Research ArticleAntithrombotic Effect and Mechanism of Radix Paeoniae Rubra

Pingyao Xie12 Lili Cui1 Yuan Shan1 and Wen-yi Kang12

1 Institute of Chinese Materia Medica Henan University Kaifeng 475004 China2Kaifeng Key Laboratory of Functional Components in Health Food Kaifeng 475004 China

Correspondence should be addressed to Wen-yi Kang kangwenyhotmailcom

Received 25 December 2016 Revised 9 January 2017 Accepted 18 January 2017 Published 16 February 2017

Academic Editor Veronika A Myasoedova

Copyright copy 2017 Pingyao Xie 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

The compounds of Radix Paeoniae Rubra (RPR) were isolated and identified by bioassay-guided method and antithromboticeffects and mechanism were investigated by the acute blood stasis rat model The RPR extract was evaluated by APTT TT PTand FIB assays in vitro Results indicated that RPR extract exhibited the anticoagulant activity In order to find active compoundssix compounds were isolated and identified and four compounds paeoniflorin (Pae) pentagalloylglucose (Pen) albiflorin (Ali)and protocatechuic acid (Pro) exhibited the anticoagulant activity in vitro Therefore the antithrombosis effects of RPR extractand four active compounds were investigated in vivo by measuring whole blood viscosity (WBV) plasma viscosity (PV) APTTPT TT and FIB Meanwhile the levels of TXB2 6-Keto-PGF1120572 eNOS and ET-1 were detected Results suggested that RPR extractand four active compounds had the inhibition effect on thrombus formation and the antithrombotic effects were associated withthe regulation of vascular endothelium active substance activating blood flow and anticoagulation effect

1 Introduction

Thrombosis the formation or presence of the thrombusin a blood vessel is a multifactorial disease induced bypromoting a combination of stasis and hypercoagulability [1]Thrombotic diseases especially heart disease and cerebrovas-cular thrombosis have become primary causes of death andtheir incidence has been increasing each year [2] It is wellknown that thrombosis is closely related to activated plateletadhesion aggregation secretion functions and activationof intrinsic and extrinsic coagulation systems which causeblood coagulation and fibrin formation Although they havemany causes [3] accumulation of thrombus is a key factor inthe final analysis

Many traditional Chinese herbal medicines have beenused for thousands of years in clinical practice because oftheir proven efficacy wide indications high safety profile andlow toxicity [4] Then Traditional Chinese Medicine (TCM)plays an important role in the clinic treatment of blood stasisin China one of which is the Radix Paeoniae Rubra (RPR)which is the dried root of Paeonia lactifloraPall and P veitchiiLynch [5] It had the function of removing pathogenic heatfrom the blood and treating blood stasis and relieving pain[6 7]

In Traditional Chinese Medicine thrombotic disordersare described as blood stasis syndrome [8] Though aspirinhas been widely used as antithrombotic medicine in clinicalpractice an increasing number of studies have indicated thatsome patients subpopulations do not respond to the anti-thrombotic effects of aspirin and may exhibit a degree ofaspirin resistance Therefore interest in TCM from naturalsources as a feasible alternative therapeutic agent for theprevention of blood stasis disorder in patients with ldquoaspirinresistancerdquo is growing [9]

RPR has some effects on activating blood circulationto dissipate blood stasis however the mechanism has beenpoorly studied In this paper antithrombotic effects andmechanism of extracts and the compounds from the RPRroot were investigated

2 Materials and Methods

21 Plant Materials TheRPR was purchased from LerentangPharmacy Kaifeng Henan Province China and a voucherspecimen was identified by Professor Chang-qin Li of HenanUniversity and deposited in the Herbarium of the Institute ofNatural Products Henan University

HindawiBioMed Research InternationalVolume 2017 Article ID 9475074 9 pageshttpsdoiorg10115520179475074

