Effects of Zanthoxylum piperitum ethanol extract on osteoarthritis inflammation and pain

Article abstract of DOI:10.1016/j.biopha.2018.05.109

Degenerative arthritis, also known as osteoarthritis (OA), is the most common type of arthritis, which is caused by degenerative damage of the cartilage, which primarily protects the joints, leading to inflammation and pain. The objective of this study was to investigate the in vivo and in vitro effects of treatment with ZPE-LR (90% EtOH extract of Zanthoxylum piperitum) on pain severity and inflammation. When using an in vivo OA model MIA (monosodiumidoacetate-induced arthritis) rats, ZPE-LR (100 mg/kg) oral-administratio significantly inhibited MIA-induced change in loaded weight ratio on the left foot, and articular cartilage thickness. To confirm the positive effects on pain relief, acetic acid, heat and formalin-induced pain were remarkably decreased by 50 and 100 mg/kg ZPE-LR oral-administration. Pain related KCNJ6 mRNA expression as well as K + current was increased after ZPE-LR treatment in BV-2 cells. To confirm the positive effects on inflammation, TPA (12-O-tetradecanoylphorbol-13-acetate) induced inflammation measured by mouse ear thickness and biopsy punch weight and TPA-induced iNOS, COX-2 mRNA and protein expression were remarkably suppressed by 50 and 100 mg/kg ZPE-LR oral-administration. In addition, TPA-induced iNOS, COX-2 mRNA level and protein expression were reduced. Acetic acid, heat and formalin-induced pain were remarkably decreased by 50 and 100 mg/kg ZPE-LR oral-administration. We examined in vitro ZPE-LR effects in LPS-induced RAW 264.7 cells. LPS-induced p65 translocation to the nucleus was prohibited by ZPE-LR 100 μg/ml oral administration. Moreover, ROS generation by LPS was significantly inhibited by ZPE-LR 50 and 100 μg/ml treatment. To investigate new ZPE-LR activating mechanisms, the gene fishing method (not a typical term, should probably use PCR based genetic screening) was used. LPS-induced HPRT1 (hypoxanthine phosphoribosyltransferase 1) was decreased by ZPE-LR. However, RPL8 (Ribosomal protein L8) which showed no change in mRNA expression due to LPS, did show increased mRNA levels after ZPE-LR treatment. Our data elucidate mechanisms underlying ZPE-LR and suggest ZPE-LR may be a potential therapeutic agent to modulate osteoarthritis inflammation and pain.

Full text of DOI:10.1016/j.biopha.2018.05.109

Biomedicine & Pharmacotherapy 105 (2018) 481–490
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Effects of Zanthoxylum piperitum ethanol extract on osteoarthritis
inflammation and pain
a,1
b,1
c,1
b
d
Kyung-A Hwang , Jeong Eun Kwon , YooHun Noh , BongKyun Park , Yong Joon Jeong ,
e
b
d,⁎
b,⁎
Sun-Mee Lee , Se-Young Kim , InHye Kim , Se Chan Kang
Department of Agrofood Resources, National Academy of Agricultural Science, Rural Development Administration, Wanju-gun, Jeolabuk-do, 55365, Republic of Korea
Department of Oriental Medicine and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin-si, 17104, Republic of Korea
Famenity Co., Ltd, Gwacheon-si, 13887, Republic of Korea
Genencell Co., Ltd, Yongin-si 16950, Republic of Korea
School of Pharmacy, Sungkyunkwan University, Suwon, 16419, Gyeonggi, Republic of Korea
a
b
c
d
e
A R T I C L E I N F O
A B S T R A C T
Keywords:
Inflammation
Neuropathic pain
Osteoarthritis
Potassium ion
Zanthoxylum piperitum
Degenerative arthritis, also known as osteoarthritis (OA), is the most common type of arthritis, which is caused
by degenerative damage of the cartilage, which primarily protects the joints, leading to inflammation and pain.
The objective of this study was to investigate the in vivo and in vitro effects of treatment with ZPE-LR (90% EtOH
extract of Zanthoxylum piperitum) on pain severity and inflammation. When using an in vivo OA model MIA
(monosodiumidoacetate-induced arthritis) rats, ZPE-LR (100 mg/kg) oral-administratio significantly inhibited
MIA-induced change in loaded weight ratio on the left foot, and articular cartilage thickness. To confirm the
positive effects on pain relief, acetic acid, heat and formalin-induced pain were remarkably decreased by 50 and
100 mg/kg ZPE-LR oral-administration. Pain related KCNJ6 mRNA expression as well as K + current was in-
creased after ZPE-LR treatment in BV-2 cells. To confirm the positive effects on inflammation, TPA (12-O-tet-
radecanoylphorbol-13-acetate) induced inflammation measured by mouse ear thickness and biopsy punch
weight and TPA-induced iNOS, COX-2 mRNA and protein expression were remarkably suppressed by 50 and
1
00 mg/kg ZPE-LR oral-administration. In addition, TPA-induced iNOS, COX-2 mRNA level and protein ex-
pression were reduced. Acetic acid, heat and formalin-induced pain were remarkably decreased by 50 and
00 mg/kg ZPE-LR oral-administration. We examined in vitro ZPE-LR effects in LPS-induced RAW 264.7 cells.
