Research Article Evaluation of Anti-Inflammatory Potential...

Research ArticleEvaluation of Anti-Inflammatory Potential ofthe New Ganghwaljetongyeum on Adjuvant-InducedInflammatory Arthritis in Rats

Wangin Kim1 Sangbin Park1 Chanhun Choi1 Youg Ran Kim2 Inkyu Park3

Changseob Seo4 Daehwan Youn1 Wook Shin1 Yumi Lee1 Donghee Choi1 Mirae Kim1

Hyunju Lee5 Seonjong Kim1 and Changsu Na1

1College of Korean Medicine Dongshin University 185 Geonjae-ro Naju-si Jeollanam-do 58245 Republic of Korea2College of Pharmacy Chonnam National University 77 Yongbong-ro Buk-gu Gwangju 61186 Republic of Korea3College of Medicine Chonnam National University 77 Yongbong-ro Buk-gu Gwangju 61186 Republic of Korea4Mibyeong Research Center Korea Institute of Oriental Medicine 1672 Yuseong-daero Yuseong-gu Daejeon 34054 Republic of Korea5School of Information and Communications Gwangju Institute of Science and Technology 123 Cheomdangwagi-ro Buk-guGwangju 61005 Republic of Korea

Correspondence should be addressed to Changsu Na nakugihanmailnet

Received 22 October 2015 Revised 22 April 2016 Accepted 24 April 2016

Academic Editor Ke Ren

Copyright copy 2016 Wangin Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ganghwaljetongyeum (GHJTY) has been used as a standard treatment for arthritis for approximately 15 years at the KoreanMedicine Hospital of Dongshin University GHJTY is composed of 18 medicinal herbs of which five primary herbs were selectedand named new Ganghwaljetongyeum (N-GHJTY) The purpose of the present study was to observe the effect of N-GHJTY onarthritis and to determine its mechanism of action After confirming arthritis induction using complete Freundrsquos adjuvant (CFA) inrats N-GHJTY (625 125 and 250mgkgday) was administered once a day for 10 days In order to determine pathological changesedema of the paws and weight were measured before and for 10 days after N-GHJTY administration Cytokine (TNF-120572 IL-1120573 andIL-6) levels and histopathological lesions in the knee joint were also examined Edema in the paw and knee joint of N-GHJTY-treated rats was significantly decreased at 6 8 and 10 days after administration compared to that in the CFA-control group whileweight consistently increased Rats in N-GHJTY-treated groups also recovered from the CFA-induced pathological changes andshowed a significant decline in cytokine levels Taken together our results showed that N-GHJTY administration was effective ininhibiting CFA-induced arthritis via anti-inflammatory effects while promoting cartilage recovery by controlling cytokine levels

1 Introduction

Arthritis collectively refers to more than 100 rheumaticdiseases that are characterized by inflammation pain andstiffness in the musculoskeletal system and that range fromlocalized self-limiting conditions to systemic autoimmuneprocesses [1] Arthritis occurs in all age groups and peaksbetween the ages of 35 and 50 affecting sim1 of the worldrsquospopulation [1 2] Rheumatoid arthritis (RA) is a systemicinflammatory disease that attacks the joints by producingproliferative synovitis leading to destruction of articularcartilage and underlying bone RA affects approximately 05

to 1 adults worldwide with women being affected two tothree times more frequently than men RA is also associatedwith a high mortality rate [1 3]

Autoimmunity and chronic inflammation are activatedby an imbalance between pro- and anti-inflammatorycytokines thereby causing joint damage in RA [4] RA ischaracterized by angiogenesis in the synovial membranewhich contributes to the advancement of the disease as wellas the production of inflammatory cells that infiltrate anddestroy the synovial tissue [1 5 6] In RA production of bothcytokines and chemokines is induced by macrophage- andfibroblast-derived cytokines [7] Of these proinflammatory

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 1230294 10 pageshttpdxdoiorg10115520161230294

2 Evidence-Based Complementary and Alternative Medicine

interleukin 1 beta (IL-1120573) and tumor necrosis factor alpha(TNF-120572) are often targeted in RA treatment strategies [8 9]

Our previous study showed that Ganghwaljetongyeum(GHJTY) can effectively attenuate RA by inhibiting synovialcell proliferation as well as the production of proinflamma-tory mediators from macrophage-like cells [10] However 18medicinal herbs are included in GHJTY making it imprac-tical for clinical application In order to improve the efficacyand convenience of pharmaceutically prescribing GHJTY weused bioinformatics (httpcombiogistackrherding) [11] toselect the five medicinal herbs with the greatest potentialto treat arthritis The selected medicinal herbs were Oster-icum koreanum Maximowicz (Osterici Radix OK) Lonicerajaponica Thunberg (Lonicerae Folium LJ) Clematis mand-shurica Ruprecht (Clematis Radix CM) Angelica gigasNakai(Angelicae Gigantis Radix AG) and Phellodendron amurenseRuprecht (Phellodendri Cortex PA)

A review of the literature revealed that each of theselected herbs is effective in targeting certain aspects ofRA pathophysiology For example OK inhibits the produc-tion of inflammatory mediators by downregulating nuclearfactor kappa beta (NF-120581B) and mitogen-activated proteinkinase (MAPK) activity in lipopolysaccharide-stimulatedRAW2647 cells [12] Additionally anti-inflammatory activityof the major constituents of LJ has been shown [13] alongwith the ability of CM extract to interact with NF-120581B TNF-120572and cyclooxygenase 2 (COX-2) in rats with collagen-inducedarthritis [14] Studies have also shown that AG inhibits focaland systemic inflammation in dinitrofluorobenzene-inducedinflammation models and that PA protects against humanosteoarthritis by regulating aggrecanases matrix metallo-proteinases (MMPs)tissue inhibitors of metalloproteinases(TIMP) proinflammatory cytokines and MAPK pathwaysignaling [15 16] However the effects of combining theseherbal medicines remain unclear

