The study of Raddeanin A cerebrovascular endothelial cell trafficking
through P-glycoprotein
Yue-yue Wang 1
, Chun-feng Jiang 1
, Xin Liu, Jian-nan Li, Guang-zhi Cai, Ji-yu Gong*
Key Laboratory of Traditional Chinese Medicine Analysis, School of Pharmaceutical Sciences, Changchun University of Traditional Chinese Medicine,
Changchun, 130117, China
article info
Article history:
Received 20 April 2021
Accepted 27 April 2021
Available online 4 May 2021
Keywords:
Raddeanin A
P-Glycolprotein
Brain-blood barrier
Cerebrovascular endothelial cell
Drug resistance
Membrane trafficking
abstract
As one of the natural triterpenoids isolated from Anemone Raddeana Regel, Raddeanin A (RA) has been
confirmed to possess therapeutic effects against multiple tumorigeneses, especially for the onset of
glioblastoma and growth in human brains. However, the mechanism by which this happens remains
poorly understood in terms of the vascular endothelium trafficking routine of RA through the brainblood barrier (BBB). To seek such answers, human brain microenvironment endothelial cells (HBMECs)
were used to stimulate the microenvironment in vitro, and to explore the intracellular accumulation of
RA. The results of this experiment illustrated that RA has a relative moderate transport affinity for such
cells. The kinetic parameter Km was 37.01 ± 2.116 mM and Vmax was 9.412 ± 0.1375 nM/min/mg of protein.
Interestingly, protein downregulation of P-glycoprotein (P-gp, ABCB1/MDR1) significantly activated RA
transmembrane activity, which proves that P-gp is responsible for RA cellular trafficking. In addition, the
selective non-specific inhibitor, LY335979 increased either RA or the classical substrate of P-gp, digoxin,
intracellular accumulation by restricting the transporter’s function but without jeopardizing cytomembrane proteins. Moreover, a decrease in the expression or activity of P-gp triggered RA drug resistance to
HBMECs. In summary, our data showed that both the expression and function of P-gp are all necessary
for RA transmembrane trafficking through cerebrovascular endothelial cells. This study provides significant evidence for the presence of a connection between RA transport and P-gp variation during drug
BBB penetration. It is also suggesting some vital guidance on the RA pharmacodynamic effect in human
brains.
© 2021 Elsevier Inc. All rights reserved.
1. Introduction
The traditional Chinese herb belonging to the Ranunculaceae
family, Anemone Raddeana Regel, exhibits analgesic, antiinflammatory, and antitumor effects [1]. As an oleanane-type triterpenoid saponin and the main bioactive constituent of this herb,
Raddeanin A (RA) is capable of preventing cell proliferation and
inducing apoptosis, and acts as a tumor antagonist for multiple
tumorigeneses such as gastric, hepatocellular carcinoma (HCC),
gastric cancer (GC), and breast cancer (BC) [ [2e4]]. It is worth
mentioning that recent studies have demonstrated that RA suppresses the glioblastoma growth by inhibiting epithelialmesenchymal transition (EMT) and angiogenesis of such carcinomas [5]. Meanwhile, it also has a curative effect for U251 glioma
xenografts in mice [6]. Together, those results imply that RA is
capable of crossing the brain blood barrier (BBB), and plays major
effect in the human brains. However, the transmembrane traf-
ficking mechanism in the brain still remains poorly understood.
As a crucial membrane transporter and one of ABC family of
transporters located in mono endotheliocytes and organelles, Pglycolprotein (P-gp, encoded by the gene MDR1/ABCB1.) mediates
multiple triterpenoid saponins membrane trafficking, and is mainly
responsible for an efflux function [ [7e9]]. It is believed that the
aberrant expression or functional elimination of P-gp can influence
its transport activities of these substrates as well as their storage
within cells, thus inducing cellular drug resistance [10]. For
Abbreviations: Raddeanin A, RA; P-Glycolprotein, P-gp; Brain-Blood Barrier,
BBB; Human Brain Microenvironment Endothelial Cells, HBMECs; Hepatocellular
Carcinoma, HCC; Gastric Cancer, GC; Breast Cancer, BC; Neuronal Glutamate, Glu;
Epithelial-Mesenchymal Transition, EMT; Cyclosporin A, CsA.
