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Heliyon 10 (2024) e28736
Available online 28 March 2024
2405-8440/© 2024 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Research article
Compounds identication and mechanism prediction of YuXueBi
capsule in the treatment of arthritis by integrating UPLC/
IM-QTOF-MS and network pharmacology
Xiaoyu Zhang
a
,
1
, Xueyuan Dong
a
,
e
,
1
, Ruihu Zhang
a
, Shufan Zhou
d
, Wei Wang
d
,
Yu Yang
d
, Yuefei Wang
a
,
b
, Huijuan Yu
a
,
b
, Jing Ma
c
,
**
, Xin Chai
a
,
b
,
*
a
National Key Laboratory of Chinese Medicine Modernization, State Key Laboratory of Component-based Chinese Medicine, Tianjin Key Laboratory
of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China
b
Haihe Laboratory of Modern Chinese Medicine, Tianjin, 301617, China
c
First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, National Clinical Research Center for Chinese Medicine Acupuncture
and Moxibustion, Tianjin, 300381, China
d
Liaoning Good Nurse Pharmaceutical (Group) Co., Ltd., Liaoning, 117201, China
e
Clinical Trials Center, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing, 100021, China
ARTICLE INFO
Keywords:
Network pharmacology
Rheumatoid arthritis
UPLC/IM-QTOF-MS
YuXueBi capsule
Mechanism
ABSTRACT
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that seriously affects the life
quality of patients. As a patent medicine of Chinese traditional medicine, YuXueBi capsule (YXBC)
is widely used for treating RA with signicant effects. However, its active compounds and
therapeutic mechanisms are not fully illuminated, encumbering the satisfactory clinical appli-
cation. In this study, we developed a method for identifying the chemical compounds of YXBC
and the absorbed compounds into blood of rats using ultra performance liquid chromatography/
ion mobility-quadrupole time-of-ight mass spectrometry (UPLC/IM-QTOF-MS) combined with
UNIFI analysis software. A total of 58 compounds in YXBC were unambiguously or tentatively
identied, 16 compounds from which were found in serum of rats after administration of YXBC.
By network pharmacology, these prototype compounds identied in serum were predicted to
regulate 30 main pathways (including HIF-1 signaling pathway, neuroactive ligand-receptor
interaction, IL-17 signaling pathway, and so on) through 146 targets, resulting in promoting
blood circulation and removing blood stasis, analgesia, and anti-inammatory activities. This
study provides a scientic basis for the clinical efcacy of YXBC in the treatment of RA.
Abbreviations: RA, rheumatoid arthritis; YXBC, YuXueBi capsule; CHMs, Chinese herbal medicines; UPLC, ultra performance liquid chroma-
tography; IM-QTOF-MS, ion mobility-quadrupole time-of-ight mass spectrometry; PBCRBS, promoting blood circulation and removing blood stasis;
AI, anti-inammation; AKBA, 3-acetyl-11-keto-beta-boswellic acid; BPI, base peak intensity.
* Corresponding author. National Key Laboratory of Chinese Medicine Modernization, State Key Laboratory of Component-based Chinese Med-
icine, Tianjin Key Laboratory of TCM Chemistry and Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China.
** Corresponding author.
E-mail addresses: majing2609@163.com (J. Ma), chaix0622@tjutcm.edu.cn (X. Chai).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2024.e28736
Received 20 October 2023; Received in revised form 21 March 2024; Accepted 22 March 2024
Heliyon 10 (2024) e28736
2
1. Introduction
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by joint swelling and progressive damage to
articular cartilage and some vital organs, such as the heart, kidney, lung, and so on [1–3]. Current research has documented that 0.5%–
1.0% of adults are suffering from RA, which poses a signicant challenge to quality of life, leading to a great burden of health care [4].
At present, long-term treatment with glucocorticoids and nonsteroidal anti-inammatory drugs is the primary means to alleviate RA
symptoms [5]. However, prolonged use of them is linked to the manifestation of several adverse side effects such as gastrointestinal
distress, high blood pressure, and cardiovascular complications [6,7]. Traditional Chinese medicine (TCM) is employed as a credible
alternative for treating RA. It is believed that the meridian paralysis and invasion of wind, dampness, or cold patterns into the human
body and blood vessels are the primary reasons for the occurrence and development of RA [8].