1
LPS-induced p65 translocation to the nucleus was prohibited by ZPE-LR 100 μg/ml oral administration.
Moreover, ROS generation by LPS was significantly inhibited by ZPE-LR 50 and 100 μg/ml treatment. To in-
vestigate new ZPE-LR activating mechanisms, the gene fishing method (not a typical term, should probably use
PCR based genetic screening) was used. LPS-induced HPRT1 (hypoxanthine phosphoribosyltransferase 1) was
decreased by ZPE-LR. However, RPL8 (Ribosomal protein L8) which showed no change in mRNA expression due
to LPS, did show increased mRNA levels after ZPE-LR treatment. Our data elucidate mechanisms underlying ZPE-
LR and suggest ZPE-LR may be a potential therapeutic agent to modulate osteoarthritis inflammation and pain.
1. Introduction
elasticity loss, reduction in water quantity, and cartilage and muscle
loss [3–5]. This disease does not only damage vasculature or the ner-
Osteoarthritis (OA) is a disease caused by aging and bone density
vous system but also metabolism which prevents surrounding tissue
damage were slowed [6]. When cartilage is acutely destroyed, damage
can occur without subjective symptom presentation. Interventions for
cartilage damage include chemical inhibitors, antibodies, and gene
therapy. These treatments ameliorate damage by suppressing in-
flammation inducing factors such as TNF-α or IL-6 [7–10]. However,
this therapy has a disadvantage, it is in re-division after first division in
decrease etc. And as a secondary change, decrease cartilage sur-
rounding joints [1]. In the past, this was believed to be a just from aging
phenomenon. But, in recently study, OA reported as a joint cartilage
disease caused by varios causes such as inflict morphological damage,
osteoporosis, inflammation and reumatoid arithris etc.[2].
Arthritis is a degenerative disease caused by combined cartilage
⁎
Corresponding authors.
E-mail addresses: ihyekim@gmail.com (I. Kim), sckang@khu.ac.kr (S.C. Kang).
These authors contributed equally to this study.
1
https://doi.org/10.1016/j.biopha.2018.05.109
Received 4 September 2017; Received in revised form 23 May 2018; Accepted 23 May 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
mesenchymal stem cells, that maintain cartilage cell traits [11,12].
There are many studies on natural substance effects on inflamma-
tion and pain amelioration for the benefits of low side-effects. These
receive much attention due to their low side-effects. Zanthoxylum pi-
peritum is a summergreen shrub from the Ructaceae and Zanthoxylum
species which grows widely throughout Eastern Asia: South Korea,
China, and Japan. Z. piperitum has been widely used as a traditional
spice, medicine, and for oil refinement because it containing many
kinds of spice and active oil ingredient [13–16]. Zanthoxylum periper-
itum has been used as a natural medicine, spice and preservation agent
due to detoxification effects, hypertension reduction, stroke, anti-bac-
terial and anti-oxidant, tyrosinase along with osteosarcoma prolifera-
tion-control [17–20]. But, Zanthoxylum piperitum’s ability to ameliorate
and treat osteoarthritis is unknown, and there are few structured stu-
dies on its biological activity.
In this study, we investigated ameliorating effects on pain and in-
flammation of. Z. piperitum extract (ZPE-LR) through tissue observation,
microbiological change, and biological marker such as p65, COX-2 and
iNOS.
2. Materials and methods
2
2
.1. Experimental design
.1.1. Fractionation scheme
Zanthoxylum piperitum DC was collected at Jeju of Korea. Z. piper-
itum DC dried leaves and ramulus were ground into a powder and
subsequently extracted with 90% EtOH at room temperature (ZPE).
Solutions were filtered and evaporated in a vacuum to give 90% EtOH
extract. Z. piperitum dried material (10 kg) was ground into a powder
and extracted with 90% EtOH for 6 h, 3 times. The 90% EtOH extracts
(
(
500 g) were evaporated in a vacuum and suspended in distilled water
800 mL). The suspension was successively extracted with n-hexane,
2 2
dichloromethane (CH Cl ), ethyl acetate (EtOAc) and n-BuOH.
2.1.2. Active component isolation from Z. piperitum
The EtOAc fraction 9.8 g was subjected to open column chromato-
graphy (elution from 40% MeOH) using a glass column (5 × 50 cm)
packed, to separate 7 fractions. Fraction 6 was again subjected to open
column chromatography (elution from 100% MeOH). Fraction 6 was
divided into 3 different samples. If 6b fractions were detected as a spot
identical to tannic acid on a TLC plate, they were concentrated and
completely dried, followed by medium-pressure liquid chromatography
(
MPLC, TELEDYNE ISCO, USA) using a RediSep (130 g vol.) flash
column (elution from 50%MeOH). The obtained 100 mg subfraction
was purified with preparative HPLC (Agilent, USA) using a reverse
phase column packed with octadecylated silica (gradient elution from
20:80 to 70:30 of MeOH:H
2
O), to separate active fractions at
10–20 minutes in which compound 1 and 2 were detected. These were
combined and dried under reduced pressure. When analyzed using af-
zelin (Sigma, USA) as a standard, the active fractions separated above
included 97.0% (g/g) afzelin based on the fraction weight.
