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Chinese Chemical Letters xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Chinese Chemical Letters

journal homepage: www.elsevier.com/locate/cclet

Original article

Screening the anti-gout traditional herbs from TCM using an in vitro

method
Wei-Jia Chen a, Yi Wu a, Xin Zhao a, Shu Liu b,*, Feng-Rui Song b, Zhong-Ying Liu a,*,
Zhi-Qiang Liu b
a
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
b
National Center of Mass Spectrometry in Changchun & Jilin Province Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun
Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

A R T I C L E I N F O A B S T R A C T

Article history: The aim of this work was to evaluate the ability of 33 herbal extracts in inhibiting the acute inflammation
Received 13 April 2016 and xanthine oxidase (XOD) activity. The anti-inflammation effects of the herbal extracts were detected
Received in revised form 6 May 2016 by an in vitro cell model, which was established by stimulating human umbilical vein endothelial cells
Accepted 12 May 2016
(HUVEC) using sodium urate (MSU). In this model, the intercellular adhesion molecule-1 (ICAM-1) and
Available online xxx
interleukin-1 beta (IL-1b) were expressed, and the anti-inflammation effects of herbal extracts were
evaluated by detecting the content changes of ICAM-1 and IL-1b in cell lysates and cell culture
Keywords:
supernates using an enzyme-linked immunosorbent assay (ELISA). Moreover, an ultrahigh performance
Gout
liquid chromatography and tandem mass spectrometry (UPLC–MS/MS) method was used for the
Inflammation
Human umbilical vein endothelial cells detection of XOD activity and the screening of XOD inhibitors in this research. The amount of uric acid
Xanthine oxidase from each analyte was directly detected using the multiple reaction monitoring mode and the uric acid
UPLC–MS/MS level could be reduced via the addition of an inhibitor. Results indicated that Salviae Miltiorrhizae Radix et
Rhizome, Rhei Radix et Rhizoma, Polygoni Cuspidati Rhizoma et Radix, Selaginellae Herba, Paeoniae Radix
Rubra, especially Ginkgo Folium seemed to be more effective in anti-inflammation and inhibiting XOD
activity. The anti-inflammation and enzyme inhibitory activities of the herbal extracts may be correlated
with their bioactive components. And the differences between the herbal extracts were correlated with
the amount of flavonoid and anthraquinone components. In our study, we have investigated the
potential anti-inflammation bioactivity of 33 herbal extracts in vitro, which could provide a reference for
further in vivo research in the prevention and treatment of gout.
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.

1. Introduction tophi depositions, and other chronic diseases, such as common

Gout is an inflammatory arthritis that is induced by the
precipitation of monosodium uratemonohydrate (MSU) crystals in
articular joints and periarticular tissues. The incidence of the
disease has increased annually, which may be associated with
dietary and lifestyle changes [1]. The global epidemiology of gout
data showed that the worldwide prevalence of gout ranges from
0.1% to approximately 10%, and developed countries tend to have a
higher burden of gout than developing countries, while gout
incidence of men is higher than women by 2–6 fold [2]. Most
frequently, gout causes recurrent attacks of acute arthritis and
* Corresponding authors.
E-mail addresses: mslab20@ciac.ac.cn (S. Liu), Liuzy@jlu.edu.cn (Z.-Y. Liu).
complications of renal [3], metabolic syndrome [4], hypertension
and cardiovascular diseases [5,6]. Therefore, the treatment of gout
has become a serious problem to solve.
New advances in gout research have revealed that there are
many complexes cellular mechanisms on the powerful inflamma-
tory response induced by MSU-stimulation, mainly involving an
early increase of pro-inflammatory cytokines, and followed in few
days by their reduction along with an increase of anti-inflamma-
tory cytokines [7]. The high levels of cytokines such as IL-1b, ICAM-
1, and tumor necrosis factor-a (TNF-a) have been detected in the
synovial fluid of gout patients [8,9]. Uric acid is also a main risk
factor for gout and hyperuricemia and formed by the oxidation of
hypoxanthine to xanthine. Xanthine can be further oxidized to uric
acid by XOD, releasing peroxide anion in the process. XOD is a key
enzyme in the occurrence and development of gout, thus, it is

