Microwave degradation of floatation-enriched ginsenoside extract from Panax quinquefolium L. Leaf

Article abstract of DOI:10.1021/jf902153a

Even though the degradation of ginsenosides has been thoroughly studied in animals and in vitro using acids, enzymes, and intestinal bacteria, a new degradation method is established for obtaining the ginsenosides Rg₃, Rh₂ and their

Full text of DOI:10.1021/jf902153a

10252 J. Agric. Food Chem. 2009, 57, 10252–10260
DOI:10.1021/jf902153a
Microwave Degradation of Floatation-Enriched Ginsenoside
Extract from Panax quinquefolium L. Leaf
Y
UPING
B
AI, LISHA
Z
HAO, CHENLING
Q
U
, XIANGZHE
MENG
,
AND
H
ANQI
Z
HANG
*
College of Chemistry, Jilin University, Changchun, 130012, P. R. China
Even though the degradation of ginsenosides has been thoroughly studied in animals and in vitro
using acids, enzymes, and intestinal bacteria, a new degradation method is established for obtaining
the ginsenosides Rg3, Rh2 and their aglycon 20(S)-protopanaxadiol. This method is based on the
microwave irradiation degradation of the major ginsenosides from the Panax quinquefolium L.
coupled with foam floatation. The results indicated that ginsenosides Rg3, Rh2 and aglycon are not
naturally present in Panax quinquefolium L., but are the products obtained simultaneously in the
microwave irradiation degradation process. The yield of Rg3 is 7.69 mg/g and 250 times as high as
that obtained from red ginseng, and the transformation rate of total ginsenosides to aglycon is
78.11%. It is important to stress that a new degradation medium, N,N-dimethylformamide, was
discovered and, when the medium was used, the transformation rate of total ginsenosides to
aglycon was 20.20%. The proposed method is simple, efficient, time saving and a noteworthy
improvement on the traditional degradation methods such as acidic hydrolysis, alkaline degradation,
enzymatic conversion or microbial degradation.
KEYWORDS: Panax quinquefolium L.; degradation; foam floatation; HPLC-APCI/MS; microwave
irradiation; ginsenoside; anticancer activities
INTRODUCTION
in vivo and in vitro which belong to rare ginsenosides are
bioactive compounds (5-8). Among the rare ginsenosides, Rg3
and Rh2 are of greatest interest because their bioactivity is higher
than others especially in the aspect of antitumor activity. It has
been reported that Rh2 can not only reduce the proliferation of a
variety of cultured cancer cells but also influence apoptosis (9). In
addition, Rg3 has been shown to possess antitumor properties
and influence drug resistant cultured cancer cells (10, 11). Bae
et al. (12) reported that ginsenoside Rg3 is metabolized to
ginsenoside Rh2 and 20(S)-protopanaxadiol when anaerobically
incubated with human fecal microflora, and the activities of
deglycosylated metabolites are comparable to or higher than that
of Rg3 (9, 13). Characteristics and mechanism of effects among
the three have been proved similar, but the sequence of functional
strength is 20(S)-protopanaxadiol, Rh2 and Rg3, which probably
has a close relation with the fact that 20(S)-protopanaxadiol is the
aglycon of Rg3 and Rh2 (14,15). Thus, preparation and determi-
nation of Rg3, Rh2 and 20(S)-protopanaxadiol are singnificant
for their biological and pharmacological activities.
The degradation of ginsenosides is to remove several or all the
glycosyl moieties in order to obtain rare ginsenosides or the
aglycon. Ginsenosides, especially malonyl ginsenosides, are ther-
mally unstable, and they may degrade during thermal extrac-
tion (16). Thermal degradation of neutral ginsenosides inaqueous
solution during conventional heating has been extensively
studied (17-19). But no or just a small amount of rare ginseno-
sides were obtained. Many degradation methods for obtaining
rare ginsenosides, including mild acid hydrolysis (20), alkaline
degradation (21), enzymatic conversion (22) and microbial de-
gradation (13), have been introduced extensively for a long time.
