Full text of DOI:10.3390/molecules23102609

molecules
Article
Quantitative Evaluation of Twelve Major
Components of Sulfur-Fumigated Astragali Radix
with Different Durations by UPLC-MS
Xiaoyan Xing †, Zhonghao Sun †, Meihua Yang, Nailiang Zhu, Junshan Yang, Guoxu Ma * and
Xudong Xu *
Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of
Education, Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural
Medicine) and Translational Medicine, Key Laboratory of Efficacy Evaluation of Chinese Medicine against
Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Institute of Medicinal
Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences,
Beijing 100193, China; xyxing@implad.ac.cn (X.X.); sun_zhonghao@126.com (Z.S.);
mhyang@implad.ac.cn (M.Y.); nlzhu@implad.ac.cn (N.Z.); jsyang@implad.ac.cn (J.Y.)
* Correspondence: mgxfl8785@163.com (G.M.); xdxu@implad.ac.cn (X.X.); Tel.: +86-010-5783-3296 (G.M.)
† These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 September 2018; Accepted: 26 September 2018; Published: 11 October 2018
Abstract: In this study, an improved UPLC-MS (Ultra-high performance liquid chromatography-tandem
mass spectrometry) method for simultaneously quantifying twelve major components belonging to
two chemical types was developed and validated, and was applied to quantitatively compare the quality
of sulfur-fumigated Astragali Radix of different durations and of the fresh reference sample. The results
showed that the contents of triterpenes astragaloside III and astragaloside IV decreased moderately,
0
0
0
while the flavonoids calycosin, formononetin, and 7,2 -dihydroxy-3 ,4 -dimethoxyisoflavane decreased
significantly. The corresponding flavonoid glycosides increased accordingly, which indicated the
occurrence of chemical transformation of flavonoids and glycosides in the process of sulfur-fumigation.
These transformations were further confirmed by the synthesis of flavonoid glycosides under
simulated sulfur-fumigation circumstances. Furthermore, the sulfur-fumigated duration varied
in proportion with the contents of compounds 7, 11, and 12. These results suggest that the
established method was precise, accurate and sensitive enough for the global quality evaluation of
sulfur-fumigated Astragali Radix. Further, sulfur-fumigation not only changes the proportions of
bioactive components, but also causes chemical transformation in Astragali Radix.
Keywords: Astragali Radix; sulfur-fumigation duration; quantification; synthesis
1. Introduction
Astragali Radix (AR), the dry roots of Astragalus membranaceus (Fisch.) Bunge. or Astragalus
mongholicus Bunge., is one of the most widely used “Qi-tonifying” Chinese herbal medicines.
In the doctrines of traditional Chinese medicines (TCM), AR is claimed to have beneficial effects,
including “tonifying the Qi” of the kidney [
yang”, inducing diuresis to alleviate edema, relieving fever with “sweet and warm-natured” drugs,
promoting wound healing and tissue regeneration [ ]. Combined with pharmacological studies,
AR has been used in clinics to treat diabetic and reduce the risk of diabetic complications [
cardio-cerebrovascular disease, respiratory disease, and digestive system disease [
immunomodulation [ ], anti-inflammation [ ], anti-tumor [ ], nerve cell protecting and recovery [
anti-aging, and cardioprotective effects [
1], arresting sweating, “tonifying Qi” and “lifting
2
3],
4], due to its
5
6
7
8
],
9]. Previous research found that the main active ingredients
Molecules 2018, 23, 2609; doi:10.3390/molecules23102609
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2609
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of AR include flavonoids and isoflavones, saponins, polysaccharides, and others [10]. Traditionally,
the post-harvest processing of the roots of AR consists of sun-drying the whole fresh root after cleaning.
Because it is prone to mildew attack, AR has been recently reported as being sulfur-fumigated during
post-harvesting handling prior to storage. So as a consequence it has become necessary to compare
the variations in AR chemical profiles after the sulfur-fumigation. Sulfur-fumigation, which is an
easy to apply and low-cost operation, has been commonly used to prevent medicinal herbs from
pest infestation, mold, and bacterial contamination [11]. However, recent studies demonstrated that
this method could leave residue of hazardous substances such as sulfur dioxide and heavy metals,
which posed a threat to human health [12]. Furthermore, sulfur-fumigation was reported to reduce
the content of the active ingredients in herbs, and influence the chemical transformation of bioactive
components and even to alleviate the pharmacological activities of edible herbs [13–15]. In 2004,
the State Food and Drug Administration of China indicated that sulfur fumigated medicinal herbs
are inferior [16]. However, the sulfur fumigation treatment of medicinal herbs and foods still prevails
all over the world, which exerts a negative impact on the safe application of edible herbs. To the
best of our knowledge, few systemic studies have been reported on the quantitative evaluation of
sulfur-fumigated AR. In particular, the durational effects of sulfur-fumigation on the proportions of
bioactive components in AR have not been quantitatively evaluated.