The purpose of the present study was to evaluate theefficacy and mechanism of action of this new GHJTY (N-GHJTY) using an animal model of RA Specifically we usedrats with complete Freundrsquos adjuvant- (CFA-) induced arthri-tis as an experimental animal model used to mimic humanRA as it produces profound systemic inflammation thatresults in severe joint swelling and remodeling [17]MoreoverCFA is typically used to investigate therapeutic agents withantiarthritic potential [18] Our specific aims were to (1)confirm the quality assessment of marker components in N-GHJTY (2) determine the antiarthritic effects of N-GHJTYby measuring paw edema and observing gross lesions of thepaw and knee joint (3) evaluate potential adverse effects ofN-GHJTY by histopathological investigation and (4) assessthe effect of N-GHJTY on proinflammatory cytokines byexamining levels of TNF-120572 IL-1120573 and IL-6

2 Materials and Methods

21 Animals Adult male Sprague-Dawley rats weighing200ndash210 g were housed in a room with constant temperature(24ndash26∘C) and humidity (40ndash60) Food (Pellet GMOKorea) and water were available ad libitum Animals were

acclimated to the laboratory environment for 1 week beforethe experiment and all procedures were approved by theInstitutional Animal Care and Use Committee of the Dong-shin University (2014-03-02)

22 CFA-Induced Arthritis and Drug Administration In theprimary adjuvant-induced arthritis model 025mL of CFA(Sigma St Louis MD USA) was injected into the left hindknee joint After 7 days secondary arthritis was inducedby injecting 005mL of CFA under the left hind knee jointand left hind sole Animals were then divided into thefollowing five groups (119899 = 6group) normal CFA arthritis(CFA-control) and CFA arthritis treated with 625 125 and250mgkg of N-GHJTY extract per day (N-GHJTY-625 N-GHJTY-125 and N-GHJTY-250 resp) Oral administrationof N-GHJTY was initiated on the 10th day after arthritisinduction and continued for 10 days thereafter Animalswere anesthetized using 25 isoflurane and volumes of thehind paw and knee joint were measured using a DigitalPlethysmometer (LE7500 Panlab Spain) 0 2 4 6 8 and 10days after oral N-GHJTY administration

23 Preparation of Herbal Materials The five herbal med-icines forming N-GHJTY were purchased from OmniherbCo (Yeongcheon Korea) and their origin was taxonomicallyconfirmed by Professor Jong-Kil Jeong in the Departmentof Herbology at the College of Oriental Medicine DongshinUniversity

The five herbs (OK LJ AG CM and PA) were combinedin a 6 4 4 4 3 ratio N-GHJTY was prepared via waterextraction at 100∘C for 3 h and concentrated using a rotaryvacuum evaporator (EYELA Japan) after filtration followedby lyophilization using a vacuum freeze drier (SamwonFreezing Engineering Co Korea)The yield was about 295

24 Reagents and High-Performance Liquid Chromatography(HPLC) Analysis Chlorogenic acid (1) and berberine chlo-ride (2) were purchased from Acros Organics (PittsburghPA USA) and Sigma-Aldrich Co LLC (St Louis MOUSA) respectively Nodakenin (3) decursin (6) and decursi-nol angelate (7) were purchased form NPC BioTechnol-ogy (Yeongi Korea) Isoferulic acid (4) and oxypeucedaninhydrate (5) were purchased from ChemFaces BiochemicalCo Ltd (Wuhan China) The purities of the seven referencecompounds were ge980 by HPLC analysis and the chemicalstructures of the seven marker compounds are shown inFigure 1 HPLC-grade solvents methanol acetonitrile andwater were obtained from JTBaker (Phillipsburg NJ USA)Analytical grade formic acid was purchased from Sigma-Aldrich (St Louis MO USA)

For quality assessment of the seven marker compoundsin N-GHJTY all experiments were conducted using a Shi-madzu Prominence LC-20A Series (Shimadzu Kyoto Japan)equipped with a solvent delivery unit (LC-20AT) onlinedegasser (DGU-20A3) column oven (CTO-20A) autosam-ple injector (SIL-20AC) and photodiode array (PDA) detec-tor (SPD-M20A) Lab solution software (version 554 SP3Kyoto Japan) was used for data acquisition and processing

Evidence-Based Complementary and Alternative Medicine 3

OH

OH

O

O

OH

OHOH

O

HO

Chlorogenic acid (1)

O

O

Berberine chloride (2)

OO O

Nodakenin (3)

O

OHO

HOOH

HO

OH

O

Isoferulic acid (4)

OH

OO O

Oxypeucedanin hydrate (5)

O

OH

OH

OO O

O

O

Decursin (6)

OO O

O

O

Decursinol angelate (7)

OCH3

OCH3

N+ Clminus

H3CO

Figure 1 Chemical structure of the seven marker compounds

Chromatographic separation of all analytes was performedusing a Waters SunFire C18 column (46 times 250mm 5 120583mMilford MA USA) The mobile phases consisted of 01(vv) formic acid in distilled water (A) and 01 (vv) formicacid in acetonitrile (B) and the gradation condition wasoptimized as follows with range of 0ndash30min 10ndash100 B 30ndash40min 100 B 40ndash50min 100ndash10 B 50ndash60min 10 BThe flow rate of mobile phase was maintained at 10mLminand the injection volume was 10mL The flow rate wasmaintained at 10mLmin and the injection volume was10mL For HPLC analysis lyophilized N-GHJTY (200mg)was dissolved in 20mL of 70 methanol and extracted for60min by sonication The N-GHJTY extract solution waspassed through a 02-120583m syringe filter (PALL Life SciencesAnn Arbor MI USA) before HPLC analysis

25 Blood and Serum Tests Blood samples were collectedand 100 120583L was used for a complete blood count (CBC) anal-ysis via a Multispecies Hematology Analyzer (950 HemavetUSA) Serum was separated from the rest of the blood usinga high-speed centrifuge (VS-600CFi Korea) at 3500 rpm

(119892 = 27391) for 20min and aspartate aminotransferase(AST) and alanine transaminase (ALT) levels weremeasured

26 Measurement of TNF-120572 IL-1120573 and IL-6 TNF-120572 wasmeasured using a Rat TNF-120572 kit (Invitrogen USA) IL-1120573wasassessed using a Rat IL-1120573 kit (RampD Systems USA) and IL-6was evaluated using a Rat IL-6 kit (Invitrogen USA) Opticaldensities (OD) of all samples were measured at 450 nm viaSpectramax (M2 Molecular Devices USA)

27 Hematoxylin and Eosin (HE) Staining The right kneejoint was removed and fixed in Bouin solution for over 24 hDecalcification was conducted in a 25 nitric acid solutionwhich was changed once a day for 7 daysThe removed tissuewas dehydrated using Tissue Processor (Tissue-Tex II Japan)deparaffinized stained with HE (Muto Japan) and observedunder an optical microscope (Nikon Japan)