As a kind of arthralgia syndrome, RA is usually treated with TCM to promote blood circulation, remove blood stasis, dredge
collaterals, and relieve pain [9–11]. YuXueBi capsule (YXBC), as a patent medicine for treating RA, has been widely employed to
alleviate and treat arthralgia syndrome, which is mainly composed of eleven Chinese herbal medicines (CHMs), including Radix et
Rhizoma Clematidis (Weilingxian, WLX), Flos Carthami (Honghua, HH), Radix et Rhizoma Salviae Miltiorrhizae (Danshen, DS),
Olibanum (Ruxiang, RX), Myrrha (Moyao, MY), Rhizoma Chuanxiong (Chuanxiong, CX), Rhizoma Curcumae Longae (Jianghuang,
JH), Radix Cyathulae (Chuanniuxi, CNX), Radix Angelicae Sinensis (Danggui, DG), Rhizoma Cyperi (Xiangfu, XF), and Radix Astragali
Praeparata Cum Melle (Zhihuangqi, HQ). Pharmacological studies have shown that YXBC can restrain angiogenesis by inhibition of
LOX/Ras/Raf-1 signaling, which decreases the disease severity of RA and reduces bone erosion [12]. YXBC has the effect of analgesic
via inhibiting the migration of macrophage to the spinal cord mediated by CCL3 [13] and plays anti-inammatory (AI) role by
regulating the phosphorylation of NF-κB p65, JNK, and p38 [14]. However, few systematic studies have been reported for unveiling the
compounds in YXBC and its potential therapeutic mechanism for RA.
In this study, we employed ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-ight mass spec-
trometry (UPLC/IM-QTOF-MS) to comprehensively analyze and identify the chemical compounds in YXBC and the absorbed com-
pounds in blood. Then, focusing on promoting blood circulation and removing blood stasis (PBCRBS), analgesia, and AI, we employed
the network pharmacology approach to explore its active compounds and clarify potential therapeutic mechanisms for treating RA by
YXBC. This strategy shows promising perspectives in illumination on active compounds and potential therapeutic mechanisms for
complex prescriptions of TCM.
2. Materials and methods
2.1. Materials and reagents
YXBCs were provided by Liaoning Good Nurse Pharmaceutical (Group) Co., Ltd. (Liaoning, China). Acetonitrile and methanol
(HPLC grade) were purchased from Sigma-Aldrich Crop. (St. Louis, MO, USA). Formic acid (HPLC grade) was obtained from Shanghai
Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). The used water was puried by a Milli-Q water purication system
(Millipore, Billerica, MA, USA). As reference compounds, danshensu, chlorogenic acid, ferulic acid, isochlorogenic acid B, rosmarinic
acid, salvianolic acid B, salvianolic acid A, dihydrotanshinone I, tanshinone IIA, and 3-acetyl-11-keto-β-boswellic acid (AKBA) were
supplied by the Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China), whose purities were all above 98%. Cryptotanshinone
(>98%) was obtained from National Institutes for Food and Drug Control (Beijing, China).
2.2. Preparation of standard solutions
Accurately weighed compounds were respectively dissolved in 50% methanol aqueous solution (v/v) to obtain 0.5020 mg/mL
danshensu, 0.1006 mg/mL chlorogenic acid, 0.05020 mg/mL ferulic acid, 0.05020 mg/mL isochlorogenic acid B, 0.2510 mg/mL
rosmarinic acid, and 2.004 mg/mL salvianolic acid B. Moreover, the other compounds were separately dissolved in methanol to
prepare 0.2008 mg/mL salvianolic acid A, 0.04980 mg/mL dihydrotanshinone I, 0.1992 mg/mL cryptotanshinone, 0.2000 mg/mL
tanshinone IIA, and 0.4980 mg/mL AKBA. By using the prepared stock solutions, the mixed solution of the tested compounds was
obtained at 4.204
μ
g/mL for danshensu, 0.7294
μ
g/mL for chlorogenic acid, 0.3891
μ
g/mL for ferulic acid, 0.7279
μ
g/mL for iso-
chlorogenic acid B, 2.698
μ
g/mL for rosmarinic acid, 44.59
μ
g/mL for salvianolic acid B, 2.510
μ
g/mL for salvianolic acid A, 0.4358
μ
g/mL for dihydrotanshinone I, 1.370
μ
g/mL for cryptotanshinone, 1.250
μ
g/mL for tanshinone IIA, and 5.042
μ
g/mL for AKBA,
respectively.
2.3. Sample preparation of YXBC
The accurately weighed YXBC powder (0.2 g) was transferred into a 50 mL conical ask and ultrasonically extracted with 25 mL
methanol at 60 ◦C for 30 min, which was centrifugated at 12700 rpm for 10 min. The supernatant was obtained for analysis.
The accurately weighed YXBC powder (6.3 g) was dissolved in 36 mL normal saline and ultrasonically mixed to obtain the sus-
pension at 0.175 g/mL for intragastric administration.
X. Zhang et al.
Heliyon 10 (2024) e28736
3
2.4. Serum sample preparation
Male Sprague-Dawley (SD) rats (200 ±10 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
(Beijing, China). Rats were fed in a satisfactory environment (12 h light-dark cycle, temperature 20–22 ◦C, and relative humidity 40%–
60%) for a week, which were fasted overnight with free access to distilled water for 12 h before the experiment. Then they were
randomly divided into blank and YXBC groups (n =3 per group). Rats in the YXBC and blank groups were respectively given at the dose
of 2.625 g/kg YXBC and the same volume of normal saline for consecutive 7 days. After the last administration, the blood was taken
from the fundus venous plexus at 15, 30, 60, and 90 min, which was placed at room temperature for 1 h and then centrifuged at 5000
rpm for 10 min at 4 ◦C. The serum was collected and frozen immediately at −80 ◦C until analysis. Blank serum samples were collected
in the same way. Animal studies were conducted according to protocols approved by the Animal Ethics Committee of Tianjin Uni-
versity of Traditional Chinese Medicine (TCM-LAEC2022105).