http://dx.doi.org/10.1016/j.cclet.2016.05.022
1001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: W.-J. Chen, et al., Screening the anti-gout traditional herbs from TCM using an in vitro method, Chin.
Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.05.022
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2 W.-J. Chen et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
important to inhibit the activity of XOD to control uric acid
accumulation and deposit.
At present, a number of anti-gout drugs including anti-
inflammatory drugs (indomethacin and colchicine) and the
urate-lowering drugs (allopurinol and benzbromarone) are used
as the primary clinical therapies to treat gout and hyperuricemia.
Allopurinol is the most widely used first-line drug to control
hyperuricemia via inhibiting XOD activity, and thus inhibiting the
generation of uric acid from hypoxanthine [10]. Indomethacin as
the most potent non-steroidal anti-inflammatory drugs (NSAIDs)
has been widely used in the treatment of acute gout to date [11,12].
However, few drugs produce satisfactory therapeutic efficacy
without causing adverse effects, including gastric damage, renal
toxicity and hypersensitivity [13–16], thus limit their clinical uses.
Consequently, new drugs lower adverse effects from natural
resources have raised hope.
Traditional Chinese medicine (TCM) is still an important
component of human health care involving a variety of herbs
and herbal combinations, in which, a large number of plant species
are used for cure of diseases, such as inflammatory, analgesic and
immune diseases [17–19]. TCM has fewer adverse effects, is safe
and has ideal effects in treating different diseases compared with
modern medicines. Previous pharmacological studies had attrib-
uted an anti-inflammatory effect of these herbal extracts to
reducing neutrophil migration and the expression of pro-
inflammatory cytokines [20]. It showed that the herbal extracts
and Chinese herbal formula have significant curative effect on
inflammation. It is beneficial to search XOD inhibitors and anti-
inflammatory herbs from TCM for the prevention of hyperuricemia
and gout.
In this study, the anti-inflammation effects of 33 herbal extracts
and 16 standards were detected by an in vitro cell model.
Moreover, the XOD inhibitory activity was measured using an
UPLC-TQ-MS method. The results would provide a reference for
screening more safe and effective herbs to prevent and treat gout.
2. Experimental
2.1. Reagents and materials
Sodium urate, XOD, xanthine and Tris were purchased from
Sigma (St. Louis, MO, USA); Allopurinol was acquired from Alfa
Aesar (Shanghai, China); Dulbecco’s modified Eagle’s medium
(DMEM) was purchased from Hyclone (USA); Fetal bovine serum
(FBS) was obtained from Hangzhou Sijiqing Co., Ltd. (Hangzhou,
China); 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoli-
um bromide (MTT), dimethyl sulfoxide (DMSO), penicillin and
streptomyc were obtained from Dingguo Changsheng Biotechnol-
ogy Co., Ltd. (Beijing, China); ELISA Kits for human ICAM-1 and IL-
1b were taken from the Cloud-Clone Corp (Houston, USA); the
herbs were provided by Jilin Pharmacy (Changchun, China); the
standards were purchased from the National Institute for the
Control of Pharmaceutical and Biological Products (Beijing, China);
HPLC grade acetonitrile was bought from Fisher Scientific
(Loughborough, UK); water was obtained from Milli-Q water
purification system (Milford, MA); all the other chemicals of
analytical grade were provided by Beijing Shiji (Beijing, China).
2.2. Preparation of MSU solution and the herbal extracts
Sodium urate solution was prepared as previously described
[21]. After sterilization by autoclaving for 1 h, 10 mg of sodium
urate was dissolved in 100 mL of DMSO and then 10 mL of serum
free sterile culture medium was added. The solution was
homogenized using an ultrasonic homogenizer (kunshangshumei,