28 Safranin O-Fast Stain After deparaffinization the rightknee joint was reacted with Weigertrsquos Iron Hematoxylin


Table 1 Changes in paw swelling after N-GHJTY administration in rats with CFA-induced arthritis (mL)

Group Before Days after CFA (days N-GHJTY administration)10 (0) 12 (2) 14 (4) 16 (6) 18 (8) 20 (10)

Normal 136 plusmn 003 147 plusmn 004 149 plusmn 004 155 plusmn 005 157 plusmn 006 163 plusmn 005 166 plusmn 006CFA-control 124 plusmn 001 377 plusmn 025 378 plusmn 026 391 plusmn 026 391 plusmn 028 396 plusmn 028 400 plusmn 030

N-GHJTY-625 126 plusmn 003 368 plusmn 033 372 plusmn 031 368 plusmn 026 353 plusmn 023 335 plusmn 021 323 plusmn 017lowast

N-GHJTY-125 124 plusmn 003 368 plusmn 015 367 plusmn 015 360 plusmn 017 336 plusmn 020 315 plusmn 020lowast 303 plusmn 020lowast

N-GHJTY-250 131 plusmn 004 363 plusmn 016 366 plusmn 016 349 plusmn 010 322 plusmn 008lowast 305 plusmn 005lowast 291 plusmn 009lowastlowast

Mean plusmn SE119875 lt 001 versus normal grouplowastlowast

119875 lt 001 and lowast119875 lt 005 versus CFA-control group

(Sigma USA) solution for 10min and stained with 0001Fast Green (Sigma USA) solution for 5min The knee jointtissue was then reacted with 1 acetate solution for 10 sand stained with 01 Safranin O (Sigma USA) solution for5min thereafter the tissue was dehydrated and observedunder an optical microscope (Nikon Japan)

29 Statistical Analysis Data were analyzed using SPSS 210version for Windows by a nonparametric Mann-Whitney 119880test A one-way analysis of variance was conducted on eachgroup and results are expressed as mean plusmn standard error(SE) Comparisons between groupswere performed using thepost hoc least squared differences (LSD) test 119875 lt 005 and119875 lt 001 were considered statistically significant

3 Results

31 Quality Assessment of Seven Marker Components inN-GHJTY HPLC was performed using the seven markercompounds in N-GHJTY for quality control The selectedcompoundswere as follows compound 1 (Lonicerae Folium)compound 2 (Phellodendri Cortex) compounds 3 6 and 7(Angelicae Gigantis Radix) compound 4 (Clematis Radix)and compound 5 (Osterici Radix) All analytes were sep-arated within 30min and the typical three-dimensionalchromatogram of the 70 methanol extract of N-GHJTY isshown in Figure 2 Quantitation was achieved by photodiodearray (PDA) detection at 310 nm (5) 325 nm (1 and 4)330 nm (6 and 7) 335 nm (3) and 340 nm (2) based onretention time and UV spectrum The retention times ofcomponents 1ndash7 were 894 1080 1200 1286 1595 2602and 2624min respectively Using a calibration curve wedetermined that correlation coefficients (1199032) of all sevencompoundswerege09996Under optimized chromatographyconditions concentrations of N-GHJTY marker compounds1ndash7 were 546 plusmn 033 187 plusmn 029 170 plusmn 018 164 plusmn 058203 plusmn 033 109 plusmn 002 and 081 plusmn 001mgg respectively

32 Effect of N-GHJTY on Gross Lesions of the Paw and KneeJoint Paw and knee joint swelling and rubefaction servedas external objective indicators for evaluating the severity ofthe inflammatory arthritic model The CFA-control groupshowed rubefaction and hind paw and knee joint swellingmdashboth of which gradually decreased following N-GHJTY

(1) (2)(3) (4)(5) (6) (7)

550minus10

200

250

300

350

400

Wav

eleng

th (n

m)

0000

5000

10000

15000

20000

25000

30000

Time (min)

500

400

300

200

100

0

Inte

nsity

(mAU

)

Figure 2 Three-dimensional chromatogram of N-GHJTY Chloro-genic acid (1) berberine chloride (2) nodakenin (3) isoferulicacid (4) oxypeucedanin hydrate (5) decursin (6) and decursinolangelate (7)

treatment at all concentrations (625 125 and 250mgkg)(Figure 3)

33 Effect of N-GHJTY on Paw and Knee Joint SwellingChanges in paw and knee joint swelling are presented inTable 1 Approximately 3 days after the second immunizationthe rat knee joint began to swell and the paw and knee jointwere observed to increase in size On the 10th day the CFA-control group showed a significant increase in both paw andknee joint swellings compared to the normal group Thisvolume significantly decreased in the N-GHJTY-250 groupon the 6th day the N-GHJTY-125 group on the 8th day andthe N-GHJTY-625 group on the 10th day compared to theCFA-control group (Table 1)

34 Effect of N-GHJTY on Body Weight Twelve days afterCFA injection statistically significant reductions in body


(a) (b) (c) (d) (e)

Figure 3 Effect on N-GHJTY in gross lesions in the hind paw and knee swelling in rats with CFA-induced arthritis (a) Normal group (b)CFA-control group (c) N-GHJTY-625 group (625mgkg) (d) N-GHJTY-125 group (125mgkg) and (e) N-GHJTY-250 group (250mgkg)

Table 2 Changes in body weight after N-GHJTY administration in rats with CFA-induced arthritis ()


Normal 1000 plusmn 00 1452 plusmn 18 1483 plusmn 24 1536 plusmn 28 1594 plusmn 32 1634 plusmn 36 1640 plusmn 37CFA-control 1000 plusmn 00 1348 plusmn 17 1395 plusmn 22 1447 plusmn 34 1489 plusmn 43 1534 plusmn 52 1540 plusmn 51N-GHJTY-625 1000 plusmn 00 1355 plusmn 22 1429 plusmn 18 1496 plusmn 19 1574 plusmn 22 1614 plusmn 16 1621 plusmn 17N-GHJTY-125 1000 plusmn 00 1360 plusmn 25 1420 plusmn 23 1486 plusmn 25 1561 plusmn 32 1599 plusmn 36 1611 plusmn 36N-GHJTY-250 1000 plusmn 00 1364 plusmn 35 1432 plusmn 37 1495 plusmn 41 1576 plusmn 48 1615 plusmn 51 1628 plusmn 54Mean plusmn SE119875 lt 005 versus normal group

