Chemical transformation of ginsenoside Re by a heteropoly acid investigated using HPLC-MS: N/HRMS

Article abstract of DOI:10.1039/c6nj01702a

The potential of heteropoly acid H₃PW₁₂O₄₀ to catalyze the chemical transformation of ginsenoside Re into rare ginsenosides was explored. This homogeneous catalyst can be recycled by extraction with diethyl ether. Eight resulting products were separated and identified through a developed high-performance liquid chromatography coupled with multistage tandem mass spectrometry and high-resolution mass spectrometry (HPLC-MSⁿ/HRMS) method. Multistage tandem mass spectrometry was employed to trace the source of fragments and determine fragmentation pathways. Also, high-resolution mass spectrometry was used for the accurate structural elucidation of fragments. Ginsenosides 25-OH-Rg₆ and 25-OH-F₄, consisting of the aglycone structures of 3β, 12β, 25-trihydroxy-dammar-20 (21/22)-ene , were obtained via chemical transformation for the first time. Chemical transformation pathways of ginsenoside Re were summarized, which involved deglycosylation, hydration, dehydration, and epimerization reactions. A carbenium ion mechanism was further employed to elucidate each transformation process, and the stability of carbenium ions was supposed to be responsible for the reaction pathways and selectivity.

Full text of DOI:10.1039/c6nj01702a

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Chemical transformation of ginsenoside Re
by a heteropoly acid investigated using
HPLC-MS /HRMS†
Cite this:DOI: 10.1039/c6nj01702a
n
a
a
a
a
ab
Yang Xiu, Huanxi Zhao, Yue Gao, Wenlong Liu and Shuying Liu*
3
The potential of heteropoly acid H PW12O40 to catalyze the chemical transformation of ginsenoside Re into
rare ginsenosides was explored. This homogeneous catalyst can be recycled by extraction with diethyl
ether. Eight resulting products were separated and identified through a developed high-performance
liquid chromatography coupled with multistage tandem mass spectrometry and high-resolution mass
n
spectrometry (HPLC-MS /HRMS) method. Multistage tandem mass spectrometry was employed to trace
the source of fragments and determine fragmentation pathways. Also, high-resolution mass spectrometry
6 4
was used for the accurate structural elucidation of fragments. Ginsenosides 25-OH-Rg and 25-OH-F ,
Received (in Montpellier, France)
3
1st May 2016,
consisting of the aglycone structures of ‘‘3b, 12b, 25-trihydroxy-dammar-20 (21/22)-ene’’, were obtained
via chemical transformation for the first time. Chemical transformation pathways of ginsenoside Re were
summarized, which involved deglycosylation, hydration, dehydration, and epimerization reactions. A carbenium
ion mechanism was further employed to elucidate each transformation process, and the stability of carbenium
ions was supposed to be responsible for the reaction pathways and selectivity.
Accepted 18th August 2016
DOI: 10.1039/c6nj01702a
www.rsc.org/njc
(
Re, Rg
1 1
, Rb , etc.). Therefore, methods including heating, acid or
Introduction
alkaline hydrolysis, and microbial and enzymatic transformation
Ginseng, the root of Panax ginseng C. A. Mayer (Araliaceae), is for the preparation of rare ginsenosides have attracted much
1
5–26
one of the most valuable medicinal herbs in traditional Chinese attention recently.
For instance, Wang et al. investigated a
medicine. It has been cultivated and taken as a medicinal resource total of 83 metabolites acquired from the transformation of
1
–3
24
in Asian countries for thousands of years. Extensive modern ginsenosides from notoginseng by artificial gastric juice. Vo
research suggests that ginseng has wide pharmacological prop- et al. developed a kinetic model and simulated the heating
erties, such as immune system modulation, antistress, anti- process with kinetic parameters for the optimum production of
2
5
hyperglycemic activities as well as potential cancer and aging ginsenoside Rg
process prevention. Ginsenosides are the main active compo-
3
by heat degradation of ginsenoside Rb
1
.
4–8
Among the conventional methods, the traditional use of
nents responsible for the pharmaceutical activities and generally homogeneous acid catalysts for ginsenoside chemical transfor-
classified into three groups according to their genuine aglycone mation exhibits high catalytic activity and low catalyst cost.
moieties, i.e. the ocotillol type, the oleanolic acid type and the Previously, we reported the studies of the hydrolysis of PPD and
dammarane type, which is subdivided into protopanaxadiol (PPD) PPT-type ginsenosides by conventional homogeneous organic
1
,9
19,20
and protopanaxatriol (PPT). To date, more than 100 ginseno- acids.
However, the large-scale use of acid suffers from
sides have been identified from ginseng and its processed catalyst recovery, unrecyclability, corrosion risk and treatment of
products, most of which have demonstrated different pharma- the acid residue. Especially, the poor selectivity of the transfor-
9
–14
cological effects with respect to individual ginsenoside.
Many mation of ginsenoside by acid catalysts strongly limits their
rare ginsenosides with significant pharmacological and biologi- application. Therefore, effective catalysts for the hydrolysis of
cal effects are scarcely present or even absent in nature and can ginsenosides are considerably in demand. Further studies are
only be prepared through a transformation of major ginsenosides sill essential to identify the mechanisms and pathways of the
chemical transformation of ginsenosides as well as those of the
side reaction to improve the conversion, selectivity and yield of
the main reaction.