Panax quinquefolium L. has been not only used as therapeutic
agents but also marketed as dietary supplements and raw materi-
als of healthy food. For example, the roots of Panax quinquefo-
lium L. have been used as additives to drinks for hundreds of
years. Ginsenosides, including neutral ginsenosides and malonyl
ginsenosides, are known to be the main bioactive components of
Panax quinquefolium L. (1, 2). Mostly derived from tetracyclic
triterpene dammarane, ginsenosides are subdivided into proto-
panaxadiol, protopanaxatriol and oleanolic acid ginsenosides.
The structures of main ginsenosides are shown in Figure 1.
The root of Panax quinquefolium L. is a typical source for use in
many traditional medicinal therapies. Compared with the root,
the leaf was less investigated. Annual recovery of ginseng leaf
could be a feasible alternative source of ginsenosides compared
with the roots requiring a long growth cycle for nutritious
products. It was found that the content of total ginsenosides in
leaf is much higher than that in root (3). The leaf of Panax
quinquefolium L. is a source of ginsenosides but has not been well
exploited up to now (3, 4).
Rare ginsenosides, including Rg2, Rg3, Rg5, Rh1, Rh2, Rh3 and
Rck, can be obtained from deglycosylation of the major ginseno-
sides. Compared with the major ginsenosides, rare ginsenosides
have higher bioactivity than major ginsenosides and many
research studies indicated that the metabolites of major ginsenosides
*Corresponding author. Mailing address: College of Chemistry,
Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R.
China. E-mail: analchem@mail.jlu.edu.cn. Fax: þ86-431-85112335.
Tel: þ86-431-85168399.
pubs.acs.org/JAFC
Published on Web 10/12/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10253
Figure 2. The setup of foam flotation.
purification unit (Millipore, Mississauga, Canada). Other reagents used in
this study were of analytical grade.
Sample Preparation. Leaf of Panax quinquefolium L. was obtained
from Jilin Province in China. The leaf was rinsed with water, dried at
30 °C, powdered in a mill, and passed through a 40-mesh sieve. Sample
powder was weighed (1.0 g) and transferred into a 150 mL conical flask.
50 mL of ethanol-water solvent (70%, v/v) was added into it. The sample
was subjected to ultrasonic extraction for 2 h. After the ultrasonic
extraction was completed, the extract was filtered. The flask and residue
were rinsed three times with ethanol. The collected extract and rinsed
solvent were combined and evaporated to dryness under vacuum at 60 °C.
Then the dried residue was dissolved in 1000 mL of water, and the resulting
aqueous solution was referred to as extraction solution.
Figure 1. Structures of main ginsenosides.
Foam Floatation of Ginsenosides. A 200 mL portion of the extraction
solution was transferred to a floatation vessel (Figure 2), and then 2.0 g of
NaCl was added into it. The first floatation for the extraction solution was
carried out when the flow rate of carrier gas was maintained at 30 mL/min
for 30 min. The foam phase obtained was diluted to 200 mL again with
water. Under the same conditions mentioned above, the second floatation
was carried out, and then the foam phase obtained was defoamed for
about 10 min. The final volume of the foam phase was 20 mL. The solution
resulting from the foam phase was referred to as floatation solution.
Microwave-Irradiation Degradation. Microwave-irradiation degra-
dation was performed on a WRT-C microwave preparation system
(Michem Technology Co. Ltd., Beijing, China) with a temperature control
system. In the degradation in neutral solution, the floatation solution was
transferred into the degradation vessel directly. The control vessel was
operated by a control terminal that could control both temperature and
irradiation time. After the degradation vessels and control vessel were put
into the microwave irradiation system, the system was turned on and the
degradation was continuously performed for 5 min under 165 °C. After the
completion of degradation, the degradation vessels were allowed to cool
down to the room temperature. Finally, the degradation products were
extracted three times with 10 mL of water-saturated n-butanol at a time.
The extracts were combined and evaporated to dryness under vacuum at
80 °C, and the residue was redissolved in 5 mL of methanol, which was
referred to as degradation solution and filtered prior to HPLC-MS
analysis.
In the degradation in acidic and alkaline solution, after the floatation
solution was transferred into the degradation vessel, HCl and NaOH were
added into it respectively. When the degradation temperature and time
were fixed at 165 °C and 5 min respectively, the effect of concentrations of
HCl ranging from 0.25 to 2 mol/L and concentrations of NaOH ranging
from 0.1 to 1 mol/L were investigated. After the completion of degrada-
tion, the subsequent steps were the same as those applied in the neutral
solution.