The serum samples were thawed and homogenized at room temperature in advance. Equal amounts (50
μ
L) of serum collected at
different times were mixed and 1 mL methanol was added, which was vortex-mixed for 5 min and centrifuged at 5000 rpm for 10 min
at 4 ◦C. The obtained supernatant was evaporated by nitrogen at room temperature. Then, the residues were dissolved in methanol
(150
μ
L) and vortexed for 1 min, which was followed by centrifugation at 12700 rpm for 10 min at 4 ◦C. The supernatant was employed
for analysis.
2.5. UPLC/IM-QTOF-MS analytical conditions
By employing ACQUITY™ UPLC system (Waters, Milford, USA), chromatographic separation was carried out on an ACQUITY™
UPLC BEH C18 column (2.1 ×100 mm, 1.7
μ
m) held at 40 ◦C. The mobile phase consisted of 0.2% formic acid aqueous solution (A) and
acetonitrile (B) with a ow rate of 0.3 mL/min using a gradient elution of 3%–10% B between 0 and 2 min, 10%–20% B between 2 and
3 min, 20%–28% B between 3 and 7 min, 28%–45% B between 7 and 7.5 min, 45%–90% B between 7.5 and 20 min, and 90%–3% B
between 20 and 21 min. The injection volume was 5
μ
L.
The MS analysis was performed on a Vion IM-Q TOF mass spectrometer (Waters Corporation, Milford, USA) using electrospray
ionization (ESI) in both positive and negative ion modes. MS conditions were as follows: the capillary voltage at 3.0 and −2.5 kV in
positive and negative ion modes, respectively; the source temperature at 120 ◦C; the temperature of the desolvation gas at 400 ◦C; the
ow rate of the desolvation gas (N
2
) at 700 L/h; the ow rate of the cone gas (N
2
) and collision gas (Ar) at 50 L/h and 0.20 mL/min,
respectively; mass range: m/z 150–1500; collision energy, 6 eV and 20–60 eV ramping. Data acquisition was controlled with UNIFI
1.8.0 informatics platform (Waters Corporation, Milford, USA).
2.6. UNIFI data processing method
The systematical information about compounds from eleven herbs of YXBC was collected by searching China National Knowledge
Infrastructure (CNKI), PubMed, PubChem, ChemSpider, and other databases. The self-built database of YXBC was constructed by
importing information, such as the compound name, molecular formula, chemical structure, and accurate molecular weight, into the
UNIFI system combining the Chinese medicine component database. The collected UPLC-Q-TOF-MS information was imported into the
UNIFI self-built database for matching compounds by the automatic matching function of UNIFI software. The acceptable error of
molecular weight was ±10 ppm. The adduct ions include +H, +Na, +K, and +NH
4
in the positive ion mode, and the adduct ions
contain +HCOO, +Cl, +CH
3
COO, and +Br in the negative ion mode. The identied compounds were checked by standards or liter-
ature reports.
2.7. Network pharmacology analysis
The structures of the focused compounds absorbed into blood from YXBC were downloaded from PubChem database (https://
pubchem.ncbi.nlm.nih.gov/) and imported into SwissTargetPrediction database (http://www.swisstargetprediction.ch/) to acquire
potential targets. PBCRBS [15], Analgesic [16], and AI [17] were selected as the main bioactivities of YXBC and RA was used as the
disease treated by YXBC to search for relevant targets from the GeneCards database (https://www.genecards.org/). The targets with a
relevance score ≥1 were collected as main targets related to bioactivities and disease. The intersection of the collected RA-related
targets and compounds-related targets was considered as the common targets, which were further overlapped with the biological
targets to obtain the shared targets of compounds-bioactivities-RA. A CHMs-compounds-targets-bioactivities-RA network was con-
structed to display relationships among them by Cytoscape (version 3.7.2). The overlapped targets of PBCRBS-compounds-RA,
Analgesic-compounds-RA, and AI-compounds-RA were imported into DAVID database (https://david.ncifcrf.gov/) for Kyoto Ency-
clopedia of Genes and Genomes (KEGG) pathway enrichment analysis, respectively.
