Caffeoylquinic acid derivatives rich extract from Gnaphalium pensylvanicum willd. Ameliorates hyperuricemia and acute gouty arthritis in animal model

Article abstract of DOI:10.1186/s12906-017-1834-9

Background: The Gnaphalium pensylvanicum willd. is used in China as a folk medicine to treat anti-inflammatory, cough and rheumatism arthritis. The aim of this study was to evaluate the potential of the extract of G. pensylvanicum to treat hyperuricemia and acute gouty arthritis in animal model. Methods: G. pensylvanicum extract was evaluated in an experimental model with potassium oxonate (PO) induced hyperuricemia in mice which was used to evaluate anti-hyperuricemia activity and xanthine oxidase (XO) inhibition. Therapies for acute gouty arthritis was also investigated on monosodium urate (MSU) crystal induced paw edema model. Results: G. pensylvanicum extract showed activity in reducing serum uric acid (Sur) through effect renal glucose transporter 9 (GLUT9), organic anion transporter 1 (OAT1) and urate transporter 1 (URAT1) mainly and inhibited XO activity in vivo of mice with PO induced hyperuricemia. The extract of G. pensylvanicum also showed significant anti-inflammatory activity and reduced the paw swelling on MSU crystal-induced paw edema model. Meanwhile, 13 caffeoylquinic acid derivatives and 1 flavone were identified by UPLC-ESI-MS/MS as the main active component of G. pensylvanicum. Conclusions: The extract of G. pensylvanicum showed significant effect on evaluated models and therefore may be active agents for the treatment of hyperuricemia and acute gouty arthritis.

Full text of DOI:10.1186/s12906-017-1834-9

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
DOI 10.1186/s12906-017-1834-9
RESEARCH ARTICLE
Open Access
Caffeoylquinic acid derivatives rich extract
from Gnaphalium pensylvanicum willd.
Ameliorates hyperuricemia and acute gouty
arthritis in animal model
Yan Jiang1, Yan Lin2, Yi-Juan Hu3, Xiao-Jun Song1, Hong-Hua Pan2 and Hong-Jian Zhang3*
Abstract
Background: The Gnaphalium pensylvanicum willd. is used in China as a folk medicine to treat anti-inflammatory,
cough and rheumatism arthritis. The aim of this study was to evaluate the potential of the extract of G.
pensylvanicum to treat hyperuricemia and acute gouty arthritis in animal model.
Methods: G. pensylvanicum extract was evaluated in an experimental model with potassium oxonate (PO) induced
hyperuricemia in mice which was used to evaluate anti-hyperuricemia activity and xanthine oxidase (XO) inhibition.
Therapies for acute gouty arthritis was also investigated on monosodium urate (MSU) crystal induced paw edema
model.
Results: G. pensylvanicum extract showed activity in reducing serum uric acid (Sur) through effect renal glucose
transporter 9 (GLUT9), organic anion transporter 1 (OAT1) and urate transporter 1 (URAT1) mainly and inhibited
XO activity in vivo of mice with PO induced hyperuricemia. The extract of G. pensylvanicum also showed significant
anti-inflammatory activity and reduced the paw swelling on MSU crystal-induced paw edema model. Meanwhile, 13
caffeoylquinic acid derivatives and 1 flavone were identified by UPLC-ESI-MS/MS as the main active component of
G. pensylvanicum.
Conclusions: The extract of G. pensylvanicum showed significant effect on evaluated models and therefore may be
active agents for the treatment of hyperuricemia and acute gouty arthritis.
Keywords: Gnaphalium pensylvanicum Willd., Caffeoylquinic acid derivatives, Hyperuricemia, Acute gouty arthritis,
Xanthine oxidase, Pro-inflammatory cytokines
Background
oxidase in liver is the key enzyme which catalyzes
Hyperuricemia is characterized by the high level of uric production of UA, and urate transport proteins in renal
acid (UA) in blood, which may derive from the increase are the main transporters for UA clearance [2, 3]. In
in purine metabolism and impairment of renal excretion addition, the crystallization of uric acid crystal within
of UA. Moreover, hyperuricemia is a key causal factor joints and tissues can drive an inflammatory response,
for the development of gout and many other diseases therefore, the goal of the clinical treatment of gout is to
such as cardiovascular and renal diseases, hypertension reduce the Sur level and inflammatory response [4].