* Corresponding author. No.1035 Boshuo Road, Changchun University of Traditional Chinese Medicine, Changchun, 130117, China.
E-mail addresses: [email protected] (G.-z. Cai), [email protected]
(J.-y. Gong).
1 These authors contributed equally to this work.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
https://doi.org/10.1016/j.bbrc.2021.04.111
0006-291X/© 2021 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications 559 (2021) 222e229
example, astragaloside IV (ASIV) can have an impact on the classical
substrate, rhodamine 123 (Rh123), aggravating its intracellular
accumulation via suppressing its extracellular efflux, which can
induce drug resistance, cytotoxicity, or apoptosis after inhibiting
the expression of P-gp [11]. Further studies have also demonstrated
that the vascular endothelial membrane surface protein restriction
of P-gp in human brains might be responsible for multiple brain
lesions such as glioblastoma [12]. These clues all imply that P-gp is
potentially the potential receptor that mediates triterpenoid
saponin and RA cerebrovascular endothelial transport, and also
intervenes in intracellular therapeutic effects and drug resistance.
To find out the mechanism behind RA brain trafficking and to
test the role that P-gp has on RA transmembrane transport in cerebrovascular endothelial cells, we hypothesized in this study that
inhibiting the function and expression of P-gp could influence RA
intracellular accumulation. With a high level of endogenous P-gp
expression, human brain microvascular endothelial cells (HBMECs)
were applied to stimulate the BBB cellular microenvironment [13].
Selective inhibitor LY335979 and siRNA were used to investigate
the effects on either the transporter function suppression or downexpression regulation of P-gp to alter the cellular RA level. Through
this study we found that RA is the substrate of P-gp, and that
function and expression suppression is linked with a reduction in
RA cellular concentration, which contributes to drug resistance in
HBMECs.
2. Materials and methods
2.1. Chemicals and chromatography reagents
Digoxin, zosuquidar trihydrochloride (LY335979) were purchased from Med Chem Express, U.S.A. Raddeanin A (RA) and glycyrrhetinic acid, as standard compounds were obtained from
National Institute for Food and Drug Control (111712e201702,
>98%, 110723e201514, >98% respectively). 3-(4,5-
dimethylthiazolyl-2)-2,5-dipheny-tetrazolium bromide (MTT)
were obtained from Sigma-Aldrich (St. Louis, MO). HPLC grade of
Methanol, acetonitrile, formic acid and hydroxyethyl piperazine
ethylsulfonic acid (HEPES) were bought from Sigma (SigmaAldrich, USA).
2.2. Cell culture
Human brain microvascular endothelial cells (HBMECs) were
purchased from the Shanghai ATCC cell bank (Shanghai, China).
Initially, cells were maintained in a monolayer culture in 95% air
and 5% CO2 at 37 C in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 100
units/ml penicillin and 100 mg/ml streptomycin which were all
purchased from Invitrogen Life Technologies (Gibco, Invitrogen, NY,
USA).
2.3. Cell viability evaluation
To ensure the appropriate cellular concentrations of RA after
uptake and intracellular transport study, the cytotoxicity was
evaluated by MTT assay. In brief, HBMECs were seeded in 96-well
plate with the density of 1.0 104 cells/well. Designated concentrations of RA were added into the culture medium following with
48 h incubation at 37 C. Serum-free medium containing MTT
(5 mg/mL) was added after original medium removal for another
4 h. Finally, DMSO replaced the MTT medium to dissolve formation
of formazan, then the absorbance at 490 nm was measured and
calculated by SpectraMax M2 microplate reader (Molecular Devices
Corporation Sunnyvale, CA, USA).
2.4. Uptake assay
Before determining the degree of uptake, cells were maintained
and cultured for 48 h on 24-well plates with a density of
2 105 cells per well. Then, they were prewarmed with Hank’s
Balanced Salt Solution (HBSS, PBS containing 25 mM HEPES, pH 7.4)
containing certain concentrations of LY335979 or digoxin for 20min
at 37 C. An assay was started by incubating the wells with HBSS
alone or mixed with different reagents after the cells were washed
three times with ice-cold PBS. Buffer removal terminated the assay
immediately after washing the cells three times with ice-cold PBS.