In an additional study, an organic medium was investigated. The
floatation solution was extracted three times with 10 mL of water-
saturated n-butanol at a time. Then the extracts were combined and
evaporated to dryness under vacuum at 80 °C, and the dried residue was
dissolved in 20 mL of DMF. After the DMF solution was transferred into
the degradation vessel, the degradation process was performed under
165 °C for 10 min. After the completion of the degradation, the subsequent
steps were the same as those applied in the neutral solution.
The degradation of ginsenosides in humans after oral adminis-
tration has been also reported (23). Though there were some
degradation methods to obtain rare ginsenosides, further efforts
devoted to method development are required. For instance, side
reactions, such as epimerization, hydration, cyclization and
hydroxylation, often occur during mild acid hydrolysis degrada-
tion (20). Most of the micro-organisms used for the degradation
of ginsenosides are not of a food-grade standard, and the
enzymatic conversion degradation process is complex and of
high cost (24). Because the alkaline degradation process is simple
and the side reactions are little, it has been mostly studied
recently. However, the yields of rare ginsenosides obtained by
all the conventional degradation methods are very low and all the
degradations are slow processes. Recently, microwave heating
has been not only employed for the extraction of major ginseno-
sides, in which faster extraction rates were observed under certain
conditions(25,26), but alsoapplied tothe degradation ofmalonyl
ginsenosides (16). Therefore, it is likely that microwave heating
may also enhance the degradation of neutral ginsenosides. Foam
floatation has been recognized as a suitable technique for the
separation and purification of surface-active substances (27).
Based on the surface-active properties of ginsenosides, the foam
floatation technique can be applied to the concentration and
purification of the ginsenosides.
The purpose of the work is to develop a new degradation
method for obtaining ginsenosides Rg3, Rh2 and 20(S)-proto-
panaxadiol. Both foam floatation and degradation conditions
were investigated and optimized. Neutral solution, acidic solu-
tion, alkaline solution and N,N-dimethylformamide (DMF) were
used as degradation media in the degradation of ginsenosides
obtained from leaf of Panax quinquefolium L., respectively.
MATERIALS AND METHODS
Materials and Reagents. Ginsenosides Rg1, Re, Rc, Rb2, Rb3, Rd,
Rg3, Rh2 and aglycon 20(S)-protopanaxadiol were purchased from
Chinese Medical and Biological Products Institute (Beijing, China).
Chromatographic grade acetonitrile was obtained from Fisher Scientific
Company (Pittsburgh, PA), and pure water was from a Milli-Q water
Determination by HPLC. The ginsenosides were quantitatively
determined by liquid chromatography using an Agilent 1100 HPLC

10254 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Bai et al.
Table 1. Calibration Curve and Concentration Range for Determination of
Seven Ginsenosides
and Mm was the total mole number of major ginsenosides, including Rc,
Rb2, Rb3 and Rd, in the floatation solution. When eq 2 was applied to
calculating the transformation rate R of ginsenoside standards, Mr was the
total mole number of the rare ginsenosides, including Rg3, Rh2 and
aglycon, in the degradation solution and Mm was the mole number of
the ginsenoside standard.