Table 3 Effect of N-GHJTY on aspartate and alanine aminotrans-ferase levels in rats with CFA-induced arthritis

Group Aspartateaminotransferase (UL)

Alanineaminotransferase (UL)

Normal 820 plusmn 50 284 plusmn 16CFA-control 937 plusmn 46 382 plusmn 29

N-GHJTY-625 860 plusmn 78 390 plusmn 21N-GHJTY-125 854 plusmn 19 287 plusmn 34N-GHJTY-250 800 plusmn 30lowast 288 plusmn 13lowast

Mean plusmn SE119875 lt 005 versus normal grouplowast

119875 lt 005 versus CFA-control group

weight were observed in the CFA-control group when com-pared to the normal group N-GHJTY treatment at all con-centrations (625 125 and 250mgkg) resulted in an increasein body weight however this change was not significant(Table 2)

35 Effect of N-GHJTY on Transaminase Levels Aspartateaminotransferase (AST) and alanine aminotransferase (ALT)levels are indicated in Table 3 As shown in the results only

Table 4 Effect of N-GHJTY on TNF-120572 IL-1120573 and IL-6 levels in ratswith CFA-induced arthritis

Group TNF-120572 (pgmL) IL-1120573 (pgmL) IL-6 (pgmL)Normal 233 plusmn 012 229 plusmn 095 43 plusmn 041CFA-control 485 plusmn 067 367 plusmn 094 180 plusmn 087

N-GHJTY-625 324 plusmn 032lowast 268 plusmn 084lowast 110 plusmn 05lowast

N-GHJTY-125 301 plusmn 018lowast 262 plusmn 052lowastlowast 104 plusmn 06lowast




the N-GHJTY-250 group showed a significant decrease inAST and ALT when compared to CFA-control group rats

36 Effect of N-GHJTY on Proinflammatory Cytokines TNF-120572 IL-1120573 and IL-6 levels are indicated in Table 4 As shownin the results TNF-120572 IL-1120573 and IL-6 in the CFA-controlgroup showed a significant increase when compared to levelsin the normal group rats A significant decrease in TNF-120572 IL-1120573 and IL-6 levels was observed in all N-GHJTY treatment


BSM

SM

ACF

ACT

JC

MH and FC

(a) (b)

(c) (d)

(e)

Figure 4 Influence of N-GHJTY on CFA-induced histopathological changes in the knee joints of CFA-induced arthritic rats Arrows (darr)indicate a damaged synovial membrane Arrow heads (998787) indicate compressed articular cartilage in the CFA control (a) Normal group (b)CFA-control group (c) N-GHJTY-625 group (625mgkg) (d) N-GHJTY-125 group (125mgkg) and (e) N-GHJTY-250 group (250mgkg)JC joint cavity ACF articular cartilage of the femur ACT articular cartilage of the tibia BSM bony spicule within themeniscus SM synovialmembrane MHampFC meniscus of hyaline and fibrocartilage HE stain scale bars = 500 nd

groups (625 125 and 250mgkg) when compared to theCFA-control group rats (Table 4)

37 Effects of N-GHJTY onHistopathological Changes Assessedwith HE Staining Representative HE stained histopatho-logical lesions in the hind knee joint of normal CFA-control N-GHJTY-625 N-GHJTY-125 and N-GHJTY-250groups are shown in Figure 4 Loose synovial membranemembrane destruction disorganized cell arrangement andcompressed cartilage were observed in the CFA-controlgroup (Figure 4(b)) Histopathological changes improved inall N-GHJTY groups (625 125 and 250mgkg) compared to

theCFA-control groupThese groups presented close synovialmembrane and regular cartilage the surface of the cartilage inthe tibia and femur was smooth and exhibited no noticeabledamage (Figures 4(c) 4(d) and 4(e))

38 Effects of N-GHJTY onHistopathological Changes Assessedwith Safranin O-Fast Staining Safranin O-fast stain wasconducted to observe histopathological changes in the kneejoint In the normal group (Figure 5(a)) a positive reactionto proteoglycans in the calcified zone was observed andthe cartilage was even The CFA-control group exhibitedlittle positive reactions and chondrocyte nuclei appeared


SB

AC

(a) (b)

(c) (d)

(e)

Figure 5 Influence of N-GHJTY on CFA-induced histopathological changes in the knee joints of CFA-induced arthritic rats A numberof shrunken nuclei (arrows darr) were observed in the CFA control (a) Normal group (b) CFA-control group (c) N-GHJTY-625 group(625mgkg) (d) N-GHJTY-125 group (125mgkg) and (e) N-GHJTY-250 group (250mgkg) AC articular cartilage SB spongy boneSafranin O-fast stain scale bars = 100 120583m

contracted when compared to nuclei in the normal group(Figure 5(b)) N-GHJTY-625 N-GHJTY-125 and N-GHJTY-250 groups exhibited a greater number of positivereactions in the calcified zone of the cartilage when comparedto the CFA-control group (Figures 5(c) 5(d) and 5(e))

4 Discussion

RA is an abnormal autoimmune disease that causes synovialinflammation and damage to joint structure One charac-teristic that is specific to RA is the network of new bloodvessels that extensively develops in the synovial membraneThis destructive vascular tissue which is called pannus

extends from the synovium to invade the junction betweenthe cartilage and subchondral bone With progression ofthe disease joint inflammation and the resulting structuralchanges caused by pannus lead to reduced joint motionpossible ankyloses joint instability muscle atrophy fromdisuse stretching of the ligaments and involvement of thetendons and muscles [1]