China) and finally, 1 mg/mL of sodium urate solution (MSU) was
Please cite this article in press as: W.-J. Chen, et al., Screening the anti-gout traditional herbs from TCM using an in vitro method, Chin.
Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.05.022
obtained. The crude extract (2 g) was extracted by immersing in
70% ethanol for 20 min and ultrasonic extraction for 40 min. The
supernatant was lyophilized. The herbal extracts were re-dissolved
with DMEM: ethanol = 1:1 (200 mg/mL); the solution of standards
was 2 mg/mL.
2.3. Cell culture and evaluate cell viability
The HUVECs were grown in DMEM culture medium containing
10% FBS, 100 units/mL penicillin and 75 units/mL streptomyc. The
environment was controlled at 37 8C in a humidified atmosphere of
5% CO2 in CO2 incubator (Therom, USA). The HUVECs were seeded
onto a 96-well culture plate at a density of 8000 cells/well and
incubated at 37 8C for 24 h. The medium was replaced with 100 mL
of DMEM culture containing 5% FBS and different concentrations of
MSU as the model group, while the control group did not contain
MSU. The medium was individually incubated for 24 h and 48 h.
The medium and MSU were respectively removed for each well
after 24 h and 48 h. MTT was dissolved in phosphate buffered
saline (PBS) at a concentration of 1 mg/mL; 100 mL of MTT was
added to cell cultures after removing medium. After 4 h, the
medium was removed and the formazan crystals were dissolved in
150 mL DMSO. The absorbance at 570 nm was read on a
microplatereader (Tecan, Austria). The absorbance of living cells
was considered as 100% to access cell viability of model group. The
percentage of cell viability was calculated as follows: cell
viability% = (model/control)  100%.
2.4. Assessment of ICAM-1 and IL-1b
The HUVECs were seeded onto a 6-well culture plate at a
density of 2  105 cells/well and incubated at 37 8C for 24 h. The
medium was replaced with 2 mL of DMEM culture containing
5% FBS and different concentrations of MSU (50, 100, 150, and
200 mg/mL) for 24 h. Then, the cell culture supernate was collected
for 10 min at 1000 r/min. Particulates was removed and assay was
carried out immediately. The cells were washed three times with
cold PBS, and then detached with trypsin (0.25%). The cells were
collected and centrifuged. The cells were re-suspended in PBS and
immediately flash-frozen in liquid nitrogen, after that, the cells
were thawed at 37 8C. After repeating the cycle for three times,
the cells were centrifuged at 1500 r/min for 10 min at 4 8C to
remove cellular debris. The productions of ICAM-1 and IL-1b were
determined by ELISA.
2.5. Screening of anti-gout of herbal extracts solution and standards
Different herbal extract solutions and the MSU (200 mg/mL)
were simultaneously added into the 96-well after 24 h. The
empirical method was mentioned in 2.3. The percentage of herbal
extract solution inhibition was calculated as follows: inhibition
(%) = (herb-model)/(control-model)  100%.
2.6. The effects of herbs of ICAM-1 and IL-1b in MSU induced HUVECs
The herbal extracts possessing better anti-inflammation
activity were selected to assess the expressions of ICAM-1 and
IL-1b after MSU-stimulation, including Ginkgo Folium (Gf), Salviae
Miltiorrhizae Radix et Rhizoma (Sm), Rhei Radix et Rhizoma (Rr),
Polygoni Cuspidati Rhizoma et Radix (Pc), Selaginellae Herba (Sh), and
Paeoniae Radix Rubra (Pr). MSU solution and corresponding herbal
extract solutions were incubated and maintained under the
conditions and time durations as mentioned in 2.3. The different
herbal extract solutions (0.5 mg/mL) and indomethacin (10 mg/mL)
as well as the control MSU (200 mg/mL) were simultaneously added
into the 6-well after 24 h. And then, the cell lysates and cell culture
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W.-J. Chen et al. / Chinese Chemical Letters xxx (2016) xxx–xxx 3