Heteropoly acids (HPAs) are a unique type of solid acid
consisting of early transition metal–oxygen anion clusters. They
a
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117,
P. R. China. E-mail: syliu@ciac.ac.cn
b
Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,
Changchun, 130022, P. R. China
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nj01702a are recyclable and environmentally benign acid catalysts due to
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their fascinating architectures and excellent physicochemical quaternary bump, an autosampler and a thermostatically controlled
properties, such as strong Brønsted acidity, easy separation column compartment. Analytes were separated by a Thermo
method, reusability, high proton mobility and regulable consti- Syncronis C18 column (100 mm  2.1 mm i.d., 1.7 mm). The gradient
tuents. Among HPAs, the Keggin-type dodeca tungstophosphoric solvent system consisted of solvent A (0.1% formic acid in water, v/v)
À1
acid, i.e. H PW O , has been extensively researched and utilized and solvent B (acetonitrile), delivered at a flow rate of 0.2 mL min
3
12 40
2
7–32
in several industrial chemical processes.
Keggin-type HPAs cannot be used as heterogeneous catalysts in as follows: 0–8 min (25–32% B); 8–10 min (32–35% B); 10–25 min
polar solvent reactions, H 40 could associate with diethyl (35–37% B); 25–28 min (37–40% B); 28–30 min (40–90% B) and
ether molecules to form an ether-complex, which is insoluble in 30–31 min (90% B). The injected sample volume was 5 mL.
diethyl ether or in water and heavier than both of them. This High-resolution mass spectrometry was carried out on a Q
ether-complex layer facilitates the extraction of H PW O from Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo
Although the and a temperature of 35 1C. The gradient elution was programmed
3
PW12O
3
12 40
aqueous reaction solution, retaining the reactant in the polar Scientific, San Jose, CA, USA), whereas multistage tandem mass
phase. After separation, the diethyl ether could be removed by spectra were acquired from an LTQ XL mass spectrometer
3
3,34
evacuation, resulting in recovered H
3
PW12O
40 for reuse.
(Thermo Scientific, San Jose, CA, USA). Both spectrometers were
However, little attention has been devoted to the application of equipped with an electrospray ionization (ESI) source. Nitrogen
HPAs in chemical transformations of the active natural product, was supplied as the sheath, auxiliary and sweep gas at flow rates of
especially in the generation of rare ginsenosides.
35, 10 and 5 a.u., respectively. The capillary temperature was set at
High-performance liquid chromatography electrospray ioni- 320 1C. Ginsenosides were detected in the negative ion mode with
zation mass spectrometry (HPLC-ESI-MS) has been proved to a spray voltage of À4000 V. The capillary voltage was optimized to
be a powerful tool for the separation and identification of À32 V. Data were collected in centroid mode. CID experiments
1
9,20,35–40
ginsenosides.
(
Multistage tandem mass spectrometry were performed on mass selected precursor ions using standard
n
MS ) analysis permits multiple isolation and fragmentation of isolation and excitation procedures (an activation q value of 0.2
precursor ions and allows the origins of the product ions to be and an activation time of 30 ms). Ions were isolated using a width
unambiguously assigned. High-resolution mass spectrometry of m/z 1.0 (Æ0.5 m/z) and then subjected to the normalized
(HRMS) analysis provides more detailed and accurate informa- collision energy. The scan range was from m/z 100 to 2000 for full
tion of molecular composition, which greatly facilitates the scan mode and from m/z 260 to 1000 for CID experiments.
structural elucidation of the analytes and differentiation of
isomers in combination with the collision induced dissociation Procedure for the transformation of ginsenoside
(
CID) technique.
Although studies on the chemical transformation of ginseno-
The standard ginsenoside Re (1.01 mg) and catalyst H
3
PW12
O
40
Á
2
6H O (0.08 mmol) were accurately weighed and dissolved in 2 mL
sides have been carried out, we herein applied HPAs as reusable
catalysts to chemically transform ginsenoside Re. Eight newly
generated ginsenosides are accurately identified via a HPLC-MS /
HRMS method. The transformation pathways and mechanisms
were further predicated and rationalized from a carbenium ion
mechanism point of view. Notably, the aglycone structures of ‘‘3b,
distilled water. The solution was introduced into a 5 mL glass vial
under air, and then heated in a shaking bath at 80 1C for 2 h. Every
n
15 min, aliquots of solution were pipetted and diluted to a constant
volume in an ice bath at 0 1C. The aqueous solution was extracted
by diethyl ether in equal volume three times to separate the catalyst.
The ether-complex layer was collected and evacuated to remove
1
2b, 25-trihydroxy-dammar-20 (21/22)-ene’’ were generated via the
diethyl ether and to obtain H
catalyst residue, the extracted solution that contained the residue of
40, unreacted ginsenoside Re and transformed products
was further allowed to react for one more hour (denoted as
h-extracted sample). For comparison, the sample that reacted
for 2 h was also prepared. As shown in Fig. S1 (ESI†), the TIC of the
h-extracted sample is similar to the TIC of the 1 h sample but
significantly different from that of the 2 h sample, which indicates
that most of the H 40 has been removed. The recovery of
40 is calculated to be 98.2%. On the other hand, the
aqueous layer was evaporated to dryness and dissolved in methanol
1 mL). Then the transformation solution was filtered through a
.45 mm membrane before being subjected to HPLC-MS analysis.
3
PW12O40 for reuse. To estimate the
chemical transformation of PPT-type ginsenosides for the first time.
3
H PW12O
Experimental
Materials
2
2
All the ginsenoside authentic standards with over 98% purity were
purchased from Beijing Wanjiashouhua Biological Technology
Co., Ltd (Beijing, China). HPLC grade methanol and acetonitrile
were acquired from Tedia (Fairfield, USA), whereas HPLC grade
formic acid was acquired from Thermo Fisher (Waltham, USA).