In this study, an improved UPLC-MS method for simultaneously quantifying twelve major
components of AR (including eight flavonoids: Calycosin (
ononin ( ), methylnissolin ( ), astraisoflavan-7-O- -D-glucoside (
isoflavane (
), and 7,20-dihydroxy-30,40-dimethoxy isoflavan-7-O-
tri-terpenoid saponins: Astragaloside I ( ), astragaloside II (10), astragaloside III (11), and astragaloside
1
), calycosin-7-glucoside (
), 7,20-dihydroxy-30,40-dimethoxy-
-D- glucopyranoside ( ); and four
2), formononetin (3),
4
5
β
6
β
7
8
9
IV (12)) (Figure 1) was developed, validated, and applied for quantitative evaluation of AR samples
sulfur-fumigated with different durations.
GluO
O
O
HO
O
GluO
OH
O
O
HO
HO
O
O
OH
OCH3
O
OCH3
OCH3
OCH3
4
1
2
3
O
GluO
O
GluO
OCH3
OCH3
O
HO
O
OH
OH
OCH3
OCH3
O
O
OCH3
OCH3
OCH3
OCH3
5
7
6
8
HO
HO
HO
O
O
O
H
O
OH
H
OH
HO
O
O
O
O
O
H
OH
O
O
OH
OH
OH
OH
O
OH
OH
OH
O
O
R1O
H
OR2
O
O
O
OH
11 R1=Xyl-Glc R2=H
12 R1=Xyl R2=Glc
OH
10
9
Figure 1. The structures of reference compounds
1
–12
:
Eight flavonoids: Calycosin (
1),
calycosin-7-glucoside (
2
), formon0onetin (
3
), ononin (
4
), methylnissolin (
5
), astraisoflavan-7-O-
0
0
0
0
0
β
-D-glucoside (
isoflavan-7-O-
astragaloside II (10), astragaloside III (11), and astragaloside IV (12).
6
), 7,2 -dihydroxy-3 ,4 -dimethoxy-isoflavane (
7), and 7,2 -dihydroxy-3 ,4 -dimethoxy
β-D-glucopyranoside (8); and four tri-terpenoid saponins: Astragaloside I (9),

Molecules 2018, 23, 2609
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2. Experimental
2.1. Chemicals and Reagents
Twelve reference compounds with purities of >98.0%, including calycosin (1), calycosin-7-glu-coside
(
2
), formononetin (
7,20-dihydroxy-30,40-dimethoxyisoflavane (
copyranoside ( ), astragaloside I ( ), astragaloside II (10), astragaloside III (11), and astragaloside
3
), ononin (
4
), methylnissolin (
5
), astraisoflavan-7-O-
β
-D-glucoside (
6),
7
), 7,20-dihydroxy-30,40-dimethoxy isoflavan-7-O-
β-D-glu-
8
9
IV (12), were purchased from the National Institute for the Control of Pharmaceutical and Biological
Products (Beijing, China). These compounds showed high stability in methanol solution, of which
their structures are shown in Figure 1. Methanol and acetonitrile were of high performance liquid
chromatography (HPLC) grade (Tedia Company, Inc., Fairfield, OH, USA). Distilled water was further
purified by Milli-Q system (Millipore, Milford, MA, USA); formic acid was purchased from the First
Chemical Company of Nanjing (Suzhou, Jiangsu, China); other chemicals were of analytical grade.
All solvents and samples were filtered through 0.22 µm membrane filters before injecting into UPLC.
The fresh reference Astragali Radix sample was collected from Inner Mongolia Autonomous
region, the indigenous cultivating region of Astragali Radix and authenticated by Prof. Rong-Tao Li.
The voucher specimen (AM171114-1) was deposited at the Institute of Medicinal Plant Development,
Beijing, China.