2 BioMed Research International

22 Animals Male and female Sprague-Dawley (SD) ratsweighing from 220 to 300 g and male New Zealand whiterabbits from 20 to 25 kg were obtained from the Experimen-tal Animal Center of Henan Province (Zhengzhou HenanChina) (12 h lightdark cycle 25∘C and humidity 45 to65) and were fed with standard rodent diet and water adlibitumThe animal procedures were approved by the ethicalcommittee in accordance with ldquoInstitute Ethical CommitteeGuidelinesrdquo for Animal Experimentation and Care Animalswere housed in standard cage

23 Chemicals The following was used PT APTT TT andFIB Assay Kits (Shanghai Sun Biotech Co Ltd) BovineFibrinogen (Sigma USA) Thrombin (Sigma USA) Uroki-nase (Biotechnology Co Ltd Hangzhou Macao and Asia)Agarose (GENE Company) Complex Tablet (HutchisonWhampoa Guangzhou Baiyunshan Chinese Medicine Com-pany Limited China) Rat Endothelin 1 (ET-1) Elisa Kit RatEndothelial Nitric Oxide Synthase (eNOS) Elisa Kit Rat 6-Keto-PGF1120572 Elisa Kit RatThromboxane B2 (TXB2) Elisa Kit(Nanjing Sengbiejia Biotech Co Ltd)

24 Extraction and Isolation Air-dried and powdered rootsof RPR (19 kg) were extracted with 70 ethanol at 60∘C fortwo times (2 h each time) and then pressure was reducedto yield a viscous gummy material (570 g) The total extractwas dissolved in 500mL of water and then extracted withpetroleum ether ethyl acetate and n-butanolThe PE EtOAcand BuOH extracts were concentrated under reduced pres-sure to give 6 g 29 g and 813 g respectively EtOAc extract(29 g) was subjected to silica gel H column chromatographyand elutedwith dichloromethane-ethyl acetate (100 0-0 100vv) to yield three fractions The first fraction (46 g) wasfurther separated on a silica gel column repeatedly to yieldcompound 1 (1231mg) The second fraction (23 g) waspurified by various column chromatography including silicagel and sephadex LH-20 to yield compound 2 (535mg)and compound 3 (365mg) n-Butanol extract (813 g) waspurified on MPLC and then further separated on silicagel H and sephadex LH-20 column chromatography to getcompounds 4 (312mg) 5 (829mg) and 6 (384mg) respec-tively The six compounds were identified as paeoniflorin(1 Pae) pentagalloylglucose (2 Pen) benzoyl paeoniflorin(3 Ben) oxypaeoniflora (4 Oxy) albiflorin (5 Ali) andprotocatechuic acid (6 Pro) by 1H NMR and 13C NMR Allthe compoundswere performed onHPLC and the purity wasover 98

25 Coagulation Time Test In Vitro Blood samples weredrawn from rabbitrsquos auricular vein After collection the bloodwas decalcified by sodium citrate to prevent blood clottingand then serums were separated from the plasma by centrifu-gation of 3000 rpm at 5∘C for 15min [10] APTT and PTweredetermined according to the literature [11] In brief serum(50120583L) was mixed with 25120583L of samples next APTT assayreagent (50 120583L) was added and incubated for 5min at 37∘Cand then 25mM CaCl2 (100 120583L) was added Clotting timeswere recorded Meanwhile in PT assays serums (50 120583L)

mixedwith 25 120583Lof sampleswere incubated for 3min at 37∘CPT assay reagent (50 120583L) which has been hatched for 10minat 37∘C was then added and clotting time was recordedDetermination of TT and FIB was performed according tothe manufacturerrsquos recommendations (Shanghai Sun BiotechCo Ltd China) In the above tests control solvent was usedas control group and breviscapine and vitamin K1 were usedas positive control group PT APTT TT and FIB assays wereconducted by Semiautomated Coagulation Analyzer

26 ExperimentalModel and Drug Administration Rats wererandomly divided into seven groups with eight animalsin each whose gender was equally distributed throughoutgroups Group 1 was the control and Group 2 was the modeland rats were given control solvent Group 3 was model +aspirin (ASP) and rats were given 100mgkg ASP Groups4ndash7 were model + drugs and rats were given 12 gkg RPRextract All treatments were performed by gavage and wereadministered seven times with an interval of 12 h