3
PW12O
3
H PW12O
(
0
Heteropoly acid H
St. Louis Missouri) and was calcinated at 300 1C to obtain
O. Distilled water was purified using a Milli-Q
3
PW12O40ÁnH
2
O was bought from Sigma-Aldrich
(
H
3
PW12
O
40Á6H
2
water purification apparatus (Millipore, Bedford, USA).
Results and discussion
HPLC-MS analysis
Determination of the molecular mass of ginsenoside by MS
The high-performance liquid chromatography analysis was All the compounds were identified through a comparison of
performed on a Dionex Ultimate 3000 system equipped with a the mass spectra and retention time with those of authentic
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standards and empirical assignment of molecular structures rearrangement and removal of two H
2
O molecules from the ion
through accurate mass of molecular ions, associated with multi- at m/z 179). Furthermore, the ions at m/z 131 and base peak
stage CID fragmentation. The identification of ginsenoside m/z 101 represented two rearrangement ions after cross-ring cleavage
isomers was performed based on the characteristic chromato- at bonds 0 and 1 as well as at bonds 2 and 5 of the glucose
graphic features of ginsenosides on the reverse-phase C18 residue, respectively.
3
column reported in the literature studies.
MS spectra on m/z 799, 783, 765, 637, 619 and 475 were
Ginsenoside molecules can be ionized through ESI in both carried out to trace the source of fragments. As shown in Fig. S3
positive and negative modes. The full-scan and CID mass (ESI†), the fragments were the same as those shown in Fig. 1.
2
spectra of the target ginsenosides were recorded in both modes. This is because in the negative ESI analysis of ginsenoside, MS
In positive ESI, a saponin molecule normally combines with an spectra could exhibit the deglycosylated fragment ions, which
+
n
alkali metal ion, such as Na , a very common impurity that are the same as most of those in MS spectra, and provide
inevitably leaches from glassware. The gas-phase positive alkali sufficient information on saccharide substitutions in ginseno-
metal ions have great binding affinity for the saccharide moiety sides. Therefore, the fragmentation correlation was concluded
of ginsenosides, and form fairly strongly bonded adducts. The to be: m/z 945 4 m/z 799 4 m/z 637 (619) 4 m/z 475 4 m/z 391
internal energy of the resulting adduct molecules usually and m/z 945 4 m/z 783 4 m/z 637 (619) 4 m/z 475 4 m/z 391.
increases via this exothermic process, and being distributed Fragment ions at m/z 799 and 783 were 146 and 162 Da less
À
over all internal degrees of freedom, which may facilitate the than the [M–H] ions at m/z 945, corresponding to the loss of
bond cleavages under CID conditions. As shown in Fig. S2 one deoxyhexose and one hexose residue, respectively. Accordingly
2
À
(
ESI†), the MS spectrum of the authentic standard of ginseno- these two ions were assignable to [M–Rha–H] (m/z 799) and
À
side Re, in positive ESI mode, presents complex and irregular [M–Glc–H] (m/z 783) ions, which proved the presence of two
structural information on the cross-ring cleavage of saccharide different saccharide moieties with terminal rhamnose and
but little direct information on aglycone and glycosidic cleavage. glucose residues individually. Notably, the peak intensity of
À
À
Correspondingly, in negative ESI, the neutral saponin molecules [M–Glc–H] ions was distinctly higher than that of [M–Rha–H]
commonly lose one proton to form deprotonated molecular ions ions, suggesting that the glucose-terminated moiety was more
À
[
M–H] , or associate loosely with one modifier ion to generate an reactive than the rhamnose-terminated one. The mass difference
À 41
adduct ion [M + HCOO] . (Formic acid herein was employed as of 308 Da between the ions at m/z 945 and m/z 637 was in
an organic modifier in the mobile phase to destroy the noncova- agreement with the total mass of one deoxyhexose and one
lent interactions and to optimize the response of deprotonated hexose residue (146 Da + 162 Da), indicating the m/z 637 ion to
À
À
À
analytes.) The formation of [M–H] and [M + HCOO] ions is be [M–Glc–Rha–H] . The ion at m/z 475, corresponding to the
À
endothermic and correlated with the acquisition of extra energy characteristic [aglycone–H] ion of PPT, was generated through
prior to fragmentation, which implies the lower internal energy further loss of one glucose residue from the [M–Glc–Rha–H] ion
of negative ions than that of their positive counterparts. There- and is denoted as [M–2Glc–Rha–H] , indicating that there were
fore, data recorded in negative ion mode exhibited more clear totally one rhamnose and two glucose residues located at the
spectra with much fewer fragments and were employed to obtain saccharide substitutions. Two dehydration ions appeared at m/z 765
the straightforward structural information about ginsenosides in and 679, having a mass 18 Da lower than the ions at m/z 783 and
À
4
2
À
this study.
637, respectively. Since the dehydration ions could not be observed
À
The molecular weight of Re was determined to be 946 Da, till the loss of one glucose residue from [M–H] ions, in combination
based on the [M + HCOO] ion at m/z 991 and the [M–H] ion at with the higher reactivity of the quaternary carbon at C-20
m/z 945. The MS spectrum of the authentic standard of ginseno- relative to the tertiary carbon at C-6 of the PPT aglycone, it is
À
À
2
side Re is shown in Fig. 1. The ions at m/z 179, 161 and 143 reasonable to assume that these two saccharide substitutions
resulting from glycosidic bond cleavage confirmed the presence consisted of one glucose residue at C-20 and one glucose connected
of hexose substitution (the ion at m/z 143 was derived from the with one terminal rhamnose residue at C-6, respectively. The
ion at m/z 205 originating from the cross-ring cleavage at bonds
0
and 2 on the glucose directly linked to aglycone provided
further evidence for the number and connection of saccharide
substitutions, as shown in the inset of Fig. 1.
