research article evaluation of a topical herbal agent for the...

Research ArticleEvaluation of a Topical Herbal Agent forthe Promotion of Bone Healing

Wing-Sum Siu123 Chun-Hay Ko12 Ka-Wing Lam4 Elaine Wat12

Wai-Ting Shum12 Clara Bik-San Lau12 Kam-Ming Ko5 Leung-Kim Hung3

David Tai-Wai Lau15 and Ping-Chung Leung123

1 Institute of Chinese Medicine 5F The CUHK Hong Kong Jockey Club School of Public Health Building Prince of Wales HospitalShatin New Territories Hong Kong2State Key Laboratory of Phytochemistry and Plant Resources in West China The Chinese University of Hong Kong ShatinNew Territories Hong Kong3Department of Orthopaedics and Traumatology The Chinese University of Hong Kong Shatin New Territories Hong Kong4Department of Chemical and Biomolecular Engineering The Hong Kong University of Science amp Technology Clear Water BayKowloon Hong Kong5Division of Life Science The Hong Kong University of Science amp Technology Clear Water Bay Kowloon Hong Kong

Correspondence should be addressed to Ping-Chung Leung pingcleungcuhkeduhk

Received 23 December 2014 Revised 10 February 2015 Accepted 13 February 2015

Academic Editor Min Li

Copyright copy 2015 Wing-Sum Siu 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

A topically used Chinese herbal paste namely CDNR was designed to facilitate fracture healing which is usually not addressed ingeneral hospital care From our in vitro studies CDNR significantly inhibited the release of nitric oxide from RAW2647 cells by 51to 77This indicated its anti-inflammatory effect CDNR also promoted the growth of bone cells by stimulating the proliferation ofUMR106 cells up to 18 It also increased the biomechanical strength of the healing bone in a drill-hole defect rat model by 165significantly This result revealed its in vivo efficacy on facilitation of bone healing Furthermore the detection of the chemicalmarkers of CDNR in the skin and muscle of the treatment area demonstrated its transdermal properties However CDNR did notaffect the bone turnover markers in serum of the rats With its anti-inflammatory and bone formation properties CDNR is foundeffective in promoting bone healing

1 Introduction

Fracture is the commonest problem in orthopaedic clinicsThe estimated world incidence of adult fractures is around90ndash228 per 1000 people per year [1] Since fractures aremore common among the elderlies the fracture incidenceis expected to further increase in the coming future due tothe increasing longevity worldwide [2] Patients with severebone fractures are usually hospitalizedThe median length ofhospital stay after fracture fixation is 133 days for men and196 days for women and themaximum could be over 30 daysfor hip fractures [3 4] Obviously fractures reduce the socialproductivities and intensify the social-economic burden [4ndash6]

Fracture management today is effectively done by sur-geons and the healing outcome can be taken for grantedHowever the healing process post-op is seldom addressedby hospital and clinical workers yet The healing of thefracture relies mostly on self-recovery Patients are usuallyleft unattended except for the pain and inflammation controlduring hospitalization In spite of many scientific researcheswhich have been conducted to find the way to promotefracture healing including using biomaterial scaffolds [7ndash9]growth factors [10 11] bone morphogenetic proteins [12ndash14]and biophysical stimulations [15 16] these interventions havenot been well accepted for routine clinical applications yet

Herbal medicine has been used in China for thousandsof years and modern researches have demonstrated its

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 905270 10 pageshttpdxdoiorg1011552015905270

2 Evidence-Based Complementary and Alternative Medicine

therapeutic effects on treating various diseases includingcancer [17]More recently its applications inmodernWesterntherapy have also been discussed [18] Facilitation of fracturehealing is one of the major areas in traditional Chinesemedicine Traditionally topical agents have been used topromote the healing of soft tissues and bone fracturesNonetheless the formulae of these herbal medicines aretoo diversified as yet More importantly the serious lack ofrelevant evidence-based scientific support and good systemicdocumentation of the clinical data makes them not wellaccepted worldwide

The aim of this study is to investigate the efficacy ofa topical herbal paste on the promotion of bone healingfrom an evidence-based scientific approach Both in vitroand in vivo biological platforms will be used to verifythe pharmacological properties essential for bone healingnamely anti-inflammation proangiogenesis and cellularregeneration The most novel component of the study lies inthe confirmation of transcutaneous transport of the chemicalcompounds via the topical application

Considering that over a hundred of medicinal herbs hadbeen used historically in China for the treatment of skeletalinjuries and bone fracture we select the herbs for the presentstudy according to the classical records and recent scientificpapers testifying the pharmacological properties of the herbsrelated to fracture healing [19]

A herbal paste for topical use (CDNR) was thus createdwith four herbs namely Carthami Flos (C) Dipsaci Radix(D) Notoginseng Rhizoma (N) and Rhei Rhizoma (R) Allof them were purchased from a reputable herbal supplier inHong Kong The identities of all herbs were authenticatedusing thin-layer chromatography with procedural referencesrecommended by the Chinese pharmacopoeia [20] Theherbarium voucher specimens of the tested herbs weredeposited in the museum bank of the Institute of ChineseMedicine at the Chinese University of Hong Kong withreference numbers as follows Dipsaci Radix 2010-3279 RheiRhizoma 2010-3280 Notoginseng Rhizoma 2010-3278 andCarthami Flos 2010-3335

2 Materials and Methods

21 Making of the Topical Agent Extracts of CDNR wereprepared via both aqueous and ethanol extractions In theaqueous extraction 250 g raw materials of each of the fourherbs were cut into small pieces (C D N R in 1 1 1 1 ww)and were soaked in 10 L distilled water for 1 hour They wereboiled twice for 2 hours under reflux and then the aqueousextract was collected and filtered through a piece of absorbentgauze The residue was further boiled with 95 ethanol for2 hours under reflux The ethanol extract was collected andfiltered again Both of the aqueous and ethanol filtrates werethen concentrated at 50∘C under reduced pressure separatelyfollowed by lyophilizing into powder forms (CDNR(aq) andCDNR(e)) in a freeze dry system (Freezone 12 LabconcoMissouri USA)The extraction yields of aqueous and ethanolextractswere 398wwand 50ww respectivelyThepasteform of CDNR for topical use was completed bymixing 195 gCDNR(aq) and 30 g CDNR(e) with 17mL 50 ethanol [19]

Table 1 Chemical composition of CDNR paste for animal studies

Herbalsource Name of chemical marker

Content(mgg of paste

mixture powder)CarthamiFlos (C)

Hydroxysafflor yellow A (HYA)Kaempferol (KAE)

2140004

DipsaciRadix (D)

Asperosaponin VI (ASP)Oleanolic acid (OA)

1923005

NotoginsengRhizoma (N)

Ginsenoside Rg1 (Rg1)Ginsenoside Rb1 (Rb1)

20055

RheiRhizoma (R)

Emodin (EMO)Rhein (RHE)

