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Quantitative LC-MS/MS analysis of seven ginsenosides and three aconitum alkaloids in Shen-Fu decoction

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  • Liaocheng People's Hospital, Shandong, China

Abstract and Figures

Shen-Fu decoction is a traditional Chinese medicine prescription with a 3:2 ratio of Radix Ginseng and Fuzi (Radix Aconiti lateralis praeparata). Ginsenosides and alkaloids are considered to be the main active components of Shen-Fu decoction. However, no analytical methods have been used to quantitatively analyse both components in Shen-Fu decoction simultaneously. We successfully developed a rapid resolution liquid chromatography coupled with tandem mass spectrometry (RRLC-MS/MS) method for the simultaneous analysis of seven ginsenosides and three aconitum alkaloids in Shen-Fu decoction, the decoction of Radix ginseng and Fuzi (Radix Aconiti lateralis praeparata). Chromatogrpahic separation by RPLC was achieved using a reversed-phase column and a water/acetonitrile mobile phase, containing 0.05% formic acid and using a gradient system. The method was optimized to allow for simultaneous analysis of all analytes in 11minutes without the need for baseline resolution of the components. Furthermore, the separation demonstrated good linearity (r > 0.9882), repeatability (RSD < 7.01%), intra- and inter-day precisions (RSD < 5.06%) and high yields of recovery (91.13-111.97%) for ten major constituents, namely ginsenoside-Re, Rg1, Rb1, Rc, Rb2, Rd, Rf, aconitine, hypacoitine and mesaconitine. The developed method could be used as a rapid and reliable approach for assessment of the quantity of the major constituents in Shen-Fu decoction.
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R E S E A R C H A R T I C L E Open Access
Quantitative LC-MS/MS analysis of seven
ginsenosides and three aconitum alkaloids in
Shen-Fu decoction
Na Guo
1
, Mingtao Liu
2
, Dawei Yang
3
, Ying Huang
1
, Xiaohong Niu
1
, Ruifan Wu
4
, Ying Liu
5
, Guizhi Ma
4*
and Deqiang Dou
6*
Abstract
Background: Shen-Fu decoction is a traditional Chinese medicine prescription with a 3:2 ratio of Radix Ginseng and
Fuzi (Radix Aconiti lateralis praeparata). Ginsenosides and alkaloids are considered to be the main active
components of Shen-Fu decoction. However, no analytical methods have been used to quantitatively analyse both
components in Shen-Fu decoction simultaneously.
Results: We successfully developed a rapid resolution liquid chromatography coupled with tandem mass
spectrometry (RRLC-MS/MS) method for the simultaneous analysis of seven ginsenosides and three aconitum
alkaloids in Shen-Fu decoction, the decoction of Radix ginseng and Fuzi (Radix Aconiti lateralis praeparata).
Chromatogrpahic separation by RPLC was achieved using a reversed-phase column and a water/acetonitrile
mobile phase, containing 0.05% formic acid and using a gradient system. The method was optimized to allow
for simultaneous analysis of all analytes in 11minutes without the need for baseline resolution of the components.
Furthermore, the separation demonstrated good linearity (r > 0.9882), repeatability (RSD < 7.01%), intra- and
inter-day precisions (RSD < 5.06%) and high yields of recovery (91.13-111.97%) for ten major constituents, namely
ginsenoside-Re, Rg
1
,Rb
1
, Rc, Rb
2
, Rd, Rf, aconitine, hypacoitine and mesaconitine.
Conclusions: The developed method could be used as a rapid and reliable approach for assessment of the
quantity of the major constituents in Shen-Fu decoction.
Keywords: Ginsenosides, Aconitum alkaloids, Shen-Fu decoction, RRLC-MS/MS
Background
Decoction is the traditional prescription of traditional
Chinese medicines (TCMs). Based on TCM theory, one
single herb or several kinds of herbs combined are
boiled in water to make the decoction. First documented
in 1465, Shen-Fu decoction is a TCM prescription with
a 3:2 ratio of Radix Ginseng and Fuzi (Radix Aconiti
lateralis praeparata). Both components have been
commonly used as herbal medicines in China for about
1800 years, predominantly used for folk treatment of dis-
eases with the sign of Yangqi decline or Yang exhaustion.
Shen-Fu decoction is also used to treat cardiovascular
diseases such as circulatory collapse, shock, thoracic ob-
struction and acute thoracic pain. Shen-Fu Injection (SFI
for intravenous medication), is a typical form of Shen-Fu
decoction, that has been used for treatment of many
kinds of diseases because of its cardiovascular protective
effectiveness [1-3]. The main active components found
in Shen-Fu decoction are ginsenosides and alkaloids.
Ginsenosides are generally classified into four groups:
protopanaxadiol, protopanaxatriol, ocotillol and oleanolic
acid type [4-6], Currently, more than 150 ginsenosides
have been isolated and identified in the literature.
Among them, ginsenosides-Rb
1
,Rb
2
, Rc, Rd, Rg
1
,Reand
Rf (Figure 1) are the most important compounds in che-
mical analysis of ginsengs. At present, about 224 alkaloids
have been isolated and identified from Aconitum [7,8].
* Correspondence: maguizhi000@sina.com;deqiangdou@126.com
4
College of Pharmacy, Xinjiang Medical University, Urumqi 830011, China
6
Department of Chinese Medicine Chemistry, Liaoning University of
Traditional Chinese Medicine, Dalian 116600, China
Full list of author information is available at the end of the article
© 2013 Guo et al.; licensee Chemistry Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Guo et al. Chemistry Central Journal 2013, 7:165
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These have been classified into four major groups, nonester
alkaloids (NEAs), monoester diterpene alkaloids (MDAs),
diester diterpene alkaloids (DDAs) and lipoalkaloids.
Aconitum alkaloids are mainly constituted of three DDAs,
diester-diterpence called aconitine (AC), measaconitine
(MA) and hypaconitine (HA) (Figure 1). They are known
for their high toxicity and pharmacological activity, as well
as being the target markers of Fuzi. In general, the cura-
tive effect of traditional Chinese medicine is an integrative
result of a number of ginsenosides and alkaloids. In order
to minimize the variability of active ingredients in the
decoction and ensure repeatable and reproducible thera-
peutic effects, it is very important to establish quality
control methodology for the decoction. To this end,
analysis of ginsenosides and Aconitum alkaloids is required
to assess the quality of Shen-Fu decoction.