Structural characterization of the transformed ginsenoside by
n
HPLC-MS /HRMS
Generally, ginsenosides could be transformed chemically by
+
active H in acidic solution through the hydrolysis of oligosac-
charide moieties as well as dehydration, addition and cyclization
reactions on the aglycone. Therefore, a certain amount of HPA
was dissolved in deionized water, resulting in an acidic aqueous
solution used for the transformation of ginsenoside Re at 80 1C
for 1 h. The total ion chromatogram of the transformed products
2
À
Fig. 1 High-resolution MS spectrum from the [M + HCOO] ion at m/z 991
and the fragmentation pathway of ginsenoside Re.
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6
the neutral loss was 84.0936 Da, corresponding to C H12 with a
mass error of 2.3 ppm, which was formed through bond cleavage
between C-20 and C-21 with the residual charge retained on
the aglycone, as illustrated in Fig. 3(b). On the other hand,
compound 4 had a neutral loss of 58.0415 Da. The molecular
3 6
mass of 58.0415 Da was determined as C H O with a mass error
of 3.4 ppm, indicating the terminal tertiary alcohol structure
bonding with two methyl groups, as shown in Fig. 3(d). There-
fore, hydroxylation was determined to occur at the C-25 position
of compounds 4 and 5. And because of the same molecular
Fig. 2 Total ion chromatogram (TIC) of the products of ginsenoside Re
mass of 4, 5 and Rg , it is reasonable to confirm the existence of
2
3
chemical transformation by H PW12O40 at 80 1C for 1 h.
unsaturated double bonds at C-20(21) or C-20(22), resulting
from the dehydration of tertiary alcohol at the C-20 position.
is shown in Fig. 2. Nine well-separated peaks were observed in the Consequently, compounds 4 and 5 could be structurally distin-
spectrum and denoted as compounds 1 to 9 in sequence. guished by the position of the double bond, which also led to
Compound 3 was unambiguously identified as the reactant their polarity differences. Since the D20(21) isomer tended to be
Re through the comparison of the retention time and molecular more polar and eluted earlier from reversed-phase columns than
mass with those of the authentic standard.
its D20(22) counterparts, compounds 4 and 5 were concluded
Compounds 4, 5, 6 and 7 were considered to be four isomers to be 25-OH-Rg and 25-OH-F , which are rarely found in
6
4
with the same molecular mass of 784 Da. Comparing to the nature.19,24,43,44
authentic standard, 6 and 7 were assignable to ginsenosides
Compounds 1 and 2 represented two Re-transformed ginseno-
0(S)-Rg and 20(R)-Rg , which were generated from the removal sides with the [M + HCOO] ion at m/z 847 and the [M–H] ion at
À
À
2
2
2
of the glucose residue at the C-20 position of Re and subsequent m/z 801, indicating a molecular mass of 802 Da (Fig. S6, ESI†).
2
isomerization. Multistage tandem mass spectrometry and high- As shown in Fig. 4, the base peak at m/z 493 in the MS spectrum
resolution mass spectrometry were performed for the structural on m/z 801 was 18 Da higher than the PPT aglycone ion at
elucidation of compound 4 as well as 20(S)-Rg
2
for comparison. m/z 475, suggesting the hydration reaction on the unsaturated
3
À
As shown in Fig. 3, the MS fragmentation on the [M–H] ions double bond at the C-24(25) position of the aglycone. Based on the
at m/z 783 provided one pair of differential ions at m/z 417 and regioselectivity of Markovnikov’s rule for electrophilic addition
3
91 besides three pairs of identical ions at m/z 637, 619 and 475, to alkenes in acid solution, the hydroxyl group was apparently
À
À
45
which were corresponding to [M–Rha–H] , [M–Rha–H O–H]
and [aglycone–H] ions. The differential ion pair at m/z 417 and The neutral loss of 308 Da between the precursor [M–H] ion at
associated with C-25, resulting in the structure of 25-OH-PPT.
2
À
À
À
391 was further confirmed to be originating from the [aglycone–H]
m/z 801 and the product ion at m/z 493 corresponded to the
4
ion of 4 and Rg
2
by the MS spectra of the ion at m/z 475 disaccharide moieties, one deoxyhexose and one hexose residue.
for the MS spectra of compound 5 and 20(R)-Rg , please see In addition, the analogous chromatographic behaviours of these
Fig. S4, ESI†). Furthermore, the accurate neutral loss between two peaks, such as the similar retention time and relative
n
(
2
À
2
the differential ions and the [aglycone–H] ion at m/z 475 was intensity as well as the identical patterns of the MS spectrum,
monitored by HRMS (Fig. S5, ESI†). For ginsenoside 20(S)-Rg
2
,
indicated the existence of C-20(S/R) epimers. The only difference
3
À
4
Fig. 3 MS spectra of the ions at m/z 783 from the [M + HCOO] ion of (a) ginsenoside 20(S)-Rg
2
and (c) 25-OH-Rg
6
. Fragmentation pathways and MS
À
spectra of the ions at m/z 475 from the [M + HCOO] ion of (b) ginsenoside 20(S)-Rg
2
and (d) 25-OH-Rg
6
.
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Fig. 4, the saccharide moiety of 8 and 9 was considered to be
identical to that of ginsenoside Rf . Comparing to the PPT
2
aglycone ion at m/z 475, the ion at m/z 457 with À18 Da mass
difference indicated the occurrence of dehydration reaction.