After the fifth administration model rats groups wereplaced in ice-cold water during the interval between twoinjections of adrenaline hydrochloride (Adr) to establishblood stasis model All other rats were subcutaneouslyinjected with Adr (08mgkg) except for the control ratswhich were injected with 09 (wv) NaCl saline solutionAfter 2 h the rats were kept in ice-cold water (0ndash2∘C) for5min and then reinjected with Adr (08mgkg) subcuta-neously 2 h after the ice-bath to obtain blood stasis modelRats were fasted overnight and administration continuedafter performing the model Blood samples were collected30min after the last administration on the following day[8]

Compoundsrsquo experiment was similar to the abovemethod Group 1 and Group 2 were the same as the aboveGroup 3 was model + xiangdan injection (Xdi) (36mLKg)Groups 4ndash7 were model + drugs and rats were given 5mgkgcompounds All treatments were performed by caudal veinand administered seven times with an interval of 24 h

After the sixth administration the model rats with bloodstasis were established by the above method

27 Collection of Blood Sample Rats were anesthetized withchloral hydrate (300mgkg) 18 h after the last injection ofAdrand blood was drawn from the abdominal aortas Blood wascollected using 8 disposable vein infusion needles 1mL ofblood was taken into centrifuge tube on standing positionuntil the serumwas fully precipitatedThe serumwas used todetermine eNOS ET-1 6-Keto-PGF1120572 and TXB2 Anotherpart of the blood was collected into vacuum tube containingheparin to detectWBV PV PT TTAPTT FIB ESR andPCVrespectively

28 Statistical Analysis All experimental results wereexpressed asmeanplusmn standard deviation (SD) Statistical anal-ysis was performed with the SPSS190 software Comparisonbetween any two groups was evaluated using one-wayanalysis of variance (ANOVA) All graphics were drawn byGraphPad Prism 5

BioMed Research International 3

Con Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben Pro Con Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben Pro

Con Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben ProCon Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben Pro

0

10

20

30

40A

PTT

(s)

0

10

20

30

40

50

TT (s

)

PT (s

)FI

B (g

L)

0

2

4

6

8

10

0

2

4

6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 1 Anticoagulation activity of RPR extract and six compounds in vitro (119899 = 4 lowastlowastlowast119901 lt 0001 or 0001 lt lowastlowast119901 lt 001 or lowast119901 lt 005 versuscontrol group 119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus breviscapine)

3 Results

31 Effects on Plasma Coagulation Parameters In Vitro Asshown in Figure 1 compared with the control group ethanolextract of RPR (Ret) petroleum extract of RPR (Rpe) ethylacetate extract of RPR (Rea) n-butyl alcohol extract of RPR(Rbu) Pae Ali Pen Oxy and Pro could significantly prolongAPTT (001 lt 119901 lt 005 0001 lt 119901 lt 001 119901 lt 0001respectively) Above all there was no difference between Rbuand breviscapine (Brv) (119901 gt 005) In PT assay comparedwith the control group all of the extracts and six compoundscould significantly prolong PT (001 lt 119901 lt 005 0001 lt119901 lt 001 119901 lt 0001 respectively) Then the prolongingPT effects of Rpe Rea and Rbu were better than that of Brv(0001 lt 119901 lt 001 119901 lt 0001 resp) and Ret Pae Pen OxyBen and Pro had no difference with the Brv (119901 gt 005) InTT assay compared with the control group the Ret Rpe ReaRbu Pae Ali Pen and Pro could significantly prolong TT(119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005 resp) For FIBassay the Rpe Pae Ali Pen Oxy and Ben could significantlydecrease the content of FIB (119901 lt 0001) while the Ret ReaRbu and Pro could significantly increase the content of FIB(119901 lt 0001)

32 The Antithrombotic Effect of RPR Extract In Vivo

321 Effects on WBV PV ESR and PCV In Vivo The effectson WBV and PV were shown in Table 1 In model groupWBV and PV significantly increased at all shear rates in theblood stasis (119901 lt 0001) which indicated the blood stasisrat model was successfully establishedThe Ret Rea and Rbugroups could significantly decrease WBV and PV at all shear

rates (119901 lt 0001) and they decreased WBV and PV effects aswell as aspirin group (119901 gt 005)