and metabolic syndrome, etc. [1]. The terminal product
Impaired renal excretion of UA rather than UA
of purine nucleotides degradation is UA. Xanthine overproduction was considered to be the major cause
of hyperuricemia. Urate transport-related proteins,
such as organic anion transporters involved in renal
* Correspondence: jian871211@sina.com
urate excretion and reabsorption, play a key role in
maintaining urate homeostasis. Renal tubular handling
of UA is dependent on urate transporters such as
3Key Laboratory of Research and Development of Chinese Medicine of
Zhejiang Province, Zhejiang Academy of Traditional Chinese Medicine, 132
Tianmushan Road, Hangzhou 310007, China
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
Page 2 of 10
URAT1, GLUT9 and OAT1. URAT1, which is located standard laboratory reagents of analytical grade and were
in the brush border membrane of the renal tubular purchased locally. Antibodies against URATl, GLUT9,
epithelial cell is responsible for reabsorption of urate OAT1 and β-actin were purchased from ProteinTech
transporter. This protein is encoded by the SLC22A12 Group (Chicago, IL), Abcam Inc. (Cambridge, MA,
gene, which is a genetic risk factor for inducing hy- USA), and Santa Cruz Biotechnology (USA). Polyvinyli-
peruricemia [5]. GLUT9 (encoded by the SLC2A9 dene fluoride (PVDF) membrane was purchased from
gene) was found to be a new target for the treatment Merck (Germany). Tissue Protein Extraction Kit was
of hyperuricemia as a high-capacity urate transporter purchased from KeyGEN Biotech. CO., Ltd. (Nanjing,
[6]. In addition, the member of organic anion trans- China). Enhanced bicinchoninic acid (BCA) protein
porter 1 (OAT1, SLC22A6) distributed at the basolat- assay kit was purchased from Tiangen Biotech (China).
eral membrane contribute to renal urate secretion by Monosodium urate (MSU) crystals were prepared
mediating urate transport across cell membranes in according to the previously described method [17].
renal proximal tubules [7]. These results suggest that Approximately 1 g of uric acid was dissolved and heated
URAT1, OAT1 and GLUT9 might play important in 200 mL of H2O with 6 mL NaOH (1 N), adjusted to
roles in regulating urate excretion, and infecting the pH 8.9, cooled overnight at 4 °C, washed, and dried.
hyperuricemia. Thus, these urate transporters have Needle-like crystals were recovered and were suspended
proved to be very important targets for hypouricemia in sterile saline and Twain 80 (20 mg/mL).
drug discovery [5–8].
Gnaphalium pensylvanicum willd. is an annual herb- Plant material
aceous plant and widely distributed in many regions of Gnaphalium pensylvanicum willd. Was collected from
China. It has been used as a folk medicine for the relief Yuhuan by Xi-Biao Zhang, Zhejiang province, PR China
of anti-inflammatory, cough and rheumatism arthritis in October 2015. It was identified by Licensed Pharmacist
[9]. Our previous study demonstrated that G. pensylva- Yi-Bo Feng, Tongde Hospital of Zhejiang Province and
nicum extract (GPE) possess many caffeoylquinic acid the voucher specimens (SYSQC20151001) was deposited
derivatives and showed potent inhibitory activity against at the Key Laboratory of Research and Development of
XO in vitro. Meanwhile, caffeoylquinic acid derivatives Chinese Medicine of Zhejiang Province, Zhejiang
had been report that they showed potential anti- Academy of Traditional Chinese Medicine.
huperuricemia and anti-gouty arthritis effect [10–13]. In
addition, previous pharmacology works on various
Preparation of plant extracts
species of the Gnaphalium genus and its components The dried G. pensylvanicum (500 g) was extracted using
possess XO inhibitory activity, showed significantly anti- 10 L 95% (v/v) ethanol for 3 times (3 × 60 min) by heat
hyperuricemia and anti-gouty arthritis [14–16]. Thus, reflux extraction. Then solvents were removed with
further studies are needed to clarify the exact mecha- vacuum rotary evaporation to yield 34.38 g of residue.
nisms of GPE in improving hyperuricemia and gout. The extraction yield of G. pensylvanicum was 6.88% (w/
Here, we assessed the effects of GPE with respect to its w) which was kept at −20 °C until used.