2.2. Sulfur-Fumigation of AR
The sulfur-fumigated AR samples were self-prepared in our lab from the non-fumigated reference
AR sample (AM171114-1) following the modified procedures similar to that by herbal farmers or
wholesalers: 50 g AR slices were moistened with 4 mL of water, and left for 0.5 h. Two grams of sulfur
powder was heated until burning, then the burning sulfur and the moistened AR slices were carefully
put into the lower and upper layer of a desiccator respectively. Seven portions (50 g each portion) were
prepared to study of the sulfur-fumigation extent at different collection points of 1, 2, 4, 6, 8, 12, 16, 24,
36, 48, 60, and 72 h, respectively. After fumigation, the AR slices were dried at 40 ◦C and ground into
fine powder.
2.3. Instruments
Analyses were carried out using an Agilent 1290 Infinity II RRLC system consisting of a quaternary
delivery system, a degasser and an auto-sampler (Agilent Technologies Inc., Santa Clara, CA, USA).
Chromatographic separation was achieved on an ACQUITY UPLCTM HSS T3 (100 mm
×
2.1 mm,
1.8
µ
m, Waters, Milford, MA, USA). The column was maintained at 40 ◦C and eluted at a flowing rate
of 0.25 mL/min. The mobile phase consisted of 0.2% formic acid water (A) and acetonitrile (B) using
a gradient elution of 1–20% B at 0–5 min, 20–25% B at 5–8 min, 25–30% B at 8–15 min, 30–40% B at
15–18 min, 40–60% B at 18–20 min, 60–90% B at 20–23 min, and washing with 99% B for 23–25 min,
equilibration with 1% B at 25–27 min.
An Applied Biosystems 3200 Q-Trap system (AB SCIEX, Framingham, MA, USA) equipped with
an electrospray ionization (ESI) source was used and the system was operated in positive and negative
mode. Optimization of multiple reaction monitoring (MRM) conditions was carried out with the
following source-dependent parameters: Gas 1 and gas 2 were set at 50 psi. The optimized ion spray
voltages were set at 5500 V and
−
4500 V in positive and negative ion mode, respectively. The optimized
◦
ion spray voltage and temperature were set at 5500 V and 700 C, respectively. The operating vaporizer
temperature, 500 ◦C. Nitrogen gas was used in all analyses, and data acquisition and processing were
performed using Analyst software version 1.6.2. The MRM parameters are outlined in Table 1.

Molecules 2018, 23, 2609
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Table 1. Multiple reaction monitoring (MRM) transitions and parameters for the detection of the
12 analytes.
Precursor
Ion (m/z)
Product Ion
CE b (V)
No.
Analyte
DP a (V)
(m/z)
1
2
3
4
5
6
Calycosin
Calycosin-7-glucoside
Formononetin
285.2
447.3
269.0
431.1
301.2
463.3
303.0
213.1
285.4
167.2
269.1
167.2
167.4
167.2
70
60
70
65
54
60
55
45
20
47
20
20
40
19
Ononin
Methylnissolin
Astraisoflavan-7-O-β-D-glucoside
0
0 0
7
7,2 -dihydroxy-3 ,4 -dimethoxyisoflavane
0
0
0
7,2 -dihydroxy-3 ,4 -dimethoxy
isoflavan-7-O-β-D-glucopyranoside
Astragaloside I
8
463.3
301.0
−74
−24
9
867.9
825.7
783.8
783.8
59.1
59.1
160.9
101.0
−250
−110
−150
−115
−83
−70
−47
−57
10
11
12
Astragaloside II
Astragaloside III
Astragaloside IV
a
declustering potential; b collision Energy.
2.4. Standard Solutions Preparation
Standard stock solution 1 and 2 consisted of 7 (
reference compounds were directly prepared in methanol, respectively. The final concentrations
of these twelve reference compounds in stock solutions were prepared to be 11.88 g/mL
for formononetin, 21.6 g/mL for calycosin, 17.41 g/mL for methylnissolin, 15.5 g/mL for
7,20-dihydroxy-30,40-dimetho-xyisoflavane, 17.44
g/mL for calycosin-7-glucoside, 38.064 g/mL
for Calycosin-7-glucoside, 6.427 g/mL for astraisoflavan-7-O- -D-glucoside, 62.4 g/mL for
7,20-dihydroxy-30,40-dimethoxyisoflavan-7-O-
-D-glucopyranoside, 214 g/mL for astragaloside I,
61.2 g/mL for astragaloside II, 28.2 g/mL for astragaloside III and 114.6 g/mL for astragaloside
1–7) and 5 (8–12) accurately weighed
µ
µ
µ
µ
µ
µ
µ
β
µ
β
µ
µ
µ
µ
IV, respectively. The working standard solutions were prepared by diluting the stock solutions with
methanol to a series of proper concentrations. The solutions were brought to room temperature and
filtered through 0.22
subsequent analysis.