The results of ESR and PCV were shown in Table 2ESR and PCV in the model group were higher than that ofcontrol group (119901 lt 0001) which indicated that the bloodstasis model was successfully established The Ret Rea andRbu groups could significantly decrease ESR and PCV (119901 lt0001) and they decreased ESR effects as well as aspirin group(119901 gt 005) The Rea and Rbu decreased PCV effects as well asaspirin group (119901 gt 005)

322 Effects on Plasma Coagulation Parameters To evaluatethe effect of the coagulation cascade on the antithromboticactivity of RPR extract the classical coagulation assays(APTT TT PT and FIB content) were determined in vivoCoagulation parameters were shown in Figure 2 APTTTT and PT were decreased and FIB content was increasedsignificantly in model group compared with control group(119901 lt 0001) RPR extracts could prolong PT APTT and TTand decrease FIB content significantly (119901 lt 0001)

323 TXB2 and 6-Keto-PGF1120572 Assay in Serum Results ofTXB2 and 6-Keto-PGF1120572 were shown in Table 3 and theplasma TXB2 level in model rats was prominently higherthan that of control group (119901 lt 0001) whereas the plasma6-Keto-PGF1120572 level in model rats was markedly lower thanthat of control group (119901 lt 005) Ret Rea and Rbu couldsignificantly counteract increase of TXB2 level (119901 lt 0001)and the effects of Ret and Rea were close to aspirin (119901 gt005) Ret and Rbu could significantly upregulate 6-Keto-PGF1120572 level in plasma compared with model rats (119901 lt 0001

4 BioMed Research International

Table 1 Effect on WBV (mPasdots) at various shear rates

Group WBV (mPasdots) PV (mPasdots)200S 30S 3S 30s

Con 434 plusmn 020 545 plusmn 033 988 plusmn 061 123 plusmn 003

Mod 544 plusmn 017lowastlowastlowast 680 plusmn 014lowastlowastlowast 1266 plusmn 025lowastlowastlowast 142 plusmn 003lowastlowastlowast

Asp 467 plusmn 047 575 plusmn 019 998 plusmn 036 127 plusmn 003

Ret 468 plusmn 017 604 plusmn 045 1039 plusmn 068 129 plusmn 001

Rea 467 plusmn 046 580 plusmn 056 1017 plusmn 041 129 plusmn 005

Rbu 464 plusmn 043 560 plusmn 061 1038 plusmn 054 126 plusmn 004

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 and 0001 lt 119901 lt 001 versus model group

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

Con Mod RetAsp Rea RbuCon Mod RetAsp Rea Rbu

0

10

20

30

40

50

APT

T (s

)

0

10

20

0 0

2

4

6

20

40

60

30

40

TT (s

)

PT (s

)FI

B (g

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779998779

998779998779

Figure 2 Anticoagulation activity of RPR extract in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group119901 lt 0001 versus aspirin 119901 lt 005 versus model group)

Table 2 Effect on ESR and PCV

Group Dose (gkg) ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 008 3715 plusmn 246

Mod mdash 1160 plusmn 114lowastlowastlowast 4683 plusmn 071lowastlowastlowast

Asp 01 740 plusmn 055 3892 plusmn 104

Ret 12 679 plusmn 084 4171 plusmn 130

Rea 12 780 plusmn 084 3973 plusmn 233

Rbu 12 680 plusmn 084 3973 plusmn 182

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 versus model group and 119901 lt 005 versus aspirin

119901 lt 005) and there were no obvious differences with aspirin(119901 gt 005) Meanwhile Ret Rea and Rbu decreased theratio of TXB26-Keto-PGF1120572 compared with model group(119901 lt 0001) The ratios of TXB26-Keto-PGF1120572 in Ret waslower than that of aspirin (119901 lt 0001) and that of Rea wasclose to those of aspirin (119901 gt 005)