3. Results and discussion
3.1. Identication of compounds in YXBC by UPLC/IM-QTOF-MS combined with UNIFI software
To ensure the comprehensiveness and accuracy of compounds identication, we established a self-built database with the aid of
UNIFI software. 381 compounds from eleven CHMs of YXBC were retrieved into the self-built database, including compound name,
X. Zhang et al.
Heliyon 10 (2024) e28736
4
(caption on next page)
X. Zhang et al.
Heliyon 10 (2024) e28736
5
molecular formulae, chemical structure, accurate molecular mass, and so on. Importantly, the comprehensive information on the
analyzed compounds was acquired in both positive and negative ion modes by UPLC/IM-QTOF-MS. The base peak intensity (BPI)
chromatograms of YXBC and the mixed standard solution are shown in Fig. 1. The acquired MS data were matched with the data from
the self-built database in UNIFI software to deduce the structures of compounds. 58 compounds in YXBC (Fig. 1 A and C), including 16
organic acids (“a”), 12 quinones (“q”), 7 terpenoids (“t”), 5 avonoids (“f”), 2 saponins (“s”), and 16 others (“o”), were tentatively
characterized. Among these, eleven compounds (peaks 4, 7, 8, 9, 13, 15, 17, 42, 47, 51, and 57) were unambiguously identied by
comparing with the retention time, quasi-molecular ions, and fragment ions of the reference standards (Fig. 1 B and D). Detailed
information on the chemical compounds was summarized in Table 1. Additionally, 58 compounds in YXBC were also traced to their
sources of CHMs, as shown in Fig. S1 and Table 1. It was found that the identied compounds were mainly originated from DS (22
compounds), HQ (eleven compounds), DG (ten compounds), CX (seven compounds), HH (three compounds), XF (three compounds),
JH (two compounds), RX (two compounds), and WLX (one compound).
According to the quasi-molecular ions, fragment ions, and adduct ions of the standard compounds, the chemical structures of
identied compounds and their fragmentation pathways were proposed. Compound 7 was identied as chlorogenic acid derived from
Honghua, which showed [M−H]
−
at m/z 353.0873 and fragment ions at m/z 191.0754, 179.0483, 135.0554, corresponded with
[M–H–Caffeoyl]
−
, [M–H–QAr]
−
, and [M–H–QAr–CO
2
]
−
[18]. The mass spectrum of compound 15 exhibited [M−H]
−
ion at m/z
717.1231, fragment ions at m/z 519.1432, 339.0831, 321.0713, 295.0894, and 293.0360 assigned to [M–H–Danshensu]
−
,
[M–H–Danshensu−CA]
−
, [M–H–2Danshensu]
−
, [M–H–Danshensu–CA–CO
2
]
−
, and [M–H–2Danshensu−CO]
−
, respectively, which
was identied as salvianolic acid B originated from Danshen [19]. Tanshinone IIA (compound 51) from Danshen, a quinone com-
pound, yielded [M+H]
+
ion at m/z 295.1230, whose product ions at 277.1157 [M +H–H
2
O]
+
and 249.1204 [M +H–H
2
O–CO]
+
were
also observed [20].
3.2. Identication of prototype compounds from YXBC in serum of rats
The identication of prototype compounds in vivo is helpful to clarify the material basis of the efcacy of Chinese Materia medica
(CMM) and its compound prescription [21]. To identify candidate therapeutic substances of YXBC, chemical proling of the rat serum
collected after administration of YXBC was conducted. On the basis of the identied compounds in YXBC, UPLC/IM-QTOF-MS
combined with UNIFI software was also employed to identify the prototype compounds absorbed into the blood from YXBC by
analyzing the dosed and blank serum. The extracted ion chromatograms of prototype compounds in the serum are displayed in Fig. 2
(A-D). 16 prototype compounds were obviously detected in rat serum, including six quinones (danshenxinkun A, danshenxinkun D,
dihydrotanshinone I, hydroxytanshinone IIA, isocryptotanshinone, and tanshinone IIA), four terpenoids (8-hydroxy-ar-turmerone,
α
-cyperone, β-boswellic acid, and AKBA), two avonoids (methylnissolin and formononetin), two phthalide (senkyunolide D and
Z-butylidenephthalide), one organic acid (9,12,13-trihydroxy-10-octadecenoic acid), and one lactone (brefeldin A), from which three
compounds were unambiguously identied by comparison with standard references.
The prototype compounds of YXBC in vivo play a key role in the prevention and treatment of arthritis. For example, tanshinone IIA
exerts AI, anticoagulant, antithrombotic, and neuroprotective effects by regulating the TLR/NF-κB and MAPKs/NF-κB pathways [22].
β-Boswellic acid and AKBA demonstrate synergistic effects in exerting AI and anti-arthritic activities by inhibiting the activity of the
5-lipoxygenase (5-LOX) pathway, resulting in improved physical and functional capacity, as well as reduced pain and stiffness [23].
Studies have shown that Z-butylidenephthalide, a phthalide compound, exhibits analgesic, AI, anti-proliferative, and antifungal
properties [24] by signicantly reducing the content of PGE2 in inammatory tissues [25]. Formononetin can protect articular
cartilage by inhibiting the expression and activation of pro-inammatory cytokine-induced cartilage degrading enzymes. Also, it can
antagonize the catabolism of proteoglycan in chondrocytes induced by IL-1β and prevent the degradation of articular cartilage under
pro-inammatory action [26].
α
-Cyperone was reported to ameliorate osteoarthritis by down-regulating NF-κB and MAPKs signaling
pathways, attenuating chondrocyte inammation, and reducing extracellular matrix degradation [27]. In summary, analgesia,
PBCRBS, and AI were considered as the characteristic activities of the 16 bioactive compounds in YXBC.