µm membrane filters, and an aliquot of 5 µL was injected into UPLC-MS for
2.5. Sample Preparation
Methanol extracts: Each AR sample was accurately weighed to approximately 1.0 g and heat
refluxed with 50.0 mL of methanol for 4 h. The extract was then filtered using a 0.22
filter before LC-MS analysis.
µm PTFE syringe
2.6. Method Validation
Method validation assays were carried out according to currently accepted Food and
Drug Administration (FDA) guidance. [Guidance for Industry, Bioanalytical Method Validation,
US Department of Health and Human Services Food and Drug Administration, Center for Drug
Evaluation and Research (CDER), 2001 Center for Veterinary Medicine (CV), 2001, http://www/fda.
gov/cder/guidance/index.htm].
2.6.1. Calibration Curves, Limits of Detection and Quantification
The calibration curves for 12 reference compounds were established by plotting peak area ratios
of each analyte using the linear regression analysis using 1/X2 as a weighting factor. Calibration curves
had to have a correlation coefficient (r) of 0.995 or better. The limit of detection (LOD) was determined
as signal-to-noise ratio >3 and the limit of quantification (LOQ) was measured as signal-to-noise ratio
>10 (Table 2).

Molecules 2018, 23, 2609
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Table 2. Validation with respect to linearity, limit of quantification (LOQ), limit of detection (LOD), precision, repeatability and stability.
Precision RSD a (%)
Regression Equation
Linear Range
(µg/mL)
LOQ
(ng/mL)
LOD
(ng/mL)
Correlation
Coefficient (R2)
Repeatability
RSD (%)
Stability
RSD (%)
Analytes
(µg/mL)
Intra-Day
Inter-Day
Calycosin
Calycosin-7-glucoside
Formononetin
y = 446765x + 322603
y = 202558x + 365088
y = 469388x + 141426
y = 51545x + 4316.5
y = 329224x + 84783
y = 130675x + 208291
y = 57437x + 11250
0.4752~11.88
0.864~21.6
0.3482~17.41
0.31~15.5
0.6976~17.44
1.5226~38.064
0.2571~6.427
0.9989
0.9992
0.9986
0.9994
0.9993
0.9991
0.9990
0.59
1.08
0.87
3.10
0.09
0.08
0.32
0.06
0.11
0.35
1.55
0.02
0.04
0.35
2.06%
1.31%
1.65%
1.42%
1.46%
2.03%
1.96%
3.16%
2.09%
1.69%
3.86%
2.53%
2.78%
3.53%
4.47%
3.43%
4.09%
4.63%
4.31%
4.49%
4.92%
3.93%
2.95%
3.37%
4.05%
2.99%
3.73%
3.77%
Ononin
Methylnissolin
Astraisoflavan-7-O-β-D-glucoside
0
0 0
7,2 -dihydroxy-3 ,4 -dimethoxyisoflavane
0
0
0
7,2 -dihydroxy-3 ,4 -dimethoxy
isoflavan-7-O-β-D-glucopyranoside
Astragaloside I
y = 127060x + 236161
2.496~62.4
0.9988
0.01
0.001
1.25%
2.37%
3.68%
1.50%
y = 1009.3x − 10607
y = 10750x + 25435
y = 12640x + 30515
y = 6748.6x + 164720
8.56~214
1.028~25.2
1.128~28.2
4.584~114.6
0.9991
0.9993
0.9993
0.9989
0.09
0.03
0.03
0.06
0.04
0.002
0.01
2.36%
1.65%
1.41%
2.18%
3.18%
3.78%
2.19%
1.98%
4.84%
2.66%
2.65%
4.67%
2.52%
2.03%
1.87%
2.06%
Astragaloside II
Astragaloside III
Astragaloside IV
0.01
a
relative standard deviation.