2.1.3. Z. piperitum active compound determination
Purified compounds 1 and 2 were dissolved in a 1.5 N hydrochloric
acid solution and subjected to hydrolysis in a 100℃ water bath for
0 min to obtain quercetin and afzelin, which were analyzed compared
to standard (Sigma, USA) by HPLC (Fig. 1B, C and D).
3
Fig. 1. Fraction and HPLC chromatogram scheme for the active fraction
obtained from Z. piperitum extract (ZPE-LR). (A) Extraction and solvent
partitions from Z. piperitum. (B) and (C) Compound 1 and 2 separation were
conducted on a Symmetry C18 column, 10 μL injection at 37℃, detection at 254
and 280 nm by HPLC. (D) and (E) Compound 1 and 2 structure.
2.1.4. Animals
Twenty 8-week-old male Sprague-Dawley rats, weighing 180–220 g,
were purchased from Raon Bio Inc, Korea. Rats were housed in solid-
bottomed plastic cages designed to allow easy access to standard la-
boratory food and water and kept in sanitary ventilated animal rooms
with a controlled temperature (25 ± 1 °C) and regular light cycle (12 h
light: 12 h dark). Animal experiments were conducted in accordance
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K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
with current ethical regulations for animal care and use at Kyung Hee
University (KHUASP-16-06).
2.1.10. mRNA level measurement by reverse transcription polymerase chain
reaction (RT-PCR)
Total RNA was extracted using a single-step guanidinium thiocya-
nate-phenol-chloroform method. RNA yield and purity were confirmed
by measuring the 260 and 280 nm absorbance ratio. PCR was per-
formed using mouse COX-2-specific primers: sense primer, 5′-AACCG
CATTGCCTCTGAAT-3′; antisense primer, 5′-ATGTTCCAGGAGGATG
GAG-3′. The following mouse iNOS-specific primers were synthesized:
sense primer, 5′-CGAAACGCTTCACTTCCAA -3′ and antisense primer,
2
.1.5. MIA-induced osteoarthritis induction in rats
Arthritis was induced by a single intra-articular 0.5 mg MIA injec-
tion into both rat knee joints as described previously [21]. Briefly, MIA
was dissolved in physiological saline at a 10 mg/mL concentration.
After being anesthetized with sodium pentobarbital (30 mg/kg), rat
knees were fixed at 90° and a 0.05 mL MIA solution was injected into
the joint cavity through the patellar ligament using a 26-gauge needle.
MIA concentration was determined based on a preliminary trial.
5′- TGAGCCTATATTGCTGTGGCT-3′. The PCR primers for the mouse β-
actin control were 5′-TGTCCACCTTCCAGCAGATGT-3′ (sense) and 5′-
AGCTCAGTAACAGTCCGCCTAGA-3′ (antisense). Contaminant absence
was routinely confirmed using negative control samples without primer
addition. Samples were stored at −20 °C after amplification.
2
.1.6. ZPE-LR treatment
Rats were randomized into four groups: (1) Saline control, (2) MIA
treatment group (negative control), (3) MIA plus celecoxib (10 mg/kg),
and (4) MIA plus ZPE-LR (100 mg/kg). Normal rat joint cavities re-
ceived a single injection of an equal volume of physiological saline
instead of MIA. ZPE-LR (100 mg/kg) was dissolved in physiological
saline and 10 ml/kg administered orally once per day for 35 days from
the day after arthritis induction. Control and negative control rats were
given an equal volume of vehicle orally. All rats were anesthetized by
sodium pentobarbital (30 mg/kg) intraperitoneal injection and killed
by cervical dislocation at 35 days after MIA injection
2
.1.11. Acetic acid-induced abdominal constrictions
The nociceptive response was evaluated after intraperitoneal (i.p.)
acetic acid injection according to the model described by Vaz et al [22].
which was a modification of the model originally described by Koster
et al [23]. Acetic acid (0.6%, 10 ml/kg) was injected intraperitoneally
and the number of abdominal constrictions associated with total hind
limb stretching was counted over 20 min.
2
.1.12. Hot-plate model
Nociceptive response was evaluated in the hot plate model as de-
scribed by Eddy and Leimbach [24]. A modification of the original
method developed by Woolfe and MacDonald [25]. Animals were
placed on a heated (56℃) metal plate. Latency for jumping or licking
paws was determined.
2
.1.7. H&E Staining
Rat articular cartilages were fixed with 10% formaldehyde for 2
days then were decalcified in 10% ethylenediamine tetra acetic acid
EDTA) for 30 days at 4 °C. Decalcified tissues were then dehydrated in
(
a gradient ethanol series (70–100%), washed twice with xylene, and
embedded in paraffin. Subsequently, 5-μm sections were stained with
hematoxylin and eosin (H&E).