To normalize the data, half of the cells were lysed with 0.2 ml 0.1%
sodium dodecyl sulfate solution to determine the cellular protein
content, which was determined through a Pierce™ BCA Protein
Assay Kit (Invitrogen). Another 40 ml aliquot of cell lysate solution
was used to determine the content of RA by LCeMS/MS.
2.5. LCeMS/MS determination
All the samples (40 ml) were added 80 ml ethyl acetate containing
glycyrrhetinic acid (internal standard, IS, 100 ng/ml). The mixtures
were vortexed for 1min and centrifugated at 12,000 rpm for 10 min.
The organic layer was dried under a reduced pressure and then
redissolved in a 100 ml solution of 0.5‰ formic acid mixed with
methanolewater (90:10, v/v). A 10 ml aliquot of the resulting solution was then injected into the LCeMS/MS system for analysis.
The sample analysis was performed on a 1260 infinity LC system
(Agilent Technologies, Waldbronn, Germany) coupled to a 6430
Triple Quad MSD (Agilent Technologies) with an electrospray
ionization source. Separation was achieved using an Agilent ZORBAX SB-C18 (2.1 50 mm,1.8 mm, Agilent Technologies). The mobile
phase consisted of 0.5‰ formic acid mixed with methanolewater
(90:10, v/v). The flow rate was 0.3 mL/min, the column temperature was 30 C, and the samples were kept at 4 C. The mass
spectrometer was operated in negative ion mode, and the major
operating parameters for the ESI were as follows: capillary voltage,
4.0 kV; nebulization pressure, 20psi; temperature of drying gas,
300 C; drying gas flow, 3L/min; sheath gas temperature, 300 C;
sheath gas flow, 3L/min. Fragment voltage and collision energy
were independently optimized for each reagent. Data was acquired
using MassHunter Software (Agilent Technologies) in multiple reaction monitoring mode (MRM) by recording ion currents for the
following transitions: m/z 895.5 > 587.6 for RA with collision energy (CE) 60eV, m/z 798.6 > 651.6 for digoxin with CE 60eV, m/z
469.4 > 425.4 for glycyrrhetinic acid (IS) with CE 45eV.
2.6. siRNA transfection
siRNA-3323, siRNA-824, siRNA-2187 targeting human P-gp
(ABCB1/MDR1) and siRNA-control were purchased from Thermo
Fisher Scientific. For the transfection method, 1 105 cells/well
HBMECs were cultured in a 24-well plate until they reached a
confluence of 70e90%, and were then transfected with the final
concentration of 50 nM RNase-free water diluted siRNAs using
Lipofectamine 3000 Reagent (L3000-015, Thermo Fisher Scientific,
Waltham, MA, U.S.A.). Proteins were collected after 48 h of transfection, efficiency was detected via an immunoblotting assay.
2.7. Immunoblotting assay
Cell lysates from HBMEC were obtained using RIPA buffer
(Pierce) containing inhibitors for proteases and phosphatases
before two-fold sonication. To quantify each sample’s protein
concentration, BCA method based on the standard curve was performed to achieve such goal by using Pierce™ BCA Protein Assay Kit
Y.-y. Wang, C.-f. Jiang, X. Liu et al. Biochemical and Biophysical Research Communications 559 (2021) 222e229
223
bought from Invitrogen Life Technologies (Gibco, Invitrogen, Grand
Island, NY, USA). After that, the protein abundance was analyzed
through SDS-PAGE, then the electrophoretic gel was transferred
onto a PVDF membrane (Millipore) followed by immunoblotting.
The membrane was then blocked with skimmed milk (5%) for 1 h
and incubated at room temperature for 1.5 h with the following
primary antibodies: P-glycoprotein Polyclonal antibody (1:500, Cat
No. 22336-1-AP, Proteintech), anti-GAPDH antibody (mouse,
1:10000, ab8245, Abcam, China). The secondary horseradish
peroxidase (HRP)-conjugated antibodies, goat anti-rabbit IgG H&L
(HRP) (1:2000, 7074, CST) and goat anti-mouse IgG H&L (HRP)
(1:2000, 7076, CST), were washed for 1 h at room temperature
through Tris-buffer saline Tween (TBST) three times. Protein bands
were then collected and developed via enhanced chemiluminescence (ECL, Thermo Fisher Scientific, MA, USA).