Molecules 2018, 23, 2609
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2.6.2. Precision, Repeatability and Stability
The intra- and inter-day precision was determined by analyzing 12 analytes from standard stock
solution in six replicates during a single day and by duplicating the experiments on three successive
days. To further evaluate the repeatability of the developed assays, samples were analyzed in six
replicates. Their criteria for acceptability of data were within
±
15% relative error (RE) from the
nominal values and a precision of within 15% relative standard deviation (RSD). Stability of AR
±
sample was tested at room temperature and analyzed at 0, 2, 4, 6, 8, 10, 12 and 24 h. The contents of
the corresponding compounds were calculated from the corresponding calibration curves.
2.6.3. Recovery Test
The measured recoveries of the compounds were determined by the method of standard addition.
Three concentration levels (low, medium, high) of the mixed standard solutions were spiked with a
sample of AR, which was analyzed previously using the above described method and the concentration
of each component was calculated according to the calibration curves (Table 3).
Table 3. Results of recovery.
Initial Amount
Added
Amount (µg)
Detected
Amount (µg)
Recovery
(%)
Analyte
Calycosin
RSD (%)
(µg)
0.61
1.90
103.50%
3.2
1.22
7.47
2.42
1.74
4.57
17.08
3.98
2.51
4.01
2.45
2.14
8.65
1.22
1.83
3.735
2.48
3.01
4.79
101.50%
98.50%
96.70%
1.5
2.3
2.7
Calycosin-7-glucoside
Formononetin
7.47
11.205
1.21
14.51
19.28
3.47
97.10%
103.20%
95.40%
0.9
1.7
1.6
2.42
3.63
0.87
5.10
6.01
2.61
105.20%
99.20%
99.60%
3.1
2.7
0.4
Ononin
1.74
2.61
2.29
3.53
4.56
7.12
101.40%
104.70%
103.90%
0.9
1.5
3.6
Methylnissolin
4.57
6.86
8.54
9.36
12.06
26.67
102.30%
105.50%
104.10%
3.7
3.0
2.6
Astraisoflavan-7-O-β-D-glucoside
17.08
25.62
1.99
34.02
42.23
6.24
99.60%
98.90%
104.50%
3.4
2.6
1.5
0
0 0
7,2 -dihydroxy-3 ,4 -dimethoxyisoflavane
3.98
5.97
1.26
8.15
10.11
3.87
102.30%
101.60%
102.70%
1.6
3.2
3.1
0
0
0
7,2 -dihydroxy-3 ,4 -dimethoxy
2.51
3.77
2.01
4.96
6.39
6.12
98.80%
101.80%
101.70%
2.3
5.7
0.6
isoflavan-7-O-β-D-glucopyranoside
Isoflavan-7-O-β-D-glucopyranoside
4.01
6.02
1.23
7.90
9.58
3.79
98.40%
95.50%
103.10%
2.3
2.6
3.1
Astragaloside I
Astragaloside III
Astragaloside IV
2.45
3.68
1.07
4.88
6.06
3.16
99.60%
98.90%
98.60%
4.1
2.8
1.5
2.14
3.21
4.33
4.40
5.30
13.50
102.90%
99.10%
104.10%
2.7
3.1
1.8
8.65
12.98
16.83
22.44
97.30%
103.80%
3.1
2.2

Molecules 2018, 23, 2609
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3. Results and Discussion
3.1. Optimization of Suitable LC-MS Conditions
We initially attempted to optimize one suitable LC-MS method to simultaneously determine
all 12 chemical marker compounds in AR. However, we could not obtain acceptable results using
one method, where the tested compounds could simultaneously achieve a good ion response in a
single ion mode. Thus, two separated batches of analysis were performed under different ion modes.
Compounds 1–7 and 8–12 were performed in positive and negative ion mode, respectively. Figure 2
presented the typical positive base peak intensity (BPI) chromatograms of plasma samples from all the
experimental groups.
Figure 2. MRM chromatograms in (
2, calycosin-7-glucoside; 3, formononetin; 4, ononin; 5, methylnissolin; 6, astraiso-flavan-7-O-
glucoside; 7, 7,20-dihydroxy-30,40-dimethoxyisoflavane; 8, 7,20-dihydroxy-30, 40-dimethoxy; 8, isoflavan-
7-O- -D-glucopyranoside; 9, astragaloside I; 10, astragaloside II; 11, astragaloside III; 12, astragaloside IV.