2
.1.13. Formaldehyde-induced nociceptive response
The nociceptive response was evaluated after subcutaneous (s.c.)
formaldehyde injection according to the model described by Vaz et al
22]. which was a modification of the original model described by
[
2.1.8. 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in mice
Hunskaar and Hole [26] for mice. The nociceptive stimulus was sub-
cutaneous formaldehyde (0.92%), 20 μl, diluted in saline injection into
the dorsum of the right hindpaw. The time the animals spent licking the
injected hindpaw was determined between 0 and 9 min (first phase)
and 30 and 39 min (second phase) after formaldehyde injection. In
some protocols, hindpaw edema was evaluated 4 h after formaldehyde
injection. After killing the animals by decapitation, both hindpaws were
cut at the knee joint and weighed in an analytical balance. Edema was
defined as the weight difference between the injected and the con-
tralateral non-injected hindpaws.
The male Institute of Cancer Research (ICR) mice used here were
maintained in accordance with the National Institute of Toxicological
Research of the Korea Food and Drug Administration guidelines for the
care and use of laboratory animals. The protocol was approved by the
Institutional Animal Care and Use Committee at Kyung Hee University.
1
2-O-tetradecanoylphorbol-13-acetate (TPA; 1 μg/ear) in acetone
(
10 μl) was applied to the right ear of ICR mice. Control mice received
acetone alone. Repeated 3 times for a day after animals were sacrificed
and the tip of the ear thickness was measured using vernier calipers
(
Mitutoyo Corporation, Kawasaki, Japan), and ear punch biopsies 6 mm
in diameter were taken and weighed. Following this, the mice were
sacrificed by cervical dislocation. The increase in thickness or weight of
the ear punches was directly proportional to the degree of inflamma-
tion.
2
.1.14. Cell culture
RAW 264.7 cells were purchased from ATCC (Rockville, MD) and
grown in DMEM medium supplemented with 100 IU/mL penicillin,
00 mg/mL streptomycin, and 10% heat-inactivated FBS in a humidi-
fied atmosphere containing 5% CO at 37 °C. BV-2 (ATCC) microglia
1
2
2
.1.9. Immunoblot analysis
Total cell lysates were prepared using RIPA buffer (150 mM NaCl,
0 mM Tris-HCl pH 8.0, 5 mM EDTA, 1% sodium deoxycholate, 0.1%
cell line is widely used to study monocyte/macrophage biology in
cultured systems. BV-2 cells were cultured in RPMI 1640 supplemented
with 2 mM L-glutamine, 100 μg/mL streptomycin, 100 IU/mL peni-
cillin, and 10% FBS.
5
SDS and 1% Triton X-100) supplemented with protease inhibitors be-
fore use. Protein concentrations were quantified using the DC protein
Assay (Bio-Rad Lab., CA, USA). Cell lysate aliquots (15–30 u g of pro-
tein) were resolved by SDS-PAGE, followed by transferring to NC/PVDF
membrane (Millipore Corp., MA, USA). After blocking with 5% skim
milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 2 h
at room temperature, the membrane was probed overnight at 4℃ with
primary antibody diluted in phosphate-buffered saline containing 0.1%
Tween-20 (PBST). After several washes with TBST, the membrane was
incubated with appropriate horseradish peroxidase-conjugated sec-
ondary antibody diluted in PBST at room temperature for 1 h. After
final washes, the immune complexes were developed using the ‘Ab
Signal’ western blotting detection reagent (AbClon, Korea).
2.1.15. Immunofluorescence
RAW264.7 cells were grown on 22-mm diameter glass coverslips
and treated with ZPE-LR (100 μg/ml), followed by LPS (1 μg/ml) ad-
dition for 4 h. Cells were washed in PBS, fixed with 3.7% formaldehyde
in PBS for 15 min at room temperature, and washed again in PBS. Ice-
cold methanol was added and cells incubated at −20 °C for 10 min,
washed in PBS, then permeabilized with 1% BSA/0.2% Triton X-100/
PBS for 1 h. Cells were then washed in PBS, incubated with antibody
against NF-κB p65 overnight at 4 °C, washed again, and incubated for
1 h with anti-rabbit IgG-FITC in 1% BSA/0.05% Triton X-100/PBS.
Coverslips were mounted on glass slides using ProLong Gold anti-fade
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K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
agent containing DAPI (Invitrogen) and photographed using a fluores-
cence microscope (BX51-Olympus Optical Co., Ltd, Center Valley, PA).
RAW 264.7 cells with p65 localization were counted per 100 cells. Data
were examined in a blind fashion by three independent reviewers to-
tally unaware of all the culture conditions to prevent observational bias.
Table 1
Inhibitory effects for each Z. piperitum part on in-
flammatory response in MIA-induced rat.
Z. piperitum
Inhibition (%)
(100 mg/kg, po)
Stalk
Root
Branchlet
Leaf
Fruit
65.0
11.8
84.7
91.6
72.8
2
.1.16. ROS production assay
ROS production was determined by H DCFDA (5,6-chloromethyl-
2
′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; Molecular
2
Probes, Eugene, OR), a redox-sensitive fluorescent dye. This was used to
evaluate intracellular ROS level by flow cytometry. VSMCs (3 × 105
cells/ml) were treated with various ZPE-LR concentrations, followed by
LPS (1 μg/ml) addition for 4 h. Cells were stained for 15 min at 37 °C
Each value represents the mean ± SD (n = 3).
Inhibition rate was relative to the control “0”.