The CFA approach developed by Pearson [19] is a widelyused arthritic model which is induced in susceptible strainsof rats via injection of heat-killed mycobacterium tubercu-losis [20] After CFA injection a rapid reliable robust andeasilymeasurable polyarthritis develops [21] Importantly thejoint pathology seen in the rat model shares the synovial


hyperplasia and cartilage degradation observed in humanarthritis particularly RA [22ndash24] CFA has recently becomea popular tool to observe the efficacy of herbal medicinesfor treating arthritis For example Zhang et al used the CFAapproach to demonstrate the antiarthritic effect of an extractfrom Dioscorea zingiberensis CH Wright [25] AdditionallyZhang et al used CFA to demonstrate antioxidant effects ofGenkwa flos flavonoids [26] while Obiri et al demonstratedthat Xylopia aethiopica (Annonaceae) fruit extract suppressesadjuvant-induced arthritis in rats [27] Several studies havealso shown that herbal medicines are effective in treatingRA in humans Shao Li et al reported that Qing-Luo-Yinextract may have a protective effect against excessive tissuebreakdown angiogenesis and degradation of extracellularmatrix in RA [28] Chi Zhang et al also reported that someherbal medicines have beneficial effects on painmanagementand swollen joint relief in individuals with RA [29] Addi-tionally Liu et al reported that Xinfeng a patent Chineseherbal medicine is effective and safe in the treating RA[30]

In the current study we found that treatment with theN-GHJTY resulted in a gradual decrease of CFA-inducedpaw and knee joint swelling as well as rubefaction Thisfinding was present in all N-GHJTY-treated groups (625125 and 250mgkg) and exhibited a dose-dependent effectmdashwith the N-GHJTY-250 group showing the greatest decreasein paw swelling (Figure 3) In terms of time course the N-GHJTY-250 group showed significantly less paw and kneejoint swelling on the 6th day after treatment the N-GHJTY-125 group on the 8th day and the N-GHJTY-625 group onthe 10th day (Table 1) Moreover rats in all N-GHJTY groups(625 125 and 250mgkg) exhibited increases in body weightwhen compared to those in the CFA-control group

Regarding proinflammatory cytokines all N-GHJTY-treated groups (625 125 and 250mgkg) displayed signifi-cant reductions in TNF-120572 IL-1120573 and IL-6 when comparedto rats in the CFA-control group RA is initiated by a Tcell-mediated immune response that stimulates the releaseof cytokines and promotes antibody formation which leadsto destruction of the joint [1] Specifically TNF-120572 inducesthe production of IL-1120573 and IL-6 [31] with the former beinga crucial mediator of the inflammatory response causingvarious autoinflammatory syndromes [32 33] and the latterbeing secreted by T cells and macrophages to stimulate theimmune response [34 35] Thus proinflammatory cytokines(particularly IL-1120573 IL-6 and TNF-120572) play an important rolein arthritis onset [36] while inhibitors of these cytokines areeffective in controlling chronic inflammation [37 38]

The protective effects of N-GHJTY were confirmed byour histopathological investigations with HE and SafraninO-fast staining Specifically rats in the CFA-control groupexhibited loose synovial membrane membrane destructiondisorganized cell arrangement and compressed cartilagemdashallof which were prevented in rats treated with N-GHJTY Asmentioned release of enzymes and inflammatory mediatorsfrom damaged tissues perpetuates the inflammatory process[1] It has also been shown that cytokines released by inflamedsynovial tissue can reach systemic circulation and act onother organs [39] Rheumatoid factor (RF) which is an

autoantibody found in RA forms an immune complex withimmunoglobulin G that contributes to RA progression byfurther triggering inflammatory responses and attractinginflammatory cells Thus the ability of N-GHJTY to protectagainst cartilage and synovial membrane destruction couldadditionally prevent systemic inflammation by preventingdamaged synovial tissue from releasing cytokines

To investigate if repeated N-GHJTY treatment couldinduce toxicity we observed transaminase levels (AST andALT) in all N-GHJTY-treated groups Our findings revealedthat N-GHJTY treatment did not alter either AST or ALTsuggesting that N-GHJTY was not toxic to rats Moreoverinvestigations of various diseases (including arthritis) havereported that AST and ALT leak into the blood stream inproportion to the extent of tissue damage [40ndash42] Indeedtransaminase levels were slightly increased in the CFA-control group in the current study Consistent with ourfindings on the anti-inflammatory effects of N-GHJTY weobserved that the highest dose of this new herbal com-bination (ie 250mgkg) resulted in a slight decrease oftransaminase levels suggesting improved liver function inthese animals

Taken together our findings suggest that N-GHJTYadministration could prevent inflammatory cells from infil-trating and destroying synovial tissue and could also suppressthe release of proinflammatory cytokines Moreover N-GHJTY was effective in preventing the destruction of jointtissue and facilitated the repair of CFA-induced injury to thejoint cartilage resulting in reduced paw and knee swellingThus the present study provides evidence supporting theclinical use of N-GHJTY for treating arthritis

5 Conclusions

N-GHJTY a new complex herbal medication was effectivein treating a rat model of inflammatory arthritis SpecificallyN-GHJTY significantly suppressed the progression of CFA-induced arthritis aswas evident from the decrease in paw andknee joint swelling and was effective in preventing articularcartilage and synovial tissue degeneration We also revealedthat the protectivemechanisms ofN-GHJTY treatment couldbe partially explained by a decrease in the proinflammatorycytokines TNF-120572 IL-1120573 and IL-6 Additional studies arerequired to determine other molecular mechanisms asso-ciated with N-GHJTY administration as well as specifictherapeutic effects

Competing Interests

The authors declare no competing interests regarding thepublication of this paper

Authorsrsquo Contributions

Wangin Kim and Sangbin Park contributed equally to thiswork


Acknowledgments

This study was supported by a Grant (no HI13C2285) fromthe Korea Healthcare Technology RampD Project Ministry forHealth ampWelfare Affairs Republic of Korea

References

[1] C M Porth Essentials of Pathophysiology Concepts of AlteredHealth States Wolters Kluwer HealthLippincott Williams ampWilkins 3rd edition 2011

[2] A J MacGregor and A J Silman ldquoRheumatoid arthritis andother synovial disorders classification and epidemiologyrdquo inRheumatology M C Hochberg A J Silman J S Smolen ME Weinblatt and M H Weisman Eds vol 1 Mosby LondonUK 2004

[3] S E Gabriel and K Michaud ldquoEpidemiological studies in inci-dence prevalence mortality and comorbidity of the rheumaticdiseasesrdquo Arthritis Research and Therapy vol 11 no 3 article229 2009

[4] I B McInnes and G Schett ldquoCytokines in the pathogenesis ofrheumatoid arthritisrdquoNature Reviews Immunology vol 7 no 6pp 429ndash442 2007