Previous methods that have been used to analyze
ginsenosides and alkaloids include HPLC-DAD (ELSD),
CE, GC-MS and LC-MS [9-19] and alkaloids [20-30]. In
comparison with traditional HPLC, RRLC provides a
higher peak capacity, greater resolution, increased sensi-
tivity and higher speed of analysis. When coupled to a
triple quadrupole tandem mass spectrometer (QQQ-MS/
MS), it can achieve high sensitivity and selectivity by using
the multiple reaction monitoring (MRM) scan mode with-
out the baseline chromatographic separation of target
analytes. This method greatly facilitates the quantification
of chemical markers in complex matrixes with only a
small amount of sample. To date, there are no studies
reporting the simultaneously quantitative determination
of ginsenosides and Aconitum alkaloids in Shen-Fu decoc-
tion. The primary aim of the present study was to develop
a direct and rapid RRLC-MS/MS method for simulta-
neously quantifying the ten constituents in Shen-Fu decoc-
tion, namely, ginsenosides-Rb1, Rb2, Rc, Rd, Rg1, Re and
Rf and Aconitum alkaloids including AC, MA and HA.
Compound R1R2R3
Protopanaxadiol-type
Ginsenoside-Rb1 -O-Glc2-1Glc -H -O-Glc6-1Glc
Ginsenoside-Rb2 -O-Glc2-1Glc -H -O-Glc6-1Arap
Ginsenoside-Rc -O-Glc2-1Glc -H -O-Glc6-1Araf
Ginsenoside-Rd -O-Glc2-1Glc -H -O-Glc
Protopanaxadiol-type
Ginsenoside-Re -OH -O-Glc2-1Rha -O-Glc
Ginsenoside-Rg1 -OH -O-Glc -O-Glc
Ginsenoside-Rf -OH -O-Glc2-1Glc -OH
R1
R2
R3
R2
H3CO
N
R1
OCH3
OCH3
OH
OR3
OH
OCH3
O O
Compound R1R2R3
AC C2H5OH acetyl
MA CH3OH acetyl
HA CH3Hacetyl
Figure 1 Chemical structures of ginsenosides and Aconitum alkaloids analyzed in Shen-Fu decoction.
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Results and discussion
Chromatographic conditions and MS/MS method
development
Different mobile phases, including acetonitrile with 0.05%,
0.1% aqueous formic acid, acetic acid, 5 mM and 10 mM
ammonium formate solutions were tested. The best peak
shape and resolution was obtained with a mixture of
acetonitrile and aqueous 0.05% formic acid solution.
Using an optimized elution gradient, the main compo-
nents were separately eluted within 11 min. The typical
RRLC-QQQ MS/MS chromatograms of the marker
chemicals in Shen-Fu decoction are shown in Figure 2.
In order to increase sensitivity and specificity of quanti-
fication, multiple reaction monitoring was performed.
All factors related with MS performance including
ionization mode, capillary voltage, fragmentor voltage,
collision energy, gas flow and desolvation temperature
were analyzed. The optimum conditions were determined
as follows: postive ion mode, capillary voltage 4000 V,
drying gas, gas temperature 350°C and nebulizer pressure
of 50 psi.
Optimization of this MS/MS method produced highest
achiveable response using the MRM pairs comprising of
the precursor and product ions, which can achieve
better quantitation than reported results using the se-
lected ion monitoring (SIM) mode. After optimization,
the precursor and product ions of the ten analytes were
recorded (Table 1). The optimum collision energy was
determined to be 50 eV for Ginsenoside Re, 40 eV for
Rg
1
, 55 eV for Rf and Rd, 65 eV for Rb
1
, Rc and Rb
2
. For
alkaloids, they required a lower collision energy of 35 eV
for MA, 40 eV for HA and 45 eV for AC (Table 1).
Method validation
To determine the reliability of the test results, the method
validation included linearity, repeatability, intra- and inter-
day precisions and recovery test. The standard calibration
curves of all compounds were shown in Table 2 with satis-
factory linearity (r > 0.9882). Aconitum alkaloids had a
linear range of 0.03 ng mL
-1
to 6.24 ng mL
-1
, whereas
ginsenosides displayed a wider linear range of 3.90 ng mL
-1
to 125.00 ng mL
-1
(Table 2). The limit of dectection (LOD)
ranged from 0.01 ng mL
-1
to 1.25 ng mL
-1
for all ten
analytes. The intra-day and inter-day with RSD less than
5.06% are demonstrated in Table 2. The repeatability was
satisfactory with RSD below 7.01%. Recovery of the ten
compounds (Table 3) was within the range of 91.13-
111.97% and showed no relevant difference in the percent
A
Re, Rg1
MA
Rf
AC, HC
Rb1 Rc Rb2
Rd
B
Re, Rg1
MA
Rf
AC, HC
Rb1
Rc
Rb2
Rd
Figure 2 Typical RRLC-QQQ MS/MS chromatograms of marker chemicals in Shen-Fu decoction (A) standard mixture (B) Shen-Fu decoction.
Guo et al. Chemistry Central Journal 2013, 7:165 Page 3 of 7
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yield recovered using with different concentrations of the
compounds. Thus, the ten analytes can be quantitatively
analyzed simultaneously in a relatively short-time using
this optimized method.
Sample analysis
The described RRLC-QQQ-MS/MS method was subse-
quently applied to the analysis of Shen-Fu decoction,
made by authenticated Radix ginseng and aconite root
(see method part). The quantitative analytical results are
shown in Table 4. The repeatability of the ten analytes in
the Shen-Fu decoction was reliable (RSD<6.28%). From
Table 4, Shen-Fu decoction showed higher amounts of
ginsenosides than alkaloids. This result meant that the
Shen-Fu decoction may have very low toxicity levels, as
aconitine, hypacoitine and mesaconitine are the main
toxicity source of some toxic herbal medicines [27]. Fur-
thermore, ginsenoside-Rb1 was the most abundant of
the ten compounds in Shen-Fu decoction. Conversely,
aconitine was shown to be the least abundant of the ten
compounds in Shen-Fu decoction. This method would
allow for comparison of the quantity of ginsenosides and
alkaloids between Shen-Fu decoction preparations and
could therefore be used as a rapid and reliable approach
for assessment of the quality of Shen-Fu decoction.
Materials and methods
Chemicals, standards and samples
HPLC grade acetonitrile was purchased from Merck (Germany)
and MS grade formic acid from Sigma-Aldrich. All other
chemicals and solvents were of an analytical grade. Ultra-
pure water (18.2MΩ) was prepared with a Milli-Q water
purification system (Millipore, Bedford, MA, USA).