Percentage of inhibition (Inh %) at 100 mg/kg.
2
with 5 μ M H DCFDA on ice in the dark. At least 10,000 cells for each
sample were analyzed using a Becton Dickinson FACS Calibur (BD
Biosciences, San Jose, CA). Changes in intracellular ROS level were
expressed as the percentage of LPS-stimulated cells without ZPE-LR
treatment.
Table 2
Z. piperitum extract inhibitory effect on inflammatory response in MIA-induced
rat.
Layers
Yield (%)
Inhibition (%)
50 mg/kg, po)
(
2
.1.17. Gene fishing annealing control primer (ACP)-based GeneFishing
PCR
Differentially expressed genes were screened using the ACP-based
n-Hexane
CH Cl
2 2
EtOAc
n-BuOH
19.6
39.6
4.8
7.4
25.0
24
48
82
27
42
PCR method with the GeneFishing DEG kits (Seegene, Seoul, Korea).
Briefly, second-strand cDNA synthesis was conducted at 50 °C during
one first-stage PCR cycle in a final reaction volume of 20 μl containing
H
2
O
3
1
–5 μl of diluted first-strand cDNA, 1 μl of dT-ACP2 (10 μM), 1 μl of
0 μM arbitrary ACP and 10 μl of 2X Master Mix (Seegene). The PCR
Each value represents the mean ± SD (n = 3). Inhibition rate was relative to
the control “0”. Percentage of inhibition (Inh %) at 50 mg/kg.
protocol for second-strand synthesis was one cycle at 94 °C for 1 min,
followed by 50 °C for 3 min and 72 °C for 1 min. After second-strand
DNA synthesis was completed, the second-stage PCR amplification
protocol was 40 cycles at 94 °C for 40 s, followed by 65 °C for 40 s and
Table 3
ZPE-LR inhibitory effect on inflammatory response in MIA-induced rat.
EtOH (%)
Yield (%)
Inhibition (%)
72 °C for 40 s and a 5-min final extension at 72 °C. Amplified PCR
(100 mg/kg, po)
products were separated on a 2% agarose gel stained with ethidium
bromide. Amplified cDNA fragments with > 2-fold differential band
intensities were re-amplified and extracted from the gel by using the
GeneClean II kit (Qbiogene, Solon, OH, USA), and directly sequenced
with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
0
1
2
30
40
5
6
7
8
7.26
2.02
2.00
4.03
3.98
3.53
6.06
4.42
5.26
3.57
2.87
30.8
31.8
36.4
38.3
40.2
41.1
55.1
55.1
57.9
71.0
91.6
0
0
0
0
0
0
2.1.18. PCR array
Pain-related gene RT2 Profiler PCR Array was purchased from
SuperArray Bioscience Corporation (Qiagen,). Following RNA extrac-
tion and cDNA synthesis, qRT-PCR was performed in an ABI Prism 7300
Sequence Detector (Applied Biosystems). Relative gene expression was
calculated using the DDCt method and was further analyzed with the
PCR Array Data Analysis Template V3.3 (Qiagen).
90
100
Each value represents the mean ± SD (n = 3). Inhibition rate was relative to
the control “0”. Percentage of inhibition (Inh %) at 100 mg/kg.
suppression was most effective with 90% EtOH (Table. 3). The 90%
EtOH extract (ZPE-LR) was used for the next experiment.
2
.1.19. Statistical analysis
Each result is reported as means ± the standard error of the mean
ZPE-LR (20 L) was extracted as 90% EtOH and the obtained con-
(
SEM). One-way analysis of variance was used to determine significance
centration solution was fractioned as n-hexane layer (98 g), CH
2 2
Cl
between groups, after which modified t –tests and two-way ANOVA
were performed by Graph pad Prism 5
(
198 g), EtOAc layer (24 g), BuOH layer (37 g), and H O layer (125 g).
2
Suppression activation for each was evaluated. The EtOAc layer showed
the most dominant activation. EtOAc fraction (9.8 g of 24 g used) se-
paration was attempted through open calumny including MM-P sup-
pressing activity-guided fractionation and isolation (Fig. 1A). Two
substances in the separated components were found to have the highest
purity after HPLC chromatogram in order (Fig. 1B and C). NMR analysis
on each component was conducted to confirm organization. Single
component of compound 1and compound 2 was confirmed. When NMR
spectra were compared with results from the initial separated compo-
nents, compound 1 was Quercetin 3-O-a-L-rhamnoside (Quercetrin),
and compound 2 was Kaempferol 3-O-a-L rhamnoside (Afzelin) (Fig. 1D
and E) [27].
3
. Results
3.1. Zanthoxylum piperitum active component isolation and determination
To confirm which Zanthoxylum piperitum component was effective in
pain suppression, the following experiments were conducted: mice
stomach was injected with 1.5% acetic acid, and writhing syndrome
occurred. Various ZPE parts were orally injected, and writhing counted.