supernates were collected. The productions of ICAM-1 and IL-1b

were detected.

2.7. Assessment of the inhibition of herbs on XOD

Xanthine was dissolved in 50 mmol/L Tris–HCl (pH 8.7) and

adjusted to 1 mmol/L. The XOD reaction was performed in a total
volume of 200 mL containing 50 mmol/L Tris–HCl, 100 mmol/L
xanthine, 20 mU/mL XOD and different concentrations of herbal
extract solutions or standards. The reaction was initiated for 5 min
at 37 8C, after that it was stopped by adding 750 mL cold
acetonitrile, and 4 mmol/L 5-fluorouracil, which is the internal
standard. The final solution volume was 1 mL. The uric acid
production was indirectly determined by using an ACQUITYTM
UPLC system (Waters Corp., Milford, MA, USA) with a thermo
stated autosampler at 4 8C. The separation was carried out on an
Agilent ZORBAX SB-C18 column (5 mm; 4.6  150 mm; USA)
maintained at 35 8C. Mobile phase component A was acetonitrile
and B was water with 0.1% formic acid, and the flow rate was set at
0.3 mL/min. The elution gradients used were: 0–4 min, 10%–30%
Fig. 1. Effect of the HUVECs viability by MSU-stimulated. Cells were cultured for
(A); 4–6 min, 30%–80% (A); 6–7 min, 80%–100% (A), and then
24 h and 48 h by MSU-stimulated in different concentrations. Cell viability was
returning to the initial condition for 2 min, followed by re- assessed by MTT assay (mean  SD, n = 6). * P < 0.05 and ** P < 0.01 were indicating a
equilibration for 5 min. The injection volume was 5 mL. highly significant discrepancy when compared with control groups for t test.
Mass spectrometric detection was carried out on Xevo TQ mass
spectrometer with an electrospray ionization (ESI) source.
Quantification analysis was performed in multiple reaction MSU and 24 h as the optimal conditions to assess the ability of drugs on
monitoring (MRM) negative mode. The following settings were inflammation.
used for MRM: capillary voltage 2.0 kV; source temperature
350 8C; cone gas flow 50 L/h; desolvation gas flow 600 L/h; cone 3.2. The expressions of ICAM-1 and IL-1b induced by MSU
and collision energy are presented in Table 1. The percentage of
herbal extract solution inhibition was calculated as follows:inhibi- The primary pathological feature of gout is generating
tion (%) = (blank-herb)/blank  100%. inflammatory response by MSU-stimulation, including activation
of nod-like receptor pyrin domain-containing-3 (NLRP3) to lead to
2.8. Statistical analysis caspase-1 and pro-IL-1b cleavage, and further result in releasing
IL-1b [23,24]. IL-1b is a pivotal mediator of gouty inflammation
The UPLC-TQ-MS data of XOD inhibitor were acquired and and pain. Endothelial cell adhesion is the essence of acute gout,
processed by Masslynx V4.1 software (Waters, USA). All data were which induced ICAM-1 expression [25] and associated with a
expressed as the mean  SD, and statistical analysis was performed variety of inflammatory cytokines [26]. Therefore, ICAM-1 and IL-
using one-way analysis of SPSS18.0 and significance t test (P < 0.01 or 1b could be used as important indicators to assess the model of
P < 0.05) was carried out. acute gout.
The results for expressions of ICAM-1 and IL-1b are respectively
shown in Tables 2 and 3, which demonstrates that both the
3. Results and discussion
expressions of ICAM-1 and IL-1b were significantly increased
(P < 0.01) in a dose-dependent manner with the different
3.1. The effect of MSU on HUVEC by MTT assay
concentrations of MSU in cell lysates and cell culture supernates.
We calculated the sum of cell lysates and cell culture supernates at
The HUVECs were damaged directly by MSU-stimulation to
different concentrations. From these tables we can see that both of
establish a cell model in vitro [21,22], and then the anti-
the expressions of ICAM-1 and IL-1b were also significantly
inflammatory ability of drugs was studied. The cell viability was
increased (P < 0.01) compared with the control group. ICAM-1 and
used as evaluation criterion by an MTT assay. This method is simple,
IL-1b could influence the cell viability under MSU-stimulation.
fast and economical on screening anti-gout drugs. In our experi-
mental in vitro study (Fig. 1), the cell viability was monotonously
decreased in a dose-dependent manner with the increases
Table 2
of MSU concentration at 24 h. The cell viability remained stable The expression of ICAM-1 in cell lysates and cell culture supermates by MSU-
at 175–200 mg/mL (175 mg/mL: 57.32%  1.81%; 200 mg/mL: stimulated (mean  SD, n = 3).
57.58%  1.27%). By contrast, the data were irregular fluctuations at
Concentration Cell lysates (ng/g) Cell culture SUM (ng/g)
48 h. Moreover, the data revealed that cells were damaged more (mg/mL) supermates
seriously by MSU-stimulation at 24 h. Thus, we chose the 200 mg/mL (ng/g)

0 290.09  8.76 – 290.09

Table 1
50 321.59  22.64** 3.37  1.01** 324.96**
Product ion pairs and parameters for MRM of compounds used in this study.
100 333.04  20.33** 3.46  1.08** 336.50**
Compounds Precursor Capillary Cone Collision 150 357.84  11.15** 6.07  1.40** 363.91**
ion ! product voltage voltage energy 200 382.92  16.87** 11.83  1.59** 394.75**
ion (m/z) (kV) (V) (V) Cells were cultured for 24 h in different MSU concentrations (0, 50, 100, 150, and
Uric acid 166 ! 96 2 20 20 200 mg/mL). ‘‘–’’ represents that the data below the detectable minimum of the
5-Fluorouracil 128 ! 42 2 26 14 kit.**P < 0.01 was indicating a highly significant discrepancy when compared with
control groups for t test.