concn range detection limit
(μg/mL)
ginsenoside regression equationa correlation coeff (μg/mL)
Rc
A = 4434.37c þ 5.223
A = 4654.77c þ 12.21
A = 4700.31c þ 33.42
A = 6103.67c - 20.81
A = 8960.69c - 7.99
A = 9025.62c þ 6.89
A = 8563.43c þ 30.82
0.99974
0.99998
0.99985
0.99974
0.99984
0.99993
0.99947
2.3-147
8.7-559
9.8-629
8.2-524
7.9-505
3.5-225
8.4-539
0.5
2.2
2.6
0.8
0.4
0.2
1.8
Rb2
Rb3
Rd
RESULTS AND DISCUSSION
The Foam Floatation of Ginsenosides. Some parameters affect-
ing the performances of the foam floatation, such as the flow
rate of carrier gas (N2), floatation time, amount of sample,
concentration of NaCl and pH value of extraction solution,
were investigated. The amount of sample in 200 mL of extrac-
tion solution has a slight effect on foam floatation. The effect of
the amount of sample ranging from 0.2 to 2.0 g on the recoveries
was evaluated. The recoveries of protopanaxadiol type ginseno-
sides decrease with increasing amount of sample. Only in the
case of Re and Rg1, no effect was obvious. Therefore, 0.2 g
sample in 200 mL of extraction solution was chosen. Sodium
chloride, which was used as salting-out agent, was added into
the extraction solution to improve the ionic strength. The
recoveries were evaluated by varying the concentration of NaCl
from 0 to 50 mg/mL. The recoveries of protopanaxatriol type
ginsenosides decrease with increasing concentration of NaCl,
and those of protopanaxadiol type are highest and constant at
the concentrations ranging from 5 to 10 mg/mL. In addition, it
can be found that when the concentration of NaCl is 10 mg/mL,
the recoveries of protopanaxadiol type ginsenosides are highest
and meanwhile there is almost no protopanaxatriol type ginse-
noside in the solution. As a result, NaCl has a significant effect
on the separation of protopanaxadiol type ginsenosides from
protopanaxatriol type ginsenosides, and the concentration of
NaCl was selected as 10 mg/mL in this work. The existing form
of ginsenosides is related to the pH value of solution. In the pH
range from 2 to 13, pH value has a significant effect on the
recoveries for ginsenosides, especially for protopanaxadiol type
ginsenosides. It is shown that the recoveries of all the ginseno-
sides are highest when the pH value is 9. However, because the
pH of water used in this study was approximately 7-8 before
floatation, no other reagents were added in order to simplify the
experimental procedure. The flow rates of N2 and the floatation
times were investigated. When the flow rate of carrier gas
increases, the amount of liquid entrained by the foam increases.
As a result, the ginsenosides adsorbed in the foam are diluted.
However, when the flow rate of carrier gas was too low, it took a
long time to complete foam floatation. The compromising
conditions selected are that the flow rate of carrier gas and
floatation time are 30 mL/min and 30 min respectively. The
foam floatation provides good recoveries (76.6-85.5%) for
protopanaxadiol type ginsenosides, and the enrichment factors
for Rc, Rb2, Rb3 and Rd are 7.66, 7.89, 8.55 and 8.50,
respectively. However, for protopanaxatriol type ginsenosides,
including Rg1 and Re, the foam floatation does not provide
good recoveries (11.1-15.3%) and enrichment factors
(1.11-1.53). The foam floatation is more favorable to obtain
Rg3, Rh2 and aglycon. Typical chromatograms of extraction
and floatation solution are shown in Figure 3.
Rg3
Rh2
aglycon
a Where A and c are the peak area and concentration of the analytes,
respectively.
system (Palo Alto, CA) equipped with a Zorbax Eclipse XDB-C18
(250 mmꢀ4.6 mm i.d., 5 μm) column (Agilent, USA). The mobile phase
was acetonitrile/water at a flow rate of 1.0 mL/min. The UV detection was
performed at 203 nm, and the column temperature was 25 °C. The
injection volume of sample solution was 20 μL. The mobile phase was a
binary solvent containing acetonitile (A) and water (B), and the gradient
condition was as follows: 0-15 min, 24% A, 76% B; 15-40 min, 24-39%
A, 76-61% B; 40-65 min, 39-53% A, 61-47% B; 65-85 min,
61-100% A, 47-0% B. The peak area was measured and used to
construct the calibration curve.
HPLC-APCI/MS Analysis. The HPLC conditions for the
HPLC-MS analysis were as described above. The effluent from chroma-
tographic column was introduced into an ABI Q-Trap Mass Spectrometer
(Applied Biosystems Sciex, Foster City, CA) equipped with atmospheric
pressure chemical ionization source. Nitrogen (99.999%) was used as
curtain gas, nebulizer gas and auxiliary gas. The curtain gas, nebulizer gas
and auxiliary gas were 30, 35, and 40 psi respectively. The ion polarity was
set to negative mode, and the ion source temperature was controlled at
350 °C. Needle current, ion spray voltage, entrance potential, declustering
potential and collision energy were -6 μA, 3000 V, -10 V, -50 V and -10 eV,
respectively.