096100

22 Chemical Composition of Topical Herbal Paste Thechemical composition of the herbal paste was determinedby high-performance liquid chromatography-electrosprayionization-mass spectrometry (HPLC-ESI-MS) whichincluded an Agilent 1290 Infinity LC System (Agilent Tech-nologies Santa Clara California USA) equipped with anonline degasser a binary pump an autosampler and Agilent6410 Triple Quad LCMS and connected with AgilentMassHunter Workstation software A Waters HSS T3 35 120583m(30mm times 150mm) HPLC column was used and kept at atemperature of 40∘C Eight chemical markers and sampleswere separated using a gradient mobile phase consisting ofwater (A) and acetonitrile (B) The gradient condition is 0 to3min 10ndash27 B 3 to 5min 27ndash33 B 5 to 12min 33-33B 12 to 13min 33ndash80 B 13 to 16min 80ndash90 B 16 to20min 90-90 B The flow rate was set at 05mLmin andthe injection volume was 20120583L

MS analysis was performed on Agilent 6410 Triple-QuadLCMS equipped with an ESI source [21] and monitored innegative ion mode and multiple reaction monitoring modeusing target ions at mz 6112rarr 3250 for hydroxysaffloryellowA (HYA)mz 2850rarr 1170 for kaempferol (KAE)mz9275rarr 6033 for asperosaponin VI (ASP)mz 4553rarr 4074for oleanolic acid (OA) mz 7995rarr 6374 for ginsenosideRg1 (Rg1) mz 11076rarr 1190 for ginsenoside Rb1 (Rb1) mz2690rarr 2410 for emodin (EMO) andmz 2830rarr 2390 forrhein (RHE) The abundance of the markers was determinedquantitatively (Table 1)

All the chemicals were purchased from Sigma-Aldrich(St Louis Missouri USA) Both methanol and acetonitrilewere HPLC-grade (CHROMASOLV Plus ge999) All thechemical markers were purchased from Tauto Biotech CoLtd (Shanghai China) Their purities were over 97

23 In Vitro Studies In vitro studies were targeting anti-inflammation and cellular proliferation

231 Cell Cultures Murine monocytemacrophageRAW2647 and rat osteosarcoma UMR-106 cells were pur-chased from American Type Culture Collection (ManassasVA USA) RAW2647 and UMR-106 cells were maintainedin high-glucose DMEM (d-glucose 3500mgL GIBCOUSA) 10 vv fetal bovine serum (FBS Gibco Gran IslandNY USA) and 1 penicillin-streptomycin (PS Gibco Gran

Evidence-Based Complementary and Alternative Medicine 3

Island NY USA) All cells were maintained at 37∘C 5 CO2

humidified incubator

(i) Nitric Oxide (NO) Inhibitory Assay Macrophage cellsRAW2647 (4 times 105 per well) were seeded With 01 120583g oflipopolysaccharide (LPS) per mL of medium CDNR(aq) orCDNR(e) was added (at concentrations ranging from 0 to400 120583gmL) and incubated for 24 hThe nitrite accumulationin the supernatant was determined by Griess reagent (SigmaSt Louis MO USA) [22 23] read at a wavelength of540 nm Cell viability of RAW2647 in various concentra-tions of CDNR(aq) or CDNR(e) was determined by MTTassay (3-[45-dimethylthiazol-2-yl]-25-diphenyltetrazoliumbromide) (Sigma St Louis MO USA) [22] The relativeamount of viable cells was determined using optical density at540 nm and expressed as the percentage of Control sampleswithout treatment

(ii) Determination of Production of Proinflammatory Cytok-ines Using ELISA Assay The RAW2647 cell culture super-natants were subjected to test for the concentrationsof cytokines TNF-120572 IL-6 and IL-1120573 by enzyme-linkedimmunosorbent assay (ELISA) [22] These assays were car-ried out according to the procedures recommended in theELISA kit manual (BD Biosciences San Jose CA USA)

(iii) Osteoblast Proliferation and Viability Assays The osteo-blast proliferation and viability were measured using 5-bromo-21015840-deoxyuridine (BrdU) [24] incorporation andMTTcell viability assay [19 25] respectively Briefly UMR-106 cellswere seeded in DMEM with 10 vv FBS After 24 h cellswere exposed to different concentrations of CDNR(aq) orCDNR(e) in DMEMwith 10 vv FBS An ELISA kit (RochePalo Alto CA USA) was used to determine the proliferationof the osteoblast in the BrdU incorporation assay For theMTT assay MTT solution (5mgmL) was added directly tothe medium in each well and the plate was then incubatedat 37∘C for 4 h All medium was then aspirated and replacedwith dimethyl sulfoxide (DMSO Sigma-Aldrich USA) Therelative amount of viable cells was determined at opticaldensity at 540 nm The osteoblast proliferation and cellviability were expressed as the percentage of Control sampleswithout treatment

24 In Vivo Study The animal model (rat) with artificiallycreated bone defects via cortical and metaphyseal drillings isa recognized model for the study of bone healing [26 27]

241 Animals Models with Bone Injury Twenty femaleSprague-Dawley rats aged 152 plusmn 141 months (mean plusmnstandard deviation) were obtained from the LaboratoryAnimal Service Centre of the Chinese University of HongKong (CUHK) All of them were housed in a temperature-controlled (25∘C) and light-controlled (12 h lightdark cycle)environment All the animals were allowed access to standardrodent chow andwater ad libitum throughout the study Afterseven days of acclimatization bone defects in the form oflarger drill holes were created in the femur and tibia of the

rats The animal study had been approved by the AnimalExperimental Ethics Committee of CUHK

The rat was firstly anesthetized using a cocktail ofketamine and xylazine intramuscularly On the left femurtwo side-by-side drill holes (2mm in diameter each) werecreated using an electric drill on the midshaft of the femurthrough an anterior-posterior approach The two holes wereconnected to form a 2mm times 4mm defect using a dentalmilling burr On the right tibia (opposite leg) a bone defect24mm in diameter was made using the electric drill at theproximal metaphysis via a medial-lateral approach All thedrilled holes were cleaned thoroughly irrigating with 09sterile saline making sure that small bone fragments wouldnot remain

The rats were divided into two groups with 10 rats eachIn the control group (Control) the left femur and the righttibia were covered with just thin self-adhesive films withoutany treatment In the herbal paste treatment group (CDNR)05mL CDNR paste was applied topically upon the left femurand right tibia covering the operated sites The paste wasprotected from falling off and drying with thin self-adhesivefilms All the films and topical treatment paste were renewedat 2-day intervals The whole treatment period lasted for 6weeks