A
) positive and (
B
) negative modes. Identification: 1, calycosin;
β-D-
β
Meanwhile, two different columns, different mobile phases and detecting ion modes were tested
during method development. The selection of UPLC columns with high separation efficiency is a
prerequisite. Here, two chromatographic columns, ethylene bridged hybrid (BEH) C18 column (2.1 mm
×
100 mm, 1.7
µ
m, Waters) and high strength silica (HSS) T3 column (2.1 mm
×
100 mm, 1.8 µm,
Waters), were utilized to investigate for the comprehensive metabolome. The BEH C18 column is one
universal column choice for UHPLC separations. While HSS T3 column with 100% silica particle,
is used to retain and separate smaller, more water-soluble polar organic compounds than the BEH C18
column (Zhao et al., 2013). The result showed that HSS T3 column could gain a better chromatographic
separation for the 12 tested analysts.
Mobile phases including acetonitrile-water and methanol-water with modifiers such as acetic
acid, formic acid, and different gradient elution modes were all investigated. The results showed that
a mobile phase consisting of water (0.2% formic acid) and acetonitrile (0.2% formic acid) gave the
best separation.
3.2. Method Validation
3.2.1. Calibration Curves, Limits of Detection and Quantification
Standard stock solutions were prepared as described and diluted to appropriate concentrations to
establish the calibration curves. At least six different concentrations were analyzed in triplicate, and the

Molecules 2018, 23, 2609
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calibration curves were then constructed by plotting the peak areas vs. the concentration of each
analyte. As shown in Table 2, all the analytes showed good linearity (R2
0.9986) in a relatively wide
≥
concentration range. The analysis of LOD and LOQ also showed a well quantification, which ranged
from 0.001–1.55 ng/mL and 0.01–3.10 ng/mL, respectively.
3.2.2. Precision, Repeatability and Stability
The precisions were determined by analyzing known concentrations of the 12 analytes from two
standard stock solutions in six replicates during a single day and by duplicating the experiments.
To further evaluate the repeatability of the developed assays, samples were analyzed in six replicates
as described above. Stability of AR samples was tested at room temperature and analyzed at different
time points within one day. The contents of the 12 analytes were calculated from the corresponding
calibration curves. Table 2 indicated that the RSD values for measurement precision, repeatability and
stability of the 12 compounds were all less than 5.0%, which demonstrates good precision, repeatability
and stability of the developed method.
3.2.3. Accuracy
Accuracy of the analytical method was evaluated by measuring percentage recovery of 12 analytes.
The results of the recovery test are shown in Table 3, which all ranged from 95–105% at three
spiked concentrations.
3.3. Quantification of the Major Components in AR with and without Sulfur-Fumigation
The validated LC-MS method was applied for quantitative determination of the 12 components
with and without sulfur-fumigation. The contents of the eight flavonoids and four triterpenoid
saponins were summarized in Table 4. From the results, it can be found that compared with the
non-fumigated sample, the contents of two flavonoids calycosin (
significantly ranging from 39.2% to 45.4% and 35.5% to 40.5%, respectively; 7,20-dihydroxy-30,
40-dimethoxyisoflavane (
) had a large fluctuation ranging from 6.5% to 39.8%; the content of
methylnissolin ( ) had no obvious change in the sulfur-fumigated samples; while the contents of
four flavonoid glycosides (compounds , and ) all increased remarkably which suggests the
1) and formononetin (3) decreased
7
5
2
,
4,
6
8
occurence of chemical transformation of flavonoids and glycosides in the sulfur-fumigated samples.
In addition, the contents of astragaloside III (11) and astragaloside IV (12) decreased moderately
ranging from 11.5% to 40.0% and 15.5% to 47.7%, respectively, when compared with the non-fumigated
sample; the content of astragaloside I (9) displayed no obvious change in the sulfur-fumigated sample;
and the content of astragaloside II (10) was not detected because of the limited detection. Furthermore,
the analyses of the detected compounds’ contents over different sulfur-fumigated times suggested
that the reduction proportions of compounds 7, 11, and 12 had a proportional relationship with
sulfur-fumigated time. All above results indicated that sulfur-fumigation can decrease the contents of
partial aglycones and triterpenoid saponins and increase the contents of flavonoid glycosides in AR
significantly. Therefore, it could be concluded that sulfur-fumigation can significantly influence the
inherent quality of the raw materials of AR.