The standards reference samples of Ginsenosides Rb
1
,
Rb
2
, Rc, Rd, Rg
1
, Re, Rf, AC, HA and MA were purchased
from the National Institute for Control of Pharmaceutical
and Biological Products (Beijing, China). The purity of the
standards was relatively high at no less than 98%. Radix gin-
seng was purchased from Liaoning luyuan Pharmaceutical
Co., Ltd. in China. The processed aconite root was pur-
chased from Tong-Ren-Tang Pharmaceutical store (Beijing,
PR China). Panax ginseng and the prepared aconite root
were authenticated by Professor Xirong, He, Insitute of
traditional Chinese medicine, China Academy of Chinese
Medical Sciences.
Sample preparation
Reference standards solutions
Stock solutions were prepared by accurate measurement
of ginsenoside Re, Rg
1
,Rf,Rb
1
, Rc, Rb
2
, Rd, aconitine,
hypacoitine and mesaconitine. They were dissolved with
methanol respectively to get ten reference standards stock
solutions (1.0 mg mL
-1
), and were stored at 4°C.
Extracts of shen-fu decoction
ShenFu Formula (SF) was prepared by combining of
Radix ginseng and the processed aconite root (at a ratio
of 3:2). Dried and pulverized white ginseng (18 g) and
Table 1 Mass spectra properties of ten compounds in
Shen-Fu decoction
Compound name Precursor ion Product ion Frag (V) CE (V)
Ginsenoside Re 969.6 789.5 150 50
Ginsenoside Rg
1
823.5 643.5 135 40
Ginsenoside Rf 823.3 365.3 140 55
Ginsenoside Rb
1
1131.6 365.0 150 65
Ginsenoside Rc 1101.7 335.0 150 65
Ginsenoside Rb
2
1101.6 334.8 150 65
Ginsenoside Rd 969.9 789.3 150 55
Aconitine 646.4 586.4 135 45
Mesaconitine 632.3 572.3 135 35
Hypacoitine 616.3 556.2 135 40
Table 2 Calibration curves, LOD, LOQ, Precision and Repeatability for ten compounds in Shen-Fu decoction
Compound name Calibration curve r Linear range
(ng·mL
-1
)
LOD
(ng·mL
-1
)
LOQ
(ng·mL
-1
)
Intra-day
(n=6)
Inter-day
(n=6)
Repeatability
(n=5)
Ginsenoside-Rb
1
Y=11.04X+181.44 0.9930 3.90~125.00 0.97 3.00 3.44 4.11 6.51
Ginsenoside-Rb
2
Y=31.68X+246.74 0.9882 3.90~125.00 0.97 3.00 2.23 3.55 7.01
Ginsenoside-Rc Y=20.13X+25.79 0.9921 3.90~125.00 1.25 3.00 2.02 3.92 4.21
Ginsenoside-Rd Y=13.60X+69.00 0.9993 3.90~125.00 0.75 1.95 4.18 4.71 3.16
Ginsenoside-Re Y=18.52X+136.68 0.9973 3.90~125.00 0.48 1.95 3.34 5.06 4.67
Ginsenoside-Rf Y=37.66X+473.14 0.9952 3.90~125.00 0.97 3.00 2.05 3.12 5.03
Ginsenoside-Rg
1
Y=52.38X+109.20 0.9994 3.90~125.00 0.48 1.50 2.29 2.49 4.12
Aconitine Y=6193.52X80.34 0.9996 0.03~1.25 0.01 0.04 2.76 3.51 4.45
Mesaconitine Y=3617.22X63.02 0.9939 0.03~1.25 0.01 0.04 3.97 3.28 4.96
Hypaconitine Y=1207.82X+180.44 0.9960 0.19~6.24 0.01 0.04 2.55 2.42 4.81
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the processed aconite root (12 g) were ground and then
refluxed three times with 300 mL of water for 60 min at
100°C. After cooling, the extracted solutions were fil-
tered under vacuum. The solutions were condensed
under decompression and finally were freeze-dried. The
decoction extract was dissolved in a measured volume of
water with a concentration equal to 10 mg of crude bo-
tanicals per milliliter. 1mL of the solution was precipi-
tated with 8 mL ethanol allowed to sit for 24 h at 4°C.
The solution was filtered under vacuum. The filtrate was
transferred to a 50 mL volumetric flask. Prior to injec-
tion, all samples were filtered through a 0.22 μm mem-
brane filter.
RRLC-MS conditions
An Agilent-1200 RRLC/6410A QQQ system (Agilent,
MA, USA) equipped with an electrospray ionization (ESI)
source and operated in positive ion mode (data analysis
software Masshunter version B.01.04) was used for the
simultaneous determination of seven ginsenosides and
Table 3 Analsysis of the recovery of ten compounds in Shen-Fu decoction
Compounds Initial amount (ng) Added amount (ng) Detected amount (ng) Recovery (%) RSD/% (n=5)
Ginsenosid-Re 2074.12 1700 3673.54 ± 230.37 94.08 6.27
2074.12 2100 3991.27 ± 117.73 91.29 2.95
2074.12 2500 4475.56 ± 236.76 96.06 5.29
Ginsenoside-Rg
1
2260.51 1800 3959.88 ± 307.37 94.41 7.76
2260.51 2250 4310.87 ± 229.80 91.13 5.33
2260.51 2700 5176.34 ± 166.16 107.99 3.21
Ginsenoside-Rb
1
2423.46 2000 4299.39 ± 196.65 93.80 4.57
2423.46 2500 4812.75 ±185.92 95.57 3.86
2423.46 3000 5271.72 ± 130.37 94.94 2.47
Ginsenoside-Rc 2231.62 1800 3933.62 ± 155.38 94.56 3.95
2231.62 2250 4644.8 ± 231.31 107.25 4.98
2231.62 2700 5043.67 ± 244.21 104.15 4.84
Ginsenoside-Rb
2
1597.95 1200 2880.31 ± 83.82 106.86 2.91
1597.95 1500 3277.51 ± 128.48 111.97 3.92
1597.95 1800 3510.26 ± 169.97 106.24 4.84
Ginsenoside-Rd 816.73 640 1416.19 ± 86.39 93.67 6.10
816.73 800 1642.99 ± 95.82 103.28 5.83
816.73 960 1700.19 ± 80.93 92.03 4.76
Ginsenoside-Rf 2000.12 1600 3563.83 ± 252.68 97.73 7.09
2000.12 2000 4173.63 ± 261.27 108.68 6.26
2000.12 2400 4549.1 ± 256.57 106.21 5.64
Aconitine 2.91 2.4 5.52 ± 0.27 108.75 4.89
2.91 3.0 5.67 ± 0.31 92.00 5.47
2.91 3.6 6.7 ± 0.34 105.28 5.07
Mesaconitine 7.12 5.6 12.98 ± 0.66 104.64 5.08
7.12 7.0 13.74 ± 0.68 94.57 4.95
7.12 8.4 15.82 ± 0.56 103.57 3.54
Hypaconitine 100.06 80 174.69 ± 7.95 93.29 4.55
100.06 100 204.9 ± 10.90 104.84 5.32
100.06 120 225.07 ± 7.81 104.18 3.47
Table 4 Contents of ten compounds in Shen-Fu decoction
Samples Content (μg/g)
Rb
1
Rd Re Rf Rg
1
Rc Rb
2
Aconitine Mesaconitine Hypaconitine
247.17±11.27 84.21±4.31 210.64±12.66 204.66±10.24 231.22±11.75 223.19±14.01 121.16±7.41 0.21±0.01 0.76±0.04 10.05±0.48
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three aconitum alkaloids in Shen-Fu decoction. The sepa-
ration was performed on an Agilent ZORBAX C18 SB co-
lumn (100 mm×2.1 mm, 1.8 μm). The gradient mobile
phases consisted of (A) water containing 0.05% formic
acid and (B) acetonitrile for gradient elution from the co-
lumn at 40°C. The linear gradient conditions assessed for
gradient optimization were as follows: 02min,28-34%B;
26min,34-35%;610 min, 35-100%; 1011 min, 100%.