Zanthoxylum piperitum leaf and small branch showed the highest pain
suppression percentage (Table 1). When Zanthoxylum piperitum leaves
and ramulus were extracted as a solvent, 82% was associated with the
most effective pain suppression (Table. 2). Furthermore, Zanthoxylum
piperitum leaves and ramulus were extracted and administered in di-
verse concentrations (0∼100%) to document pain suppression. Pain
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K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
performed a loaded weight ratio between left and right legs after ar-
thritis inducement. Celecoxib (10 mg/kg) [28] and ZPE-LR (100 mg/kg)
were injected for five weeks and results observed. Negative control
showed a change in left foot loaded weight ratio that was kept above
60%. However, for positive controls treated with ZPE-LR and celecoxib,
loaded weight ratio decreased compared to negative control, and a
th
significant drug effect was observed on the 14 day compared to the
negative control (Fig. 2A). Also, right cartilage inflammation in the
induced knee was histologically observed. For negative control, ar-
ticular cartilage thickened significantly whereas in the celecoxib and
ZPE-LR orally injected group, joint cartilage thickness thinned (Fig. 2B).
To confirm ZPE-LR pain-amelioration in the central nervous system,
ZPE-LR 25, 50 and 100 mg/kg was administered orally an hour before
acetic acid 1.5% was injected inside mice stomach, and abdominal
writhing syndrome was induced. Writhing episodes with ZPE-LR 50 and
100 mg/kg decreased similarly those seen in the celecoxib positive
control group (Fig. 3A). Consequently to confirm ZPE-LR pain-ameli-
oration in the central nervous system, hot plate and formalin tests were
used. For the hot plate method, ZPE-LR 25, 50 and 100 mg/kg was
orally injected an hour before hot plate treatment. Seconds till mice
flicked their hind-foot or licked was recorded. ZPE-LR 25, 50 and
1
00 mg/kg treated groups showed 5.2%, 34.7% and 74% pain sup-
Fig. 2. ZPE-LR ability to reduce pain in OA-induced rats. (A) Comparison of
change in hind paw weight for the ZPE-LR treatment group. (B) H&E stained
sections representative of osteoarthritis. Saline control group; no stimulation,
MIA treatment group ; control group, celecoxib group; stimulation with MIA
and celecoxib (10 mg/kg) by oral administration (p.o.) and ZPE-LR group; sti-
mulation with MIA and ZPE-LR (100 mg/kg) by p.o.
pression effects, and significance was observed in 50 and 100 mg/kg
treated groups (Fig. 3B). To confirm ZPE-LR pain suppression effects, a
formalin test was conducted. Celecoxib 10 mg/kg and ZPE-LR 25, 50
and 100 mg/kg were orally injected into mice, and phase 1 (0∼9 min),
phase 2 (30∼39 min) was measured under 10% formalin injection, and
response number was recorded. Formalin test results yielded 34% in
phase 1 for the ZPE-LR 100 mg/kg group, and 56.8% in phase 2 for the
ZPE-LR 100 mg/kg group (Fig. 3c).
3.2. ZPE-LR effect on analgesic activity during osteoarthritis inducement
To measure ZPE-LR ability to reduce the pain in OA rats, we
Fig. 3. ZPE-LR effect on analgesic activity in
osteoarthritis inducement. (A) Mice were
treated with ZPE-LR (25, 50 and 100 mg/kg)
and celecoxib (10 mg/kg) for 1 h before 0.6%
aqueous solution of acetic acid (10 ml/kg, i.p.)
injection. Number of writhes was counted for
20 min after acetic acid administration. (B)
Mice were placed on a heated (56℃) metal
plate. Latency for jumping or licking paws was
determined. (C) ZPE-LR-treated mice were in-
jected with 2% formalin in 0.9% saline into the
dorsal surface of the right hind paw. Time spent
licking and biting the injected paw was mea-
sured with a hand-stop watch from 0 to 9 min
(
first phase) and from 30 to 39 min (second
phase) after formalin injection. Values are ex-
pressed as the mean ± SEM (n = 6).
*p < 0.05, indicates statistically significant
differences in the vehicle control group.
485

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Biomedicine & Pharmacotherapy 105 (2018) 481–490
Fig. 4. Expression changes in genes related
to pain assessed by PCR arrays. (A) BV-2
mouse microglia cells were treated with ZPE-
LR (100 μg/ml). Gene fold changes indicate
relative expression compared to control. Kcnj6
potassium inwardly-rectifying channel, sub-
family J, member 6; Kcnq2 potassium voltage-
gated channel, subfamily Q, member 2; Kcnq3
potassium voltage-gated channel, subfamily Q,
member 3; Scn10a sodium channel, voltage-
gated, type X, alpha; Scn11a sodium channel,
voltage-gated, type XI, alpha; Scn3a sodium
channel, voltage-gated, type III, alpha; Scn9a
sodium channel, voltage-gated, type IX, alpha.
(B) Inwardly rectifying K + currents increased
by ZPE-LR in BV-2 mouse microglia cells. I–V
relationship plotted from average currents re-
corded using step pulses from −0.16
to + 0.06 mV during 400–500 ms (5, 60, and
140 mM KCl). Data points are mean ± SEM
from 5 to 7 separate bilayers. control group;
stimulation with 0.5% DMSO and ZPE-LR
group; stimulation with DMSO and ZPE-LR
(100 mg/kg) by p.o.