possible mechanisms by determining the XO, Sur,
interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), UPLC-ESI-MS/MS analysis of G. pensylvanicum
and the expression level of mUART1, mOAT1 and Sample preparation
mGLUT9 in renal were also carried out to comprehen- The G. pensylvanicum extracts were prepared by
sively study the mechanism of G. pensylvanicum against ultrasonic extraction of 1.0 g of powdered dried plant
hyperuricemia and gout.
material in 20 mL methanol for 1.5 h at room
temperature. The extract was filtered and evaporated in
a rotary evaporator. The final extracts were dissolved
with 2.0 mL and filtered through a 0.25 mm fluorpore
Methods
Chemicals and reagents
Potassium oxonate, allopurinol and uric acid were pur- membrane (Millipore, MA, USA) prior to injection into
chased from Sigma-Aldrich Chemical Company. Colchi- the UPLC-ESI-MS/MS system.
cine tablets (each tablet contains colchicine in a dosage
of 0.5 mg) were purchased from Kunming Pharmaceut- UPLC-ESI-MS/MS analyses
ical Co., Ltd. (Yunnan, China). Commercial kit used for The Acquity Ultra Performance LC-system (Waters,
determining uric acid and XO activity were obtained Milford, MA) with TQ detector was employed to
from the Jiancheng Institute of Biotechnology (Nanjing, analyses. The UPLC analyses were performed using an
China). Enzyme-linked immunosorbent assay (ELISA) Acquity UPLC BEH column (2.1 × 100 mm i.d., 1.7 μm)
kits for IL-1β and TNF-α assay were purchased from with a binary mobile phase. Solvetn A was 0.1% formic
Assay R&D Systems. All other reagents used were acid aqueous and B was acetonitrile.

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
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The gradient elution at room temperature was as 15– ICR mice were divided into 6 groups, and each
60% (v/v) B at 15 min. The flow rate was 0.25 mL/min group including 10 animals. They were blank, model,
and the sample volume injected was 1 μL. The detection colchicine (1 mg/kg/day), the GPE (100, 200 and
wavelength was 250 nm. LC-MS analysis was carried out 400 mg/kg/day) groups. Drugs were intragastricly
using a Waters TQ Detector equipped with electrospray feed once daily for 7 days. On the 7th day, the MSU
ionization (ESI) source. The MS was operated in negative crystal was injected 1 h later after drugs were given.
mode, and the data were acquired in scan mode using a Drugs continued to be given for 1 day after injection
m/z range of 150 to 1000. The data acquisition and ana- of MSU crystal. Paw swelling was expressed as thick-
lyses was carried out with Masslynx Workstation Software ness variation. Paw thickness was measured with
(version 4.1.896).
digital display micrometer gauge (Shanghai ChuanLu
Measuring Tool Co., Ltd) at 0, 1, 4, 12 and 24 h
after MSU injection. All doses were expressed as mg
per kg body weight of the respective drugs.
Preparation of drugs and test solutions for in vivo
methods
All solution and potassium oxonate suspension were
prepared dependent on the medium weight of each
group. Allopurinol (4 mg/mL), colchicine (100 mg/L),
low content G. pensylvanicum (LGP) (20 mg/mL),
middle content G. pensylvanicum (MGP) (40 mg/mL)
and high content G. pensylvanicum (HGP) (80 mg/mL)
were solubilized in 0.5% DMSO and physiological saline.
Potassium oxonate was prepared in suspension with
0.9% NaCl solution (30 mg/mL). Monosodium urate
crystals were suspended in 0.9% sterile saline (25 mg/
mL) and twain 80.
Blood and tissue processing
Models of hyperuricemia
On the 7th day, 1 h after final administration, blood
samples were collected and allowed to clot for 1 h at
room temperature, and centrifuged at 3000 rpm for
10 min to obtain serum. Serum samples were used
for the estimation of uric acid level and stored at
−20 °C until testing. The liver tissues were also dis-
sected quickly on an ice-plate for the XO assay. The
renal were cut into tubes for histological and western
blotting analysis. All the samples were frozen at −80 °
C until used for assays.
Experimental animals
60 male Kun-Ming mice (18 ~ 22 g) and 60 male ICR
mice (18 ~ 22 g) were obtained from Experimental Ani-
mal Center of Zhejiang Academy of Medical sciences.