324 Effect of RPR on ET and eNOS In Figure 3 the plasmaET-1 level in blood stasis model rats (6590 plusmn 424 120583gL)was prominently higher than that of control group (4675 plusmn254 120583gL) (119901 lt 0001) whereas the plasma eNOS level in

BioMed Research International 5

Table 3 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose (gkg) 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 9179 plusmn 738 8336 plusmn 675 0911 plusmn 008

Mod mdash 8126 plusmn 205lowast 10878 plusmn 379lowastlowastlowast 134 plusmn 006lowastlowastlowast

Asp 01 9058 plusmn 456 8190 plusmn 438 0910 plusmn 007

Ret 12 9627 plusmn 813 8352 plusmn 374 0874 plusmn 010

Rea 12 8681 plusmn 610 8442 plusmn 796 0976 plusmn 011

Rbu 12 9124 plusmn 536 9924 plusmn 693 109 plusmn 009

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 or lowast119901 lt 005 versus control group 119901 lt 0001 or 001 lt 119901 lt 0001 or 119901 lt 005 versus model group119901 lt 0001 versus aspirin

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

ET-1

(120583g

L)

eNO

S (U

L)

0

20

40

60

80

100

0

5

10

15

20

25

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779

998779

Figure 3The effects of RPR treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus aspirin 119901 lt 005)

model rats (1896 plusmn 127UL) was markedly lower than thatof control group (2261 plusmn 073UL) (119901 lt 0001) Ret and Reacould effectively downregulate of ET-1 level and upregulateeNOS level in plasma compared with model rats (119901 lt 0001119901 lt 005 resp) and Rea downregulation of ET-1 level effectwas better than that of aspirin (119901 lt 0001) and the effect ofRet downregulation of ET-1 level had no obvious differencecompared to aspirin group (119901 gt 005) Ret Rea and Rbucould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 119901 lt 005 resp) and the effects of Rbuupregulation of eNOS level were better than that of aspirin(119901 lt 005) the effects of Rea upregulation of eNOS level hadno obvious difference compared to aspirin (119901 gt 005)

33 The Antithrombotic Effect of Active Compounds In Vivo

331 Effects onWBV PV ESR and PCV Theeffects onWBVand PV were shown in Table 4 In model group WBV andPV significantly increased at all shear rates in the blood stasis(119901 lt 0001) which indicated the blood stasis rat modelwas successfully established Pro Ali Pae and Pen couldsignificantly decrease WBV and PV at all shear rates (119901 lt0001 0001 lt 119901 lt 001 119901 lt 005) and they decreased WBVeffects as well as xiangdan injection (Xdi) (119901 gt 005) at lowshear rate

The results of ESR and PCV for each group are shown inTable 5 ESR and PCV in the model group were higher than

that of control group (119901 lt 0001) Pro Ali Pae and Pen couldsignificantly decrease ESR and PCV (119901 lt 0001 0001 lt 119901 lt001 119901 lt 005) Effects on ESR and PCV of Ali were equal toXdi (119901 gt 005) Effect on ESR of Pro was equal to Xdi (119901 gt005) The effect on PCV of Pae was equal to Xdi (119901 gt 005)

332 Effects on PlasmaCoagulation Parameters Coagulationparameters were shown in Figure 4 APTT TT and PT weredecreased and FIB content was increased very significantlyin model group compared with control group (119901 lt 0001)Pro Ali Pae and Pen could prolong PT APTT and TT anddecrease FIB content effectively (119901 lt 0001 119901 lt 005)

333 TXB2 and 6-Keto- PGF1120572 in Serum In Table 6 theplasma TXB2 level in blood stasis model was prominentlyhigher than that of control group (119901 lt 0001) whereas theplasma 6-Keto-PGF1120572 level in model group was markedlylower than that of control group (119901 lt 0001)

Ali and Pen could significantly counteract increase ofTXB2 level (119901 lt 0001) and they were close to those of Xdigroup (119901 gt 005) Pro Ali Pae and Pen could effectivelyupregulate 6-Keto-PGF1120572 level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and Pae andPen had no obvious difference compared to Xdi (119901 gt 005)Meanwhile Pro Ali Pae and Pen could decrease the ratioof TXB26-Keto-PGF1120572 compared with model group (119901 lt0001) Pro and Ali ratios of TXB26-Keto-PGF1120572 in the rat

