Comparison of Chemical Constituents in Pseudostellariae Radix with Different Dosage Forms Based on HPLC-Q-Exactive Orbitrap/MS Combined with ...
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Hindawi
Evidence-Based Complementary and Alternative Medicine
Volume 2021, Article ID 6644127, 10 pages
https://doi.org/10.1155/2021/6644127
Research Article
Comparison of Chemical Constituents in Pseudostellariae
Radix with Different Dosage Forms Based on HPLC-Q-Exactive
Orbitrap/MS Combined with Multivariate Statistical Analysis
Yujiao Hua , Xiaoyuan Liu , and Fen Xie
Department of Clinical Pharmacy, Affiliated Hospital of Jiangnan University, Wuxi 214000, China
Correspondence should be addressed to Fen Xie; 303991002@qq.com
Received 15 December 2020; Revised 6 April 2021; Accepted 27 April 2021; Published 10 May 2021
Academic Editor: Amir Syahir
Copyright © 2021 Yujiao Hua et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background. Pseudostellariae Radix (PR) is an important traditional Chinese herbal medicine with vast clinical consumptions,
which has two different dosage forms, PR decoction pieces and PR formula granules. However, these two forms are bound to have
an impact on the accumulation of the effective components in PR, so the effectiveness of clinical use cannot be guaranteed.
Objective. To determine the effective composition of PR. Methods. In this research, PR decoction pieces and formula granules were
collected, and their composition was detected by HPLC-Q-Exactive Orbitrap/MS; multivariate statistical analysis was used to
distinguish differential metabolites between PR decoction pieces and formula granules. Results. A clear cut difference in the
composition of the two groups was observed. 98 differential chemical constituents could be identified in the positive mode, while
52 differential chemical compositions could be identified in the negative mode. The differential chemical compositions were
mainly concentrated in flavonoids, organic acids, fatty acids, and amino acids and present different change rules, mainly involved
in the isoquinoline alkaloid biosynthesis metabolic pathways. Conclusions. This study provides basic information to reveal the
influence law of different dosage forms on the metabolite synthesis and quality formation mechanism of PR.
1. Introduction a potent drug with high traditional following. On the other
hand, the advantages of PR formula granules lie in small
Pseudostellariae Radix (PR) is an arid tuberous root of package, multiple varieties, and multiple specifications, and
P. heterophylla (Miq.) Pax ex Pax et Hoffm [1]. It is a kind of the relevant parameters of drugs are clearly marked, which is
important traditional Chinese herbal medicine which has conducive to the identification and selection of medicinal
the functions of promoting immunity, relieving cough, materials by patients; also, the formula granules are easy for
antitumor, antioxidation, and protecting myocardial func- patients to take home [10, 11].
tion [2]. This herbal medicine is used wildly for its positive The different dosage forms are bound to have an impact
effects. It is reported that PR can be used for inappetence [3], on the accumulation of its effective constituents, so the
thirst [4], debility [5], diabetes [6], and weakness after illness effectiveness of clinical use cannot be guaranteed, and the
[7]. Currently, PR decoction pieces and formula granules are active components of these two dosage forms have not been
utilized in a Chinese hospital; among them, PR formula studied in depth. In order to have a more comprehensive
granules is a novel form of decoction pieces, which is understanding on the synthesis and accumulation of me-
prepared by water extraction, concentration, drying, and tabolites in PR with different dosage forms, as well as the
granulation of the PR decoction pieces [8, 9]. These two difference in quality between PR decoction pieces and
types of PR used have their own advantages and disad- formula granules, it is necessary to build a quality analysis
vantages. One of the advantages of PR decoction pieces method for the overall chemical constituents of PR with
being that it has been prescribed since thousands of years as different dosage forms.