242 Assessments

(i) Change in Microarchitecture Using Microcomputed Tomog-raphy (Micro-CT) In vivo longitudinal changes in bone vol-ume (BV) of the drill-hole bone defect at the proximal tibia ofthe rat weremonitored usingmicro-CT (MicroCT 40 ScancoMedical Bassersdorf Switzerland) within the experimentalperiod The rat was anesthetized using a cocktail of ketamineand xylazine intramuscularly and then fixed on a plasticholder to ensure a repeatable positioningThe right proximaltibia was scanned for 700 slices with energy 70 kVp andintensity 114 120583A The isotropic resolution was 125 120583m pervoxel The volume of interest was determined within 250continuous slices covering the whole drill hole Segmentationparameters to define BV were fixed at sigma = 05 support= 10 and threshold = 245 for all analyses These in vivomeasurements were performed one day before the surgicaldrill-hole operation (Day minus1) right after the operation (Day0) and on 14 28 and 42 days post-op (Day 14 Day 28 andDay 42 resp)

(ii) Bone Turnover Monitoring by Measuring BiochemicalMarkers Prior to microarchitecture measurement usingmicro-CT bloodwas collected from the orbital venous plexusafter the rat was anesthetized Sera were obtained aftercentrifugation at 3000 rpm for 15 minutes at 4∘C They werethen aliquoted and stored at minus80∘C before the biochemicalmarkers were measured Bone-specific alkaline phosphatase(BALP) was measured following the wheat germ lectin(WGL) (Sigma St Louis MO USA) precipitation method[28 29] Deoxypyridinoline (Dpd) was also measured usingcommercial available ELISA kit (MyBioSource San DiegoUSA)


(iii) Biomechanical Bending Tests for Bone Strength Assess-mentThe rats were euthanized onDay 42Their left and rightfemora were harvested Excessive soft tissue was removedbut the periosteum was preserved Four-point bending testswere performed using a Hounsfield material testing machine(KM25 Redhill United Kingdom) A load cell with maxi-mum 2500NwasmountedThe spans of the upper and lowersupports were 80 and 20mm respectively The drill-holebone defect at the midshaft of the femur was located in themiddle of the two upper supports and then the whole femurwas loaded at a constant speed of 5mmmin in posterior-anterior approach until failure Load and work done at yieldwere recorded for analysis All the data of the drilled femur(left) were normalized with the normal femur (right) Theresults were expressed as the normalized percentage based onthe normal femur

25 Testing for Transcutaneous Transport of the Topical AgentIn order to study whether the topical herbal materialsgo across the skin barrier the presence and quantity ofessential herbal chemical markers of the medicinal herbsused (CDNR) in the rat skin and underneath muscle weremeasured 01 g of rat skin and muscle was collected fromthe region of the topical paste application after the rat wassacrificed The tissues were rinsed with PBS gently and thenhomogenized with 1mL of homogenization buffer (50mMTris 1mM DTT and pH 75) using a homogenizer on iceThen 1mL of acetonitrile was added to the homogenate andfiltered for analysis The presence of the chemical markersof the herbal compounds was revealed using the LC-MSequipmentThe abundance of eachmarker was quantitativelydetermined

26 Statistical Analysis For the multiple group comparisonsof cell proliferation viability NO and cytokine productionthe significance of the differences among the treatmentgroups and their respective control groups were tested byone-way ANOVAwithDunnettrsquos post hoc test For the in vivostudies the differences between Control and the treatmentgroup (CDNR) were analyzed with nonparametric Mann-Whitney 119880 test The results of bone microarchitecture (frommicro-CT) and biochemical markers (BALP and Dpd) wereanalyzed following repeated measure ANOVA Data werepresented as mean plusmn standard deviation (SD) unless other-wise specified All the statistical analyses were carried outusing GraphPad Prism version 50 for Windows (GraphPadSoftware SanDiego CAUSA) and testswere two-sided with119875 lt 005 being considered as statistical significant

3 Results

31 In Vitro Anti-Inflammatory Effects of CDNR Resultsdemonstrated that CDNR(aq) did not affect the NO pro-duction in LPS-induced RAW2647 cells (Figure 1(a)) Incontrast CDNR(e) at 100 120583gmL and 200120583gmL signifi-cantly suppressed the NO production in RAW2647 cellsafter LPS induction by 51 and 77 respectively (119875 lt0001) when compared to the control group without herbal

Table 2 Change of bone volume of the drill-hole bone defect

Control (mm3) CDNR (mm3) 119875 valueDay minus1 20223 plusmn 1589 19509 plusmn 2111 0457Day 0 15045 plusmn 1696 15427 plusmn 2247 0707Day 14 18249 plusmn 1766 18091 plusmn 2413 0883Day 28 17582 plusmn 1801 17231 plusmn 2180 0730Day 42 18020 plusmn 1949 17111 plusmn 1826 0352There was no significant difference in the bone volume (mm3) of the drill-hole bone defect in tibial metaphysis measured by using micro-CT betweenControl andCDNRat each time point Dayminus1 andDay 0measurement priorto and right after the drill-hole bone defect creation respectively Data areexpressed as mean plusmn standard deviation

treatment (0 120583gmL) (Figure 1(b)) However CDNR(e) wasnontoxic to the cells because it did not affect the normalcellular activity at concentration lower than 200 120583gmL (datanot shown) CDNR(e) also suppressed the proinflammatorycytokines production in RAW2647 cells after LPS inductionAt 200120583gmL it effectively downregulated the productionof TNF-120572 (by 252 119875 lt 005 Figure 1(c)) IL-6 (by352 119875 lt 0001 Figure 1(d)) and IL-1120573 (by 209 119875 lt005 Figure 1(e)) when compared with its correspondingcontrols which further indicated that CDNR(e) was anti-inflammatory

32 In Vitro Osteogenic Effect of CDNR As shown inFigure 2(a) after treatment of CDNR(aq) for 24 h significantproliferative effects were observed in UMR-106 cells from 25to 100 120583gmL (119875 lt 0001) with increments of 108 to 22Similar significant cell viability-enhancement effects wereobserved in UMR-106 from 625 to 100 120583gmL (Figure 2(b))CDNR(e) did not affect UMR-106 cells after 24 h of treatmentand up to 100 120583gmL in both assays

33 In Vivo Effect of CDNR on the Microarchitecture of DrilledBone From the quantitative micro-CT analysis at the drill-hole bone defects over the proximal tibial metaphyses of therats bone volume (BV) (from Day 0 to Day 42) was changedsignificantly (119875 lt 0001) depending on the time (number ofdays post-op) in both Control and CDNR groups It changedfrom 1543mm3 to 1711mm3 in CDNRwhile from 1505mm3to 1802mm3 in Control (Table 2) However there was nosignificant difference in the BV between the CDNR and thecontrol group when it was compared at each specific timepoint throughout the study Since the femur was designed forbiomechanical tests nomicro-CTdatawas obtained from thefemur

34 In Vivo Effects of Topical CDNR Application on SystemicBone Remodeling There was a significant overall increase inthe bone-specific alkaline phosphatase (BALP) level in serumin both control and CDNR groups of the rats after the drill-hole bone defect had been created (119875 lt 0001) Howeverthere was no significant difference between the two groupsat each time point throughout the study (Table 3) On theother hand an overall significant descending trend of serumdeoxypyridinoline (Dpd) was observed after the surgery in