The deglycosylation of the glucose moiety at C-20 generated a
tertiary alcohol group, which was responsible for the dehydration.
Therefore, a new double bond was expected to be located at C-20(21)
or C-20(22) on the aglycone, resulting in two Re-transformed
ginsenoside isomers. Consequently, it is concluded that com-
pounds 8 and 9 originated from Re through deglycosylation
followed by dehydration at the C-20 position and were assigned
as ginsenoside Rg and F , respectively. It is worth noting that,
6
4
under the same CID conditions, there was no fragment further
generated from the aglycone ions at m/z 457, since these two double
bonds led to a relatively stable rigid aglycone. The spectral informa-
tion on all the transformed products is summarized in Table 1.
Generally, the ionized ginsenoside molecules usually lose
saccharide substitutions in sequence to generate an aglycone
moiety in CID experiment in negative ESI mode. Through the
observed neutral loss, the number and connection of saccharide
substitutions as well as the structural difference to PPT aglycone
could be directly identified. And the aglycone moiety could be
2
À
Fig. 4 High-resolution MS spectra from the [M–H] ion and the fragmen-
tation pathways of ginsenoside (a) 20(S)-Rf and (b) 20(R)-Rf
2
2
.
between the epimers was the configuration of the chiral carbon at further fragmented to generate specific ions used for structural
C-20. Because the 20(S)-epimer eluted earlier than the correlative discrimination. In this study, the aglycone moieties of all the 8
2
2
2
0(R)-epimer, compounds 1 and 2 were identified as ginsenoside transformed compounds can be classified into four types, i.e.
2
0,44
0(S)-Rf
2
2
and 20(R)-Rf , respectively.
2
Moreover, ginsenoside (a) 3b, 12b, 20-trihydroxy-dammar-24-ene (Re and 20(S/R)-Rg );
5-OH-PPT is expected to represent safe and effective therapeutic (b) 3b, 12b-dihydroxy-dammar-20(21/22), 24-diene (Rg and F );
6
4
agents, since its 25-OH-PPD counterpart has already exhibited (c) 3b, 12b, 20, 25- tetrahydroxy-dammarane (20(S/R)-Rf ); and
2
46
potent anticancer activity.
(d) 3b, 12b, 25-trihydroxy-dammar-20 (21/22)-ene (25-OH-Rg and
6
4
Similarly, structural elucidation for the isomers of compounds 8 25-OH-Rg ). All the aglycone moieties exhibited absolutely different
and 9 with a molecular mass of 766 Da was performed (Fig. 5 and fragmentation pathways. In CID experiments, aglycone (a) pro-
Fig. S7, ESI†). Owing to the same neutral loss and fragmentation duced ions at m/z 475 and 391. The ion at m/z 475 is the typical
patterns of the precursor ions at m/z 765 in Fig. 5 and m/z 801 in deprotonated ion of PPT aglycone, whereas m/z 391 resulted from
the bond cleavage between C-20 and 22 with 84 Da neutral loss.
Only one ion at m/z 457, 18 Da lower than PPT aglycone, was
obtained from aglycone (b). This is due to the rigid framework
derived from the dehydration at C-20 of PPT and hence difficulty in
dissociation. Aglycone (c) produced three ions at m/z 491, 417 and
391, which correspond to the hydrated ion of PPT aglycone, the ion
with bond cleavage at C-24(25) and following dehydration of one
water molecule, and the ion with bond cleavage at C-20(22). For
aglycone (d), which is the isomer of aglycone (a), the fragmentation
of m/z 417 is conducive to the isomer differentiation. This ion is
considered to have resulted from the bond cleavage between C-24
and 25, with a neutral loss of 58 Da. Furthermore, based on the
n
developed HPLC-MS /HRMS method, precise molecular weight
information could be obtained, which was useful for the accurate
structural elucidation of fragments, and the origins of fragments
were recognized, which was the foundation of structure analysis.
n
The combined use of HRMS and MS analyses provides reliable
results of structural elucidation for unknown ginsenosides.
Pathways and mechanisms of the chemical transformation of
Re in acid solution
2
À
2
In summary, a total of eight ginsenosides, 20(S/R)-Rf , 20(S/R)-
Fig. 5 High-resolution MS spectra from the [M–H] ion and the frag-
mentation pathways of ginsenoside (a) Rg and (b) F
6
4
.