2 Evidence-Based Complementary and Alternative Medicine
Metabolomics technology is a new omics technology PR decoction pieces and formula granules were pulverized
developed in the mid-1990s, among which plant metab- into homogeneous powders (80 mesh). Powder samples
olomics is a high-throughput, unbiased, and comprehensive (0.1 g) were accurately weighed out and transferred to a
analysis technology for metabolomics in plant extracts, and 25 mL conical flask equipped with a stopper. Then, samples
it is especially fit for the analysis of multicomponent were applied to extraction procedure, extracted with 800 μL
complex systems of TCM [12–14]. In recent years, liquid- of methanol and 10 μL of internal standard (2.8 mg/mL, DL-
mass coupling technology is widely used in the qualitative o-chlorophenylalanine). All samples were grinded to fine
and quantitative research of complex TCM systems [15, 16], powder using a grinding mill at 65 Hz for 90 s, and then, they
and the commonly used mass spectrometry techniques were ultrasonicated for 30 min, by 40 KHz and let stand for 1
include quadrupole mass spectrometry, ion trap mass hour at −20°C. The samples were centrifuged at 12000 rpm
spectrometry, time-of-flight mass spectrometry, and com- and 4°C for 15 min, and the supernatants were filtered
pound tandem mass spectrometry [17–20]. The structure of through a 0.22 μm membrane before injection into the
the compounds can be rapidly identified based on the HPLC system.
fragmentation information of the multistage mass spec-
trometry compounds and related database. Liquid phase
2.4. HPLC-Q-Exactive Orbitrap/MS Conditions. The HPLC
with high efficiency and rapid separation performance
analysis was performed on the Ultimate 3000 HPLC system
combined with sensitive and accurate mass spectrometry is
(Thermo, Waltham, MA, USA). A hyper gold C18 column
one of the most promising metabolomics technologies and
(100 mm × 2.1 mm × 1.9 μm) (Thermo, Waltham, MA, USA)
has been widely used in the analysis of polar compounds,
was used to carry out the chromatographic separation at
thermal unstable compounds, and macromolecular
40°C. The mobile phase consisted of eluent A (water + 5%
compounds.
acetonitrile + 0.1% formic acid, v/v) and eluent B
In this study, an accurate and reliable method based on
(acetonitrile + 0.1% formic acid, v/v) with a flow rate of
HPLC-Q-Exactive Orbitrap/MS coupled with multivariate
0.35 mL/min with a liner gradient program: 100%–80% A
statistical analysis and signal pathway analysis has been
from 0 to 1.5 min, 80%–0% A from 1.5 to 9.5 min, 0% A from
developed to analyze the differential chemical compositions
9.5 to 14.5 min, 0%–100% A from 14.5 to 14.6 min, and 100%
and major metabolic pathways in PR decoction pieces and
A from 14.6 to 18 min.
formula granules, exploring the dynamic change law of
Mass spectrometry detection was performed on
different dosage forms of PR. It is expected to provide basic
Q-Exactive Orbitrap/MS (Thermo, Waltham, MA, USA)
data for revealing the influence of different dosage forms on
equipped with an electrospray ionization (ESI) source op-
the synthesis and accumulation of metabolites of PR and for
erating in the positive and negative ion modes. The scan
discussing the formation mechanism of the quality in PR
range was m/z 50–1000 and m/z 50–1100 in positive and
decoction pieces and formula granules.
negative ion modes, respectively. The optimized instru-
mental parameters were as follows: heater temperature was
2. Materials and Methods 300°C, sheath gas flow rate was 45 arb, Aux gas flow rate was
2.1. Chemicals and Reagents. Formic acid and acetonitrile of 15 arb, sweep gas flow rate was 1 arb, and capillary tem-
HPLC grade were purchased from Sigma-Aldrich (St. Louis, perature was 350°C. The spray voltage was floating at 3.0 KV
MO, USA) and Merck (Darmstadt, Germany), respectively. (ESI+) or 3.2 KV (ESI−). S-lens RF level was 30% (ESI+) or
Ultrapure water was prepared using a Milli-Q purifying 60% (ESI−).