0

10

20

30

40

400 200 100 50 0 No LPS(120583gmL)

lowastlowastlowastNO

conc

entr

atio

n (120583

M)

(a)

0

10

20

30

40

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast


conc

entr

atio

n (120583

M)

(b)

0

50

100

150TNF-120572

lowast lowastlowast

200 100 50 0 No LPS(120583gmL)

25

C

ontro

l (ve

rsus

no

treat

men

t)

(c)

0

50

100

150

Con

trol (

vers

us n

o tre

atm

ent)

IL-6

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast

lowast

(d)

0

50

100

150

C

ontro

l (ve

rsus

no

treat

men

t)

IL-1120573

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowast

(e)

Figure 1 Anti-inflammatory effects of CDNR (a b) Effect of aqueous (CDNR(aq)) and ethanol (CDNR(e)) component of CDNR on nitricoxide (NO) production by RAW2647 induced by LPS (c d and e) Effect of CDNR(e) on TNF-120572 IL-6 and IL-1120573 production by RAW2647Data are expressed as mean and standard deviation (error bar) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus Control (0 120583gmL)


0

20

40

60

80

100

120

140Pr

olife

ratio

n (

of C

ontro

l)

lowastlowastlowast lowastlowastlowast lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(a)

0

20

40

60

80

100

120

140

Cel

l via

bilit

y (

of C

ontro

l)

lowastlowastlowastlowastlowast lowastlowastlowast lowastlowastlowast

lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(b)

Figure 2 Osteogenic effect of CDNR on bone cells (a) UMR106 proliferation at different concentration of CDNR assessed by BrdU assay(b) UMR106 viability at different concentration of CDNR assessed by MTT assay (CDNR(aq) CDNR(e)) Aqueous and ethanol componentof CDNR Data are expressed as mean and standard deviation (error bar) lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus Control (0 120583gmL)

Table 3 Change of biochemical markers in serum after drill-hole bone defects had been created

BALP (UL) Dpd (nmolL)Control CDNR 119875 value Control CDNR 119875 value

Day minus1 3582 plusmn 1207 3473 plusmn 1391 0882 3665 plusmn 1421 2862 plusmn 0577 0229Day 14 4887 plusmn 1610 4606 plusmn 1649 0762 2964 plusmn 1118 2614 plusmn 0710 0532Day 28 5909 plusmn 2235 5120 plusmn 2458 0557 2761 plusmn 1657 2303 plusmn 0624 0540Day 42 4991 plusmn 1605 5283 plusmn 2153 0785 2791 plusmn 1586 1891 plusmn 0411 0208There was no significant difference in the serum biochemical markers between Control and CDNR at each time point Day minus1 blood was collected prior to thedrill-hole bone defect creation BALP bone-specific alkaline phosphatase Dpd deoxypyridinoline Data are expressed as mean plusmn standard deviation

both groups (119875 = 0003) Similar to BALP no difference ofDpd between the two groups at each time point was foundneither (Table 3)

35 In Vivo Effect of CDNR on the Bone Strength (Biome-chanical Properties) after Healing Femora with cortical drillholes showed 165higher normalized yield strength ( fromthe contralateral normal femur) in the CDNR treated groupwhen compared with the Control (119875 lt 005) after 42 daysof treatment (Figure 3(a)) Similar findings were observedwhen the total work done at yield point was analyzed CDNRtreated bone showed 138 higher energy than the Control toreach the yield point (119875 lt 005) (Figure 3(b)) The 4-pointbending tests illustrated that the biomechanical propertiesof the healing at the drill-hole bone defects were improvedsignificantly when the topical herbal paste was applied

36 Transdermal Transport of the Herbal Compounds throughthe Rat Skin and Muscle The transdermal transport ofCDNR was determined by identifying the relevant chemicalmarker compounds in the skin and muscle after 6 weeksof herbal treatment using Q-TOF LCMS technique As

shown in Figure 4(a) all the chemical markers of CDNRexcept oleanolic acid (OA) were detected in the rat skinwith asperosaponin VI (ASP) showing the most abundanceSimilarly those 7 chemical markers detected in the skinexcept kaempferol (KAE) could also be found in the muscleOA was nondetectable neither in the muscle With a little bitdifference from skin rhein (RHE) but not the ASP was foundto be the most abundant in the muscle (Figure 4(b))

4 Discussion

Scientific studies on topical TCM treatment for fracturehealing are seldom reported in international journals As aresult there is lack of scientific evidence to support the useof topical herbal paste to facilitate fracture healing clinicallyThis study is one of the few scientific reports exploringthe pharmacological activities of a topical agent from twostandard directions namely direct cellular influences usingstandard cell lines for the study of anti-inflammation andcellular proliferation (in vitro) and life-animal (in vivo)situation of bone regeneration

In the in vitro studies the aqueous component ofCDNR promoted the UMR-106 proliferation significantly


0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)

Figure 3 Biomechanical properties of femurwith drill-hole bone defect (a) Yield strength (b) work done at yield strengthData are expressedas mean and standard error of mean (error bar) lowast119875 lt 005 versus Control

00

01

02

03

04

Rg1 Rb1 ASP OA EMO RHE HYA KAE

(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)

Figure 4 Concentration of the chemical markers of CDNR remained in the soft tissues of the rat (a) Skin (b) muscle Data are expressedas mean and standard deviation (error bar) Rg1 ginsenoside Rg1 Rb1 ginsenoside Rb1 ASP asperosaponin VI OA oleanolic acid EMOemodin RHE rhein HYA hydroxysafflor yellow A KAE kaempferol

This observation indicated that CDNR could promote osteo-genesis which must be an important mechanism duringbone repair particularly during the reparative phase offracture healing when endochondral ossification takes placesand osteoblasts start to form new lamellar bone on thecartilaginous callus [30]

Anti-inflammation is one of the key principles in thetreatment of fractures as well as traditional bone settersrsquoadvocations It controls the swelling and relieves the painfrom the fracture site and the surrounding soft tissue whichpromotes overall healing During inflammation there is anincreased production of various mediators such as arachi-donic acid metabolites and cytokines Besides there areconfirming evidences stating that nitric oxide (NO) producedby inducible NO synthase (iNOS) plays one of the importantroles in inflammatory disorders such as rheumatoid arthritis[31 32] Therefore chemicals that inhibit the NO productionby iNOS in macrophages would be the potential treatmentsto reduce inflammatory responses [22] In this regard it

is meaningful to examine the effects of CDNR on NOproduction One of our in vitro studies demonstrated thatthe ethanol component of CDNR suppressed LPS-inducednitric oxide and cytokines production significantly Theseobservations indicated that there are active ingredients inCDNR that can suppress inflammation and thus controls theswelling and relieves the pain during fracture healing