Animals were housed in plastic cages and maintained on
a 12/12 h light/dark cycle and had free access to
standard pellet diet and tap water. All experimental pro-
cedures were approved by the Guidance Suggestions for
the Care and Use of Experimental Animals in Zhejiang
University.
Models of acute gouty arthritis
Blood samples were collected 24 h after MSU injection
and allowed to clot for approximately 1 h at room
temperature and then centrifuged to obtain the serum.
Serum samples were stored at −20 °C until testing for
cytokines levels were assayed. The mice were then
sacrificed and paw surrounding tissues were collected
for histological analyses and frozen at −80 °C.
Models of hyperuricemia and drug treatment
Uricase inhibitor PO was used to induce the hyperurice-
mia model in mice, as described previously. Sixty male
Kun-Ming mice were divided into six groups: blank,
model, allopurinol (40 mg/kg/day), and the GPE (100,
200 and 400 mg/kg/day) groups. The GPE and allopur-
inol were given continuously every 24 h six days before
hyperuricemia was induced. Normal saline was given to
the blank group. The dosages of the GPE were based on
the effective dosages in the clinic [9]. Mice were injected
Uricosuric activity in mice
Sur was measured by the enzymatic colorimetric method
using a standard diagnostic kit (Jiancheng, Nanjing),
according to manufacturer’s instructions.
Assays inhibition of liver XO activity
intraperitoneally with PO (300 mg/kg) to induce hyper- The livers were homogenized in ice-cold saline (9
uricemia one hour after the last intragastric administra- volumes), then centrifuged at 8000 rpm for 10 min
tion of GPE and allopurinol on the 7th day.
at 4 °C. After centrifugation, the lipid layer was care-
fully removed and the final supernatant fraction was
used for XO activity assays. The XO activity in liver
Models of acute gouty arthritis and drug treatment
The model of acute gouty arthritis was induced by was determined using a standard diagnostic kits
hypodermic injection of 0.1 mL (10 mg) of MSU (Jiancheng, Nanjing). The liver XO enzyme activity
crystal suspension into the right foot pad. The male is expressed as mol/min per g protein (U/g).

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
Page 4 of 10
Western blotting analysis of mGLUT9, mOAT1 and
mURAT1 in kidney tissues
solution, embedded in paraffin and sectioned at 5 μm,
followed by staining with H & E and examined under
Total protein was obtained from the renal using the the light microscope at a 200× magnification.
Tissue Protein Extraction Kit (KeyGEN Biotech. CO.,
Ltd., China). Briefly, kidney were collected and lysed
Statistical analysis
in appropriate cold lysis buffer contained 1 mM phe-
nylmethylsulfonyl fluoride (PMSF). After centrifuga-
tion (15,000 g for 15 min at 4 °C), the total protein
was obtained in the supernatant. The protein level
was quantified of the BCA protein assay kit (Tiangen
Biotech Co., Ltd., China), according to the manufac-
turer’s instructions. Protein sample (7 mg/mL) was
denatured by mixing with an equal volume of 2 sam-
ple loading buffer, and boiling at 100 °C for 5 min.
Samples were resolved by electrophoresis with 10%
SDS-PAGE and transferred onto a PVDF membrane
(Merk, Germany). After blocking non-specific binding
sites for 3 h with 5% dried skim milk dissolved in
PBST (0.05% Tween 20), the membranes were indi-
vidually incubated for overnight with anti-URAT
(1:500 dilution), anti-GLUT9 (1:500 dilution), anti-
OAT1 (1:500 dilution) as well as anti-β-actin (1:500
dilution). Develop the color of the blot rocking in 3,
3′-diaminobenzidine (DAB) substrate solution. Stop
the reaction by pouring out the substrate after the ex-
pected band appears, then well rinsed with distilled
water repeatedly. Dry the membrane and store it in
the dark place. The data were analyzed via densitom-
etry using Molecular Analyst software (Bio-Rad La-
boratories, Hercules, CA) and quantitated levels were
normalized to their respective blotting from β-actin.
All results were expressed as the mean standard error
of the mean (SEM). The one-way analysis of variance
(ANOVA) was used to determine the level of signifi-
cance followed by using GraphPad Prism 5.0 Software
(GraphPad Software, San Diego, CA, USA).