[5] G S Firestein ldquoEvolving concepts of rheumatoid arthritisrdquoNature vol 423 no 6937 pp 356ndash361 2003

[6] Y Okada D Wu G Trynka et al ldquoGenetics of rheumatoidarthritis contributes to biology and drug discoveryrdquoNature vol506 no 7488 pp 376ndash381 2014

[7] D E Furst and P Emery ldquoRheumatoid arthritis pathophysiol-ogy update on emerging cytokine and cytokine-associated celltargetsrdquo Rheumatology vol 53 no 9 pp 1560ndash1569 2014

[8] B Bresnihan J M Alvaro-Gracia M Cobby et al ldquoTreatmentof rheumatoid arthritis with recombinant human interleukin-1receptor antagonistrdquo Arthritis and Rheumatism vol 41 no 12pp 2196ndash2204 1998

[9] Y Zhuang S Lyn Y Lv et al ldquoPharmacokinetics and safetyof golimumab in healthy Chinese subjects following a singlesubcutaneous administration in a randomized phase I trialrdquoClinical Drug Investigation vol 33 no 11 pp 795ndash800 2013

[10] B-R Jeoung K D Lee C-S Na Y-E Kim B Kim andY R Kim ldquoGanghwaljetongyeum an anti-arthritic remedyattenuates synoviocyte proliferation and reduces the productionof proinflammatory mediators in macrophages the therapeuticeffect of GHJTY on rheumatoid arthritisrdquoBMCComplementaryand Alternative Medicine vol 13 article 47 2013

[11] W Choi C Choi Y R Kim S Kim C Na and H LeeldquoHerDing herb recommendation system to treat diseases usinggenes and chemicalsrdquo Database vol 2016 Article ID baw0112016

[12] HW Jung R Mahesh J H Park Y C Boo K M Park and Y-K Park ldquoBisabolangelone isolated from Ostericum koreanuminhibits the production of inflammatory mediators by down-regulation of NF-120581B and ERK MAP kinase activity in LPS-stimulated RAW2647 cellsrdquo International Immunopharmacol-ogy vol 10 no 2 pp 155ndash162 2010

[13] S J Lee E J Shin K H Son H W Chang S S Kang and HP Kim ldquoAnti-inflammatory activity of themajor constituents ofLonicera japonicardquo Archives of Pharmacal Research vol 18 no2 pp 133ndash135 1995

[14] C Peng P K Perera Y-M Li W-R Fang L-F Liu and F-W Li ldquoAnti-inflammatory effects of Clematis chinensis Osbeck

extract(AR-6) may be associated with NF-120581B TNF-120572 andCOX-2 in collagen-induced arthritis in ratrdquo RheumatologyInternational vol 32 no 10 pp 3119ndash3125 2012

[15] S S Joo D S Park S H Shin et al ldquoAnti-allergic effectsand mechanisms of action of the ethanolic extract of Angelicagigas in dinitrofluorobenzene-induced inflammation modelsrdquoEnvironmental Toxicology and Pharmacology vol 30 no 2 pp127ndash133 2010

[16] J-H Kim J-E Huh Y-H Baek J-D Lee D-Y Choi andD-S Park ldquoEffect of Phellodendron amurense in protectinghuman osteoarthritic cartilage and chondrocytesrdquo Journal ofEthnopharmacology vol 134 no 2 pp 234ndash242 2011

[17] R Roubenoff L M Freeman D E Smith L W Abad C ADinarello and J J Kehayias ldquoAdjuvant arthritis as a model ofinflammatory cachexiardquo Arthritis and Rheumatism vol 40 no3 pp 534ndash539 1997

[18] A Omoto Y Kawahito I Prudovsky et al ldquoCopper chelationwith tetrathiomolybdate suppresses adjuvant-induced arthri-tis and inflammation-associated cachexia in ratsrdquo ArthritisResearch ampTherapy vol 7 no 6 pp R1174ndashR1182 2005

[19] C M Pearson ldquoDevelopment of arthritis periarthritis andperiostitis in rats given adjuvantsrdquo Experimental Biology andMedicine vol 91 no 1 pp 95ndash101 1956

[20] M Durai H R Kim and K D Moudgil ldquoThe regulatory C-terminal determinants within mycobacterial heat shock pro-tein 65 are cryptic and cross-reactive with the dominant selfhomologs implications for the pathogenesis of autoimmunearthritisrdquoThe Journal of Immunology vol 173 no 1 pp 181ndash1882004

[21] S S Patel and P V Shah ldquoEvaluation of anti-inflammatorypotential of the multidrug herbomineral formulation in maleWistar rats against rheumatoid arthritisrdquo Journal of Ayurvedaand Integrative Medicine vol 4 no 2 pp 86ndash93 2013

[22] M L Andersen E H R Santos M D L V Seabra A A Bda Silva and S Tufik ldquoEvaluation of acute and chronic treat-mentswithHarpagophytumprocumbens on Freundrsquos adjuvant-induced arthritis in ratsrdquo Journal of Ethnopharmacology vol 91no 2-3 pp 325ndash330 2004

[23] A Bendele J Mccomb T Gould et al ldquoAnimal models ofarthritis relevance to human diseaserdquo Toxicologic Pathologyvol 27 no 1 pp 134ndash142 1999

[24] J D Taurog D C Argentieri and R AMcReynolds ldquoAdjuvantarthritisrdquoMethods in Enzymology vol 162 pp 339ndash355 1988

[25] X-X Zhang Y Ito J-R Liang J-L Liu J He and W-JSun ldquoTherapeutic effects of total steroid saponin extracts fromthe rhizome of Dioscorea zingiberensis CHWright in Fre-undrsquos complete adjuvant induced arthritis in ratsrdquo InternationalImmunopharmacology vol 23 no 2 pp 407ndash416 2014

[26] C-F Zhang S-L Zhang X He et al ldquoAntioxidant effects ofGenkwa flos flavonoids on Freundrsquos adjuvant-induced rheuma-toid arthritis in ratsrdquo Journal of Ethnopharmacology vol 153 no3 pp 793ndash800 2014

[27] D D Obiri N Osafo P G Ayande and A O Antwi ldquoXylopiaaethiopica (Annonaceae) fruit extract suppresses Freundsadjuvant-induced arthritis in Sprague-Dawley ratsrdquo Journal ofEthnopharmacology vol 152 no 3 pp 522ndash531 2014