The ultimate goal of bone repair is to restore the bonestrength of the injured bone to its original level withoutfracture or defect [33] Assessment on the bone strengthduring fracture or defect healing in preclinical studies isessential to evaluate the effectiveness of an intervention onbone repair The biomechanical tests of the current studyshowed strong evidences of healing promotion from thetopical use of the herbal paste The higher bending yieldstrength and work load ability of the femur in the CDNRgroup than in the control group revealed that the treatmentelevated the strength of the bone that can tolerate muchmore stress and energy before permanent deformation which


could be caused by microfractures Perhaps the topical useof CDNR may reduce the risk of refracture during fracturehealing due to insufficient structural remodeling [34] CDNRtherefore might act like some antiresorptive pharmaceuticalagents when they help building up bone density and bonestrength in the prevention of refracture [35]

Nowadays microstructure of the trabecular bone is oneof the most important parameters in orthopaedic researchesThe microarchitecture of trabecular bone was highly cor-related with bone strength [36 37] Therefore assessmenton microarchitecture gives a new dimension to researchersin terms of clinical observations of bone In the micro-CTanalysis of the current study no difference in BV in thetrabecular region of the tibia was found between CDNR andControl It might be due to the fact that the bone healingprocess in trabecular metaphysis is different from that incortical diaphysis Healing of bone lesions involves manyfactors including different processes according to the typeof bone (cortical or cancellous) position and size of thewound and the biomechanical environment provided ontothe injured bone [38] Diaphyseal defect on femur of micewas rapidly repaired while the epimetaphyseal defect failedto bridge the cortical gap even 13 weeks after surgery [39]Another study reported that when the defect with the samesize was located at the metaphysis of distal femur of mice itbridged completely within 6 weeks [40]

Bone formation and bone resorption are two tightlycoupled activities in a continuous and dynamic process ofbone remodeling During bone resorption calcium and othermatrix constituents are released into the bloodstream Whenin bone formation skeletal-specific proteins (enzymes ormatrix components) can leach into the circulation Thesebiochemical markers can be measured by a noninvasivesystemic and sensitive method to detect changes of boneturnover in a short interval of time [41]These measurementshave also been used to predict bone fracture [42 43] Thebone formationmarker BALP increased in both groups of ratsin this studyThis observation illustrated that the osteoblasticactivity of the rats whether Control or CDNR was increasedafter the drill-hole bone defect had been createdThe data wascoherent with the results of a previous study on human inwhich the increase of BALP started to be observed from the7th day after fracture elevated by nearly 200 to week 12 andwas maintained at high level thereafter [44] In the fractureof the tibial shaft BALP increased at 4 weeks [45] On theother hand the serum concentration of the bone resorptionmarker Dpd was found reduced in both groups of rats post-op It indicated that bone resorption activity was retardedduring bone repair Similar findings were also reported inthe studies using bovine [46] and canine [47] model of bonefracture healing They showed that the serum TRAP activityanother bone resorption biochemical marker was low duringthe postfracture period On the whole there was a net bonegain after the systemic bone remodeling process of the rats inthe processes of drill-hole bone repair Apparently the topicalCDNR treatment did not alter this process systematically Itspromotional effect on bone healing could possibly be a localresponse

The essential factor required for the efficacy of any topicaltreatment is its transdermal transport The efficiency of thetransport of the active ingredients of the drug depends onthe physicochemical properties of the molecules includingthe size (molecular weight) charge and hydrophilicity (watersolubility) [48ndash50] In the transdermal experiment of thisstudy almost all the chemical markers of CDNR weredetected by LCMS in both the skin and muscle at thetreatment sites of the rats This observation illustrated thateach herbal component of CDNR did pass through the skinbarrier to cast its therapeutic effect It further supports thehypothesis that the herbal components of CDNR influencebone healing via local therapeutic responses

Topical agents have been popular before the presentera of fracture management Now that fracture treatment iswell developed clinicians have isolated themselves from thetraditional treatment applications As a matter of fact sinceno scientific records are available neither with the biologicalactivities nor with the more basic transdermal transport theold practice might not deserve much attention

However since topical herbal agents are still widely usedin the households and there might be special indications ofutilization it would be necessary to start serious scientificexplorations Indeed some difficult fracture healings mightstill benefit from simple harmless topical applications as longas toxicity and sensitivity could be ruled out Moreover theanti-inflammatory effects could also help with the recoveringof soft tissue injuries [51]

5 Conclusions

The present study demonstrated for the first time that a4-herb formula (CDNR) was effective in the control ofinflammation and facilitation of bone regeneration whenused topically Its efficacy on the promotion of cortical bonerepair was also revealed in the rat experiments This studygave us sufficient scientific evidences that our novel herbalpaste is a safe and promising supplement agent to facilitatebone healing under special circumstances

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Wing-Sum Siu and Chun-Hay Ko equally contributed to thepaper

Acknowledgment

This work was supported by Research Fund for Innovationand Technology Support Programme (ITP01410NP) Inno-vation and Technology Commission Hong Kong


References

[1] C M Court-Brown and B Caesar ldquoEpidemiology of adultfractures a reviewrdquo Injury vol 37 no 8 pp 691ndash697 2006

[2] O Johnell C Cooper S Cummings C Slemenda and ESeeman ldquoThe socioeconomic burden of fractures today andin the 21st centuryrdquoThe American Journal of Medicine vol 103supplement 2 pp 20Sndash25S 1997

[3] R S Morrison J Magaziner M A McLaughlin et al ldquoTheimpact of post-operative pain on outcomes following hipfracturerdquo Pain vol 103 no 3 pp 303ndash311 2003

[4] K Lippuner J von Overbeck R Perrelet H Bosshard and PJaeger ldquoIncidence and direct medical costs of hospitalizationsdue to osteoporotic fractures in Switzerlandrdquo OsteoporosisInternational vol 7 no 5 pp 414ndash425 1997

[5] M L Kilgore M A Morrisey D J Becker et al ldquoHealth careexpenditures associated with skeletal fractures amongmedicarebeneficiaries 1999ndash2005rdquo Journal of Bone andMineral Researchvol 24 no 12 pp 2050ndash2055 2009

[6] National Osteoporosis Society 25th anniversary reportmdashafragile future 2011 httpwwwnosorgukdocumentdocid=904

[7] D J Hak ldquoThe use of osteoconductive bone graft substitutesin orthopaedic traumardquo Journal of the American Academy ofOrthopaedic Surgeons vol 15 no 9 pp 525ndash536 2007

[8] G M Calori E Mazza M Colombo and C Ripamonti ldquoTheuse of bone-graft substitutes in large bone defects any specificneedsrdquo Injury vol 42 supplement 2 pp S56ndashS63 2011

[9] K A Hing L F Wilson and T Buckland ldquoComparative per-formance of three ceramic bone graft substitutesrdquo The SpineJournal vol 7 no 4 pp 475ndash490 2007

[10] A Lode C Wolf-Brandstetter A Reinstorf et al ldquoCalciumphosphate bone cements functionalized with VEGF releasekinetics and biological activityrdquo Journal of Biomedical MaterialsResearch Part A vol 81 no 2 pp 474ndash483 2007