3.3. Construction of network for CHMs-preparation-compounds-targets-bioactivities-RA
In recent years, network pharmacology analysis has been successfully applied to prediction of therapeutic mechanisms in various
CMM [28]. As a particularly effective web-based tool, SwissTargetPrediction can accurately predict potential targets for the reported
bioactive compounds and novel synthetic analogs, thereby providing reliable predictions and identifying more targets [29,30]. Po-
tential targets of the prototype compounds in serum from YXBC were retrieved by SwissTargetPrediction. After removing duplicate
targets, a total of 695 targets were acquired, in which hydroxytanshinone IIA, 8-hydroxyarturmerone, Z-butylidenephthalide, dan-
shenxinkun D, and brefeldin A were found to target the most proteins, and the number of their targets was 274, 148, 130, 124, and 122,
respectively. By GeneCards, 2875, 97, 375, and 1560 targets associated with RA and the bioactivities of analgesia, PBCRBS, and AI
were obtained, respectively. Using the venny (2.1.0) website, 26 targets from the intersection of compounds-RA-analgesia, 85 targets
from compounds-RA-PBCRBS, and 230 targets from compounds-RA-AI were acquired after merging and removing the duplicate
targets, respectively (Fig. S2). The intricate relationship among eleven CHMs, 16 absorbed compounds in blood, 3 bioactivities, and
Fig. 1. BPI chromatograms of YXBC in the positive (A) and negative (C) ion modes and mixed standards in the positive (B) and negative (D)
ion modes.
X. Zhang et al.
Heliyon 10 (2024) e28736
6
Table 1
Analysis of chemical compounds from YXBC by UPLC/IM-QTOF-MS.
Serial
No.
Identication
(compounds category
#
)
t
R
/
min
Formula Quasi-molecular
ion/adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
1 arginyl-fructose (o) 0.75 C
12
H
24
N
4
O
7
[M+H]
+
337.1716 ﹘0.5 319.1516,
175.1198
– DG
2 guanosine (o) 1.11 C
10
H
13
N
5
O
5
[M −H]
–
282.0843 ﹘0.3 150.0426 – HQ, DG
3 citric acid (a) 1.12 C
6
H
8
O
7
[M −H]
–
191.0194 ﹘1.6 173.0090 – DS
4* danshensu (a) 2.05 C
9
H
10
O
5
[M −H]
–
197.0451 ﹘2.2 179.0346,
151.0393
– DS
5 succinyladenosine (o) 2.06 C
14
H
17
N
5
O
8
[M+H]
+
384.1164 3.6 252.0746,
162.0788,
188.0580
– DG, HQ
6 indole-3-acrylic acid (a) 3.48 C
11
H
9
NO
2
[M+H]
+
188.0713 3.6 170.0613 – DG, HQ
7* chlorogenic acid (a) 4.04 C
16
H
18
O
9
[M −H]
–
353.0873 ﹘1.4 191.0754,
179.0483,
135.0554
– HH
8* ferulic acid (a) 5.45 C
10
H
10
O
4
[M −H]
–
193.0502 ﹘2.4 177.0917,
162.8387
– HQ
9* isochlorogenic acid B
(a)
5.72 C
25
H
24
O
12
[M −H]
–
515.1171 ﹘4.7 191.0551,
173.0450
– WLX
10 lithospermic acid (a) 5.80 C
27
H
22
O
12
[M −H]
–
537.1020 ﹘3.5 339.0493,
295.0596,
185.0236
– DS
11 salvianolic acid D (a) 5.83 C
20
H
18
O
10
[M −H]
–
417.0808 ﹘4.6 339.0493,
197.0454,
179.0344,
157.0289
– DS
Serial
No.
Identication
(compounds category
#
)
t
R
/
min
Formula Quasi-molecular
ion/adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
12 cartormin (f) 6.20 C
27
H
29
NO
13
[M+H]
+
576.1729 2.9 414.1197,
249.0553
– HH
13* rosmarinic acid (a) 6.45 C
18
H
16
O
8
[M −H]
–
359.0764 ﹘2.5 197.0455,
179.0350,
161.0242,
151.0398
– DS
14 salvianolic acid G (a) 6.53 C
18
H
12
O
7
[M+H]
+
341.0658 0.6 295.0608,
279.0660,
187.0399
– DS
15* salvianolic acid B (a) 7.01 C
36
H
30
O
16
[M −H]
–
717.1231 ﹘3.8 519.1432,
339.0831,
321.0713,
295.0894,
293.0360
– DS
16 baicalin (f) 7.43 C
21
H
18
O
11
[M −H]
–
445.0786 2.3 269.0458,
241.1204,
223.0393
– HQ
17* salvianolic acid A (a) 7.77 C
26
H
22
O
10
[M −H]
–
493.111 ﹘6.2 295.0595,
267.0647,
185.0237
– DS
18 isosalvianolic acid C (a) 8.71 C
26
H
20
O
10
[M −H]
–
491.0984 0.1 311.0556,
293.0449
– DS
19 senkyunolide D (o) 9.23 C
12
H
14
O
4
[M −H]
–
221.0816 ﹘1.5 203.0712,
177.0919,
173.0227
+CX
20 calycosin (f) 9.34 C
16
H
12
O
5
[M −H]
–
283.0608 ﹘1.5 161.0242 – HQ
21 methylnissolin (f) 9.67 C
17
H
16
O
5
[M −H]
–
299.0921 ﹘1.4 167.0718 +HQ
22 9,12,13-trihydroxy-10-
octadecenoic acid (a)
9.68 C
18
H
34
O
5
[M −H]
–
329.2319 ﹘4.3 311.2222,
229.1442,
171.1021
+HH
Serial
No.