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Table 4. The contents of twelve reference compounds in AR with and without sulfur-fumigation (mg/g, n = 3).
AR with and without Sulfur-Fumigation.
Compounds
1 h
2 h
4 h
6 h
8 h
12 h
16 h
24 h
36 h
48 h
60 h
72 h
Non-Fumigated
1
2
3
4
5
6
7
8
9
7.49 ± 0.03
4.24 ± 0.05
0.59 ± 0.01
0.81 ± 0.02
0.30 ± 0.01
0.55 ± 0.00
4.18 ± 0.06
0.86 ± 0.00
0.71 ± 0.01
ND
5.04 ± 0.04
5.45 ± 0.02
0.59 ± 0.03
1.19 ± 0.09
0.25 ± 0.00
0.90 ± 0.01
2.61 ± 0.01
1.59 ± 0.00
0.74 ± 0.02
ND
6.40 ± 0.02
5.10 ± 0.00
0.75 ± 0.02
1.04 ± 0.01
0.27 ± 0.02
0.69 ± 0.01
2.99 ± 0.05
1.38 ± 0.05
0.70 ± 0.00
ND
7.36 ± 0.07
4.83 ± 0.01
0.78 ± 0.00
1.05 ± 0.04
0.27 ± 0.00
0.64 ± 0.02
3.44 ± 0.00
1.20 ± 0.07
0.71 ± 0.05
ND
4.91 ± 0.00
4.61 ± 0.04
0.50 ± 0.02
0.88 ± 0.05
0.21 ± 0.00
0.59 ± 0.00
2.93 ± 0.02
1.13 ± 0.06
0.70 ± 0.00
ND
5.97 ± 0.01
4.27 ± 0.02
0.64 ± 0.01
0.80 ± 0.07
0.22 ± 0.01
0.58 ± 0.05
3.29 ± 0.04
1.16 ± 0.02
0.70 ± 0.02
ND
7.44 ± 0.10
4.07 ± 0.05
0.50 ± 0.03
1.06 ± 0.02
0.35 ± 0.02
0.53 ± 0.07
3.06 ± 0.01
0.95 ± 0.01
0.74 ± 0.04
ND
5.37 ± 0.02
4.39 ± 0.03
0.62 ± 0.00
0.89 ± 0.00
0.22 ± 0.05
0.58 ± 0.00
2.51 ± 0.00
1.27 ± 0.00
0.69 ± 0.07
ND
5.42 ± 0.05
4.33 ± 0.04
0.60 ± 0.02
0.91 ± 0.02
0.23 ± 0.01
0.52 ± 0.02
3.17 ± 0.02
1.08 ± 0.04
0.70 ± 0.00
ND
4.86 ± 0.01
4.48 ± 0.01
0.56 ± 0.01
0.95 ± 0.01
0.22 ± 0.02
0.67 ± 0.00
2.60 ± 0.00
1.42 ± 0.06
0.68 ± 0.01
ND
5.28 ± 0.06
3.92 ± 0.02
0.61 ± 0.02
0.76 ± 0.02
0.23 ± 0.00
0.47 ± 0.00
2.54 ± 0.00
0.97 ± 0.00
0.67 ± 0.02
ND
6.03 ± 0.03
3.87 ± 0.02
0.63 ± 0.01
0.75 ± 0.01
0.22 ± 0.01
0.44 ± 0.01
3.24 ± 0.01
0.75 ± 0.00
0.70 ± 0.04
ND
13.55 ± 0.68
3.21 ± 0.01
1.37 ± 0.04
0.40 ± 0.02
0.24 ± 0.01
0.32 ± 0.02
4.47 ± 0.10
0.68 ± 0.00
0.73 ± 0.04
ND
10
11
12
0.78 ± 0.06
0.84 ± 0.02
0.79 ± 0.01
0.70 ± 0.04
0.71 ± 0.00
0.73 ± 0.01
0.75 ± 0.00
0.59 ± 0.02
0.60 ± 0.00
0.64 ± 0.01
0.57 ± 0.02
0.58 ± 0.01
0.95 ± 0.03
3.97 ± 0.00
4.17 ± 0.02
3.73 ± 0.01
3.38 ± 0.01
3.59 ± 0.00
3.53 ± 0.02
3.61 ± 0.01
2.68 ± 0.05
2.81 ± 0.02
3.03 ± 0.04
2.62 ± 0.00
2.58 ± 0.01
4.94 ± 0.02

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3.4. General Procedure for the Synthesis of Flavonoid Glycosides
The variation of flavonoid and glycoside content after the sulfur-fumigation of AR compared with
the reference sample suggest that the flavonoids may have a reaction with glucoses under the high
temperature and acidic conditions that occur during the sulfur-fumigation process. In order to confirm
the deduction, we further designed a procedure for the synthesis of flavonoid glycosides which was
similar to the sulfur-fumigation circumstances.