system (Millipore, Bedford, MA, USA) under a resistivity of
18.2 MΩ/cm, and other reagent solutions such as methanol 2.5. Data Analysis. The data were performed feature ex-
and 2-chloro-L-phenylalanine were analytical grade (Merck, traction and preprocessed with Compound Discoverer soft-
Darmstadt, Germany). ware (Thermo, Waltham, MA, USA) and then normalized
and edited into a two-dimensional data matrix by Excel 2010
2.2. Plant Materials. Five PR decoction pieces were pur- software (Redmond, WA, USA), including retention time
chased from Suzhou Tianling Chinese Herbal Medicine Co. (RT), compound molecular weight (compMW), observations
Ltd., Jiangsu Province, China. Five PR formula granules (samples), and peak intensity. The edited data matrix was
were purchased from Jiangyin Tianjiang Pharmaceutical Co. imported into SIMCA-P 13.0 software (Umetrics, Umea,
Ltd., Jiangsu Province, China. The botanical origin of the Sweden) for multivariate statistical analysis. Principal com-
materials was identified by Professor Xunhong Liu (De- ponent analysis (PCA) was performed to intuitively express
partment for Authentication of Chinese Medicines, Nanjing the difference in chemical compositions between PR decoc-
University of Chinese Medicine, China), and the voucher tion pieces and PR formula granules by observing the ag-
specimens were deposited at TCM pharmacy of Affiliated gregation of each sample. Partial least squares discriminant
Hospital of Jiangnan University. analysis (PLS-DA) and orthogonal least squares discriminant
analysis (OPLS-DA) were performed to further classify the
samples. Two parameters, R2Y and Q2, were used to evaluate
2.3. Sample Preparations. PR decoction pieces and PR the model, the closer R2Y is to 1, the more stable the model is,
formula granules were naturally put at room temperature and Q2 > 0.5 indicates a high prediction rate. Variable im-
prior to HPLC-Q-Exactive Orbitrap/MS analysis. The dried portance in the projection (VIP) > 1 via OPLS-DA analysis
Evidence-Based Complementary and Alternative Medicine 3
RT: 0.00–18.00 ESI+ RT: 0.00–18.00 ESI–
16.31 0.88
100 100
95 95
90 2.78
90
85 85
80 80
75 75 0.95
Relative abundance
Relative abundance
70 0.86 70
65 65
60 60
55 55
50 50
45 16.56 45 3.10
40 40
35 3.11
3.48
11.63 35 5.06
30 30 1.15 3.47
10.55 10.69
16.34
25 10.09 25 5.31 9.61 16.47
20 1.15
5.08 7.88 8.63 20 7.99 9.12 11.28
10.89 16.21 4.71 5.58 11.93 12.67
15 7.07 9.42
15 2.78
7.62
12.75
10 4.71 5.32 13.25 10 16.19
1.34 16.03 1.56 14.90
5 5
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (min) Time (min)
(a) (b)
RT: 0.00–18.00 ESI+ RT: 0.00–18.00 ESI–
16.29 0.89
100 100
95 95
90 90
85 0.87 85
80 80 0.98
75 75
70 70
Relative abundance
Relative abundance
65 1.16 65
60 60
55 2.78 3.09 55
50 50 1.15
3.48
45 45
40 2.72 16.49 40 3.10
35 35 5.07
11.63
30 5.06 30 3.47 5.32
25 25 11.27
20 1.28 20 3.97
1.83 3.61 10.09 16.20 10.57 16.34
15 5.31 15 2.72 5.59
5.58 5.84 6.93 9.93 1.39 5.67 16.43
10 12.45 12.59 16.06 10 7.10 7.20 10.10 11.64
12.25 13.08 16.26
16.78
5 5
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (min) Time (min)
(c) (d)
Figure 1: BPC of PR decoction pieces (a-b) and PR formula granules (c-d) in both positive and negative modes.
and the p value of the t-test (p < 0.05) were used to find for this experiment. Different mobile phases (including
potential metabolites that significantly contributed to the methanol-water, acetonitrile-water, methanol-0.1% formic
difference among the groups [21]. Biochemical databases, acid water, acetonitrile-0.1% formic acid water, acetonitrile
METLIN (http://metlin.scripps.edu/), HMDB (http://www. with 0.1% formic acid water-acetonitrile with 0.1% formic
hmdb.ca/), KEGG (http://www.kegg.com/), and Metab- acid, and 5% acetonitrile with 0.1% formic acid water-ace-
oAnalyst (http://www.metaboanalyst.ca/), were used to tonitrile with 0.1% formic acid), flow rate (0.25, 0.35, and
identify potential metabolites. According to the data of 0.45 mL/min), and column temperature (35, 40, and 45°C)
MetaboAnalyst, the impact value threshold was set at 0.6, and were examined and compared. As a result, a 5% acetonitrile
therefore, the most important potential metabolic pathways with 0.1% formic acid water-acetonitrile with 0.1% formic
were filtered out [22]. acid at a flow rate of 0.35 mL/min and a column temperature
of 40°C was found satisfactory for separation of molecules in
a short time.