The flow rate was 0.35 ml/min. The column temperature
was 40°C. The conditions for MS analysis were as follows:
drying gas N
2
flow rate 12 L min
-1
, gas temperature 350°C
and nebulizer pressure was 50 psi. The capillary voltage was
set to 4000 V. MRM was employed for quantification. The
precursor-to-product ion pair, fragmentor voltage (Frag V)
and collision energy (CE) for each analyte are described
(Table 1). The dwell time of each ion pair was 200 ms.
Method validation
An external calibration method was used for quantitative
analysis with the linear calibration curves constructed
using six different concentrations of the ten compounds.
Each concentration was analyzed in triplicate and then the
calibration curves were constructed by plotting the peak
areas versus the concentrations of each analyte. The LOD
and limit of quantification (LOQ) were measured with the
signal-to-noise ratios of 3:1 and 10:1, respectively. The
intra-day precision was determined by analysis of the
standard solution at six times within 1 day. Inter-day preci-
sion on other hand, was determined by repeated analysis of
the sample for three consecutive days. For the assessment
of experimental repeatability test, five independent sample
solutions were prepared by the procedures noted in
Extracts of Shen-Fu decoction. The recovery of this method
was determined using the standard addition method. Three
different concentration levels (approximately equivalent
to 0.8, 1.0 and 1.2 times of the concentration of the ori-
ginal amount in the matrix) of the references standards were
added into the sample in triplicate. The average recoveries
were determined by the following equation: Recovery(%)=
(Observed amount Original amount)/Spiked amount ×
100%,RSD(%) = (SD/mean) × 100%.
Conclusions
This is the first report of the simultaneous determination
of the major compounds in Shen-Fu decoction. By using
RRLC coupled with an ESI triple quadrupole tandem spec-
trometer, we developed and validated a rapid, simple
and reliable method to simultaneously determine ten
marker chemicals (ginsenoside Re, Rg
1
,Rb
1
,Rc,Rb
2
,
Rd, Rf, aconitine, hypacoitine and mesaconitine) in the
Shen-Fu decoction. This method provides an excellent
quantitative tool for the quality assessments of TCM
formulae because of its high capacity, high sensitivity,
high selectivity and short analysis time.
Abbreviations
RRLC-MS/MS: Rapid resolution liquid chromatography coupled with tandem
mass spectrometry; QQQ-MS/MS: Triple quadrupole tandem mass
spectrometer; RSD: Relative standard deviations; TCMs: Traditional Chinese
medicines; SFI: Shen-Fu Injection; SIM: Selected ion monitor; MRM: Multiple
reaction monitor; ESI: Electrospray ionization; Frag V: Fragmentor voltage;
CE: Collision energy; LOD: Limit of detection; LOQ: Limit of quantification;
NEAs: Nonester alkaloids; MDAs: Monoester diterpene alkaloids; DDAs: Diester
diterpene alkaloids; AC: Aconitine; MA: Measaconitine; HA: Hypaconitine.
Competing interests
The authors declare that they have no competing interests.
Authorscontributions
GN, M-GZ and D-DQ conceived of the study, participated in its design and
coordination, and drafted the manuscript. GN, L-MT and Y-DW performed
experiments and analyzed results and helped to draft the manuscript.
HY, N-XH, W-RF and LY helped to do experiments. All authors read and
approved the manuscript.
Acknowledgments
This work was financially supported by 2013 Program for Liaoning Innovative
Research Team in University (LT2013020 the Autonomic Project of China
Academy of Chinese Medicine Sciences (project number zz2012011) and the
National Natural Science Foundation of China (Grant 81001597 and 81370095).
Author details
1
Experimental Research Center, China Academy of Chinese Medical Sciences,
Beijing 100700, China.
2
SRI International, Menlo Park, CA 94025, USA.
3
Key
Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess
Technology, Chinese Academy of Sciences, Songling road 189, Qingdao
266101, China.
4
College of Pharmacy, Xinjiang Medical University, Urumqi
830011, China.
5
Key Laboratory of Bioactive Substances and Resource
Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China.
6
Department of Chinese Medicine
Chemistry, Liaoning University of Traditional Chinese Medicine, Dalian
116600, China.
Received: 9 July 2013 Accepted: 9 September 2013
Published: 10 October 2013
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doi:10.1186/1752-153X-7-165
Cite this article as: Guo et al.:Quantitative LC-MS/MS analysis of seven
ginsenosides and three aconitum alkaloids in Shen-Fu decoction.
Chemistry Central Journal 2013 7:165.
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... Many techniques including HPLC [11][12][13], GC-MS [14,15], and LC-MS [16][17][18][19][20] were used to analyze ginsenosides. Ginsenoside-Rb2 Among them, the LC-MS and tandem mass spectrometry (MS/MS) techniques have been widely used for analysis of ginsenosides, because of their high dynamic range of detection, high sensitivity, and specificity [3,21]. ...