Identication (compounds
category
#
)
t
R
/
min
Formula Quasi-
molecular ion/
adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
23 β -rotunol (t) 9.79 C
15
H
22
O
2
[M+H]
+
235.1699 2.9 197.0606 – XF
24 Z-butylidenephthalide (o) 9.86 C
12
H
12
O
2
[M+H]
+
189.0917 3.5 159.0810 +DG
25 3-butylphthalide (o) 9.94 C
12
H
14
O
2
[M+H]
+
191.1074 3.7 161.0609 – DG
26 formononetin (f) 9.98 C
16
H
12
O
4
[M −H]
–
267.0652 ﹘4.0 252.0416 +HQ
(continued on next page)
X. Zhang et al.
Heliyon 10 (2024) e28736
7
Table 1 (continued )
Serial
No.
Identication (compounds
category
#
)
t
R
/
min
Formula Quasi-
molecular ion/
adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
27 3- butyl-4-hydroxyphthalide
(o)
10.06 C
12
H
14
O
3
[M −H]
–
205.0867 ﹘1.6 161.0692 – CX
28 astragaloside II (s) 10.20 C
43
H
70
O
15
[M+H]
+
827.4793 0.7 571.2917,
419.2423,
353.2316
– HQ
29 salvianolic acid F (a) 10.36 C
17
H
14
O
6
[M+H]
+
315.0870 2.0 295.1343,
243.0680,
203.1074
– DS
30 β -cyperone (t) 10.54 C
15
H
22
O [M+H]
+
219.1747 6.4 159.0816 – XF
31 curcumin (o) 11.10 C
21
H
20
O
6
[M −H]
–
367.1178 ﹘2.4 285.1129,
245.0833,
175.0763
– JH
32 senkyunolide (o) 11.18 C
12
H
16
O
2
[M+H]
+
193.1227 1.8 177.1285,
175.1127,
163.1137
– CX
33 8-(3-((3-pentyloxiran-2-yl)
methyl)oxiran-2-yl)octanoic
acid (a)
11.21 C
18
H
32
O
4
[M −H]
–
311.2219 ﹘2.7 293.2119,
223.1702
– HQ
34 danshenxinkun A (q) 11.36 C
18
H
16
O
4
[M −H]
–
295.0970 ﹘1.8 277.0859,
262.0641
+DS
35 1,2,5,6-tetrahydrotanshinone
I (q)
11.52 C
18
H
16
O
3
[M +NH
4
]
+
298.1449 3.7 253.1237,
215.1081
– DS
Serial
No.
Identication (compounds
category
#
)
t
R
/
min
Formula Quasi-
molecular ion/
adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
36 isoastragaloside I (s) 11.68 C
45
H
72
O
16
[M+H]
+
869.4903 1.1 851.4818,
689.4253,
671.4164,
473.3637
– HQ
37 danshenxinkun D (q) 11.69 C
21
H
20
O
4
[M+H]
+
337.1417 ﹘5.2 309.1145,
279.1028
+DS
38 dehydromiltirone (q) 12.00 C
19
H
20
O
2
[M+H]
+
281.1547 4.1 266.1315,
251.1090,
239.1081
– DS
39 8-hydroxyarturmerone (t) 12.01 C
15
H
20
O
2
[M+H]
+
233.1531 ﹘2.4 191.1081,
173.0976
+JH
40
α
-cyperone (t) 12.07 C
15
H
22
O [M+H]
+
219.1752 3.8 203.1444,
187.1122
+XF
41 2-methoxy-5-acetoxy-
fruranogermacr-1(10)-en-6-
one (o)
12.09 C
18
H
24
O
5
[M+H]
+
321.1709 3.8 292.1344,
229.1242
– DG
42* dihydrotanshinone I (q) 12.15 C
18
H
14
O
3
[M+H]
+
279.1017 0.5 261.0914,
233.0963,
205.1015
+DS
43 hydroxytanshinone IIA (q) 12.18 C
19
H
18
O
4
[M+H]
+
311.1288 3.3 293.1183,
278.0946
+DS
44 sedanolide (o) 12.20 C
12
H
18
O
2
[M+H]
+
195.1390 5.3 161.0975 – CX
45 Z-ligustilide (o) 12.26 C
12
H
14
O
2
[M+H]
+
191.1070 1.5 173.0972,
163.1129
– CX
46 danshenxinkun B (q) 12.62 C
18
H
16
O
3
[M+H]
+
281.1172 1.6 253.0876,
235.1123
– DS
Serial
No.