Degradation of Ginsenosides Standards. Ginsenoside standards
Rc, Rb2, Rb3 and Rd (1.0 mg each) were placed in the degradation vessels
and dissolved with 20 mL of sodium hydroxide solution (0.5 mol/L).
Subsequently, the degradation was performed at 165 °C for 5 min. The
degradation solution obtained was extracted three times with 10 mL of
water-saturated n-butanol at a time. The extracts were combined and
evaporated to dryness under vacuum at 80 °C. The residue was redissolved
in 5 mL of methanol, and filtered prior to detection by HPLC-MS.
For comparison, the ginsenoside standards were directly detected by
APCI/MS.
Standard Curve for Quantitative Analysis. As shown in Table 1,
standard curves were constructed for each of the ginsenoside standards
(Rc, Rb2, Rb3, Rd, Rg3, Rh2 and aglycon) and used to determine the
analytes in the degradation solution. Some ginsenosides in the degradation
solution were identified by comparison of the retention times with those
obtained from the mixed ginsenoside standards. The other ginsenosides
were identified by matching of their mass spectral fragments with MS data
reported in previously studies (28, 29).
Statistical Analysis of Experimental Data. In the study, each value
reported is the mean of three replicate measurements.
The degradation yield Y of rare ginsenosides in microwave degradation
is defined as follows:
Y ¼ mg=ms
ð1Þ
where mg is the mass of rare ginsenosides obtained and ms is the mass of
sample powder.
Equation 2 was applied to calculating the transformation rate R of
major ginsenosides to rare ginsenosides in microwave degradation:
The Degradation of Ginsenosides. The main source of both
ginsenosides Rg3 and Rh2 has been reported to be exclusively
Korean red ginseng root which is steam-processed (30, 31). In this
study, microwave irradiation degradation was applied in order to
obtain rare ginsenosides. Both time and temperature of degradation
have a significant effect on yields of rare ginsenosides, and different
degradation media, including neutral solution, acidic solution,
alkaline solution and DMF, were also investigated. The highest
R ¼ Mr=Mm
ð2Þ
When eq 2 was applied to calculating the transformation rate R of major
ginsenosides in practical samples, Mr was the mole number of rare
ginsenosides, including Rg3, Rh2 and aglycon, in the degradation solution

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10255
Figure 3. The chromatograms of extraction solution (a) and floatation solution (b): (1) Rg1þRe; (2) Rc; (3) Rb2; (4) Rb3; (5) Rd.
Table 2. The Transformation Rates of Total Ginsenoside to Rare Ginseno-
sides
When the degradation temperature was kept at 165 °C, the
effect of degradation times was investigated. From the Figure 4b,
it can be seen that the yield of Rg3 reaches the highest when the
degradation time is 15 min. However, the yields of Rh2 and
aglycon increase withthe increase ofthe degradation time ranging
from 0 to 5 min, and no significant change was observed when the
degradation time was longer than 5 min. Because the activity of
aglycon is stronger than that of Rg3 and Rh2 (9, 13), the
degradation time of 5 min was chosen to save time.
transformation rate (%)
ginsenoside
neutral solution
acidic solution
4.83
alkaline solution
DMF
3.54
Rg3
Rh2
47.98
6.43
9.14
6.94
aglycon
14.98
26.92
78.11
20.20
yield of aglycon obtained under optimal conditions in alkaline
medium was 4.83 mg/g, and the transformation rate of degradation
of major ginsenosides to aglycon was 78.11% (Table 2).
In the degradation in acidic solution, the degradation yield of
aglycon decreases obviously with the increase of concentration of
HCl ranging from 1.5 mol/L to 2.0 mol/L, and no significant
change (0.40 mg/g) was observed when the concentration of HCl
was lower than 1.5 mol/L (Figure 4c). In addtition, ginsenoside
Rh2 was not obtained. Therefore, the concentration of HCl
chosen was 1.5 mol/L in the present work.