0
0 0
Calycosin (1), formononetin (3), and 7,2 -dihydroxy-3 ,4 -dimethoxyisoflavane (7) (10 mg each) were
dissolved with mixed solvent DMSO and H2O (2 mL each) in the sealing tubes, respectively. Then 1 mL of
concentrated hydr◦ochloric acid and D-glucose (10 mg, 0.055 mmol) were added. The reaction mixture
was heated to 80 C. After 12 h, the four reaction mixtures were analyzed by HPLC, respectively.
The results showed that calycosin-7-glucoside (
2
), ononin (
4
), astraisoflavan-7-O-
β-D-glucoside (6), and
0
0
0
7,2 -dihydroxy-3 ,4 -dimethoxy isoflavan-7-O-
β
-D-glucopyranoside (8
) were generated by comparison
with the standard materials (Figure 3). This experiment further confirmed that sulfur-fumigation can
increase the extent of transformation of flavonoids to flavonoid glycosides.
HO
O
GluO
O
HO
80℃
OH
O
OH
OH
OH
+
OH
H+
O
O
OCH3
OH
OCH3
2
1
HO
O
GluO
O
HO
80℃
O
OH
OH
+
OH
H+
O
OCH3
O
OH
OCH3
3
4
HO
O
GluO
O
HO
OH
OH
80℃
O
OCH3
OCH3
OCH3
OCH3
OH
OH
+
OH
H+
OH
7
8
HO
O
HO
dehydrogenate
O
GluO
O
80℃
H+
O
O
HO
OCH3
OCH3
OCH3
OCH3
OCH3
5
OCH3
6
Figure 3. The synthesis of flavonoid glycosides.
4. Conclusions
In the present study, a LC-MS method was established for simultaneous quantification of
twelve major components in AR, and successfully applied for quantitatively evaluating the effects
of sulfur-fumigation on the quality of AR. Compared with previously reported methods, the newly
developed method used MRM mode of LC-MS which was the first application to simultaneously
detect flavonoids and triterpenoid saponins in A. mongholicus.

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In this study, it was observed that the content of the major flavonoids decreased significantly,
while the corresponding glycosides increased accordingly when compared with non-fumigated AR.
The contents of the major triterpene glycosides also decreased in the sulfur-fumigation samples,
but the degree of reductions were limited. Sulfur-fumigation can influence not only the content of the
components in AR, but also the chemical transformation of flavonoids and glycosides. It was suggested
that sulfur-fumigation should be forbidden for processing and conservation of Chinese medicinal herbs
before the efficacy and safety of sulfur-fumigated herbs are systematically investigated. Alternatives
to sulfur-fumigation for processing and conservation of AR should also be further developed.
Author Contributions: G.M. and X.X. conceived and designed the experiments; X.X. and Z.S. performed the
experiments; M.Y., N.Z., and J.Y. given the help of data analyses.
Funding: This research was funded by the CAMS Innovation Fund for Medical Sciences (CIFMS) (No.
2017-I2M-1-013), National Science and Technology Major Project for ‘Significant New Drugs Development’
(2018ZX09721004-010), and Beijing Natural Science Foundation (7172138).
Acknowledgments: The work was financially supported by the CAMS Innovation Fund for Medical Sciences
(CIFMS) (No. 2017-I2M-1-013) and the Technological Large Platform for Comprehensive Research and
Development of New Drugs in the Twelfth Five-Year “Significant New Drugs Created” Science and Technology
Major Projects (No. 2012ZX09301-002-001-026).
Conflicts of Interest: There is no conflict of interest associated with the authors of this paper.
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Sample Availability: Samples of the compounds 1–12 are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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