3
.3. ZPE-LR pain inhibitory mechanism investigation
K + currents.
.4. ZPE-LR effects on inflammatory responses in osteoarthritis mice and
To confirm ZPE-LR’s pain inhibitory mechanism, pain-related gene
3
expression including membrane protein pain genes involved in pain
suppression, were evaluated. RNA was isolated from BV-2 cell treated
with ZPE-LR (100 μg/ml) and a non-treated group, then PCR array was
conducted. Results confirmed changes in K + ion gate controlling
membrane protein (Kcnj, Kcnq) and sodium channel voltage-gated
LPS-induced RAW 264.7 cell
To verify in vivo ZPE-LR effects on inflammation, TPA (12-O-tetra-
decanoylphorbol-13-acetate) was topically applied to rat right ear for
three days and sub-chronic edema occurred. ZPE-LR (25, 50 and
(
Scn) gene expression. When the cells were treated with ZPE-LR, KCNJ6
100 mg/kg), celecoxib for the positive control group (10 mg/kg) and
mRNA expression increased 12 times (Fig. 4A). Based on this data, in-
wardly rectifying K + currents were analyzed in BV-2 mouse microglia
cells, the current was increased about two times when ZPE-LR treated
dexamethasone (1 mg/kg) were orally injected. On day 3, rats were
sacrificed and ear thickness was assessed with a thickness gauge, then a
biopsy punch was used to measure weight per ear. Ear thickness and
weight increased significantly after TPA intervention in the two positive
control groups. ZPE-LR 50 and 100 mg/kg orally injected groups
showed significant increased ear thickness and weight (Fig. 5A and B).
Also, to confirm suppression effects on iNOS, and COX-2 which induce
inflammation related chronic pain, mRNA and protein was derived
from mice ear tissue. iNOS and COX-2 mRNA expression confirmation
through RT-PCR indicated that iNOS and COX-2 mRNA expression was
(
Fig. 4B).
There are many cases where clear reasons for chronic pain are
elucidated. Osteoarthritis is known to induce pain through peripheral
nerve inflammation or repetitive stimuli to damage tissue, and to inflict
pain through inflammation and infection in the central nervous system
[
29,30]. Taken together these results suggest ZPE-LR can exert both
peripheral as well as central analgesic effects possibly by blocking
486

K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
Fig. 5. ZPE-LR effects on inflammatory response in vivo and in vitro. The right ear of each mouse was topically treated with ZPE-LR (25, 50 and 100 mg/ear) in
0 μl, prior to 1 μg/ear TPA application in 50 μl. The left ears were treated with vehicle alone. 24 h later, edema was measured as thickness (A) and right ear weight
B) over left ear weight. (C and D) iNOS and COX-2 expression level in TPA-induced ear tissue was determined by RT-PCR or western blot analysis, respectively. β-
actin was used as a loading control. Data are the mean ± S.E.M for three independent experiments. * p < 0.05, significantly different from TPA group.
5
(
significantly suppressed in a concentration-dependent manner post
ZPE-LR treatment (Fig. 5C) TPA-induced COX-2 protein level decreased
in a ZPE-LR concentration-dependent manner (Fig. 5D).
showed that LPS induced p65 translocation into the nucleus. When the
highest ZPE-LR concentration (100 μg/ml) was applied, p65 did not
translocate into the nucleus, but remained in the cytoplasm. ZPE-LR
interference on p65 intracellular translocation was confirmed (Fig. 6B).
In addition, ROS (reactive oxygen species) are known to regulate LPS
induced p65 intracellular translocation [31]. To investigate whether
3.5. ZPE-LR anti-inflammatory effects on cell-activation mechanisms in
LPS-induced RAW 264.7 and BV-2 cells
ZPE-LR suppressed LPS-induced ROS generation, H DCFDA was used.
2
LPS-induced ROS generation was confirmed, and ZPE-LR 25, 50 and
To investigate the cell-activating mechanism underlying anti-in-
flammatory effects, Raw 264.7 cells were used. ZPE-LR 25, 50 and
00 μg/ml were applied for 24 h, and MTT was used for cellular toxi-
city; no cellular toxicity was found at any concentration level (data not
shown). Next, we examined ZPE-LR effects on iNOS and COX-2 ex-
pression in LPS-treated Raw 264.7 cells. ZPE-LR 25, 50 and 100 μg/ml
were applied for an hour, and LPS 1 μg/ml was applied for 12 h to in-
crease iNOS and COX-2 manifestation. iNOS and COX-2 protein of
which increased by LPS were decreased concentration dependently
100 μg/ml decreased a ROS generation in a concentration dependent
manner (Fig. 6C and D). Moreover, to investigate new ZPE-LR acti-
vating mechanisms involved in osteoarthritis, the gene fishing method
was used. Gene fishing was a method to compare the expression of
genes when a substance was treated and when it was not treated with a
universal primer. Through this method, we found genes that were in-
creased and suppressed when ZPE-LR was treated. Various gene li-
braries showed increased by LPS (data not shown). Among them, the
HPRT1 (hypoxanthine phosphoribosyltransferase 1) was decreased by
ZPE-LR. On the other hand, in case of RPL8 (Ribosomal protein L8),
when ZPE-LR treated, it was increased (Fig. 6E).