[11] E Wernike M-O Montjovent Y Liu et al ldquoVEGF incorpo-rated into calcium phosphate ceramics promotes vascularisa-tion and bone formation in vivordquo European Cells and Materialsvol 19 pp 30ndash40 2010

[12] J C Y Cheng X Guo L P Law K M Lee D H K Chow andR Rosier ldquoHow does recombinant human bonemorphogeneticprotein-4 enhance posterior spinal fusionrdquo Spine vol 27 no 5pp 467ndash474 2002

[13] G Schmidmaier R Capanna B Wildemann T Beque and DLowenberg ldquoBonemorphogenetic proteins in critical-size bonedefects what are the optionsrdquo Injury vol 40 supplement 3 ppS39ndashS43 2009

[14] N Mokbel N Naaman J Nohra and N Badawi ldquoHealingpatterns of critical size bony defects in rats after grafting withbone substitutes soaked in recombinant human bone mor-phogenetic protein-2 histological and histometric evaluationrdquoBritish Journal of Oral and Maxillofacial Surgery vol 51 no 6pp 545ndash549 2013

[15] O Akkus F Korkusuz S Akin and N Akkas ldquoRelationbetween mechanical stiffness and vibration transmission offracture callus an experimental study on rabbit tibiardquo Proceed-ings of the Institution of Mechanical Engineers Part H vol 212no 5 pp 327ndash336 1998

[16] K Shakouri B EftekharsadatM R Oskuie et al ldquoEffect of low-intensity pulsed ultrasound on fracture callus mineral densityand flexural strength in rabbit tibial fresh fracturerdquo Journal ofOrthopaedic Science vol 15 no 2 pp 240ndash244 2010

[17] C-Q Ling X-Q Yue and C Ling ldquoThree advantages ofusing traditional Chinese medicine to prevent and treat tumorrdquoJournal of Integrative Medicine vol 12 no 4 pp 331ndash335 2014

[18] C Q Ling L N Wang Y Wang et al ldquoThe roles of tradi-tional Chinese medicine in gene therapyrdquo Journal of IntegrativeMedicine vol 12 no 2 pp 67ndash75 2014

[19] L H Peng C H Ko S W Siu et al ldquoIn vitro amp in vivoassessment of a herbal formula used topically for bone fracturetreatmentrdquo Journal of Ethnopharmacology vol 131 no 2 pp282ndash289 2010

[20] Chinese Pharmacopoeia Commission Pharmacopoeia of thePeoplersquos Republic of China Chinese Medical Science and Tech-nology Press Beijing China 2010

[21] J Li X-L Wu Y Chen et al ldquoAntidiarrheal properties ofdifferent extracts of Chinese herbal medicine formula Bao-Xie-Ningrdquo Journal of Chinese Integrative Medicine vol 11 no 2 pp125ndash134 2013

[22] G Chen K K Li C H Fung et al ldquoEr-Miao-San a tra-ditional herbal formula containing Rhizoma Atractylodis andCortex Phellodendri inhibits inflammatory mediators in LPS-stimulated RAW2647 macrophages through inhibition of NF-120581B pathway and MAPKs activationrdquo Journal of Ethnopharma-cology vol 154 no 3 pp 711ndash718 2014

[23] R Karki C-H Park and D-W Kim ldquoExtract of buckwheatsprouts scavenges oxidation and inhibits pro-inflammatorymediators in lipopolysaccharide-stimulated macrophages(RAW2647)rdquo Journal of Chinese Integrative Medicine vol 11no 4 pp 246ndash252 2013

[24] Y-X J Wang C H Ko J F Griffith et al ldquoOrganic nitratemaintains bone marrow blood perfusion in ovariectomizedfemale rats a dynamic contrast-enhanced magnetic resonanceimaging (MRI) studyrdquo Pharmaceutics vol 5 no 1 pp 23ndash352013

[25] K-W Luo J-G Sun J Y-W Chan et al ldquoAnticancer effectsof imperatorin isolated from Angelica dahurica induction ofapoptosis in HepG2 cells through both death-receptor- andmitochondria-mediated pathwaysrdquo Chemotherapy vol 57 no6 pp 449ndash459 2012

[26] S Wingerter M Tucci J Bumgardner and H BenghuzzildquoEvaluation of short-term healing following sustained deliveryof osteoinductive agents in a rat femur drill defect modelrdquoBiomedical Sciences Instrumentation vol 43 pp 188ndash193 2007

[27] A Obenaus and P Hayes ldquoDrill hole defects induction imag-ing and analysis in the rodentrdquo Methods in Molecular Biologyvol 690 pp 301ndash314 2011

[28] W EHoffmannN EverdsM Pignatello andP F Solter ldquoAuto-mated and semiautomated analysis of rat alkaline phosphataseisoenzymesrdquo Toxicologic Pathology vol 22 no 6 pp 633ndash6381994

[29] C E Wiedmeyer D G Morton P K Cusick T Wright P FSolter and W E Hoffmann ldquoSemiautomated analysis of alka-line phosphatase isoenzymes in serum of normal cynomolgusmonkeys (Macaca fascicularis)rdquo Veterinary Clinical Pathologyvol 28 no 1 pp 2ndash7 1999

[30] S B Kessler S Deiler M Schiffl-Deiler H K Uhthoff andL Schweiberer ldquoRefractures a consequence of impaired localbone viabilityrdquoArchives ofOrthopaedic andTraumaSurgery vol111 no 2 pp 96ndash101 1992

[31] S Moncada R M J Palmer and E A Higgs ldquoNitric oxidephysiology pathophysiology and pharmacologyrdquo Pharmaco-logical Reviews vol 43 no 2 pp 109ndash142 1991


[32] D O Stichtenoth and J C Frolich ldquoNitric oxide and inflamma-tory joint diseasesrdquo British Journal of Rheumatology vol 37 no3 pp 246ndash257 1998

[33] M B Habal ldquoControlled bone regeneration the ultimateprocess in bone repairrdquo Clinics in Plastic Surgery vol 29 no 1pp 15ndash27 2002

[34] L D Esposti L Sinigaglia M Rossini et al ldquoAdherence totherapeutic and diagnostic recommendations in patients withfemur fracture and at risk of re-fracture or death results of ananalysis of administrative databasesrdquo Reumatismo vol 64 no1 pp 18ndash26 2012

[35] F Shapiro ldquoBone development and its relation to fracture repairThe role of mesenchymal osteoblasts and surface osteoblastsrdquoEuropean Cells and Materials vol 15 pp 53ndash76 2008

[36] J C M Teo K M Si-Hoe J E L Keh and S H TeohldquoRelationship between CT intensity micro-architecture andmechanical properties of porcine vertebral cancellous bonerdquoClinical Biomechanics vol 21 no 3 pp 235ndash244 2006

[37] A Syahrom M R Abdul Kadir J Abdullah and A OchsnerldquoMechanical and microarchitectural analyses of cancellousbone through experiment and computer simulationrdquo Medicaland Biological Engineering and Computing vol 49 no 12 pp1393ndash1403 2011