Rg
2
, 25-OH-Rg
/F
6 4
, Rg
6
and F
4
, are available from the chemical
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Table 1 Major ions (m/z) observed in the HRMS spectra of the chemical transformation products of ginsenoside Re
Molecular
Mass
Theoretical error
Measured formula
À
2
Peak Identification mass (m/z) [M–H]
mass
(ppm) MS fragment ions
À
À
À
1
2
3
20(S)-Rf
2
801.4997
801.4999
945.5401
C
C
C
42
42
48
H
H
H
73
73
81
O
O
O
14 801.5000
14 801.5000
18 945.5423
À0.4
À0.4
À2.3
847.5044 [M + HCOO] , 655.4426 [M–Rha–H] , 637.4322 [M–Rha–H
2
O–H] ,
À
À
4
3
93.3894 [M–Rha–Glc–H] , 417.3372 [M–Rha–Glc–C
3 8 2
H O –H] ,
À
91.2852 [M–Rha–Glc–C
6
H14O–H]
À
À
À
20(R)-Rf
2
847.5044 [M + HCOO] , 655.4425 [M–Rha–H] , 637.4322 [M–Rha–H
2
O–H] ,
À
À
4
3
93.3897 [M–Rha–Glc–H] , 417.3374 [M–Rha–Glc–C
3 8
H
O
2
–H] ,
À
91.2853 [M–Rha–Glc–C
6
H14O–H]
À
À
À
Re
991.5477 [M + HCOO] , 799.4830 [M–Rha–H] , 783.4878 [M–Glc–H] ,
À
À
7
6
65.4778 [M–Glc–H
2
O–H] , 637.4301 [M–Glc–Rha–H] ,
À
À
19.4194 [M–Glc–Rha–H
2
O–H] , 475.3777 [aglycone–H]
À
À
4
5
6
7
8
9
25-OH-Rg
25-OH-F
20(S)-Rg
20(R)-Rg
Rg
6
783.4885
783.4887
783.4879
783.4881
765.4790
765.4791
C
42
C
42
C
42
C
42
C
42
C
42
H
H
H
H
H
H
71
O
71
O
71
O
71
O
69
O
69
O
13 783.4895
13 783.4895
13 783.4895
13 783.4895
12 765.4789
12 765.4789
À1.2
À1.0
À2.0
À1.8
0.1
637.4314 [M–Rha–H] , 619.4203 [M–Rha–H
2
O–H] ,
À
À
À
4
75.3781 [M–Rha–Glc–H] , 417.3361 [M–Rha–Glc–C
3
H
6
6
O–H]
À
À
4
637.4315 [M–Rha–H] , 619.4207 [M–Rha–H
2
O–H] ,
À
4
75.3785 [M–Rha–Glc–H] , 417.3367 [M–Rha–Glc–C
3
H
O–H]
À
À
2
637.4308 [M–Rha–H] , 619.4198 [M–Rha–H
2
O–H] ,
À
À
4
75.3780 [aglycone–H] , 391.2844 [aglycone–C
6
H
12–H]
À
À
2
637.4308 [M–Rha–H] , 619.4197 [M–Rha–H
2
O–H] ,
À
À
4
75.3780 [aglycone–H] , 391.2845 [aglycone–C
6
H
12–H]
À
À
6
619.4212 [M–Rha–H] , 601.4091 [M–Rha–H
2
O–H] ,
À
4
57.3687 [M–Rha–Glu–H]
À
À
F
4
0.3
619.4212 [M–Rha–H] , 601.4101 [M–Rha–H
2
O–H] ,
À
4
57.3689 [M–Rha–Glu–H]
3
transformation of Re in the H PW12O40 dissolved strongly acidic attack in two non-selective avenues on either side of the plane,
aqueous phase. Therefore, the observed transformation pathways yielding a mix of epimers in equal proportion, as illustrated in
4
7
involve deglycosylation at the C-20 position prior to hydration, Fig. 6.
dehydration and epimerization reaction occurring on the side
chain, as demonstrated in Scheme 1.
After deglycosylation at the C-20 position, the active tertiary
alcohol could be further eliminated in the acidic aqueous phase
Deglycosylation of Re primarily occurred to yield the main through an E1 mechanism, resulting in ginsenosides Rg and F .
6
4
2
products of ginsenoside 20(S/R)-Rg through the hydrolysis of the As shown in Scheme 2, the protonation of the alcoholic oxygen
saccharide moiety at C-20 rather than the one at C-6. This can be firstly generates an oxonium ion, providing a leaving group of a
rationalized by the carbenium ion mechanism: the cleavage of neutral water molecule, which is ideal to start the elimination
the glycosidic bond at C-20 tends to generate the tertiary carbe- reaction. The loss of the leaving group generates a carbenium
nium ion with more stability on the chiral carbon atom in a much intermediate followed by the removal of the hydrogen from the
lower activation energy pathway compared to the formation of the b-carbon to result in the creation of the double bond at C20(21)
secondary carbenium ion at C-6 on the rigid backbone. Therefore, and C20(22). The generation of carbenium is the rate-determining
the deglycosylation reactivity of C-20 is distinctly higher than that step and related to the stability of the carbenium intermediate.
of C-6, leading to the preferential hydrolysis of the saccharide Therefore, the tertiary hydroxyl at C-20 showed higher reactivity in
moiety and hence the formation of 20(S/R)-Rg
alcohol at the C-20 position. Intriguingly, the generated epimers, the secondary carbenium ion. Moreover, the content ratio of
0(S)-Rg and 20(R)-Rg , are present in almost equal amounts. Rg /F is equal to 3 : 7 in accordance with Zaitsev’s rule which
This is due to the trigonal planar molecular geometry of the sp predicts that the most substituted alkene will be typically stable
hybridized carbenium intermediate, which allows the nucleophilic and favoured in elimination reactions.
2
with tertiary the dehydration reaction than those at C-3 and C-12 which bear
2
2
2
6
4
2
4
8,49
3
Scheme 1 Chemical transformation pathway of ginsenosides Re catalyzed by H PW12O40. Glc, b-D-glucopyranosyl; Rha, a-L-rhamnopyranosyl.
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forming a tetravalent tertiary carbenium intermediate. This is the
rate-determining step. The more stable the carbenium inter-
mediate is, the easier it is to form. Afterwards, the lone-pair
electrons of water molecules further attack on the carbenium ion
to hydroxylate by leaving the catalytic hydrogen cations, resulting
in the hydrated products. This reaction pathway is thermodyna-
mically favoured compared to the hydroxylation on the C-24 atom,
which undergoes the generation of a secondary carbenium ion
50,51
with less stability and higher activation energy.