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Table 3 Table 5
The expression of IL-1b in cell lysates and cell culture supermates by MSU- Anti-inflammatory activities of standards (mean  SD, n = 6).
stimulated (mean  SD, n = 3).
Name The inhibition of 0.2 mg/mL (%) IC50 (mg/mL)
Concentration Cell lysates Cell culture SUM
Catechin 95.02  2.31 0.47  0.04
(mg/mL) (ng/g) supermates (ng/g)
Luteolin 86.96  3.56 1.50  0.05
(ng/g)
Amentoflavone 84.75  2.01 2.86  0.16
0 11.72  3.80 25.34  6.67 37.06 Indomethacin 84.72  2.47 2.87  0.11
50 24.50  4.66** 54.60  9.37** 79.10** Tanshinone 79.95  3.52 1.01  0.12
100 26.71  4.36** 78.58  2.34** 105.28** Quercetin 75.25  1.32 0.94  0.09
150 28.38  3.68** 94.09  5.46** 122.47** Rutinum 58.45  1.89 0.69  0.07
200 34.84  2.02** 120.98  10.38** 155.82** Emodin 54.02  2.53 1.45  0.09
Rhein 40.7  1.07 1.35  0.08
Cells were cultured for 24 h in different MSU concentrations (0, 50, 100, 150, and
Aloeemodin 34.96  2.30 0.30  0.01
200 mg/mL). **P < 0.01 was indicating a highly significant discrepancy when
Kaempferide 32.65  3.63 0.27  0.02
compared with control groups for t test.
Jatrorrhizine 14.31  2.16 0.08  0.01
Palmatine chloride 13.01  1.84 0.99  0.06
This might be due to the fact that the intracellular substances were Resveratrol 5.99  1.93 5.90  0.07
activated after stimulating with MSU to produce ICAM-1 and IL-1b Quercetin-3-rhamnoside 4.84  1.40 2.08  0.14
Berberine – –
and they could damage cells as inflammatory cytokines do. IL-1b
Chlorogenic acid – –
could promote the recruitment of neutrophils through the up-
‘‘–’’ represents that the standard had no activity on anti-inflammation.
regulations of E-selectin and ICAM-1 expressions by the endothe-
lial cells [27]. Therefore, this model is useful for studying the
pathological mechanism of acute gout on anti-inflammation anti-inflammatory activity at 0.2 mg/mL. The inhibition rate of
induced by MSU. To screen the anti-gout traditional herbs from Ginkgo Folium, Salviae Miltiorrhizae Radix et rhizome, Rhei Radix et
TCM, in this research, the anti-inflammatory mechanism of herbal Rhizoma, Polygoni Cuspidati Rhizoma et Radix, Selaginellae Herba,
extracts was observed through the expressions of ICAM-1 and IL-1b. and Paeoniae Radix Rubra was all more than 90%. Moreover,
Scutellarlae Radix, Lonicerae Japonicae Flos, Bupleuri Radix, Glycyr-
3.3. The effects of herbs and standards on anti-gout in vitro rhizae Radix et Rhizoma and Schisandrae Chinensis Fructus also
showed anti-inflammatory activity with lower IC50. The anti-
We detected the anti-inflammatory ability of herbal extracts inflammation effect was evaluated using indometacin as a
using the convenient method, i.e., in vitro cell model to find out comparator. As shown in Table 5, the standards possessed similar
effective herbs in the treatment and prevention of gout. In this ability of anti-inflammation. Catechin, luteolin and amentoflavone
study, 33 herbal extracts and 16 standards were detected. were better than the other standards on inflammation at
From Table 4 we can see that most herbal extracts have the 0.2 mg/mL. Catechin, quercetin, rutinum, aloeemodin, palmatine
chloride, jatrorrhizine and kaempferide possessed lower IC50
Table 4 compared with indometacin. In short, the anti-inflammatory
Anti-inflammatory activity of herbal extracts (mean  SD, n = 6). performance of most flavonoid standards was better than that of