... Many works have been carried out for the identification, quantification, and quality control of ginsenosides in raw plant materials, extracts, and marketed products [22][23][24]. Ginsenosides-Rb1, Rb2, Rc, Rd, Rg1, Re, and Rf ( Figure 1) have been identified in Dushen Tang and Shenfu Tang in the previous studies [19,25]. But simultaneous comparatively determination of twelve ginsenosides in Dushen Tang and Shenfu Tang has not been studied until now. ...
... Shenfu Tang is a TCM prescription with a 3:2 ratio of Radix Ginseng and Fuzi (Radix Aconiti lateralis praeparata). Dried and pulverised white ginseng (1200 g) and the processed aconite root (800 g) were ground and then refluxed three times with 20 L of water for 60 min at 100 ∘ C based on our published papers [19,25]. After cooling, the extracting solutions were filtered and condensed under decompression and finally were freeze-dried. ...
Article
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Shenfu Tang and Dushen Tang (one of the composite medicines for Shenfu Tang) are widely used Traditional Chinese herbal formulations and ginsenosides are their main bioactive components. However, there are rare studies about simultaneous analysis of ginsenosides in Shenfu Tang and Dushen Tang. In order to identify ginsenosides in Shenfu Tang and Dushen Tang and to explore law of compatibility of medicines in the decoction, a method for simultaneous determination of twelve ginsenosides in Shenfu Tang and Dushen Tang was developed by ultraresolution liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The method showed satisfactory linearity (r > 0.9915), repeatability (RSD < 9.58%), intra- and interday precisions (RSD<11.90%), and high yields of recovery (92.26-113.20%) for twelve major constituents, namely, ginsenosides-Rb1, Rb2, Rb3, Rc, Rd, Rg1, Re, Rf, Rg2, Rg3, Rh1, and F2. Furthermore, the concentration of twelve ginsenosides in Dushen Tang and Shenfu Tang was also simultaneously analyzed. Most of ginsenosides except Rg1 and Rb1 showed higher contents in Shenfu Tang compared to Dushen Tang. The compatibility of the formula had the effect of promoting or inhibiting the dissolution of some major components. The present research provided a reliable evidence for the illustration of chemical basis and compatibility regularity of Shenfu Tang. This study demonstrated the utility of the developed method for assessment of the quantity of the major constituents in Dushen Tang and Shenfu Tang.
... In addition, the amounts of R 1 , Rb 1 and Rg 1 that were detected from the rat plasma samples were 2.5 ng·mL −1 , 4.0 ng·mL −1 and 4.6 ng·mL −1 , respectively. Finally, the target analytes in the elution solution obtained from MISPE sorbents have been structurally identified based on HPLC-MS and the experimental results were listed in Table 5 [55] 2 Rg 1 C 42 H 72 O 14 24.34 824.5 [56] 3 Rb 1 C 54 H 92 O 23 36.34 1109.31 ...
... These results demonstrated that the MISPE coupled with HPLC offers a suitable method for selective separation and determination of trace saponins in biological samples. m/z) Reference 1 R1 C47H80O18 16.88 932.5 [55] 2 Rg1 C42H72O14 24.34 824.5 [56] 3 Rb1 C54H92O23 36.34 1109.31 [57] As could be observed from the chromatograms, the sensitivities of Rb1, Rg1 and R1 in plasma samples were greatly enhanced with the MISPE coupled with HPLC-UV analysis. ...
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The feasible, reliable and selective multi-template molecularly imprinted polymers (MT-MIPs) based on SBA-15 ([email protected]) for the selective separation and determination of the trace level of ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1) and notoginsenoside R1 (R1) simultaneously from biological samples were developed. The polymers were constructed by SBA-15 as support, Rb1, Rg1, R1 as multi-template, acrylamide (AM) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The new synthetic [email protected] were satisfactorily applied to solid-phase extraction (SPE) coupled with high performance liquid chromatography (HPLC) for the separation and determination of trace Rb1, Rg1 and R1 in plasma samples. Under the optimized conditions, the limits of detection (LODs) and quantitation (LOQs) of the proposed method for Rb1, Rg1 and R1 were in the range of 0.63-0.75 ng·mL⁻¹ and 2.1-2.5 ng·mL⁻¹, respectively. The recoveries of R1, Rb1 and Rg1 were obtained between 93.4% and 104.3% with relative standard deviations (RSDs) in the range of 3.3-4.2%. All results show that the obtained [email protected] could be a promising prospect for the practical application in the selective separation and enrichment of trace Panax notoginseng saponins (PNS) in the biological samples.
... Several methods for component analysis and determination of major constituents in ShenFu injection and their serum pharmacochemistry have been reported by using HPLC-PAD [15], UPLC-Q-TOF-MS [16,17], and HILIC-RPLC-MS/MS technologies [14]. Few papers have focused specifically on the decoction of ShenFu, either investigating pharmacochemistry [12] or just quantitative analysis for several constituents [18]. Therefore, it is still necessary to develop a set of comprehensive and rapid quality evaluation methods for qualitative analysis and quantitative analysis of the major components in SFPD simultaneously. ...
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Full-text available
Decoction is one of the oldest forms of traditional Chinese medicine and it is widely used in clinical practice. However, the quality evaluation and control of traditional decoction is a challenge due to the characteristics of complicated constituents, water as solvent, and temporary preparation. ShenFu Prescription Decoction (SFPD) is a classical prescription for preventing and treating many types of cardiovascular disease. In this article, a comprehensive and rapid method for quality evaluation and control of SFPD was developed, via qualitative and quantitative analysis of the major components by integrating ultra-high-performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry and ultra-fast-performance liquid chromatography equipped with triple quadrupole mass spectrometry. Consequently, a total of 39 constituents were tentatively identified in qualitative analysis, of which 21 compounds were unambiguously confirmed by comparing with reference substances. We determined 13 important constituents within 7 min by multiple reaction monitoring. The validated method was applied for determining five different proportion SFPDs. It was found that different proportions generated great influence on the dissolution of constituents. This may be one of the mechanisms for which different proportions play different synergistic effects. Therefore, the developed method is a fast and useful approach for quality evaluation of SFPD.
... According to the structures, the ginsenosides could be classified into two major groups, that is, 20(S)-protopanaxadiol type (ppd-type) and 20(S)-protopanaxatriol type (ppt-type). The major ginsenosides present in Hongshen include the ppdtype Rb 1 , Rb 2 , Rc, and Rd and the ppt-type Re, Rf, and Rg 1 [12]. The structures are shown in Figure 2. The publications report the bioactivity of ginsenosides including cardiovascular protection, oxidized free radicals scavenging, and cell immunity regulation [13][14][15]. ...