3. Results and Discussion According to the set conditions of sample treatment,
liquid chromatography and mass spectrometry were de-
3.1. Based Peak Chromatogram. The extraction conditions,
termined. The based peak chromatogram (BPC) of PR de-
including extraction (ultrasonic and refluxing extraction),
coction pieces and formula granules obtained from the
extraction solvent (100% methanol, 70% methanol, 50%
analysis in both positive and negative modes is shown in
methanol, and 30% methanol), and extraction time (15, 30,
Figure 1.
45, 60, and 75 min) were optimized to acquire the most
outstanding extraction efficiency. The results showed that
the chromatographic peak shape and relative peak area were
3.2. Multivariate Statistical Analysis
superior to the others under ultrasonic extraction with 100%
methanol for 30 min. 3.2.1. Principal Component Analysis. Based on LC-MS
In order to gain fast effective analysis, a hyper gold C18 spectra, chemical classification of all samples was carried out
column (100 mm × 2.1 mm × 1.9 μm) column was employed by multivariate data analysis, which aims to evaluate the
4 Evidence-Based Complementary and Alternative Medicine
80 80
60 60
40 40
20 20
0 0
t (2)
t (2)
–20 –20
–40 –40
–60 –60
–80 ESI+ –80 ESI–
–100 –100
–150 –100 –50 0 50 100 –150 –100 –50 0 50 100
t (1) t (1)
PR decoction pieces PR decoction pieces
PR formula particles PR formula particles
(a) (b)
Figure 2: The scores plot obtained from PCA analysis of PR decoction pieces and formula granules. The scores plot of PCA revealed that the
10 samples were classified into two major groups in the positive mode (a) and negative mode (b).
80
0.8
60
40 0.6
20 0.4
t (2)
0
–20 0.2
–40 0
–60
–0.2
–80 ESI+ ESI+
–100 –0.4
–150 –100 –50 0 50 100 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
t (1) R2
Q2
PR decoction pieces
PR formula particles
(a) (b)
1
100
0.8
50
0.6
t (2)
0 0.4
0.2
–50
0
–100
–0.2
ESI– ESI–
–150 –0.4
–100 –80 –60 –40 –20 0 20 40 60 80 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
t (1)
R2
PR decoction pieces Q2
PR formula particles
(c) (d)
Figure 3: PLS-DA scores plot and the permutation test with 200 permutations of PR decoction pieces and formula granules. The scores plot
of PLS-DA revealed that PR decoction pieces and PR formula granules could be clearly distinguished into two groups in the positive mode
(a) and negative mode (c). The permutation test with 200 permutations indicated that PLS-DA is effective and can be used for subsequent
differential component analysis in the positive mode (b) and negative mode (d).
Evidence-Based Complementary and Alternative Medicine 5
100 100
50 50
1.14041 ∗ to (1)
1.02796 ∗ to (1)
0 0
–50 –50
–100 –100
ESI+ ESI–
–150 –150
–100 –80 –60 –40 –20 0 20 40 60 80 –100 –80 –60 –40 –20 0 20 40 60 80
t (1) 1.00028 ∗ t (1)
PR decoction pieces PR decoction pieces
PR formula particles PR formula particles
(a) (b)
0.8
1
0.6
0.4
0.5
p (corr) (1)
p (corr) (1)
0.2
0 0
–0.2
–0.5
–0.4
–0.6
–1
–0.8 ESI+ ESI–
–1 –1.5
–0.2 –0.15 –0.1 –0.05 0 0.05 0.1 0.15 –0.25 –0.2 –0.15 –0.1 –0.05 0 0.05 0.1 0.15 0.2
p (1) p (1)
(c) (d)
Figure 4: OPLS-DA scores plot and S-plot of PR decoction pieces and formula granules. The scores plot of OPLS-DA revealed that PR
decoction pieces and PR formula granules were significantly different in the two classes in the positive mode (a) and negative mode (b). S-
plots indicated that the farther the distance from the central region represented the metabolites contributing more to the separation between
groups in the positive mode (c) and negative mode (d).