[28] S Li A-P Lu Y-Y Wang and Y-D Li ldquoSuppressive effectsof a Chinese herbal medicine Qing-Luo-Yin extract on theangiogenesis of collagen-induced arthritis in ratsrdquo The Ameri-can Journal of ChineseMedicine vol 31 no 5 pp 713ndash720 2003

[29] C Zhang M Jiang and A Lu ldquoChinese herbal medicinesversus disease modifying antirheumatic drugs for management


of rheumatoid arthritis a systematic reviewrdquo European Journalof Integrative Medicine vol 3 no 3 pp e219ndashe231 2011

[30] J Liu C-B Huang Y Wang et al ldquoChinese herbal medicineXinfeng Capsule in treatment of rheumatoid arthritis studyprotocol of a multicenter randomized controlled trialrdquo Journalof Integrative Medicine vol 11 no 6 pp 428ndash434 2013

[31] F M Brennan and I B McInnes ldquoEvidence that cytokines playa role in rheumatoid arthritisrdquo Journal of Clinical Investigationvol 118 no 11 pp 3537ndash3545 2008

[32] RefSeq ldquoRefSeq NCBI Reference Sequence Databaserdquo 2008httpwwwncbinlmnihgovgeneDb=geneampCmd=ShowDe-tailViewampTermToSearch=3553

[33] S L Masters A Simon I Aksentijevich and D L KastnerldquoHorror autoinflammaticus the molecular pathophysiology ofautoinflammatory diseaserdquo Annual Review of Immunology vol27 pp 621ndash668 2009

[34] T Van Der Poll C V Keogh X Guirao W A Buurman MKopf and S F Lowry ldquoInterleukin-6 gene-deficient mice showimpaired defense against pneumococcal pneumoniardquo Journal ofInfectious Diseases vol 176 no 2 pp 439ndash444 1997

[35] M A Febbraio and B K Pedersen ldquoContraction-inducedmyokine production and release is skeletal muscle anendocrine organrdquo Exercise amp Sport Sciences Reviews vol 33no 3 pp 114ndash119 2005

[36] E H S Choy and G S Panayi ldquoCytokine pathways and jointinflamation in rheumatoid arthritisrdquo The New England Journalof Medicine vol 344 no 12 pp 907ndash916 2001

[37] K J Tracey ldquoThe inflammatory reflexrdquo Nature vol 420 no6917 pp 853ndash859 2002

[38] V A Pavlov and K J Tracey ldquoControlling inflammation thecholinergic anti-inflammatory pathwayrdquo Biochemical SocietyTransactions vol 34 no 6 pp 1037ndash1040 2006

[39] N Sattar D W McCarey H Capell and I B McInnesldquoExplaining how lsquohigh-gradersquo systemic inflammation acceler-ates vascular risk in rheumatoid arthritisrdquo Circulation vol 108no 24 pp 2957ndash2963 2003

[40] E G Giannini R Testa and V Savarino ldquoLiver enzymealteration a guide for cliniciansrdquo CanadianMedical AssociationJournal vol 172 no 3 pp 367ndash379 2005

[41] C I BWalker G TrevisanM F Rossato et al ldquoAntinociceptiveeffect of Mirabilis jalapa on acute and chronic pain models inmicerdquo Journal of Ethnopharmacology vol 149 no 3 pp 685ndash693 2013

[42] A A Adeneye A I Oreagba I O Ishola and H A KalejaiyeldquoEvaluation of the anti-arthritic activity of the hydroethanolicleaf extract of Alchornea cordifolia in ratsrdquo African Journal ofTraditional Complementary and Alternative Medicines vol 11no 2 pp 402ndash410 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


interleukin 1 beta (IL-1120573) and tumor necrosis factor alpha(TNF-120572) are often targeted in RA treatment strategies [8 9]

Our previous study showed that Ganghwaljetongyeum(GHJTY) can effectively attenuate RA by inhibiting synovialcell proliferation as well as the production of proinflamma-tory mediators from macrophage-like cells [10] However 18medicinal herbs are included in GHJTY making it imprac-tical for clinical application In order to improve the efficacyand convenience of pharmaceutically prescribing GHJTY weused bioinformatics (httpcombiogistackrherding) [11] toselect the five medicinal herbs with the greatest potentialto treat arthritis The selected medicinal herbs were Oster-icum koreanum Maximowicz (Osterici Radix OK) Lonicerajaponica Thunberg (Lonicerae Folium LJ) Clematis mand-shurica Ruprecht (Clematis Radix CM) Angelica gigasNakai(Angelicae Gigantis Radix AG) and Phellodendron amurenseRuprecht (Phellodendri Cortex PA)

A review of the literature revealed that each of theselected herbs is effective in targeting certain aspects ofRA pathophysiology For example OK inhibits the produc-tion of inflammatory mediators by downregulating nuclearfactor kappa beta (NF-120581B) and mitogen-activated proteinkinase (MAPK) activity in lipopolysaccharide-stimulatedRAW2647 cells [12] Additionally anti-inflammatory activityof the major constituents of LJ has been shown [13] alongwith the ability of CM extract to interact with NF-120581B TNF-120572and cyclooxygenase 2 (COX-2) in rats with collagen-inducedarthritis [14] Studies have also shown that AG inhibits focaland systemic inflammation in dinitrofluorobenzene-inducedinflammation models and that PA protects against humanosteoarthritis by regulating aggrecanases matrix metallo-proteinases (MMPs)tissue inhibitors of metalloproteinases(TIMP) proinflammatory cytokines and MAPK pathwaysignaling [15 16] However the effects of combining theseherbal medicines remain unclear

The purpose of the present study was to evaluate theefficacy and mechanism of action of this new GHJTY (N-GHJTY) using an animal model of RA Specifically we usedrats with complete Freundrsquos adjuvant- (CFA-) induced arthri-tis as an experimental animal model used to mimic humanRA as it produces profound systemic inflammation thatresults in severe joint swelling and remodeling [17]MoreoverCFA is typically used to investigate therapeutic agents withantiarthritic potential [18] Our specific aims were to (1)confirm the quality assessment of marker components in N-GHJTY (2) determine the antiarthritic effects of N-GHJTYby measuring paw edema and observing gross lesions of thepaw and knee joint (3) evaluate potential adverse effects ofN-GHJTY by histopathological investigation and (4) assessthe effect of N-GHJTY on proinflammatory cytokines byexamining levels of TNF-120572 IL-1120573 and IL-6