Results
Screening of chemical compounds in the G.
pensylvanicum
The base peak chromatogram that resulted for the GPE
is depicted in Fig. 1, where the peaks are numbered ac-
cording to their elution order. Totally 14 metabolites
present in G. pensylvanicum were identified by interpret-
ation of their DAD, MS and MS/MS spectra found by
UPLC-ESI-MS/MS (Table 1) combined with the data
provided in the literature [18–22]. The analysis of the
methanol extract revealed that caffeoylquinic acid
derivatives were the major classes of secondary metabo-
lites in G. pensylvanicum.
Effects of G. pensylvanicum on uric acid levels in serum
As shown in Table 2, compared with blank group
(5606.7
1564.9 μmol/L), uric acid level of serum in
3423.7 μmol/L) was
model group mice (10,675.5
significantly increased after PO administration.
Allopurinol, as a positive control drug, significantly
reduced uric acid level of serum compared with
model group mice (p < 0.01). All doses of G. pen-
sylvanicum markedly decreased uric acid level a dose-
dependent, compared with model group mice.
Meanwhile, as shown in Fig. 2, all doses of G. pensyl-
vanicum used in this study did not significant effect
weight compared with blank group mice. But in the
allopurinol group the allopurinol significantly
inhibited the weight growth.
Assessment of paw swelling
The inflammation was quantified by measuring the
thickness of the paw at 0, 1, 4, 12 and 24 h after MSU
crystal injections.
Cytokines assay
The levels of IL-1β and TNF-α in the serum were deter-
mined using ELISA kits (Assay, R&D Systems), accor-
ding to manufacturer’s instructions.
Histological analyses
Models of hyperuricemia
Mice’s kidneys were fixed for 24 h at room temperature
in 4% form paraformaldehyde. Paraffin sections (4 μm Effect of G. pensylvanicum on XO activity in liver
thick) were prepared from each kidney, stained with As shown in Table 2, PO administration induced a
hematoxylin and eosin (H & E) and examined under significant advancement of XO activity in liver of
light miscroscope (Eclipse TI-SR, Nikon Instruments model group mice compared with blank group mice
Inc., Japan) at a 200× magnification.
(p < 0.01). Allopurinol significantly attenuated the XO
activity in liver (5.34 0.65 U/g) of hyperuricemia
mice model (54.28 8.17 U/g). GPE also significantly
Models of acute gouty arthritis
The paw region of MSU induced gouty arthritis mice exhibited a dose-dependent XO activity in liver of hy-
was isolated and decalcified in 10% formaldehyde peruricemia mice model.

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
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Fig. 1 Base peak chromatogram of G. pensylvanicum extract by UPLC-ESI-MS/MS in the negative ion mode. Peak labelling represents the
compounds identified
Effects of G. pensylvanicum on expressions of mGLUT9,
mURAT1 and mOAT1 in kidney
model group. Allopurinol, could also regulated the
expression levels of renal mGLUT9, mOAT1 and mU-
As shown in Fig. 3, compared with blank group, PO RAT1 protein in hyperuricemia mice compared with
administration induced a significant down-regulation of model group as showed in Fig. 3.