2.8. Data analysis
Statistical analyses were carried out and performed using Prism
7 (GraphPad), Image J (Java, National Institutes of Health), and
Image Pro Plus 6.0 Software. Data was obtained from three or six
independent experiments. Significant statistical observations were
analyzed using ordinary one-way ANOVA with a subsequent Dunnett’s test or student’s t-test. P < 0.05 was considered statistically
significant. Error bars were shown as standard errors of the
mean ± S.D.
Fig. 1. Raddeanin A (RA) transmembrane trafficking in human brain microvascular endothelial cells. (A) The different extraction solvents including methanol, acetonitrile and
ethyl acetate for sample repreparation mediated RA intracellular accumulation variation in HBMECs. (B) Gradient pH values (pH ¼ 4.0, 6.0 and neutral 7.4) mediated RA intracellular
accumulation variation in HBMECs. (C) The time consuming ranged from 0 to 60min of HBSS solvent mediated RA intracellular accumulation variation in HBMECs under optimal
extraction solvent, ethyl acetate and pH ¼ 4 value condition. (D) Uptake kinetics analysis of RA intracellular accumulation in HBMECs under 20 min incubation and optimal
conditions. The final concentration of RA in the incubation system for each of (A, B, C) was 4 mM, Km and Vmax values for (D) were calculated based on the Michaelis-Menten curve.
****P < 0.0001, compared with each group. All the results are counted as Mean ± S.D. from six independent experiments according to the ordinary one-way ANOVA.
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3. Results
3.1. RA transcellular transport across HBMEC monolayers
To determine whether RA can be transported across HBMEC
monolayers, the LC-MS/MS method was applied to measure the
intracellular accumulation of RA inside of cells. The subsequent
results illustrated that both the incubation microenvironment pH
and extraction solvents can influence the intracellular accumulation of RA. Ethyl acetate solvent and acid condition (pH ¼ 4) were
shown to facilitate RA transmembrane trafficking in HBMECs
(Fig. 1A and B). Meanwhile, the appropriate incubation time for RA
was then measured. According to the data, the RA cellular
concentration was initially boosted then alleviated along with the
incubation time under optimal extraction solvent and pH values.
After 20min, the concentration of RA reached a steady state
(Fig. 1C). According to the appropriate aforementioned conditions,
the kinetic parameters for membrane transport of RA in consecutive gradient concentrations were calculated using the values of
Km ¼ 37.01 ± 2.116 mM and Vmax ¼ 9.412 ± 0.1375 nM/min/mg
protein. It is worth mentioning that the reaction rate did not change
much as the RA concentrations changed between 100 and 300 mM
(Fig. 1D). These results indicate that a certain concentration of RA
can be transported across the cell membrane and accumulate
within cells.
Fig. 2. Cellular concentration increase of Raddeanin A (RA) under P-gp protein expression inhibition. (A) Immunoblot analysis of P-gp protein expression in HBMECs after
transfected with siRNA-control/siRNA-824/siRNA-2187/siRNA-3322 for 48 h. Optical density value for each stripe was quantified and normalized to GAPDH expression. (B) P-gp
protein inhibition mediated 4 mM RA intracellular accumulation variation in HBMECs. (C) Uptake kinetics analysis of RA intracellular accumulation in HBMECs after P-gp protein
inhibition. Km and Vmax values for (C) were calculated based on the Michaelis-Menten curve. ****P < 0.0001, compared with negative control group. All the results are counted as
Mean ± S.D. from three (A) and six (B, C) independent experiments according to the student’s t-test.
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3.2. RA intracellular accumulation alteration under P-gp expression
inhibition
As we outlined above, P-gp is involved in the multiple triterpenoid saponins membrane efflux process. To investigate
whether RA membrane trafficking is related to the performance of
P-gp, we wanted to determine how the intracellular concentration
of RA was affected by P-gp down-regulation. Initially, the siRNAs
were transfected and reduced the P-gp in the HBMECs. The data
illustrated that the protein level of endogenous P-gp was downregulated (Fig. 2A). The siRNA-3322 was responsible for the
optimal reduction efficiency and was thus chosen for the following
purpose. After the P-gp was inhibited, the intracellular accumulation of RA was then measured. The data illustrated that the intracellular concentrations of RA significantly increased compared to
the control group under suitable conditions (Fig. 2B), which proved
our hypothesis that P-gp expression is connected with RA membrane trafficking in HBMECs. In addition, we found the Km value
under such conditions underwent a significant decrease, which
illustrated that blocking the P-gp expression could drive the
reduction in RA transport activity and affinity (Fig. 2C).