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Full-text available
A sensitive and reliable LC-MS/MS method has been developed and validated for simultaneous determination of six Aconitum alkaloids (aconitine, hypaconitine, mesaconitine, benzoylaconitine, benzoylhypacoitine, and benzoylmesaconine) and seven ginsenosides (Rb 1 , Rb 2 , Rc, Rd, Re, Rf, and Rg 1 ) in rat plasma after oral administration of Shen-Fu prescription. Psoralen was selected as internal standard (IS). Protein precipitation with methanol was used in sample preparation. The chromatographic separation was achieved on a CORTECS™ C18 column with 0.1% formic acid aqueous solution and acetonitrile as mobile phase. The flow rate was 0.3 mL/min. The detection was performed on a tandem mass system with an electrospray ionization (ESI) source in the positive ionization and multiple-reaction monitoring (MRM) mode. The calibration curves of six Aconitum alkaloids and seven ginsenosides were linear over the range of 0.1-50 and 1-500 ng/mL, respectively. The extraction recoveries of the analytes in plasma samples ranged from 64.2 to 94.1%. Meanwhile, the intra- and interday precision of the analytes were less than 14.3%, and the accuracy was in the range of −14.2% to 9.8%. The developed method was successfully applied to the pharmacokinetics of six Aconitum alkaloids and seven ginsenosides in rat plasma after oral administration of Shen-Fu prescription.
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Introduction: Aconitum spp. are prime medicinal plants rich in alkaloids and have been used as the main constituents of traditional medicine in India and China. The whole plant can be toxic and creates pathophysiological conditions inside the human body. Therefore, simultaneous quantification of alkaloids within plant parts and herbal medicines associated with this genus is essential for quality control. Objective: We aimed to develop and validate methods using ultra-high-performance liquid chromatography-diode array detector-quadrupole time-of-flight ion mobility mass spectrometry (UHPLC-DAD-QTOF-IMS) and to develop an analytical strategy for the identification and quantification of alkaloid compounds (aconitine, hypaconitine, mesaconitine, aconine, benzoylmesaconitine, benzoylaconine, bulleyaconitine A, and deoxyaconitine) from Aconitum heterophyllum. Methodology: We developed a simultaneous identification and quantification method for eight alkaloids using UHPLC-DAD-QTOF-IMS. The method was validated as per International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and also in IMS mode. Results: The developed method has good linearity (r2 = 0.997-0.999), LOD (0.63-8.31 μg/mL), LOQ (0.63-2.80 μg/mL), recovery (86.01-104.33%), reproducibility, intra- and inter-day variability (<3.25%), and stability. Significant qualitative and quantitative variations were found among different plant parts (flower, leaf, stem, root, and tuber) and five market products of A. heterophyllum. Furthermore, a total of 21 metabolites were also profiled based on the fragmentation pattern of MS2 using the validated method. Conclusion: An appropriate mobile phase using acetonitrile and water in a gradient elution gave a satisfactory chromatographic separation of eight Aconitum alkaloids with their adjacent peaks. Therefore, this method could provide a scientific and technical platform for quality control assurance.
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There is no effective method for unified quality evaluation for the same medicinal materials in similar prescriptions. In this study, we used Ginseng-Fuzi (Aconitum carmichaelii) proprietary Chinese medicines (GFPCMs), with different dosage forms, as examples to develop a new unified quality control strategy. Thus, using a high performance liquid chromatography-tandem mass spectrometry method, coupled with unified quality evaluation strategies to comprehensively characterize the contents of 12 aconitines and 25 ginsenosides containing eight groups of isomers in 10 GFPCMs. Quality control was carried out for the first time for aconitines and ginsenosides in Jianbuqiangshen Pill, Nanbao Capsule, Nvbao Capsule, Pingfengshengmai Capsule, Wumei Pill, and Xinbao Pill. Furthermore, 4 aconitines and 16 ginsenosides were firstly quantified in all 10 GFPCMs. Subsequently, there was a comprehensive comparison of the universality and accuracy of the quality control strategies based on absolute, normalized, and unit content. It was indicated the strategy-based unit content was more practical and effective. As for Fuzi, the total unit content of diester alkaloids should be less than 0.020%, while monoester alkaloids should be more than 0.010%. As for ginseng, the total unit content of Rb1, Rb2, Rc, Rg5, and Re should be higher than 2.0%, and the unit content of Rg5 was a good indicator for the classification of the dosage forms and processing methods. In summary, this strategy showed great potential for supervising the quality of Chinese medicine.
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Shenfu Injection (SFI), derived from the classical traditional Chinese medicine formula “Shenfu Decoction”, is a modern preparation used to treat heart failure and shock in clinic. In this study, an ultra-high performance liquid chromatography-triple quadruple tandem mass spectrometry (UHPLC-QQQ MS) method was established to simultaneously quantify twenty-eight main active components of SFI in rat plasma, including eighteen ginsenosides and ten aconite alkaloids. Multi-reaction monitoring in positive and negative ionization switching modes is used for mass spectrometry analysis, and the whole analysis process was within 14 min. The developed method was well validated and successfully applied to the pharmacokinetic study of multiple components of SFI in rat plasma. Eight PPD-type ginsenosides Ra2, Ra3, Rb1, Ra1, Rc, Rb2, Rb3 and Rd presented relative high systemic exposure levels among ginsenosides with AUC0-t larger than 10000 μg h/L, while mesaconine and hypaconine possessed relative high plasma abundance among aconite alkaloids with AUC0-t at 142.50 ± 17.42 μg h/L and 40.65 ± 5.61 μg h/L, respectively. Several PPT-type ginsenosides had obviously higher AUC0-t levels (e.g. 639.70 ± 134.61 μg h/L for ginsenoside Re and 874.79 ± 188.87 μg h/L for ginsenoside Rg1) than alkaloids but similar t1/2 levels (0.14 ± 0.03 h for ginsenoside Re, 0.16 ± 0.03 h for ginsenoside Rg1, 0.04–0.33 h for aconite alkaloids), indicating their quick elimination. Collectively, the pharmacokinetic research of ginsenosides and aconite alkaloids in SFI would provide a scientific basis for its clinical use and drug-drug interactions.