[38] LMonfoulet B RabierOChassande and J C Fricain ldquoDrilledhole defects in mouse femur as models of intramembranouscortical and cancellous bone regenerationrdquo Calcified TissueInternational vol 86 no 1 pp 72ndash81 2010

[39] H Uusitalo J Rantakokko M Ahonen et al ldquoA metaphysealdefect model of the femur for studies of murine bone healingrdquoBone vol 28 no 4 pp 423ndash429 2001

[40] S W Veitch S C Findlay A J Hamer A Blumsohn R Eastelland B M Ingle ldquoChanges in bone mass and bone turnoverfollowing tibial shaft fracturerdquo Osteoporosis International vol17 no 3 pp 364ndash372 2006

[41] B L Riggs and S Khosla ldquoRole of biochemical markers inassessment of osteoporosisrdquo Acta Orthopaedica ScandinavicaSupplementum vol 66 no 266 pp 14ndash18 1995

[42] P Garnero E Hausherr M-C Chapuy et al ldquoMarkers of boneresorption predict hip fracture in elderly women the EPIDOSProspective Studyrdquo Journal of Bone and Mineral Research vol11 no 10 pp 1531ndash1538 1996

[43] S Vasikaran R Eastell O Bruyere et al ldquoMarkers of boneturnover for the prediction of fracture risk and monitoringof osteoporosis treatment a need for international referencestandardsrdquo Osteoporosis International vol 22 no 2 pp 391ndash420 2011

[44] K Stoffel H Engler M Kuster and W Riesen ldquoChangesin biochemical markers after lower limb fracturesrdquo ClinicalChemistry vol 53 no 1 pp 131ndash134 2007

[45] P Seebeck H J Bail C Exner et al ldquoDo serological tissueturnover markers represent callus formation during fracturehealingrdquo Bone vol 37 no 5 pp 669ndash677 2005

[46] C Sousa H Abreu C Viegas et al ldquoSerum total and bonealkaline phosphatase and tartrate-resistant acid phosphataseactivities for the assessment of bone fracture healing in dogsrdquoArquivo Brasileiro de Medicina Veterinaria e Zootecnia vol 63no 4 pp 1007ndash1011 2011

[47] R K Schenk ldquoBiology of fracture repairrdquo in Skeletal TraumaBasic Science Management and Reconstruction B D BrownerJ B Jupiter A M Levine and P G Trafton Eds pp 29ndash73Saunders Philadelphia Pa USA 2003

[48] J Hadgraft and R H Guy Transdermal Drug Delivery Develop-mental Issues andResearch InitiativesMarcelDekkerNewYorkNY USA 1989

[49] G E Bast ldquoInfluence of solubility and permeant size onabsorption and metabolism of xenobiotics in rabbit skinrdquoHuman amp Experimental Toxicology vol 16 no 8 pp 435ndash4401997

[50] R K Subedi S Y Oh M-K Chun and H-K Choi ldquoRecentadvances in transdermal drug deliveryrdquo Archives of PharmacalResearch vol 33 no 3 pp 339ndash351 2010

[51] M J Kruger and C Smith ldquoPostcontusion polyphenol treat-ment alters inflammation and muscle regenerationrdquo Medicineand Science in Sports and Exercise vol 44 no 5 pp 872ndash8802012

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


therapeutic effects on treating various diseases includingcancer [17]More recently its applications inmodernWesterntherapy have also been discussed [18] Facilitation of fracturehealing is one of the major areas in traditional Chinesemedicine Traditionally topical agents have been used topromote the healing of soft tissues and bone fracturesNonetheless the formulae of these herbal medicines aretoo diversified as yet More importantly the serious lack ofrelevant evidence-based scientific support and good systemicdocumentation of the clinical data makes them not wellaccepted worldwide

The aim of this study is to investigate the efficacy ofa topical herbal paste on the promotion of bone healingfrom an evidence-based scientific approach Both in vitroand in vivo biological platforms will be used to verifythe pharmacological properties essential for bone healingnamely anti-inflammation proangiogenesis and cellularregeneration The most novel component of the study lies inthe confirmation of transcutaneous transport of the chemicalcompounds via the topical application

Considering that over a hundred of medicinal herbs hadbeen used historically in China for the treatment of skeletalinjuries and bone fracture we select the herbs for the presentstudy according to the classical records and recent scientificpapers testifying the pharmacological properties of the herbsrelated to fracture healing [19]

A herbal paste for topical use (CDNR) was thus createdwith four herbs namely Carthami Flos (C) Dipsaci Radix(D) Notoginseng Rhizoma (N) and Rhei Rhizoma (R) Allof them were purchased from a reputable herbal supplier inHong Kong The identities of all herbs were authenticatedusing thin-layer chromatography with procedural referencesrecommended by the Chinese pharmacopoeia [20] Theherbarium voucher specimens of the tested herbs weredeposited in the museum bank of the Institute of ChineseMedicine at the Chinese University of Hong Kong withreference numbers as follows Dipsaci Radix 2010-3279 RheiRhizoma 2010-3280 Notoginseng Rhizoma 2010-3278 andCarthami Flos 2010-3335

2 Materials and Methods

21 Making of the Topical Agent Extracts of CDNR wereprepared via both aqueous and ethanol extractions In theaqueous extraction 250 g raw materials of each of the fourherbs were cut into small pieces (C D N R in 1 1 1 1 ww)and were soaked in 10 L distilled water for 1 hour They wereboiled twice for 2 hours under reflux and then the aqueousextract was collected and filtered through a piece of absorbentgauze The residue was further boiled with 95 ethanol for2 hours under reflux The ethanol extract was collected andfiltered again Both of the aqueous and ethanol filtrates werethen concentrated at 50∘C under reduced pressure separatelyfollowed by lyophilizing into powder forms (CDNR(aq) andCDNR(e)) in a freeze dry system (Freezone 12 LabconcoMissouri USA)The extraction yields of aqueous and ethanolextractswere 398wwand 50ww respectivelyThepasteform of CDNR for topical use was completed bymixing 195 gCDNR(aq) and 30 g CDNR(e) with 17mL 50 ethanol [19]

Table 1 Chemical composition of CDNR paste for animal studies

Herbalsource Name of chemical marker

Content(mgg of paste

mixture powder)CarthamiFlos (C)

Hydroxysafflor yellow A (HYA)Kaempferol (KAE)

2140004

DipsaciRadix (D)

Asperosaponin VI (ASP)Oleanolic acid (OA)

1923005

NotoginsengRhizoma (N)

Ginsenoside Rg1 (Rg1)Ginsenoside Rb1 (Rb1)

20055

RheiRhizoma (R)

Emodin (EMO)Rhein (RHE)