Identication
(compounds category
#
)
t
R
/
min
Formula Quasi-molecular
ion/adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
47* cryptotanshinone (q) 13.73 C
19
H
20
O
3
[M+H]
+
297.1485 2.9 279.1298,
282.1185,
254.0855,
251.1362
– DS
48 brefeldin A (o) 13.74 C
16
H
24
O
4
[M+K]
+
319.1311 1.4 178.0580 +DG
49 isocryptotanshinone (q) 13.75 C
19
H
20
O
3
[M+H]
+
297.1494 2.9 279.1386,
251.1436
+DS
50 dan shen spiroketal
lactone (o)
14.82 C
18
H
22
O
3
[M+H]
+
287.1648 2.1 269.1546,
241.1605,
199.0768
– DS
(continued on next page)
X. Zhang et al.
Heliyon 10 (2024) e28736
8
RA-related targets was clearly illustrated by Cytoscape 3.7.2 in Fig. 3. The results indicated that the absorbed compounds in blood
primarily exhibited anti-RA biological activity by targeting proteins such as ALB, ILB, PTGS2, and MMP9. Specically, 8-hydroxyar-
turmerone demonstrated AI effect by targeting HMOX1, OPRM1, CYP19A1, CASR, and LTA4H proteins. Z-Butylidenephthalide played
analgesic role by targeting P2RX7, PTGS1, PTGS2, FAAH, and ABCB1 proteins. Formononetin was found to mainly affect PBCRBS
through its interaction with IL2, ALOX12, EGFR, XDH, and ESR1 proteins. Especially, dihydrotanshinone I was found to play a key role
in PBCRBS, AI, and analgesia through ACHE, HTR3A, TSPO, KDR, ERBB2, EGFR, ADAM17, and PLAUR targets. These ndings suggest
that YXBC contains multiple active compounds with different biological activities, which might be the effective compounds for YXBC
in treating RA.
3.4. KEGG enrichment analysis of core targets
The intersected targets for the absorbed compounds into blood-RA-bioactivities were imported into DAVID for KEGG enrichment
analysis to respectively obtain the 10 shared signaling pathways associated with analgesic, PBCRBS, and AI. As shown in Fig. 4, the
signaling pathways related to analgesic mainly include neuroactive ligand-receptor interaction, serotonergic synapse, retrograde
endocannabinoid signaling, and calcium signaling pathway. The signaling pathways related to PBCRBS primarily involve rapl
signaling pathway, lipid and atherosclerosis, and HIF-1 signaling pathway. Furthermore, the signaling pathways associated with AI
mainly consist of virus infection-related pathways, immune regulation-related pathways, and phospholipase D signaling pathway. To
further predict the potential therapeutic mechanisms of the absorbed compounds from YXBC for the treatment of RA, the enriched
representative pathways were analyzed in detail. In the neuroactive ligand-receptor interaction signaling pathway, the compounds
absorbed in the blood can regulate the pathway by interacting with targets such as P2RX7, OPRD1, MC1R, CNR1, HTR1A, and TSPO,
leading to analgesic effects. Similarly, in the HIF-1 signaling pathway, the active compounds from YXBC can target IL6, FLT1, PIK3CA,
NOS2, NOS3, and ERBB2 to regulate the pathway and achieve the purpose of PBCRBS. In the IL-17 signaling pathway, the prototype
compounds from YXBC play a regulatory role by acting on GSK3B, MMP1, MMP3, PTGS2, and MAPK1 targets to achieve the effect of
AI. For example, dihydrotanshinone I achieves analgesic by regulating the target HTR1A in neuroactive ligand-receptor interaction,
which is associated with the balance of neuro function [31] (Fig. 4A). HIF-1 signaling pathway is involved in angiogenesis and
erythropoiesis [32], dihydrotanshinone I may perform PBCRBS by exerting impacts on the ERBB2 and EGFR in HIF-1 signaling
pathway (Fig. 4B). Recent research has revealed that IL-17 and its related cytokines lead to the activation of the transcription factors
NF-κB, AP1, and C-EBP, which induce transcription of multiple genes in a tissue-specic fashion, including major inammatory cy-
tokines (TNF and IL-6) [33]. For IL-17 signaling pathway, dihydrotanshinone I probably plays an important role in AI via MAPK8
(Fig. 4C). Therefore, YXBC may play an anti-RA role by regulating signaling pathways related to cardiovascular, inammatory re-
sponses, viral infections, cancer, immunity, and central nervous system. These results indicated that 16 prototype compounds in vivo
exert pharmacological activity in the treatment of RA, which provided evidence for the clinical application of YXBC.