Since there is chemical equilibrium between alcohols and
alkenes in acid environments, both of the hydrations on the
double bond at C-24(25) atoms and the dehydration on C-20(21/22)
atoms of PPT type ginsenosides are considered to be reversible
reactions, which could be rationalized from the carbenium ion
mechanism point of view. Therefore, the shift of both reversible
transformation pathways depends on the reaction temperature
and catalytic activity. In our previous studies on the chemical
transformation of PPT and PPD type ginsenosides using a volatile
organic acid, such as formic acid and acetic acid, no aglycone
structures of 3b, 12b, 25-trihydroxy-dammar-20 (21/22)-ene are
Fig. 6 Representation of the epimerization of C-20 resulting from the
non-selective attack of the hydroxyl group on the trigonal planar sp
2
hybridized carbon.
19,20
observed.
In the present study, 25-OH-Rg /F could be produced
6 4
through further dehydration of 20(R/S)-Rf
2
6 4
or hydration of Rg /F .
This is attributed to the strong acidity of HPA in aqueous solutions
which promote the loss of the leaving group and are conductive to
the formation of stable carbenium ions at C-20 and C-25 of the
aglycone to obtain dynamic equilibrium of those generated
ginsenosides in the transformation process.
Conclusions
Scheme 2 The proposed carbenium ionic route of dehydration of
2
ginsenoside Rg .
The heteropoly acid H PW O was developed for the chemical
3
12 40
transformation of ginsenoside Re and recycled through simple
extraction by diethyl ether. Eight products were obtained and
identified based on a HPLC-MS /HRMS method. Particularly,
The stability of the carbenium ion also plays a decisive role in
the hydration on the double bond at C-24(25) to yield 20(S/R)-Rf2
and 25-OH-Rg /F . As shown in Scheme 3, the electrophilic attack
of hydrogen cations gives rise to the break of the double bond,
protonation of C-24 and creation of a positive charge on C-25,
n
four isomeric products of 784 Da could be easily identified
through the developed method. The transformation pathway
involves the acid-catalyzed deglycosylation, hydration, dehydra-
tion and isomerization reactions, all of which proceed through
the carbenium ion mechanism. 25-OH-Rg6 and 25-OH-F4,
consisting of the aglycone structures of ‘‘3b, 12b, 25-trihydroxy-
dammar-20 (21/22)-ene’’, were generated via chemical transfor-
mation for the first time, which provides a prospective feasibility
to transform certain effective and rare ginsenosides. Although
there is still a need to improve the selectivity of the transforma-
tion reaction, which is precisely what we are focusing on, HPA
catalysts open up a clean, economical and environmentally
benign process in the chemical transformation of active natural
products.
6
4
Acknowledgements
This work was financially supported by the Science and Technology
Development Plan Project of Jilin Province (20160520123JH) and
Special Fund for Agro scientific Research in the Public Interest
(201303111).
Scheme 3 The proposed carbenium ionic route of hydration on the
double bond between C-24 and C-25.
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Notes and references
25 H. T. Vo, J. Y. Cho, Y. E. Choi, Y. S. Choi and Y. H. Jeong,
J. Ginseng Res., 2015, 39, 304–313.
1
2
D. H. Kim, J. Ginseng Res., 2012, 36, 1–15.
L. Jia, Y. Zhao and X. J. Liang, Curr. Med. Chem., 2009, 16,
26 Y. Yuan, Y. B. Hu, C. X. Hu, J. Y. Leng, H. L. Chen,
X. S. Zhao, J. Gao and Y. F. Zhou, J. Mol. Catal. B: Enzym.,
2924–2942.
2015, 120, 60–67.
3
4
5
6
Y. Z. Xiang, H. C. Shang, X. M. Gao and B. L. Zhang, Phytother.
Res., 2008, 22, 851–858.
A. S. Attele, J. A. Wu and C. S. Yuan, Biochem. Pharmacol.,
2
2
2
7 I. V. Kozhevnikov, Chem. Rev., 1998, 98, 171–198.
8 I. V. Kozhevnikov, J. Mol. Catal. A: Chem., 2007, 262, 86–92.
9 I. V. Kozhevnikov, J. Mol. Catal. A: Chem., 2009, 305,
1
999, 58, 1685–1693.
M. S. Lee, E. J. Yang, J. I. Kim and E. Ernst, J. Alzheimers Dis.,
009, 18, 339–344.
J. M. L u¨ , Q. Yao and C. Chen, Curr. Vasc. Pharmacol., 2009, 7,
93–302.
1
04–111.
30 N. Mizuno, K. Yamaguchi and K. Kamata, Coord. Chem.
Rev., 2005, 249, 1944–1956.
2
3
3
1 Y. B. Huang and Y. Fu, Green Chem., 2013, 15, 1095–1111.
2 W. Deng, Q. Zhang and Y. Wang, Dalton Trans., 2012, 41,
2
7
8
H. J. Kim, P. Kim and C. Y. Shin, J. Ginseng Res., 2013, 37, 8–29.
J. Y. Park, P. Choi, H. K. Kim, K. S. Kang and J. Ham,
J. Ginseng Res., 2016, 40, 62–67.
9817–9831.
3
3
3 H. Wu, J. Biol. Chem., 1920, 43, 189–220.
4 J. Tian, J. Wang, S. Zhao, C. Jiang, X. Zhang and X. Wang,
Cellulose, 2010, 17, 587–594.