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Ethnopharmacological relevance Shen-Fu Decoction (SFD), a classic Traditional Chinese paired herb formulation, has been widely used for the treatment of sepsis in China. This study was carried out to assess the effects of SFD in sepsis-induced intestinal permeability and intestinal epithelial tight junction damage in rats with sepsis. Materials and methods A rat model of sepsis was created by cecal ligation and puncture (CLP). Rats in Sham and CLP + vehicle groups received equal distilled water, while rats in SFD group were treated by gavage of SFD (3 mg/kg, twice a day) for 72h. Mortality, sepsis-induced peritoneal inflammation, intestinal and liver histopathology damage, intestinal permeability (serum FITC-dextran and D-lactate), serum LPS, serum inflammation (PCT, TNF-α, and IL-6), and liver function (AST and ALT) were evaluated. The levels of zonula occluden (ZO-1), Occludin, Claudin-1 were analyzed by Real-time quantitative PCR and Western blotting (WB) respectively. Vasodilator-stimulated phosphoprotein (VASP) and p-VASP in intestinal epithelium were analyzed by WB. Results Our study showed that SFD markedly reduced the mortality rate of CLP rats, prevented intestine and liver damage, relieved sepsis-induced intestinal permeability and inflammation elevation, ameliorated sepsis-induced impaired intestinal permeability by regulating the expression of ZO-1, Occludin, Claudin-1 and p-VASP. Conclusions The herbal formula SFD may be useful for reducing sepsis-induced organic damage and mortality by ameliorating the condition of sepsis-induced intestinal permeability by regulating tight junction proteins and p-VASP.
Preprint
The feasible, reliable and selective multi-template molecularly imprinted polymers (MT-MIPs) based on SBA-15 (SBA-15@MT-MIPs) for the selective separation and determination of the trace level of ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1) and notoginsenoside R1 (R1) (Panax notoginseng saponins, PNS) from biological samples were developed. The polymers were constructed by SBA-15 as support, Rb1, Rg1, R1 as multi-template, acrylamide (AM) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The new synthetic SBA-15@MT-MIPs were satisfactorily applied to solid-phase extraction (SPE) coupled with high performance liquid chromatography (HPLC) for the separation and determination of trace PNS in plasma samples. Under the optimized conditions, the limits of detection (LODs) and quantitation (LOQs) of the proposed method for Rb1, Rg1 and R1 were in the range of 0.63-0.75 ng mL-1 and 2.1-2.5 ng mL-1, respectively. The recoveries of R1, Rb1 and Rg1 were obtained between 93.4% and 104.3% with relative standard deviations (RSDs) in the range of 3.3-4.2%. All results show that the obtained SBA-15@MT-MIPs could be a promising prospect for the practical application in the selective separation and enrichment of trace PNS in the biological samples.
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For identification of ginsenoside enantiomers, electrospray ionization mass spectrometry (ESI-MS) was used to generate silver complexes of the type [ginsenoside + Ag](+) . Collision induced dissociation of the silver-ginsenoside complexes produced fragment ions by dehydration, allowing differentiation of ginsenoside enantiomers by the intensity of [M + Ag - H(2) O](+) ion. In the meanwhile, an approach based on the distinct profiles of enantiomer-selective fragment ion intensity varied with collision energy was introduced to refine the identification and quantitation of ginsenoside enantiomers. Five pairs of enantiomeric ginsenosides were distinguished and quantified on the basis of the distribution of fragment ion [M + Ag - H(2) O](+) . This method was also extended to the identification of other type of ginsenoside isomers such as ginsenoside Rb2 and Rb3. For demonstrating the practicability of this novel approach, it was utilized to analyze the molar ratio of 20-(S) and 20-(R) type enantiomeric ginsenosides in enantiomer mixture in red ginseng extract. The generation of characteristic fragment ion [M + Ag - H(2) O](+) likely results from the reduction of potential energy barrier of dehydration because of the catalysis of silver ion. The mechanism of enantiomer identification of ginsenosides was discussed from the aspects of computational modeling and internal energy. Copyright © 2012 John Wiley & Sons, Ltd.
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North American ginseng (Panax quinquefolius) has a long history of use and is currently a commercially reliable natural health commodity. Ginsenosides or triterpene saponins are generally regarded as bioactive constituents for several observed health effects associated with ginseng. North American ginseng was dried using 3 different drying techniques to assess the ginsenoside content of prepared extracts. Drying methods included freeze-drying (FD), air-drying (AD), and vacuum microwave-drying (VMD) of ginseng root. High-performance liquid chromatography (HPLC) analysis showed that FD ginseng processing gave greater (P≥ 0.05) amounts of the fingerprint ginsenosides Rg1 (28 ± 0.9 mg/g, dry weight) and Re (45 ± 0.1) compared with AD (Rg1 19 ± 0.7, Re 29 ± 0.1) and VMD (Rg1 22 ± 0.8, Re 24 ± 0.1); whereas, VMD produced greater amounts of Rb1 (83 ± 0.1) and Rd (13 ± 0.0) than FD (Rb1 62 ± 0.1, Rd 9 ± 0.1) and AD (Rb1 69 ± 0.1, Rd 5 ± 0.0), respectively. Total ginsenoside content was similar for FD and VMD and was the lowest (P≥ 0.05) for AD. Electrospray mass spectrometry (ESI-MS) analysis showed a total of 12 compounds detected in FD ginseng compared with 10 compounds in ginseng dried by both VMD and AD. Our results support the fact that FD and VMD drying methods of North American ginseng can improve both extraction efficiency and actual retention of individual ginsenoside in root material.
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This study systematically investigated the retention behavior of seven neutral ginsenosides Rg1, Re, Rf, Rb1, Rb2, Rc, Rd, and an acidic ginsenoside R0, the major pharmacologically active components of Radix Ginseng with RP-HPLC. The effects of solvent, pH value, ionic strength of the mobile phase, and column temperature were investigated using an octadecylsiloxanebonded silica gel column. Based on the ginsenosides’ retention characteristics, the concentration of acetonitrile and the gradient of the mobile phase needed to maintain the baseline separation of the major neutral ginsenosides in Radix Ginseng were theoretically predicted. Furthermore, the ionic strength of mobile-phase necessary to achieve good resolution of the neutral ginsenosides and acidic ginsenosides was carefully investigated. According to the results of the quantitative analysis of ginsenosides in eight batches of ginseng samples from different sources, the developed HPLC technique may be a valuable tool for the quality assessment of Radix Ginseng. Key wordsGinsenoside-HPLC-Retention behavior-Radix Ginseng-Quantitation- Panax
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As one of the most commonly used medicinal plants, ginseng has been an attractive model herb for study. A wide range of analytical methods has been used to characterize its constituents. However, less effort has been devoted to the rare ginseng saponins, especially their isomers and sugar linkages. In this study, we used segmental monitoring and diagnostic ion filtering to characterize ginseng saponins by rapid liquid chromatography with time-of-flight mass spectrometry (LC-TOF-MS). By using selected diagnostic ions, specific groups of ginseng saponins were readily extracted from the complicated matrix. 20(R) and 20(S) stereo-saponins were differentiated using the peak abundance ratio of [M-H(2)O+H](+) to [M-2H(2)O+H](+). The fragmentation behavior of ginsenosides was first reported in negative ion mode by MS/MS with high-energy collision-induced dissociation, producing rules to determine sugar numbers, positions and linkages. Using the rules, we identified and compared the nontarget ginseng saponin profiling of raw and steamed American ginseng roots and berries. We characterized 70 saponins in the samples. Our strategy can be extended to screen and characterize other rare ginseng saponins and their metabolites.