the mOAT1 protein expressions in mice kidney, as well
as a significant up-regulation of the mGLUT9 and mUR- Effect of G. pensylvanicum on improving renal
ATl protein expression. All doses of G. pensylvanicum dysfunction
significantly inhibited the mURATl and mGLUT9 As results above, levels of Sur could be restored by all
protein expression, and 400 mg/kg G. pensylvanicum doses of G. pensylvanicum in hyperuricemia mice. Our
significantly elevated the mOAT1 protein expressions in histological data support the observations of uric acid
hyperuricemia mice kidney, respectively, compared with level changes after treatment, and is consistent with the
Table 1 Proposed compounds detected in G. pensylvanicum obtained by UPLC-ESI- MS/MS
Peak
Rt (min)
UV
m/z
MS/MS fragments
Proposed compound
Reference
λ
max (nm)
1
2.41
2.64
4.69
5.31
5.71
5.89
6.34
6.61
6.68
7.03
7.54
8.01
8.21
8.26
213.7, 325.7
322.7
352.9
352.9
367.0
447.1
515.2
515.3
515.2
515.2
601.0
528.7
529.2
529.0
557.0
615.4
146.1
3-caffeoylquinic acid
[18–22]
[18–21]
[20, 21]
[22]
2
249.2
4-caffeoylquinic acid
3
235.7, 325.7
252.7, 345.7
219.7, 242.7, 324.7
218.7, 242.7, 326.7
218.7, 243.7, 326.7
244.7, 326.7
243.7, 327.7
243.7, 325.7
243.7, 326.7
244.7, 328.7
243.7, 327.7
243.7, 326.7
308.9, 201.9, 175.3
435.7, 293.5, 106.6
428.8, 334.7, 94.9
490.9, 352.0, 179.1
352.9, 173.0
feruloylquinic acid
4
luteolin-7-O-glucoside
5
1, 3-di-caffeoylquinic acid
1, 4-di-caffeoylquinic acid
3, 4-di-caffeoylquinic acid
4, 5-di-caffeoylquinic acid
3-methoxyoxaloyl-1, 5-di-caffeoylquinic acid
1-caffeoyl-3-feruloylquinic acid
4-caffeoyl-3-feruloylquinic acid
4-caffeoyl-5-feruloylquinic acid
acetyldi-caffoylquinic acids
succinoyl dicaffeoylquinic acids
[19–21]
[19, 20]
[18–21]
[18–21]
[22]
6
7
8
434.1, 346.3, 178.8
288.4, 240.9
9
10
11
12
13
14
367.4, 172.8, 149.0
362.7, 349.2, 179.2
374.0, 179.0, 155.6
395.4, 233.3, 173.0
453.5, 360.9, 235.8
[21]
[18, 21]
[18, 21]
[22]
[20]

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
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Table 2 Effects of G. pensylvanicum and Allopurinol on Sur and XO active in Liver of PO-Induced Hyperuricemic Mice
Groups
Dose (mg/kg)
Sur(μmol/L)
Liver XO (U/g protein)
Blank
-
5606.7 1564.9
10,675.5 3423.7**
4280.1 2229.5##
8388.6 1546.3**
7288.3 4615.0**
6012.9 783.6**
11.14 1.06
Model
-
54.28 8.17##
5.34 0.65**
10.85 1.53**
8.48 0.85**
Allopurinol
G. pensylvanicum extract
40
100
200
400
6.09 0.45**
Data represent mean SEM for 10 mice; #p < 0.05, ##p < 0.01 compared with Blank group (independent samples t-test); *p < 0.05, **p < 0.01 compared with
Model group
Sur observations in hyperuricemia mice model. Histo- 200, and 400 mg/kg) and colchicine (1 mg/kg) treatment
logical examination of renal tissue from the normal mice decreases the paw diameter significantly in MSU crystal-
(Fig. 4a) showed normal renal architecture. Histological induced mice.
analyses showed that the renal tubules were shrank in
Joints synovial and surrounding tissues were obtained
the model mice (Fig. 4b), compared with the normal from the knee joint 2 days after the MSU injection. In the
mice. These pathological states were ameliorated in blank control group showed normal and no inflammatory
some degree by treating with G. pensylvanicum (Fig. 4.). cell infiltration. Model group showed apparent joint in-
However, it is worth noting that allopurinol treatment flammation, a number of inflammatory cells infiltration,
induced the surface of kidney in allopurinol group and hyperplasia synovial. These pathological states were
showed white were different from the other groups. ameliorated in some degree by treating with colchicine
These results suggested that allopurinol may have the and G. pensylvanicum dose (Fig. 6). The results demon-
renal toxicity. These results suggested that allopurinol strated that G. pensylvanicum and colchicine could
may have the renal toxicity.
improve the symptoms of MSU crystal-induced mice, and
they could decrease the infiltration of inflammatory cells.