9
L. P. Christensen, Adv. Food Nutr. Res., 2009, 55, 1–99.
1
1
1
1
0 S. F. Nabavi, A. Sureda, S. Habtemariam and S. M. Nabavi,
J. Ginseng Res., 2015, 39, 299–303.
1 S. J. Lee, W. J. Lee, S. E. Chang and G. Y. Lee, J. Ginseng Res.,
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5 S. Li, S. Lai, J. Song, C. Qiao, X. Liu, Y. Zhou, H. Cai, B. Cai
and H. Xu, J. Pharm. Biomed. Anal., 2010, 53, 946–957.
6 N. Fuzzati, B. Gabetta, K. Jayakar, R. Pace and F. Peterlongo,
J. Chromatogr. A, 1999, 854, 69–79.
7 M. Cui, F. Song, Y. Zhou, Z. Liu and S. Liu, Rapid Commun.
Mass Spectrom., 2000, 14, 1280–1286.
2015, 39, 238–242.
2 G. C. Han, S. K. Ko, J. H. Sung and S. H. Chung, J. Agric. Food
Chem., 2007, 55, 10641–10648.
3 Y. Usami, Y. N. Liu, A. S. Lin, M. Shibano, T. Akiyama,
H. Itokawa, S. L. Morris-Natschke, K. Bastow, R. Kasai and
K. H. Lee, J. Nat. Prod., 2008, 71, 478–481.
8 F. Song, Z. Liu, S. Liu and Z. Cai, Anal. Chim. Acta, 2005, 531,
6
9–77.
9 S. Liu, M. Cui, Z. Liu and F. Song, J. Am. Soc. Mass Spectrom.,
004, 15, 133–141.
1
4 J. S. Jung, J. A. Shin, E. M. Park, J. E. Lee, Y. S. Kang,
S. W. Min, D. H. Kim, J. W. Hyun, C. Y. Shin and H. S. Kim,
J. Neurochem., 2010, 115, 1668–1680.
2
0 F. Wu, H. Sun, W. Wei, Y. Han, P. Wang, T. Dong, G. Yan
and X. Wang, J. Sep. Sci., 2011, 34, 3194–3199.
1 Z. Wu, W. Gao, M. A. Phelps, D. Wu, D. D. Miller and
J. T. Dalton, Anal. Chem., 2004, 76, 839–847.
2 R. B. Cole, Electrospray and MALDI Mass Spectrometry, Wiley
John & Sons, 2010.
3 S. W. Kwon, S. B. Han, I. H. Park, J. M. Kim, M. K. Park and
J. H. Park, J. Chromatogr. A, 2001, 921, 335–339.
1
1
1
5 B. H. Han, M. H. Park, Y. N. Han, L. K. Woo, U. Sankawa,
S. Yahara and O. Tanaka, Planta Med., 1982, 44, 146–149.
6 P. Pietta, P. Mauri and A. Rava, J. Chromatogr., 1986, 362,
291–297.
7 I. H. Park, N. Y. Kim, S. B. Han, J. M. Kim, S. W. Kwon,
H. J. Kim, M. K. Park and J. H. Park, Arch. Pharmacal Res.,
2002, 25, 428–432.
4 H. Yang, Y. D. Lee, K. B. Kang, J. Y. Kim, S. O. Kim, Y. H. Yoo
and S. H. Sung, J. Pharm. Biomed. Anal., 2015, 109, 91–104.
5 S. Liu, C. Rong and T. Lu, J. Phys. Chem. A, 2014, 118,
1
1
2
2
2
8 E. A. Bae, M. J. Han, E. J. Kim and D. H. Kim, Arch.
Pharmacal Res., 2004, 27, 61–67.
9 X. Zhang, F. Song, M. Cui, Z. Liu and S. Liu, Planta Med.,
3698–3704.
2007, 73, 1225–1229.
6 W. Wang, E. R. Rayburn, Y. Zhao, H. Wang and R. Zhang,
Cancer Lett., 2009, 278, 241–248.
7 Q. Xiang, Y. Y. Lee, P. O. Petterson and R. Torget, Appl.
Biochem. Biotechnol., 2003, 105, 505–514.
8 C. M. Rosado-Reyes, W. Tsang, I. M. Alecu, S. S. Merchant
and W. H. Green, J. Chromatogr. A, 2013, 117, 6724–6736.
9 M. M. Toteva and J. P. Richard, J. Am. Chem. Soc., 1996, 118,
0 W. Wu, Q. J. Qin, Y. Y. Guo, J. H. Sun and S. Y. Liu, J. Agric.
Food Chem., 2012, 60, 10007–10014.
1 D. Wang, P. Y. Liao, H. T. Zhu, K. K. Chen, M. Xu, Y. J.
Zhang and C. R. Yang, Food Chem., 2012, 132, 1808–1813.
2 N. Yamabe, K. I. Song, W. Lee, I. H. Han, J. H. Lee, J. Ham,
S. N. Kim, J. H. Park and K. S. Kang, J. Ginseng Res., 2012, 36,
256–262.
1
1434–11445.
0 P. K. Chattaraj, U. Sarkar and D. R. Roy, Chem. Rev., 2006,
06, 2065–2091.
2
2
3 J. Y. Wan, P. Liu, H. Y. Wang, L. W. Qi, C. Z. Wang, P. Li and
C. S. Yuan, J. Chromatogr. A, 2013, 1286, 83–92.
4 J. R. Wang, L. F. Yau, R. Zhang, Y. Xia, J. Ma, H. M. Ho,
P. Hu, M. Hu, L. Liu and Z. H. Jiang, J. Agric. Food Chem.,
1
1 A. Aizman, R. Contreras, M. Galv ´a n, A. Cedillo, J. C. Santos
and E. Chamorro, J. Phys. Chem. A, 2002, 106, 7844–7849.
2014, 62, 2558–2573.
New J. Chem.
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