the other standards.
Name The inhibition of IC50 (mg/mL)
0.2 mg/mL (%) These herbs were classified according to the main ingredients.
For example, Ginkgo Folium and Selaginellae Herba were classified
Ginkgo Folium 113.25  2.53 0.04  0.01
Salviae Miltiorrhizae Radix et Rhizoma 105.24  1.62 0.09  0.01
as flavonoids, Rhei Radix et Rhizoma and Polygoni Cuspidati Rhizoma
Rhei Radix et Rhizoma 99.54  3.92 0.01  0.00 et Radix were classified as anthraquinones, Phellodendri Chinensis
Selaginellae Herba 95.80  4.03 2.25  0.05 Cortex and Coptidis Rhizoma were classified as alkaloids. Modern
Paeoniae Radix Rubra 95.53  3.18 0.01  0.00 pharmacological studies have demonstrated that the herbs and
Polygoni Cuspidati Rhizoma et Radix 95.12  5.28 0.02  0.01
standards of flavonoids [28–32], anthraquinone [33–35], and
Forsythiae Fructus 87.22  4.45 0.09  0.01
Achyranthis Bidentatae Radix 84.02  6.12 1.21  0.02 alkaloids [36,37] could inhibit the inflammatory cytokine expres-
Crataegi Fructus 79.03  4.74 0.28  0.03 sion and suppress nuclear transcription factor-kB (NF-kB) activa-
Gentianae Macrophyllae Radix 77.77  5.17 0.29  0.05 tion. Besides, glycosides are the main ingredients of Paeoniae Radix
Scutellariae Radix 75.35  2.52 0.01  0.00 Rubra. Paeoniflorin is a potent anti-inflammatory agent that can
Coicis Semen 68.16  3.93 8.33  0.10
Lonicerae Japonicae Flos 66.12  3.60 0.02  0.01
ameliorate the leukocyte infiltrates and adhesion molecules
Bupleuri Radix 58.03  2.54 0.04  0.01 expression [38,39]. These data suggest that most herbs from
Andrographis Herba 57.82  6.78 2.22  0.07 TCM have a better therapeutic effect on inflammation. The capacity
Astragali Radix 53.68  1.93 2.14  0.01 of herbal extracts on inflammation may depend upon their
Atractylodis Rhizoma 44.59  3.49 0.95  0.06
bioactive ingredients.
Glycyrrhizae Radix et Rhizoma 43.12  3.24 0.05  0.01
Schisandrae Chinensis Fructus 42.59  5.28 0.01  0.00
Stephaniae Tetrandrae Radix 42.06  4.80 0.11  0.01 3.4. The effects of herbal extracts on expressions of ICAM-1 and IL-1b
Cassiae Semen 40.28  3.74 1.09  0.04
Poria 31.87  2.67 1.65  0.03 The stronger anti-inflammatory herbs, including Ginkgo Folium
Paeoniae Radix Alba 30.91  1.37 0.15  0.01
Dioscoreae Hypoglaucae Rhizoma 30.24  4.21 0.04  0.01
(Gf), Salviae Miltiorrhizae Radix et Rhizoma (Sm), Rhei Radix et
Corydalis Rhizoma 30.00  2.13 0.04  0.01 Rhizoma (Rr), Polygoni Cuspidati Rhizoma et Radix (Pc), Selaginellae
Euodiae Fructus 25.64  2.69 – Herba (Sh), and Paeoniae Radix Rubra (Pr), were used as examples to
Clematidis Radix et Rhizoma 25.15  3.81 0.83  0.06 assess the effects of them on the expressions of ICAM-1 and IL-1b
Lysimachiae Herba 23.64  3.64 2.97  0.04
in cell lysates and cell culture supernates. As shown in Table 6, the
Artemisiae Scopariae Herba 21.59  4.01 0.54  0.02
Plantaginis Semen 20.72  1.29 0.04  0.01 expression of ICAM-1 on model group was significantly increased
Alismatis Rhizoma 9.38  2.61 1.38  0.15 compared with the control group (P < 0.01). And all the herbal
Phellodendri Chinensis Cortex – – extracts were significantly decreased compared with the model
Coptidis Rhizoma – – group (P < 0.01) in cell lysates. This indicates that the herbal
‘‘–’’ represents that the herbal extract had no activity on anti-inflammation. extracts could reduce the intracellular expressions of ICAM-1.

