G, Pfeiffer K (2001) J Liq Chromatogr Relat Technol
24:2415–2427
19. Choi MPK, Chan KKC, Leung HW, Huie CW (2003) J Chro-
matogr A 983:53–162
20. Kwon JH, Belanger JMR, Pare JRJ, Yaylayan VA (2003) Food
Res Int 36:491–498
21. Shu YY, Ko MY, Chang YS (2003) Microchem J 74:131–139
22. Cui J (1995) Eur J Pharm Sci 3:77–85
23. Shangguan D, Han H, Zhao R, Zhao Y, Xiong S, Liu G (2001)
J Chromatogr 910:367–372
24. Park MK, Park JH, Han SB, Shin YG, Park IH (1996) J Chro-
matogr A 736:77–81
25. Park MK, Park JH, Lee MY, Kim SJ, Park IJ (1994) J Liq
Chromatogr 17:1171–1178

26. Cai Z, Qian T, Wong RNS, Jiang ZH (2003) Anal Chim Acta
492:283–293
27. Nicol RW, Yousef L, Traquair JA, Bernards MA (2003)
Phytochemistry 64:257–264
28. Fuzzati N (2004) J Chromatogr 812:119–133
29. Wang X, Sakuma T, Asafu-Adjaye E, Shiu GK (1999) Anal
Chem 71:1579–1584
1105
30. Ackloo SZ, Smith RW, Terlouw JK, McCarry BE (2000)
Analyst 125:591–597
31. van Breeemen RB, Huang CR (1995) Anal Chem 67:3985–
3989
32. Song F, Liu Z, Liu S, Cai Z (2005) Anal Chim Acta 531:69–77