In the degradation in alkaline solution, the effect of the
concentration of NaOH is very significant in the generation of
aglycon, with the yields changing from 0.09 to 4.83 mg/g
(Figure 4d). The yield of Rg3 first decreases with the increase of
the concentration of NaOH ranging from 0.1 to 0.25 mol/L, and
In the degradation in neutral solution, the effect of the
degradation temperatures ranging from 120 to 190 °C was
investigated. The experimental results showed that the tempera-
ture had a significant effect on the yield of aglycon, which ranges
from 0 to 0.43 mg/g. In the degradation solution, ginsenoside Rg3
Rh2 and aglycon were all detectable. As shown in Figure 4a, the
yields of the three rare ginsenosides are highest at 165 °C. Too
high or too low temperature is not favorable for the degradation
yield, so 165 °C was chosen as the degradation temperature.

10256 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Bai et al.
Figure 4. Influence of degradation temperature (a), degradation time (b), concentration of HCl (c) and concentration of NaOH (d) on yields of rare
ginsenosides in H2O.
then does not significantly change. The concentration of NaOH
has a slight effect on the yield of Rh2. As a result, 0.5 mol/L was
selected as the concentration of NaOH.
Temperature has a significant effect on the yields of rare
ginsenosides instead of pressure (30). Increasing temperature
has been made at the expense of increasing pressure in water,
so it is essential to introduce a suitable medium with high boiling
point to accept microwave energy in which the ginsenosides can
be degradable. Unlike studies reported (32, 33), we discovered
that the ginsenosides were also degradable in N,N-dimethylfor-
mamide, which is a proton inert solvent with lower toxicity than
pyridinium and lower pressure than water.
In the degradation in DMF, different degradation tempera-
tures ranging from 150 to 170 °C were examined (Figure 5a). The
results show that the temperature has a significant effect on the
yield of aglycon which ranges from 0.003 to 0.44 mg/g. In the
degradation solution, Rh2 was not obtained and the yield of Rg3
was quite low. So, 165 °C, which is the same as the temperature in
degradation in neutral solution, was chosen.
The effect of degradation times ranging from 5 to 20 min was
investigated when the degradation temperature was maintained
at 165 °C. As shown in Figure 5b, degradation time has a
significant effect on the yield of Rg3 and aglycon. The yield of
aglycon reaches the highest (0.44 mg/g) when the degradation
time is 10min. As a result, 10min of degradation time was chosen.
HPLC-APCI/MS Analysis of Degradation Products. In order
to identify ginsenosides in the degradation products, the degrada-
tion solutions were analyzed by HPLC-APCI/MS. APCI that
could provide explicit information for identifying and distin-
guishing protopanaxadiol and protopanaxatriol type ginseno-
sides (26) has been considered to be mainly applicable to the assay
of weakly polar compounds. Figure 6 shows the negative-ion
Figure 5. Influence of degradation temperature (a) and degradation time
(b) on yields of rare ginsenosides in DMF.
HPLC-APCI/MS traces of ginsenosides in the degradation
solution. It can be seen that the target compounds in the

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10257
Figure 6. HPLC chromatogram for degradation solutions obtained in neutral solution (a), acidic solution (b), alkaline solution (c) and DMF (d): (1) Rb1; (2)
Rc; (3) Rb2; (4) Rb3; (5) Rd; (6) Rd2; (7) F2; (8) 20(S)-Rg3; (9) 20(R)-Rg3; (10) not reported; (11) not reported; (12) Rg5; (13) 20(S)-Rh2; (14) 20(R)-Rh2;
(15) Rk1/Rs4; (16) Rk1/Rs4; (17) not reported; (18) not reported; (19) 20(S)-protopanaxadiol; (20) 20(R)-protopanaxadiol.

10258 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Bai et al.