096100

22 Chemical Composition of Topical Herbal Paste Thechemical composition of the herbal paste was determinedby high-performance liquid chromatography-electrosprayionization-mass spectrometry (HPLC-ESI-MS) whichincluded an Agilent 1290 Infinity LC System (Agilent Tech-nologies Santa Clara California USA) equipped with anonline degasser a binary pump an autosampler and Agilent6410 Triple Quad LCMS and connected with AgilentMassHunter Workstation software A Waters HSS T3 35 120583m(30mm times 150mm) HPLC column was used and kept at atemperature of 40∘C Eight chemical markers and sampleswere separated using a gradient mobile phase consisting ofwater (A) and acetonitrile (B) The gradient condition is 0 to3min 10ndash27 B 3 to 5min 27ndash33 B 5 to 12min 33-33B 12 to 13min 33ndash80 B 13 to 16min 80ndash90 B 16 to20min 90-90 B The flow rate was set at 05mLmin andthe injection volume was 20120583L

MS analysis was performed on Agilent 6410 Triple-QuadLCMS equipped with an ESI source [21] and monitored innegative ion mode and multiple reaction monitoring modeusing target ions at mz 6112rarr 3250 for hydroxysaffloryellowA (HYA)mz 2850rarr 1170 for kaempferol (KAE)mz9275rarr 6033 for asperosaponin VI (ASP)mz 4553rarr 4074for oleanolic acid (OA) mz 7995rarr 6374 for ginsenosideRg1 (Rg1) mz 11076rarr 1190 for ginsenoside Rb1 (Rb1) mz2690rarr 2410 for emodin (EMO) andmz 2830rarr 2390 forrhein (RHE) The abundance of the markers was determinedquantitatively (Table 1)

All the chemicals were purchased from Sigma-Aldrich(St Louis Missouri USA) Both methanol and acetonitrilewere HPLC-grade (CHROMASOLV Plus ge999) All thechemical markers were purchased from Tauto Biotech CoLtd (Shanghai China) Their purities were over 97

23 In Vitro Studies In vitro studies were targeting anti-inflammation and cellular proliferation

231 Cell Cultures Murine monocytemacrophageRAW2647 and rat osteosarcoma UMR-106 cells were pur-chased from American Type Culture Collection (ManassasVA USA) RAW2647 and UMR-106 cells were maintainedin high-glucose DMEM (d-glucose 3500mgL GIBCOUSA) 10 vv fetal bovine serum (FBS Gibco Gran IslandNY USA) and 1 penicillin-streptomycin (PS Gibco Gran












242 Assessments







3 Results









0

10

20

30

40

400 200 100 50 0 No LPS(120583gmL)


conc

entr

atio

n (120583

M)

(a)

0

10

20

30

40

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast


conc

entr

atio

n (120583

M)

(b)

0

50

100

150TNF-120572

lowast lowastlowast

200 100 50 0 No LPS(120583gmL)

25

C

ontro

l (ve

rsus

no

treat

men

t)

(c)

0

50

100

150

Con

trol (

vers

us n

o tre

atm

ent)

IL-6

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast

lowast

(d)

0

50

100

150

C

ontro

l (ve

rsus

no

treat

men

t)

IL-1120573

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowast

(e)



0

20

40

60

80

100

120

140Pr

olife

ratio

n (

of C

ontro

l)


CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(a)

0

20

40

60

80

100

120

140

Cel

l via

bilit

y (

of C

ontro

l)


lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(b)









4 Discussion




0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)


00

01

02

03

04


(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)













5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























242 Assessments







3 Results









0

10

20

30

40

400 200 100 50 0 No LPS(120583gmL)


conc

entr

atio

n (120583

M)

(a)

0

10

20

30

40

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast


conc

entr

atio

n (120583

M)

(b)

0

50

100

150TNF-120572

lowast lowastlowast

200 100 50 0 No LPS(120583gmL)

25

C

ontro

l (ve

rsus

no

treat

men

t)

(c)

0

50

100

150

Con

trol (

vers

us n

o tre

atm

ent)

IL-6

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast

lowast

(d)

0

50

100

150

C

ontro

l (ve

rsus

no

treat

men

t)

IL-1120573

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowast

(e)



0

20

40

60

80

100

120

140Pr

olife

ratio

n (

of C

ontro

l)


CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(a)

0

20

40

60

80

100

120

140

Cel

l via

bilit

y (

of C

ontro

l)


lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(b)









4 Discussion




0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)


00

01

02

03

04


(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)













5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results









0

10

20

30

40

400 200 100 50 0 No LPS(120583gmL)


conc

entr

atio

n (120583

M)

(a)

0

10

20

30

40

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast


conc

entr

atio

n (120583

M)

(b)

0

50

100

150TNF-120572

lowast lowastlowast

200 100 50 0 No LPS(120583gmL)

25

C

ontro

l (ve

rsus

no

treat

men

t)

(c)

0

50

100

150

Con

trol (

vers

us n

o tre

atm

ent)

IL-6

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast

lowast

(d)

0

50

100

150

C

ontro

l (ve

rsus

no

treat

men

t)

IL-1120573

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowast

(e)



0

20

40

60

80

100

120

140Pr

olife

ratio

n (

of C

ontro

l)


CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(a)

0

20

40

60

80

100

120

140

Cel

l via

bilit

y (

of C

ontro

l)


lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(b)









4 Discussion




0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)


00

01

02

03

04


(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)













5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

400 200 100 50 0 No LPS(120583gmL)


conc

entr

atio

n (120583

M)

(a)

0

10

20

30

40

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast


conc

entr

atio

n (120583

M)

(b)

0

50

100

150TNF-120572

lowast lowastlowast

200 100 50 0 No LPS(120583gmL)

25

C

ontro

l (ve

rsus

no

treat

men

t)

(c)

0

50

100

150

Con

trol (

vers

us n

o tre

atm

ent)

IL-6

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowastlowastlowast

lowast

(d)

0

50

100

150

C

ontro

l (ve

rsus

no

treat

men

t)

IL-1120573

200 100 50 0 No LPS(120583gmL)

25

lowastlowastlowast

lowast

(e)



0

20

40

60

80

100

120

140Pr

olife

ratio

n (

of C

ontro

l)


CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(a)

0

20

40

60

80

100

120

140

Cel

l via

bilit

y (

of C

ontro

l)


lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(b)









4 Discussion




0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)


00

01

02

03

04


(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)













5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

140Pr

olife

ratio

n (

of C

ontro

l)


CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(a)

0

20

40

60

80

100

120

140

Cel

l via

bilit

y (

of C

ontro

l)


lowastlowastlowast

CDNR(aq)CDNR(e)

100500 25625 125

(120583gmL)

(b)









4 Discussion




0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)


00

01

02

03

04


(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)













5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

from

nor

mal

fem

ur

CDNRControl

lowast

(a)

fr

om n

orm

al fe

mur

0

10

20

30

40

CDNRControl

lowast

(b)


00

01

02

03

04


(120583g

g)

(a)

00

02

04

06


(120583g

g)

(b)













5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























5 Conclusions






Acknowledgment



References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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 a topical herbal agent for the...

Documents