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Table 6 Miltiorrhizae Radix et Rhizoma could effectively inhibit the releases

Effect of herbal extracts on ICAM-1 level (mean  SD, n = 6).
of ICAM-1 and IL-1b. Ginkgo Folium has been reported that
Group Cell lysates Cell culture SUM possessed activity on inflammation via inhibiting the production of
(ng/g) supermates (ng/g) pro-inflammatory cytokines IL-1b and TNF-a [29,30].
(ng/g)

Control 260.09  8.76** Null 260.09** 3.5. The XOD inhibitors of herbal extracts and standard compounds
Model 372.92  16.87 11.83  4.59 384.75
Indometacin 178.39  20.68** 6.74  3.34** 185.14**
XOD is a key enzyme for the treatments of gout and
Ginkgo Folium 174.45  28.73** 3.94  2.71** 178.39**
Salviae Miltiorrhizae 288.39  22.52** 22.82  4.18 311.22** hyperuricemia. A large number of the plant species are potential
Radix et Rhizoma sources of natural XOD inhibitors [40,41] which could decrease
Rhei Radix et Rhizoma 267.45  3.85** 25.56  3.45 293.01** XOD activity in serum or liver of the animal models of
Selaginellae Herba 245.25  33.73** 9.10  1.62* 254.36**
hyperuricemia and gout [42,43]. To date, TCM has been used for
Paeoniae Radix Rubra 220.40  22.36** 15.44  1.61 235.85**
Polygoni Cuspidati Rhizoma 281.90  37.91** 24.26  3.53 306.16**
inhibiting XOD activity and decreasing the level of uric acid. It can
et Radix be used as alternatives to allopurinol because of fewer adverse
effects [44]. In this research, the UPLC–MS/MS method was used
Cells were cultured in MSU (200 mg/mL) and different herbal extracts (0.2 mg/mL)
for 24 h. ‘‘–’’ represents that the data below the detectable minimum of the kit. for the detection of XOD activity and screening XOD inhibitors by
*P < 0.05 or **P < 0.01 were indicating a highly significant discrepancy when quantifying products of enzymatic reaction. This method is
compared with model groups for t test. sensitive, accurate, rapid, and can eliminate false positive and
negative results.
The representative chromatograms of samples are showed in
In cell culture supernates, In, Gf, and Sh were decreased (P < 0.05 Fig. 2. The black represents the enzyme reaction without inhibitor
or P < 0.01) compared with the model group, but in contrast, Sm, and red represents the enzyme reaction with allopurinol. From this
Rr, Pr and Pc were increased and even more than model group. The figure we can see that the peaks of uric acid (TR = 5.13 min) and
sum of ICAM-1 of cell lysates and cell culture supernates showed internal standard (TR = 6.04 min) were separated. As shown in
that each herbal extract was significantly decreased compared Table 8, the XOD inhibitory activity of Rhei Radix et Rhizoma,
with the model group (P < 0.01), and the order of total expression Paeoniae Radix Rubra, Salviae Miltiorrhizae Radix et rhizoma,
level was In, Gf, Pr, Sh, control, Rr, Pc, Sm, and model. Selaginellae Herba, Paeoniae Radix Alba, Polygoni Cuspidati Rhizoma
The expression of IL-1b was similar to ICAM-1, thus from et Radix, Ginkgo Folium, and Glycyrrhizae Radix et Rhizoma was over
Table 7 we can see that the model group was significantly 25% at 5 mg/mL. The activity of the standards ininhibiting XOD is
increased compared with the control group (P < 0.01) in cell shown in Table 9. Allopurinol is a powerful XOD inhibitor, since it
lysates. In comparing with the model group, the herbal extracts could significantly inhibit the enzyme reaction (IC50 0.06 mg/mL).
group was significantly decreased (P < 0.01). In cell culture Luteolin and quercetin, which belong to flavonoids were stronger
supernates, the herbal extracts group decreased (P < 0.05 or than the other standards. Flavonoids and anthraquinone can
P < 0.01) except Pc compared with the model group. On the other inhibit XOD and reduce serum uric acid levels in vitro and in vivo
hand, Pc obviously decreased IL-1b in cell lysates that might be [45–49], but the exact mechanism is not known and should be
released into cell supernates. From Table 7, it can be seen that for further investigated.
the sum of IL-1b expression level of all the herbal extracts was In short, we demonstrated that the herbal extracts and
significantly decreased compared with the model group (P < 0.01), standards could ameliorate the MSU induced cellular damage by
of which, the order of total expression level was Sm, Con, In, Gf, Rr, inhibiting the release of inflammatory cytokines including ICAM-1
Sh, Pr, Pc, and Mod. These data indicate that the herbal extracts and IL-1b and suppress the oxidation of xanthine to uric acid. Our
could reduce the release of inflammatory cytokines and protect the results support that the compounds of flavonoid and anthraqui-
cell viability. Especially, Ginkgo Folium seemed to be more effective none seem to be more effective on inflammation and inhibiting
in inhibiting the expression of ICAM-1 and IL-1b. XOD. However, further investigation is needed to probe the
In conclusion, herbal extracts possess anti-inflammatory possible mechanism. The results obtained in this research provide
bioactivity to protect HUVECs. The possible reason was the active a theoretical basis for TCM on inflammation and lowering uric acid.
components in the herbal extracts inhibited ICAM-1 and IL-1b
expression. Compared with indometacin, Ginkgo Folium and Salviae