Table 3. HPLC-APCI/MS Data of Ginsenosides in Degradation Solutions
main fragmentation ion m/z
others
peak
identification
retention time (min)
[M - H]-
Neutrala
[M þ HCOO]-
1
Rb1
Rc
34.97
36.56
38.18
38.67
41.36
48.28
52.60
57.60
59.59
60.47
71.14
72.65
73.19
76.11
77.84
79.65
80.53
86.07
1077.9
1123.8
1123.8
1123.8
1124.0
991.9
961.6
829.5
829.5
829.5
765.5
765.5
765.5
765.5
765.5
683.7
649.8
699.5
699.5
699.5
699.7
699.7
667.8
667.6
475.4
475.4
475.4
667.6
667.9
649.7
493.5
373.5
373.5
373.5
621.7
621.7
621.7
2
1077.9
1077.9
1078.0
945.8
915.8
783.9
783.9
783.9
3
Rb2
Rb3
Rd
4
5
6
Rd2
F2
7
8
20(S)-Rg3
20(R)-Rg3
not reported
Rg5
9
10
12
13
14
15
16
17
18
20
765.4
621.5
621.5
621.5
621.5
765.4
649.4
459.7
621.7
493.6
493.6
459.7
459.7
459.7
475.8
475.8
20(S)-Rh2
20(R)-Rh2
Rk1/Rs4
667.5
667.5
667.5
667.5
Rk1/Rs4
not reported
not reported
20(R)-protopanaxadiol
668.7
505.4
545.9
Acidica
5
Rd
F2
41.36
52.60
57.60
59.59
71.14
77.84
79.65
80.53
85.30
86.07
945.8
783.9
783.9
783.9
765.4
621.5
765.4
649.4
459.7
459.7
991.9
829.5
829.5
829.5
765.5
649.8
699.7
667.6
667.9
493.5
621.7
7
8
20(S)-Rg3
20(R)-Rg3
Rg5
9
12
16
17
18
19
20
621.7
459.7
459.7
Rk1/Rs4
667.5
not reported
not reported
20(S)-protopanaxadiol
20(R)-protopanaxadiol
668.7
505.4
505.4
545.9
Alkalinea
1
Rb1
Rc
34.97
36.56
38.18
38.67
41.36
48.28
52.60
57.60
60.47
71.14
72.65
76.11
77.84
79.65
80.53
85.30
1107.9
1077.9
1077.9
1078.0
945.8
1123.8
1123.8
1123.8
1124.0
991.9
765.5
765.5
765.5
765.5
765.5
683.7
649.8
699.5
699.5
699.5
699.7
699.7
667.8
667.6
475.4
475.4
475.4
667.6
667.9
649.7
493.5
373.5
373.5
373.5
621.7
621.7
621.7
2
3
Rb2
Rb3
4
5
Rd
Rd2
6
915.8
783.9
961.6
829.5
7
F2
8
20(S)-Rg3
not reported
Rg5
783.9
829.5
10
12
13
15
16
17
18
19
765.4
621.5
621.5
621.5
765.4
649.4
459.7
621.7
493.6
459.7
459.7
459.7
475.8
20(S)-Rh2
Rk1/Rs4
Rk1/Rs4
not reported
not reported
20(S)-protopanaxadiol
667.5
667.5
667.5
668.7
505.4
545.9
DMFa
1
Rb1
Rc
34.97
36.56
38.18
38.67
41.36
48.28
52.60
57.60
60.47
71.14
76.11
77.84
79.65
80.53
85.30
1077.9
1077.9
1077.9
1078.0
945.8
1123.8
1123.8
1123.8
1124.0
991.9
765.5
765.5
765.5
765.5
765.5
683.7
649.8
699.5
699.5
699.5
699.7
699.7
667.8
667.6
475.4
475.4
475.4
667.6
667.9
649.7
493.5
373.5
373.5
373.5
621.7
621.7
621.7
2
3
Rb2
Rb3
4
5
Rd
Rd2
6
915.8
783.9
961.6
829.5
7
F2
8
20(S)-Rg3
not reported
Rg5
783.9
829.5
10
12
15
16
17
18
19
765.4
621.5
621.5
765.4
649.4
459.7
621.7
459.7
459.7
459.7
Rk1/Rs4
Rk1/Rs4
667.5
667.5
not reported
not reported
20(S)-protopanaxadiol
668.7
505.4
545.9
a Degradation solution.

Article
J. Agric. Food Chem., Vol. 57, No. 21, 2009 10259
degradation solution were well separated under the HPLC con-
ditions adopted. Based on retention times and the m/z values of
both intact molecular ions and the product ions, ginsenosides
labeled on the reconstructed ion chromatograms were identified.