differences in chemical constituents of PR decoction pieces In this research, the model parameters were R2X � 0.799,
(TY) and formula granules (TK). Principal component R2Y � 0.840, Q2 � 0.452 in ESI+ (Figure 3(a)); R2X � 0.884,
analysis (PCA) was utilized to reduce the dimensions of R2Y � 0.993, and Q2 � 0.885 in ESI− (Figure 3(c)). The PLS-
multivariate problems. After Vilfredo Pareto with mean DA scores plot showed that TY and TK were clearly isolated
centering, the data were showed as scores in a coordinate into two groups, and the interclass differences were less than
system of latent variables, which resulted from the above that in the PCA model, so the PCA model was more effective
samples. The PCA score plot (R2X � 0.805, Q2 � 0.686 in to ensure the differences in two different dosages of PR. The
ESI+, Figure 2(a); R2X � 0.886, and Q2 � 0.654 in ESI−, PLS-DA model was further validated by a permutation test
Figure 2(b)) showed clear separation in different dosage with 200 permutations (Figures 3(b) and 3(d)). The R2 and
forms of PR in both ESI+ and ESI−. This indicated that there Q2 values generated by the random permutation at the left
was a significant difference in chemical constituents between end are both smaller than the original values at the right end,
TY and TK. indicating that the predictive power of the original model is
greater than that of the random permutation y variables, so
the model is effective and can be used for subsequent dif-
3.2.2. Partial Least Square Discriminant Analysis. Partial ferential component analysis [24].
least square discriminant analysis (PLS-DA) is a supervised
analysis, which extends a regression of PCA and uses class
information to maximize the separation between groups of 3.2.3. Orthogonal Least Squares Discriminant Analysis.
observations [23]. This frequently used classification method In order to further identify the significant metabolites
is categorical (categories described with dummy variables) contributing to distinction in the two forms of PR, or-
and expressed the class membership of the statistical units. thogonal least squares discriminant analysis (OPLS-DA), a
6 Evidence-Based Complementary and Alternative Medicine
Group
Guanosine Group
Choline 2
Plaunol B L-Glutamate 2
Isoformononetin
LysoPE (0:0/16:0) Thymidine
LysoPC (16:0)
Formononetin 1 Apiin
1
Diosmin Sucrose
Spinosic acid A
Soyasapogenol C Raffinose
Vitamin D2 0 0
Sphinganine L-Ascorbic acid
Phytosphingosine
Camellenodiol Citric acid
Terminaline –1
Sphingosine D-Glucarate –1
Formononetin 7-(6″-malonylglucoside)
α-Tocotrienol Succinic acid
Pfaffic acid
Glutinone –2 Uridine –2
Phosphocholine Hesperetin 7-O-glucuronide
Sophoraflavanone B
Sophoraisoflavanone A (-)-Jasmonic acid
Uracil
L-asparagine LPA(0:0/16:0)
Epinephrine
5′-deoxyadenosine Diosmin
Indoleacrylic acid
L-glutamine Linoleic acid
2-furoic acid
L-tryptophan 6″-Malonylapiin
5′-methylthioadenosine
Pantothenic acid lsoformononetin
Cinnamic acid
p-coumaroyl quinic acid LysoPE(0:0/16:0)
Thymol Fomiononetin
Adenine
Genistin 3′-Sialyllactose
6-gingerol
Tangeritin Pantothenic acid
Histamine
S-adenosylhomocysteine Auxin a
cis-jasmone
Pyroglutamic acid Tropic acid
Trigonelline
Quercetin Nonanedioic acid
Glutinosone
DL-pipecolic acid Hexadecanedioic acid
Coumarin Traumatic acid
(-)-jasmonic acid
L-dopa Abscisic acid
Valeric acid
p-hydroxyphenylacetic acid Tangeritin