1
(
Fig. 6A).
The expression of iNOS and COX-2 requires the activation of NF-κB,
which is an important mechanism for the overproduction of the in-
flammatory mediators in macrophages in response to LPS and cytokines
[
47,48]. NF-κB is located in the cytosol and is bound to the inhibitory
4. Discussion
IκB protein under unstimulated conditions. The activation of NF-κB in
response to LPS stimulation leads to an increase in nuclear translocation
and DNA binding ability.
Next, when iNOS and COX-2 expression was increased by LPS, im-
munofluorescence assay was conducted. Immunofluorescence assay
In this study, we conducted an experiment to investigate ZPE-LR
effects in arthritis-induced rat. In conclusion, ZPE-LR suppression ef-
fects on osteoarthritic pain and inflammatory generation by 90% EtOH
extract of leaf and ramulus of Zanthoxylum piperitum (ZPE-LR) was
487

K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
Fig. 6. ZPE-LR anti-inflammatory effects on cell-activa-
tion mechanisms in LPS-induced RAW 264.7. RAW 264.7
cells were pre-treated with ZPE-LR for 1 h and then treated
with LPS for 12 h. (A) iNOS and COX-2 expression levels were
determined by western blot analysis (B) RAW 264.7 cells were
pre-treated with ZPE-LR for 1 h and then treated with LPS for
30 h. p65-FITC was added to RAW 264.7 cells for 12 h and
DAPI was added for 20 min prior to observation. p65-FITC
translocation from cytosol to nucleus was observed under a
fluorescent microscope at 400
Representative images of intracellular ROS levels by
DCFDA fluorescence (green). (D) H DCFDA fluorescence
x
magnification. (C)
H
2
2
relative quantification was determined by flow cytometry. (E)
Agarose gel electrophoresis for gene fishing PCR products.
mRNA was obtained from RAW264.7 cells following LPS
treatment of (1 μg/ml). HPRT1 and RPL8 mRNA levels were
determined. Data are the mean ± S.E.M for three in-
dependent experiments. * p < 0.05, significantly different
from LPS-treated cells.
confirmed.
mechanisms underlying osteoarthritis intervention and amelioration
are not known. In this study, we conducted an experiment to investigate
ZPE-LR effects Raw 267.4 cells, and its effect in arthritis-induced rat
and central and peripheral nervous system pain-induced mice. It was
Z. piperitum is already known to be useful in for anti-bacterial, anti-
oxidant, and tyrosinase interference activation effects as well as in os-
teosarcoma cell proliferation suppression. However, Z. piperitum
488

K.-A. Hwang et al.
Biomedicine & Pharmacotherapy 105 (2018) 481–490
confirmed that ZPE-LR oral-administration reduced loaded weight
change ratio in arthritis-induced rats. H&E staining in MIA-induced
osteoarthritis showed decreased cartilage thickness.
to elucidate the role of HPRT1 and R8L8 in osteoarthritis.
In summary, in vitro treatment with ZPE-LR at each concentration
significantly inhibited COX-2 and iNOS expression, p65 translocation
into the nucleus and ROS generation in LPS-induced RAW 264.7 cells.
In vivo, suppression of pain and inflammation in MIA-induced os-
teoarthritis and several in vitro cell lines by ZPE-LR suggests it may have
a curing effect on degenerative process.
COX-2 is an enzyme protein that changes arachidonic acid to
prostaglandin, thromboxane and plays an important role in regulating
inflammatory responses and nerve pain [32–34]. COX-2 expression
inhibition is known to cause pain and ameliorate inflammation. In
addition, NO (Nitric oxide) is formed from L-arginine by nitric oxide
synthase (NOS) [35,36], and contributes to inflammatory function,
blood coagulation, and blood regulation [37]. NO is also a neuro-
messenger that sends signals between synapses in the central and per-
ipheral nervous systems [38]. NOS (nitric oxide synthase) is mostly
concentrated on the surface area of olfactory sense in spinal cord [39].
According to previous studies, when chemical stimuli are applied to the
central nervous system, NO increases in responsive nerve cell of ol-
factory nerve of spinal cord, which causes neurological pain [40]. We
confirmed that ZPE-LR inhibits COX-2 and iNOS mRNA and protein
expression which were increased in MIA-induced osteoarthritis rats. For
central nervous and peripheral nervous pain caused by osteoarthritis,
an in vivo experiment was conducted to determine the pain relief ef-
fects. An acetic acid-induced writhing syndrome test (peripheral ner-
vous system), a hot plate test (central nervous system) and a formalin
test (both systems) was conducted. ZPE-LR suppressed pain not only in
the peripheral, but also the central nervous system. ZPE-LR treatment
inhibited COX-2 and iNOS mRNA and protein levels and resulted in
reduced pain in both the central and peripheral nervous systems and
showed anti-inflammatory, pain-relief effects.
Conflict of interest
The authors declare that there are no conflicts of interest.
Funding
This work was supported by Korea Institute of Planning and
Evaluation for Technology in Food, Agriculture, Forestry and Fisheries
(IPET) through Agri-Bio industry Technology Development Program,
funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA)
(314069-3).
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