2 Materials and Methods

21 Animals Adult male Sprague-Dawley rats weighing200ndash210 g were housed in a room with constant temperature(24ndash26∘C) and humidity (40ndash60) Food (Pellet GMOKorea) and water were available ad libitum Animals were

acclimated to the laboratory environment for 1 week beforethe experiment and all procedures were approved by theInstitutional Animal Care and Use Committee of the Dong-shin University (2014-03-02)

22 CFA-Induced Arthritis and Drug Administration In theprimary adjuvant-induced arthritis model 025mL of CFA(Sigma St Louis MD USA) was injected into the left hindknee joint After 7 days secondary arthritis was inducedby injecting 005mL of CFA under the left hind knee jointand left hind sole Animals were then divided into thefollowing five groups (119899 = 6group) normal CFA arthritis(CFA-control) and CFA arthritis treated with 625 125 and250mgkg of N-GHJTY extract per day (N-GHJTY-625 N-GHJTY-125 and N-GHJTY-250 resp) Oral administrationof N-GHJTY was initiated on the 10th day after arthritisinduction and continued for 10 days thereafter Animalswere anesthetized using 25 isoflurane and volumes of thehind paw and knee joint were measured using a DigitalPlethysmometer (LE7500 Panlab Spain) 0 2 4 6 8 and 10days after oral N-GHJTY administration

23 Preparation of Herbal Materials The five herbal med-icines forming N-GHJTY were purchased from OmniherbCo (Yeongcheon Korea) and their origin was taxonomicallyconfirmed by Professor Jong-Kil Jeong in the Departmentof Herbology at the College of Oriental Medicine DongshinUniversity

The five herbs (OK LJ AG CM and PA) were combinedin a 6 4 4 4 3 ratio N-GHJTY was prepared via waterextraction at 100∘C for 3 h and concentrated using a rotaryvacuum evaporator (EYELA Japan) after filtration followedby lyophilization using a vacuum freeze drier (SamwonFreezing Engineering Co Korea)The yield was about 295

24 Reagents and High-Performance Liquid Chromatography(HPLC) Analysis Chlorogenic acid (1) and berberine chlo-ride (2) were purchased from Acros Organics (PittsburghPA USA) and Sigma-Aldrich Co LLC (St Louis MOUSA) respectively Nodakenin (3) decursin (6) and decursi-nol angelate (7) were purchased form NPC BioTechnol-ogy (Yeongi Korea) Isoferulic acid (4) and oxypeucedaninhydrate (5) were purchased from ChemFaces BiochemicalCo Ltd (Wuhan China) The purities of the seven referencecompounds were ge980 by HPLC analysis and the chemicalstructures of the seven marker compounds are shown inFigure 1 HPLC-grade solvents methanol acetonitrile andwater were obtained from JTBaker (Phillipsburg NJ USA)Analytical grade formic acid was purchased from Sigma-Aldrich (St Louis MO USA)

For quality assessment of the seven marker compoundsin N-GHJTY all experiments were conducted using a Shi-madzu Prominence LC-20A Series (Shimadzu Kyoto Japan)equipped with a solvent delivery unit (LC-20AT) onlinedegasser (DGU-20A3) column oven (CTO-20A) autosam-ple injector (SIL-20AC) and photodiode array (PDA) detec-tor (SPD-M20A) Lab solution software (version 554 SP3Kyoto Japan) was used for data acquisition and processing


OH

OH

O

O

OH

OHOH

O

HO


O

O


OO O

Nodakenin (3)

O

OHO

HOOH

HO

OH

O

Isoferulic acid (4)

OH

OO O


O

OH

OH

OO O

O

O

Decursin (6)

OO O

O

O


OCH3

OCH3

N+ Clminus

H3CO



















3 Results



(1) (2)(3) (4)(5) (6) (7)

550minus10

200

250

300

350

400

Wav

eleng

th (n

m)

0000

5000

10000

15000

20000

25000

30000

Time (min)

500

400

300

200

100

0

Inte

nsity

(mAU

)






(a) (b) (c) (d) (e)
























BSM

SM

ACF

ACT

JC

MH and FC

(a) (b)

(c) (d)

(e)







SB

AC

(a) (b)

(c) (d)

(e)



4 Discussion












5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OH

OH

O

O

OH

OHOH

O

HO


O

O


OO O

Nodakenin (3)

O

OHO

HOOH

HO

OH

O

Isoferulic acid (4)

OH

OO O


O

OH

OH

OO O

O

O

Decursin (6)

OO O

O

O


OCH3

OCH3

N+ Clminus

H3CO



















3 Results



(1) (2)(3) (4)(5) (6) (7)

550minus10

200

250

300

350

400

Wav

eleng

th (n

m)

0000

5000

10000

15000

20000

25000

30000

Time (min)

500

400

300

200

100

0

Inte

nsity

(mAU

)






(a) (b) (c) (d) (e)
























BSM

SM

ACF

ACT

JC

MH and FC

(a) (b)

(c) (d)

(e)







SB

AC

(a) (b)

(c) (d)

(e)



4 Discussion












5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























3 Results



(1) (2)(3) (4)(5) (6) (7)

550minus10

200

250

300

350

400

Wav

eleng

th (n

m)

0000

5000

10000

15000

20000

25000

30000

Time (min)

500

400

300

200

100

0

Inte

nsity

(mAU

)






(a) (b) (c) (d) (e)
























BSM

SM

ACF

ACT

JC

MH and FC

(a) (b)

(c) (d)

(e)







SB

AC

(a) (b)

(c) (d)

(e)



4 Discussion












5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c) (d) (e)
























BSM

SM

ACF

ACT

JC

MH and FC

(a) (b)

(c) (d)

(e)







SB

AC

(a) (b)

(c) (d)

(e)



4 Discussion












5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BSM

SM

ACF

ACT

JC

MH and FC

(a) (b)

(c) (d)

(e)







SB

AC

(a) (b)

(c) (d)

(e)



4 Discussion












5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SB

AC

(a) (b)

(c) (d)

(e)



4 Discussion












5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























5 Conclusions


Competing Interests





Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Evaluation of Anti-Inflammatory Potential...

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Transcript of Research Article Evaluation of Anti-Inflammatory Potential...