Effects of G. pensylvanicum on the MSU crystal-induced
mice
MSU crystals injection caused a significant increase in paw
Effects of G. pensylvanicum extracts on the levels of IL-1β
and TNF-α in MSU crystal-induced mice
swelling compared with the model group. As shown in Fig. To investigate the mechanism of G. pensylvanicum in the
5, the paw volume of MSU-induced mice revealed an in- improvement of paw swelling of MSU crystal-induced mice,
crease in ankle diameter, whereas G. pensylvanicum (100, the levels of two pro-inflammatory cytokines in the serum
Fig. 2 Effects of G. pensylvanicum on body weights of hyperuricemia mice. Data are expressed as mean SEM (n = 10)

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
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Fig. 3 Effects of G. pensylvanicum and Allopurinol on protein expression of mGLUT9, mOAT1 and mURAT1 in renal tissues in hyperuricemia mice
by Western blot analysis. N = 10 per group. ##p < 0.01, ###p < 0.001 compare with blank; **p < 0.01, ***p < 0.001 compare with model
were determined. As shown in Fig. 7, MSU crystal induced conditions such as gout. Although the disease afflicts
a significant increase of TNF-α and IL-1β levels in serum humans for years, there were a limited number of drugs
(p < 0.01, p < 0.01, respectively.). Post-hoc analysis indicated currently used in clinical for treating, but there are side
that G. pensylvanicum extracts decreased TNF-α (100, 200, effects associated with its use [23, 24]. Natural plant
400 mg/kg: p < 0.01) and IL-1β (100 mg/kg: p < 0.05; 200
products were the most popular source of complemen-
mg/kg: p < 0.05; 400 mg/kg: p < 0.01, respectively.) levels. In tary and alternative medicines for novel anti-
addition, the positive drug colchicine (1 mg/kg) also reduced hyperuricemia agents [25].
the TNF-α and IL-1β levels in serum (p < 0.01, p < 0.01, re-
spectively.) compared to the model group.
Gout is a form of inflammatory arthritis character-
ized by recurrent attacks. And then inflammation is
one of the major problems of gout patients has been
evaluated [26]. It is well accepted that pro-
Discussion
Hyperuricemia is a disorder of uric acid metabolism dis- inflammatory cytokines such as TNF-α and IL-1β help
ease associated with the development of pathological to propagate the extension of a local or systemic
Fig. 4 Histology of the kidney, H & E Stain, 200×. (a) Blank group, (b) Model group with PO, (c) Allopurinol 40 mg/kg, (d) G. pensylvanicum
100 mg/kg, (e) G. pensylvanicum 200 mg/kg and (f) G. pensylvanicum 400 mg/kg

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
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Fig. 5 Effect of G. pensylvanicum and colchicine on MSU crystal-induced paw edema in mice. Data are expressed as mean SEM (n = 10).
*
##p < 0.01 compare with Blank group; **p < 0.01, p < 0.05 compare with Model group
inflammatory process [27]. So, anti-inflammatory was may represent efficacy in the treatment of hyperurice-
considered as the main method for the treatment of gout.
mia and gout. In addition, G. pensylvanicum extracts
In this study, G. pensylvanicum extracts were able showed renal protection which different from the
to inhibit the residual activity of xanthine oxidase in allopurinol has renal toxicity.
vivo and improvement MSU crystal-induced mice paw
In the phytochemical investigations of G. pensylva-
swelling. Meanwhile, administration of the G. pensyl- nicum resulted suggested this plant was rich in caf-
vanicum extracts caused a reduction in Sur level in feoylquinic acid derivatives. Among these compounds,
hyperuricemia mice by regulating the abnormal ex- chlorogenic acid [13], 3,5-dicaffeoylquinic acid [11],
pression levels of mURAT1, mOAT1 and mGLUT9. 4,5-dicaffeoylquinic acid methyl ester [10] and 1,5-
Thus, G. pensylvanicum extracts were able to reduce dicaffeoylquinic acid [12] showed significant inhibition
hyperuricemia and against gouty arthritis through of xanthine oxidase activity and anti-hyperuricemia.
these pathways: increasing uric acid excretion, inhibit- Thus, anti-hyperuricemia effects by uricosuric or
ing xanthine oxidase activity and pro-inflammatory inhibition of xanthine oxidase way by the G. pensylva-
cytokines secretion, indicating that G. pensylvanicum nicum extracts can be partially attributed to
Fig. 6 Effects of G. pensylvanicum on pathological changes in paw surrounding tissue of mice. All sections were stained with H & E Stain
(magnification A-F, ×200). a Blank group, (b) Model group with MSU, (c) Colchicine 1 mg/kg, (d) G. pensylvanicum 100 mg/kg, (e) G. pensylvanicum
200 mg/kg and (f) G. pensylvanicum 400 mg/kg

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
Page 9 of 10
Fig. 7 Effects of G. pensylvanicum and colchicine on the levels of IL-1β (a), and TNF-α (b) in the serum were measured 24 h after hypodermic
injection of MSU crystals. Results were shown as mean SEM (n = 10). ##p < 0.01 vs. Blank group; *p < 0.05, **p < 0.01 vs. Model group
caffeoylquinic acid derivatives. It could also effectively
regulate PO-induced changes of renal mURAT1,
mGLUT9 and mOAT1 expressions, resulting in the
enhancement of uric acid excretion. However, further
investigation is needed to evaluate potential clinical
using of G. pensylvanicum and its bioactive compo-
nents. These insights are relevant to further develop
the new drugs from G. pensylvanicum for the
treatment of gouty.