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The mother and lateral root of Aconitum carmichaelii Debx, named "Chuanwu" (CW) and "Fuzi", respectively, has been used to relieve joint pain and treat rheumatic diseases for over 2000 years. However, it has a very narrow therapeutic range, and the toxicological risk of its usage remains very high. The traditional Chinese processing approach, Paozhi (detoxifying measure),can decompose poisonous Aconitum alkaloids into less or nontoxic derivatives and plays an important role in detoxification. The difference in metabolomic characters among the crude and processed preparations is still unclear, limited by the lack of sensitive and reliable biomarkers. Therefore, this paper was designed to investigate comprehensive metabolomic characters of the crude and its processed products by UPLC-Q-TOF-HDMS combined with pattern recognition methods and ingenuity pathway analysis (IPA). The significant difference in metabolic profiles and changes of metabolite biomarkers of interest between the crude and processed preparations were well observed. The underlying regulations of Paozhi-perturbed metabolic pathways are discussed according to the identified metabolites, and four metabolic pathways are identified using IPA. The present study demonstrates that metabolomic analysis could greatly facilitate and provide useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Aconite roots in the clinic.
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The lateral root of Aconitum carmichaelii Debx is named "Fuzi" which is widely distributed across Asia and North America and has been used to relieve joint pain and treat rheumatic diseases for over two thousand years. However, it has very narrow therapeutic ranges and despite the toxicological risk, its usage remains very high. A traditional Chinese processing approach (Paozhi, detoxifying measure) is necessary to remove the poisonous Aconitum alkaloids mainly deriving from the diester diterpene alkaloids (DDAs) including aconitine, mesaconitine and hypaconitine. They can be decomposed into less or non-toxic derivatives through Paozhi that plays an essential role in detoxification. Processed Fuzi is mainly focused on the three main forms of Yanfuzi (YFZ), Heishunpian (HSP) and Baifupian (BFP) which are highly desirable in order to guarantee the clinical safety and their low toxicity in decoctions. The difference in metabolomic characters between Fuzi and its processed preparations is still completely unclear. Therefore, this paper was designed to investigate a comprehensive metabolome of Fuzi and its processed products by ultra-performance liquid-chromatography/electrospray-ionization synapt high-definition mass spectrometry (UPLC-Q-TOF-HDMS) combined with pattern recognition methods. The difference in metabolic profiles between Fuzi and its processed preparations was well observed by the principal component analysis (PCA) of the MS spectra. Significant changes of 19 metabolite biomarkers were detected in the Fuzi samples and three preparations. The underlying regulations of Paozhi-perturbed metabolic pathways were also discussed according to the identified metabolites. The present study proves that UPLC-Q-TOF-HDMS based metabolomic analysis greatly contributes to the investigation of Fuzi metabolism through Paozhi techniques, and provides useful information to further comprehensively understand the pharmacological activity and potential toxicity of processed Fuzi in a clinical environment.
Article
Fuzi, which is the processed lateral roots of Aconitum Carmichaeli. Debx and is widely distributed over the southwest provinces of China, is recognised for its anti-inflammatory and analgesic effects. The pharmacokinetic properties of Fuzi are inadequately understood. Aconitine, the primary highly toxic ingredient of Fuzi, is well known as the target marker of Fuzi. The purpose of the present study is to investigate the pharmacokinetic behaviours of aconitine in vivo following single and multiple administrations of processed Fuzi extracts and to compare the pharmacokinetic characteristics of aconitine after administrations of pure aconitine or Fuzi extracts as well as compare the difference at single dose and multiple doses. The in vitro aconitine protein binding in plasma through equilibrium dialysis was also examined. A high performance liquid chromatography (HPLC) method was developed for the determination of aconitine in Fuzi crude extracts and a fast ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) was developed to investigate the pharmacokinetic behaviour of aconitine as the targeted marker of Fuzi. The absolute bioavailability (F %) after the administration of 0.5 mg/kg aconitine and Fuzi extract (0.118 mg/kg aconitine) in rat was 8.24±2.52% and 4.72±2.66%, respectively. Aconitine absorption was very fast at the t(max) 30.08±9.73 min for pure aconitine and 58.00±21.68 min for Fuzi extract administration. Aconitine was also eliminated rapidly with a short half-life (i.v., 80.98±6.40 min) and a low rate of protein bounding (23.9-31.9%). No significance was observed on all the pharmacokinetics parameters following the single and multiple doses of pure aconitine (ANOVA, p>0.05). However, the absorption of aconitine after multiple administrations of Fuzi extract was much faster than that of a single dose (t(max): 58.00±21.68 vs. 20.00±8.66 min, p<0.05), and the area under the plasma concentration-time curve (AUC) was higher than that of a single dose. The pharmacokinetic behaviour of processed Fuzi was determined in this paper. The aconitine has low bioavailability. No variation in the pharmacokinetic behaviours of pure aconitine was observed after single and multiple administrations. In contrast, multiple administrations of processed Fuzi extract could result in variations in its pharmacokinetic behaviour in AUC and t(max) indicating that multiple dose might increase the bioavailability of aconitine, which may result in its toxicity. In addition, aconitine has a low protein bounding (23.9-31.9%), resulting in its rapid elimination.
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Ginseng occupies a prominent position in the list of best-selling natural products in the world. Because of its complex constituents, multidisciplinary techniques are needed to validate the analytical methods that support ginseng's use worldwide. In the past decade, rapid development of technology has advanced many aspects of ginseng research. The aim of this review is to illustrate the recent advances in the isolation and analysis of ginseng, and to highlight new applications and challenges. Emphasis is placed on recent trends and emerging techniques.