However, the use of network pharmacology approach has certain limitations in identication of active ingredients and clarication
of potential mechanisms, which overlooks the impact of the compounds’ content in TCM on the activity. Therefore, it is crucial to
conduct further experimental analysis and verication to validate the active ingredients and related targets regarding the mechanism
Table 1 (continued )
Serial
No.
Identication
(compounds category
#
)
t
R
/
min
Formula Quasi-molecular
ion/adduct ion
Observed
value (m/z)
Mass
error
(ppm)
Fragment
ions (m/z)
Absorbed
compounds in
serum
Source
&
51* tanshinone IIA (q) 15.55 C
19
H
18
O
3
[M+H]
+
295.1230 1.7 277.1157,
249.1204,
262.0922,
262.0988
+DS
52 rosmariquinone (q) 16.13 C
19
H
22
O
2
[M+H]
+
283.1697 1.5 267.1408,
241.1227,
225.0920
– DS
53 senkyunone (q) 16.80 C
22
H
30
O
2
[M −H]
–
325.2189 5.0 241.1223,
297.1873
– CX
54 stigmasterol (o) 19.28 C
29
H
48
O [M+H]
+
413.3782 1.0 233.1929,
226.1700
– DG
55 ethyl linoleate (o) 19.63 C
20
H
36
O
2
[M+H]
+
309.2791 1.0 263.2388 – CX
56 β-boswellic acid (t) 19.74 C
30
H
48
O
3
[M −H]
–
455.3521 ﹘2.2 385.2735,
373.2739,
339.2687
+RX
57* AKBA (t) 19.86 C
32
H
48
O
5
[M+H]
+
513.3567 ﹘1.5 407.3297,
173.1329
+RX
58 ursolic acid (t) 19.97 C
30
H
48
O
3
[M+H]
+
457.3668 ﹘1.7 413.2691,
259.2429,
203.1803
– DG
“*” Compared with the reference standard; “#” Compounds category; “–” Not detected; “+” Detected.
“
&
” Source of compounds; “a” organic acids; “q” quinones; “t” terpenoids; “f” avonoids; “s” saponins; “o”others.
“DG” Danggui; “HQ” Zhihuangqi; “DS” Danshen; “HH” Honghua; “WLX” Weilingxian; “CX” Chuanxiong; “JH” Jianghuang; “XF” Xiangfu; “RX”
Ruxiang.
X. Zhang et al.
Heliyon 10 (2024) e28736
9
(caption on next page)
X. Zhang et al.
Heliyon 10 (2024) e28736
10
of YXBC in the treatment of RA.
4. Conclusions
This research developed an integrative approach integrated with UPLC/IM-QTOF-MS, serum pharmacochemistry, and network
pharmacology to investigate the active compounds and action mechanisms of YXBC in the treatment of RA. A total of 58 compounds in
YXBC were unambiguously or tentatively identied, and 16 compounds were found in rat serum after administration of YXBC, sug-
gesting their potential biological activity. The network pharmacology analysis revealed that the therapeutic effects of YXBC against RA
may be attributed to analgesia, PBCRBS, and AI bioactivities of the active compounds that could exert regulatory effects on signicant
signaling pathways including the IL-17 signaling pathway, neuroactive ligand-receptor interaction, and HIF-1 signaling pathway. This
study aims to elucidate the potential molecular mechanisms of YXBC in the treatment of RA, thereby enhancing the effectiveness and
specicity for YXBC clinical treatment.
Ethics statement
The animal study protocol was approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine
(TCM-LAEC2022105).
Data availability statement
Data will be made available on request.
CRediT authorship contribution statement
Xiaoyu Zhang: Writing – original draft, Visualization, Validation, Methodology, Formal analysis, Conceptualization. Xueyuan
Dong: Writing – original draft, Validation, Methodology, Conceptualization. Ruihu Zhang: Visualization, Investigation, Formal
analysis. Shufan Zhou: Visualization, Investigation, Formal analysis. Wei Wang: Visualization, Investigation, Formal analysis, Data
curation. Yu Yang: Visualization, Resources, Formal analysis, Data curation. Yuefei Wang: Project administration, Funding acqui-
sition, Formal analysis. Huijuan Yu: Resources, Project administration, Funding acquisition, Formal analysis. Jing Ma: Writing –
Fig. 2. The extracted ion chromatograms of prototype compounds from the dosed serum in the positive (A) and negative (C) ion modes and blank
serum in the positive (B) and negative (D) ion modes.
Fig. 3. The network of “CHMs-compounds-targets-bioactivities-RA”.
X. Zhang et al.
Heliyon 10 (2024) e28736
11
(caption on next page)
X. Zhang et al.
Heliyon 10 (2024) e28736
12
review & editing, Supervision. Xin Chai: Writing – review & editing, Supervision, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to
inuence the work reported in this paper.
Acknowledgments
This work was supported by grants from the Science and Technology Project of Haihe Laboratory of Modern Chinese Medicine
(22HHZYSS00007), Shandong Provincial Natural Science Foundation, China (ZR2021LZY035), and State Key Laboratory of
Component-based Chinese Medicine (QMJJ202102).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e28736.
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