levels of uric acid; TNF-α: Tumor necrosis factor-α; UA: Uric acid; URAT1: Urate
transporter 1; XO: Xanthine oxidase
Acknowledgments
The present study was supported by Key Laboratory of Research and
Development of Chinese Medicine of Zhejiang Province, Zhejiang Institute of
Traditional Chinese Medicine for providing all the necessary facilities to carry
out this work.
Funding
This work was financially supported by the Program of Zhejiang Provincial
Department of Science and Technology (2016C31001), Project of
Administration of Traditional Chinese Medicine of Zhejiang Province
(2A21624).
Conclusions
The present results clearly demonstrated that the G.
pensylvanicum showed remarkable anti-hyperuricemia
and anti-gouty arthritis activity. The tested extract pos-
sesses potent uricosuric effect in hyperuricemia mice
through effect renal mGLUT9, mOAT1 and mURAT1
mainly and inhibiting XO activity in a certain extent,
which are attributable to the enhancement of uric acid
excretion and protect hyperuricemia-induced renal
dysfunction. This beneficial anti-gouty arthritis effect
may be mediated, at least in part, by inhibiting the pro-
duction of IL-1β and TNF-α. Meanwhile, the caffeoyl-
quinic acid derivatives were identified by UPLC-ESI-MS/
MS as the main active ingredients of G. pensylvanicum.
Therefore, G. pensylvanicum extracts can be considered
promising in the treatment of diseases like hyperurice-
mia and gout. The safety and potential clinical utility of
the G. pensylvanicum in the uric acid excretion and
renal dysfunction reduction needs to be confirmed and
evaluate by future studies. In addition, the active
contents of G. pensylvanicum are engaged to explain.
Availability of data and materials
The datasets supporting the conclusions of this article are presented in this
main paper. Plant material used in this study was identified at Tongde
Hospital of Zhejiang Province and voucher specimens are deposited at the
Key Laboratory of Research and Development of Chinese Medicine of
Zhejiang Province, Zhejiang Academy of Traditional Chinese Medicine.
Authors’ contributions
HJZ designed the experiments, and was a major contributor in writing the
manuscript. YJ, YL and YJH carried out the laboratory experiments. XJS and
HHP analyzed the data, interpreted the results and prepared figures. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval
All animal studies were performed using protocols approved by the Animal
Care and Use Committee of Zhejiang University.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Abbreviations
ANOVA: Analysis of variance; BCA: Bicinchoninic acid; ELISA: Enzyme-linked
immunosorbent assay; GLUT9: Glucose transporter 9; GPE: Gnaphalium
pensylvanicum extract; H & E: Hematoxylin and eosin; HGP: High content of
G. pensylvanicum extract; IL-1β: Interleukin-1β; LGP: Low content of G.
pensylvanicum extract; MGP: Middle content of G. pensylvanicum extract;
MSU: Monosodium urate; OAT1: Organic anion transporter 1;
PMSF: Phenylmethylsulfonyl fluoride; PO: Potassium oxonate;
PVDF: Polyvinylidene fluoride; SEM: Standard error of the mean; Sur: Serum
Author details
1Dispensary of Traditional Chinese Medicine, Hangzhou First People’s
Hospital, 261 Huansha Road, Hangzhou 310006, China. 2Department of
Pharmacy, Tongde Hospital of Zhejiang Province, 234 Gucui Road, Hangzhou
310012, China. 3Key Laboratory of Research and Development of Chinese
Medicine of Zhejiang Province, Zhejiang Academy of Traditional Chinese
Medicine, 132 Tianmushan Road, Hangzhou 310007, China.

Jiang et al. BMC Complementary and Alternative Medicine (2017) 17:320
Page 10 of 10
Received: 28 March 2017 Accepted: 8 June 2017
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