3.3. RA intracellular accumulation alteration combining the usage
of P-gp selective functional inhibitors
To further investigate whether functional changes of P-gp could
affect the accumulation of intracellular RA, the selective efflux
functional inhibitor of P-gp, LY335979, was applied to test it. The
classical substrate of P-gp, digoxin, was used as a positive control.
The data showed that adding 0.5 mM of LY335979 to either the
Fig. 3. Raddeanin A (RA) accumulation augment under chemical selective inhibitor mediated P-gp function suppression. (A) digoxin or (B) RA intracellular accumulation
determination in HBMECs incubated with 4 mM RA and 0.5 mM LY335979 to the contrasts of P-gp classical substrate, digoxin under same condition. (C) Immunoblot analysis of P-gp
protein expression in HBMECs after treatment with 0.5 mM LY335979 for 48 h. Optical density value for each stripe was quantified and normalized to GAPDH expression. **P < 0.01,
***P < 0.001 compared with digoxin or RA single treated group. All the results are counted as Mean ± S.D. from six (A, B) and three (C) independent experiments according to the
student’s t-test.
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digoxin or RA treated group presented a significant increase in the
intracellular accumulation compared to each of them being separately treated (Fig. 3A). Interestingly, LY335979 did not impact the
protein expression of P-gp (Fig. 3B), which strengthened the
conclusion that reducing the expression or function of P-gp also
restricts RA efflux, and increases its intracellular accumulation.
3.4. Cellular drug resistance for RA under the expression and
function inhibition of P-gp
To investigate how the RA cellular pharmacodynamic effect was
altered by impairing P-gp protein expression inhibition and functional loss, cell viability was determined to estimate its cellular
drug resistance in HBMECs. The data showed that either inhibition
or LY335979 mediated functional loss of P-gp can raise the half
maximal inhibitory concentration (IC50) of RA in HBMECs (Fig. 4A
and B). Moreover, such value was even up-regulated after inhibiting
both of the P-gp protein level and function to its substrates at the
same time (Fig. 4C). All of these factors that indicate that both the
expression and function of P-gp are connected with RA cerebrovascular endothelial trafficking, and the creation of intracellular
accumulation induced drug resistance.
4. Discussion
As an important effective factor of Anemone Raddeana Regel, RA
was not considered to be a cause of brain inflammation until a
recent report suggested that it can be used to repress the growth of
brain glioblastomas. To further investigate its membrane trafficking
mechanism across the BBB, HBMECs were applied to stimulate the
brain microenvironment, and then RA intracellular accumulation
was determined. In this study, we discovered that RA could penetrate the membrane and accumulate in the HBMECs. Meanwhile,
inhibiting the surface protein expression or transport function of
ABC transporter, P-gp jeopardizes the intracellular accumulation of
RA then induces its drug resistance. This means that P-gp participated in the trafficking of RA across membranes and is responsible
for its pharmacodynamic effect in cerebrovascular endothelial cells.
From the findings we obtained, we were able to infer that acidic
surroundings with lower pH values such as 4 or 6 facilitated the
movement of RA across the membranes of cerebrovascular
endothelial cells. It is worth mentioning that brain diseases or tumorigeneses such as epilepsy, cerebral ischemic strokes, and glioblastomas could produce an acidic microenvironment and facilitate
the efflux of drugs or small molecules across the BBB which lead to
their deteriorations [ [14e16]]. For instance, neuronal glutamate
(Glu) intracellular concentrations can be up-regulated in the hippocampus of epileptic rats by using pentylenetetrazol; saikosaponin, which ameliorates epilepsy, can also suppress the intracellular
Glu concentration [17]. Further study has confirmed that pH and
variability in passive permeability significantly influences the performance of ABC transporters, such as P-gp mediated protic substrate efflux [18]. Mitra et al. also came to a major conclusion that
low pH and acidic conditions leads to an increased apical-tobasolateral flux of colchicine, and reduces the bidirectional flux in
MDCK-MDR1 overexpressed cells. This means that the efflux
function of P-gp was jeopardized after microenvironmental acidic
pH change [19]. Moreover, the altered pH values outside the cellular
trigger drug resistance [20,21]. All of these factors indicate that low
pH increases the intercellular concentration of RA, and that the
drug resistance must be caused by this condition blocking the
efflux of RA.