L-leucine
Adenosine Sebacic acid
L-proline
Guanine Chrysin
L-histidine
Benzoic acid Daidzein
L-Phenylalanine
L-valine Gallocatechin
Norepinephrine
Chrysin Citramalic acid
Niacinamide L-Dopa
Tropic acid
Anthranilic acid Chlorogenic acid
Caprolactam
Isokaempferide Glycitein
L-nicotine
4-hydroxybenzaldehyde Liquiritigenin
L-tyrosine
L-methionine Keioside
Hesperidin
Glabrolide Glutaric acid
Gentisin
Naringin Taxifolin
Salicylic acid
Jasmolone Eriodictyol
α--ketoisovaleric acid Riboflavin
Niacin
3-indoleacetic acid α-D-Glucose
Ferulic acid
Naringenin-7-0-glucoside Astragalin
Pyridoxal
Dopamine Quinic acid
Cirsilineol
Glycitein Luteolin
Genipin
Hydrouracil Genistein
Malvidin
Puerarin Naringenin-7-O-Glucoside
Luteone 7-glucoside p-Coumaroyl quinic acid
Luteolin
Pyridoxamine Malvidin
Phenylacetic acid
Naringenin 3″,4″-Diacetylcosmosiin
Sinapic acid
Liquiritigenin Hesperetin
TYS1
TYS2
TYS3
TYS4
TYS5
TYS6
TYS7
TYS8
TYS9
TYS10
TYS1
TYS2
TYS3
TYS4
TYS5
TYS6
TYS7
TYS8
TYS9
TYS10
Group Group
TK TK
TY TY
(a) (b)
Figure 5: Hierarchical clustering analysis of PR decoction pieces and formula granules. Clear differentiation was observed in the heat map,
colors varying from green to red graphically indicated that the relative contents of metabolites were from low to high. 98 and 52 differential
chemical compositions were identified in the positive mode (a) and negative mode (b).
supervised pattern recognition approach was performed Q2 � 0.984 in ESI− (Figure 4(b)), which indicated good
[25]. In OPLS-DA scores plot, each spot represents a sample. ability of prediction and reliability of the model. To identify
As shown in Figure 4, the TY group can be clearly separated the metabolites contributing to the discrimination, S-plots
from the TK group in both positive and negative modes, and were generated (Figure 4(c) and 4(d)). Each spot in OPLS-
the model parameters were R2X � 0.933, R2Y � 1, Q2 � 0.673 DA scores plot and S-plots represents a variance. Farther the
in ESI+ (Figure 4(a)); R2X � 0.884, R2Y � 0.993, and distance from the central region, higher is the contribution
Evidence-Based Complementary and Alternative Medicine 7
1% 1% 1%
4% 1%
4%
4%
3%
4% 3%
3%
4% 31% 4% 24%
5% 4%
5%
13%
10% 23%
31%
17%
Fatty acids Alkaloids
Flavonoid Terpenoids
Organic acids Vitamins Organic acids Alkaloids
Nucleosides Steroids Flavonoid Active amines
Phenols Amino acids Phenols
Nucleosides Coumarins
Vitamins Ketone
Terpenoids Fatty acids
Catecholamines Aldehyde
(a) (b)
5% 3% 3%
3%
5% 3%
6%
11% 26%
6%
49%
11%
21%
27%
21%
Flavonoid Carbohydrate Flavonoid Fatty acids
Organic acids Vitamins Organic acids Phenylpropanoids
Fatty acids Amino acids Carbohydrate Catecholamines
Nucleosides Vitamins Terpenoids
(c) (d)
Figure 6: Comparative analysis of contents of differential chemical compositions in PR decoction pieces and formula granules. (a) The
relative contents of differential chemical compositions in PR decoction pieces > PR formula granules in the positive mode. (b) The relative
contents of differential chemical compositions in PR decoction pieces < PR formula granules in the positive mode. (c) The relative contents
of differential chemical compositions in PR decoction pieces > PR formula granules in the negative mode. (d) The relative contents of
differential chemical compositions in PR decoction pieces < PR formula granules in the negative mode.