The retention times and the set of ions for individual ginsenosides
in the four degradation solutions are listed in Table 3. From
Figure 6, it can be seen that ginsenoside Rg3 and aglycon are
detectable in all the degradation solutions and ginsenoside Rh2,
another rareginsenoside, is detectableonlyinalkaline and neutral
degradation solutions. In the neutral degradation solution, the
isomeric compounds of Rg3 and Rh2, including 20(R) and 20(S)
configuration, are generated simultaneously at almost the same
concentration. Furthermore, the configuration of aglycon ob-
tained in neutral degradation solution was only 20(R) (Figure 6a).
In the acidic degradation, compared with the result of earlier
study (20), the ginsenosides were completely degradated and only
a small amount of aglycon, of which the concentration of 20(R) is
more than that of 20(S), was obtained (Figure 6b). On the
contrary, impurities in alkaline and DMF degradation solution
are less than those obtained in the other two media and the
configuration of degradation products is only 20(S), which has
stronger pharmacutical activity than 20(R) (Figures 6c and 6d).
Degradation Products of Ginsenoside Standards. The degrada-
tion yields of rare ginsenosides including Rc, Rb2, Rb3 and Rd are
shown in Table 4. Unlike the results reported (29), ginseno-
side Rh2 is the only degradation product of Rc with yield of
0.19 mg/mg. Moreover, ginsenoside Rg3 is the only degradation
product of Rd with the yield of 0.13 mg/mg. However, aglycon
was generated from all four major ginsenosides but with the
highest yield from Rd. The difference may be due to the difference
in heating modes and degradation media.
Degradation Mechanism. For the protopanaxadiol type ginse-
nosides with β-OH moiety at C3, C12 and C20, when the glycosidic
bond at the C-20 position is hydrolyzed, the glycosidic bond at the
C-3 position is also attacked (34, 35).
During the degradation of ginsenosides in the strongly acidic
solution, noaglyconbut some byproductcan beobtained, which is
mainly because the side chain moiety of the ginsenoside molecular
structure is easily cyclized in the strongly acidic solution.
When the four degradation media were applied in the degrada-
tion of ginsenosides, the generation reactions of rare ginsenosides
were as shown in Figure 7, taking the neutral solution as an
example. The increase of Rh2 and aglycon usually goes with the
decreaseofRg3, but the results obtained inthispaper indicatethat
the generation tendency of three rare ginsenosides is the same, so
it can be speculated that the three ginsenosides are generated
simultaneously in the method used in this paper. The possible
reason is that the energy offered from microwave irradiation is so
intensive that the hydrolyzation of glycosidic bonds at the C3, C12
and C20 positions is accelerated and occurs simultaneously.
Conclusion. Microwave irradiation degradation coupled with
foam floatation was developed to prepare the rare ginsenosides
Rg3, Rh2 and aglycon. Foam floatation prior to degradation of
ginsenosides is favorable to the concentration and purification of
the ginsenosides of interest. Owing to its simplicity, low operation
cost, few impurities and particularly high yield and the saving in
time, the proposed method can be applied successfully to the
degradation of major ginsenosides in order to obtain rare
Table 4. Yields for Rg3, Rh2 and Aglycon Obtained from Ginsenoside
Standards
target degradation
product
yield
(mg/mg)
transformation rate
(%)
ginsenoside
Rc
Rg3
Rh2
0.00
0.19
0.03
0.00
0.00
0.02
0.00
0.00
0.02
0.20
0.00
0.35
39.96
4.69
aglycon
Rg3
Rh2
Rb2
Rb3
Rd
aglycon
Rg3
Rh2
4.69
aglycon
Rg3
Rh2
96.11
aglycon
Figure 7. The generation of rare ginsenosides Rg3 (a), Rh2 (b) and aglycon (c) in neutral solution.

10260 J. Agric. Food Chem., Vol. 57, No. 21, 2009
Bai et al.
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entire plant cycle, it is an important finding that the leaf of Panax
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coupled with foam floatation is a new excellent source of rare
ginsenosides. This method is therefore valuable for the generation
of rare ginsenosides and may be a good alternative to the
traditional techniques.
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Received June 23, 2009. Revised manuscript received September 20,
2009. Accepted September 22, 2009.