The surface protein expression of P-gp in the cellular membrane
and part of its functional loss are mutually independent. In our
study, we found that the selective functional inhibitor, LY335979
(zosuquidar trihydrochloride), did not threaten P-gp protein
expression in HBMECs. In contrast, some selective inhibitors such
as cyclosporin A (CsA) or verapamil not only ameliorated P-gp activity, but also inhibited the protein level in some cell lines [
[22,23]]. However, the inhibition efficiency of LY335979 was found
to be more potent than cyclosporine A or other inhibitors of P-gp,
especially as it increased the oral uptake of paclitaxel in mice
[24,25]. It is worth mentioning that unlike protein degradation
mediated functional loss, such activity inhibition of P-gp regulated
by these inhibitors is reversible. Compared to cyclosporin A (CsA) or
verapamil, LY335979 can quickly recover the P-gp efflux function
from inhibition [26]. Further studies showed that using LY335979
as a treatment for human non-small cell lung carcinoma xenografted mice could increase the Taxol sensitivity when a dose of
20 mg/kg was used. Combining the usage of LY335979 and Taxol
was able to suppress tumor growth [27]. This example indicates
that a certain concentration of this inhibitor can also be used as a
Fig. 4. The drug Raddeanin A (RA) intracellular resistance investigation under protein expression or function inhibition of P-gp. (A) Cell viability analysis of HBMECs treated
with different concentrations of RA for another 48 h after siRNA targeting to P-gp transient transfection for 48 h or (B) 0.5 mM LY335979 pre-treatment for 48 h or (C) both of siRNA
targeting to P-gp transient transfection and 0.5 mM LY335979 pre-treatment for 48 h. IC50 values of RA in HBMECs for each group of (A, B, C) were measured and counted through
Log (inhibitor) vs. response from variable slope. All the results are counted as Mean ± S.D. from three independent experiments according to the student’s t-test.
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potential agonist to enhance intracellular RA pharmacodynamic
effects.
RA was usually applied to prevent the cellular resistance toward
other anti-tumor drugs from building up, such as 5-fluorouracil in
cholangiocarcinoma cells or doxorubicin in osteosarcoma cells.
However, no reports suggest that increased RA within cells could
induce resistance [9,28]. Our findings proved that inhibiting either
the expression or substrate transport of P-gp can enhance RA
cellular IC50, which contributes to the reduction in its cellular
pharmacodynamic effect. Meanwhile, cell viability is also hindered
as more RA accumulates. Interestingly, inhibiting the P-gp expression and function at the same time exacerbated the RA-caused
intracellular drug resistance. That is to say, using some agonists
such as dihydroxylated xanthone to either up-regulate the protein
expression of P-gp, or stimulate its efflux function to substrates
may have the potential to upgrade the RA effects [29]. Further assays still need to be performed to verify whether combining usage
of RA and P-gp agonists could strengthen RA’s roles in the brain.
In conclusion, our study supports the hypothesis that regulating
the expression and transport function of P-gp is connected with RA
intracellular accumulation and drug resistance in cerebrovascular
endothelial cells. These findings provide crucial bases for RA’s
trafficking across the BBB.
Author contributions
Y.W. and C.J. have designed and performed the integral experiments. G.C. and J.G. drafted this manuscript. G.C. and J.L. have
provided the editing and writing assistance and suggestions. X.L.
have critically revised it for important intellectual content. G.C. and
J.G. have approved the final version for publication. All the authors
have made contributions to the procedures of either the manuscript’s initiation or submission.
Declaration of competing interest
The authors have no conflicts of interest to declare.
Acknowledgments
This work was supported by the National Major Project of
“Traditional Chinese Medicine Standardization: Construction of
Seven Traditional Chinese Medicine Decoction Pieces” (Project
Approve Number: ZYBZH-Y-JL-25).
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