of the metabolites. The importance of each variance to 3.3. Identification of Differential Compositions with Relative
classification was determined by the value of variable in the Contents Analysis. VIP >1 of OPLS-DA combined with the
projection (VIP). Metabolites with the VIP value above 1.0 t-test (p < 0.05) was used to discover the significantly dif-
and P value below 0.05 were considered as potential met- ferential metabolites in TY and TK. METLIN and HMDB
abolic markers. databases are used to search the accurate mass-to-charge
8 Evidence-Based Complementary and Alternative Medicine
ratio to identify the differential chemical compositions. As
seen from hierarchical clustering analysis (Figures 5(a) and
5(b)), a total of 98 and 52 differential chemical compositions 4
were identified in the positive mode and negative mode,
respectively, and the detailed information is given in
Table S1 and Table S2. 3
Clear differentiation was observed in the heat map based
on differential chemical compositions, and colors varying
–log (p)
from green to red graphically indicate that the relative 2
contents of metabolites are from low to high. In the positive
mode, there were 23 differential chemical compositions that
had the relative contents of TY greater than that in TK, while 1
there were 75 differential chemical compositions that the
relative contents of TY were less than that in TK. In the
0
negative mode, there were 19 differential chemical com-
positions that the relative contents of TY were greater than 0.0 0.2 0.4 0.6 0.8 1.0
that in TK, while there were 33 differential chemical com- Pathway impact
positions that the relative contents of TY were less than that Figure 7: Analysis of pathway impact. Isoquinoline alkaloid
in TK. biosynthesis which was the potential key metabolic pathway.
As shown in Figures 6(a) and 6(b), in the positive mode,
the relative contents of differential chemical compositions in
TY above that in TK were distributed in nine kinds of the larger the radius. The color of dots represents the p value
compositions. Flavonoids and fatty acids had the highest of the metabolic pathway, and the lower the p value, the
proportion, accounting for 31%. The relative contents of redder the color. The threshold value for the effect of the
differential chemical compositions in TY below that in TK metabolic pathway was set to 0.6 with topological Fenix,
were distributed in fourteen varieties of compositions. greater than which will be selected as potential key metabolic
Among them, 18 organic acids accounted for the largest pathways. Finally, one metabolic pathway, isoquinoline al-
proportion (24%), followed by flavonoids (17, 23%) and kaloid biosynthesis (A), was coenriched (Figure 7).
amino acids (13, 17%). In the negative mode, the relative
contents of differential chemical compositions in TY above 4. Conclusions
that in TK were mainly involved in seven varieties of
compositions. Flavonoids (5, 26%) were the most abundant, In this research, the difference of chemical compositions in
followed by organic acids (4, 21%) and fatty acids (4, 21%) PR with different dosage forms was analyzed by HPLC-Q-
(Figure 6(c)). The relative contents of differential chemical Exactive Orbitrap/MS combined with multivariate statistical
compositions in TY below that in TK belonged to eight analysis. Results showed that the chemical compositions
varieties of compositions. Among them, flavonoids exited great differences in TY and TK. A total of 98 dif-
accounted for the highest proportion (16, 49%), followed by ferential chemical compositions were found in ESI+, while
organic acids 9, 27% (Figure 6(d)). The results showed that 52 were found in ESI−. These constituents were mainly
differential chemical compositions of PR with different concentrated in flavonoids, organic acids, fatty acids, and
dosage forms were mainly concentrated in flavonoids, or- amino acids, mainly involved in the isoquinoline alkaloid
ganic acids, amino acids, and fatty acids. In additional, in biosynthesis metabolic pathways. In addition, both in
both positive and negative modes, the amounts and types of positive and negative modes, the amounts and types of
differential chemical compositions with higher relative differential chemical compositions with higher relative
contents in TK were both more than those with higher contents in TK were both more than those with higher
relative contents in TY, for example, flavonoids with anti- relative contents in TY, including flavonoids with the an-
tumor effects, including chrysin, naringin, luteolin, and titumor effect, organic acids containing the antibacterial
genistin; antimicrobial resistance to oxidation of organic antioxidant effect, and amino acids which can boost
compounds, including cinnamic acid, ferulic acid, and immunity.
salicylic acid; and amino acid compounds with immune- This study will provide the basic information for ex-
promoting effects, including L-tryptophan, L-phenylalanine, ploring the influence law of different dosage forms on the
and L-tyrosine. synthesis and accumulation of metabolites in PR and its
quality-forming mechanism. It will also provide a reliable
and accurate approach for the analysis of complex samples
3.4. Metabolic Pathway Analysis. The metabolic pathway and identification of differential chemical compositions.
was analyzed by MetaboAnalyst website, and the Kyoto
Encyclopedia of Genes and Genomes (KEGG) of the above Data Availability
identified compounds was introduced to MetaboAnalyst for
pathway analysis. The radius of dots represents the impact The data used to support the findings of this study are
value of the metabolic pathway. The larger the impact value, available from the corresponding author upon request.
Evidence-Based Complementary and Alternative Medicine 9
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