Table 7
Effect of herbal extracts on IL-1b level (mean  SD, n = 6).

Group Cell lysates Cell culture SUM (ng/g)

(ng/g) supermates
(ng/g)

Control 11.72  2.80** 25.34  6.67 37.06**

Model 33.84  2.01 120.98  10.38 154.82
Indometacin 9.68  2.66** 79.06  4.75** 88.74**
Ginkgo Folium 14.64  2.09** 76.46  5.29** 91.10**
Salviae Miltiorrhizae Radix 6.35  1.33** 22.37  4.98** 28.72**
et Rhizoma
Rhei Radix et Rhizoma 9.21  0.78** 92.13  1.39** 101.33**
Selaginellae Herba 15.42  0.23** 90.18  8.73** 105.60**
Paeoniae Radix Rubra 15.96  2.02** 104.80  6.37* 120.76**
Polygoni Cuspidati Rhizoma 8.38  2.11** 121.69  10.86 130.07**
et Radix

Cells were cultured in MSU (200 mg/mL) and different herbal extracts (0.2 mg/mL) Fig. 2. The total ion chromatograms of uric acid and 5-fluorouracil. Black represents
for 24 h.*P < 0.05 or **P < 0.01 was indicating a highly significant discrepancy when the enzyme reaction without inhibitor. Red represents the enzyme reaction with
compared with model groups for t test. inhibitor (allopurinol).

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Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.05.022
G Model
CCLET-3711; No. of Pages 7

6 W.-J. Chen et al. / Chinese Chemical Letters xxx (2016) xxx–xxx

Table 8 XOD inhibitory activity were stronger than that of the other
The herbal extracts for inhibition of XOD (mean  SD, n = 3).
compounds. In short, the compounds of flavonoid and anthraqui-
Name The inhibition of 5 mg/mL (%) none seem to be more effective on inflammation and inhibiting
Rhei Radix et Rhizoma 43.90  0.72 XOD and they could be widely used in the treatment of gout. Since
Paeoniae Radix Rubra 40.96  1.23 the inhibitory ability of natural products should relate to their
Salviae Miltiorrhizae Radix et Rhizoma 31.24  0.99 bioactive components, so the results could provide a theoretical
Selaginellae Herba 31.21  1.52 basis for the further development of natural medicines to treat
Paeoniae Radix Alba 29.75  1.21
gout.
Polygoni Cuspidati Rhizoma et Radix 27.60  0.92
Ginkgo Folium 25.91  0.51
Glycyrrhizae Radix et Rhizoma 25.83  0.57 Acknowledgments
Lonicerae Japonicae Flos 24.58  1.02
Scutellarlae Radix 24.47  1.37
Atractylodis Rhizoma 23.55  0.82
The authors would like to thank the National Natural Science
Coicis Semen 22.59  0.69 Foundation of China (Nos. 21175128, 81303280, 81573574).
Clematidis Rsdix et Rhizoma 19.16  1.05
Forsythiae Fructus 18.95  1.33
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