6 BioMed Research International

Table 4 Effect on WBV (mPasdots) at various shear rates

Group Dose WBV (mPasdots) PV (mPasdots)200S 30S 3S 30S

Con mdash 396 plusmn 011 526 plusmn 009 1006 plusmn 021 127 plusmn 004

Mod mdash 529 plusmn 018lowastlowastlowast 660 plusmn 023lowastlowastlowast 1255 plusmn 043lowastlowastlowast 141 plusmn 003lowastlowastlowast

Xdi 36mLKg 414 plusmn 004 528 plusmn 019 1049 plusmn 086 127 plusmn 001

Pro 50mgKg 477 plusmn 009 582 plusmn 011 1110 plusmn 018 136 plusmn 003

Ali 50mgKg 437 plusmn 017 539 plusmn 026 1027 plusmn 047 133 plusmn 003

Pae 50mgKg 446 plusmn 011 558 plusmn 012 1062 plusmn 023 135 plusmn 003

Pen 50mgKg 463 plusmn 005 569 plusmn 099 1070 plusmn 039 134 plusmn 002

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 0001 lt 119901 lt 001 and 119901 lt 005 versus model group 119901 lt 00010001 lt 119901 lt 001 and 119901 lt 005 versus Xdi

0

20

40

60

APT

T (s

)

0

10

20

30

PT (s

)

0

20

40

60

TT (s

)

0

2

4

6

FIB

(gL

)

Con Mod Xdi Pro Ali Pae PenCon Mod Xdi Pro Ali Pae Pen

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779

998779

998779

998779998779

998779998779998779

Figure 4 Anticoagulation activity of active compounds in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or 119901 lt 005 versusmodel group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt 119901 lt 001)

were lower compared to the Xdi (119901 lt 0001) and those of thePae and Pen were close to those of Xdi (119901 gt 005)

334 Effect of Compounds on ET and eNOS As shown inFigure 5 the plasma ET-1 level in blood stasis model rats(9482 plusmn 419 120583gL) was prominently higher than that ofcontrol group (8708 plusmn 334 120583gL) (119901 lt 0001) whereasthe plasma eNOS level in model rats (2283 plusmn 145UL) wasmarkedly lower than that of control group (2677 plusmn 098UL)(119901 lt 0001) Ali and Pae could effectively downregulate ET-1 level and upregulate eNOS level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and the Pae group

downregulation of ET-1 level effect had no obvious differencecompared to Xdi group (119901 gt 005) Pro Ali Pae and Pencould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005) and Ali andPen upregulation of eNOS level effect was better than aspiringroup (119901 lt 0001 119901 lt 005)

4 Discussion

APTT PT TT and FIB are four indicators related toanticoagulant in clinical practice In our study Rpe PaeAli and Pen could significantly extend TT APTT and TT

BioMed Research International 7

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

ET-1

(ng

L)

eNO

S (U

L)

0

50

100

150

0

10

20

30

40

lowastlowast

lowastlowastlowast

998779998779998779

998779

998779

998779

Figure 5 The effects of active compounds treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 or 0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt lowastlowast119901 lt 001 versus control group)

Table 5 Effect on ESR and PCV

Group Dose ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 082 3604 plusmn 123

Mod mdash 1050 plusmn 058lowastlowastlowast 4580 plusmn 130lowastlowastlowast

Xdi 36mLKg 725 plusmn 050 3711 plusmn 173

Pro 5mgKg 850 plusmn 058 4073 plusmn 116

Ali 5mgKg 85 plusmn 129 3756 plusmn 237

Pae 5mgKg 875 plusmn 17 3868 plusmn 148

Pen 5mgKg 890 plusmn 084 3955 plusmn 148

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group and119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus model group and0001 lt 119901 lt 001 or 119901 lt 005 versus Xdi

and reduced the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways and hinder fibrin formation Ret Rea Rbu andProcould significantly extend TT APTT and PT but they couldnot reduce the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways Then the Oxy and Ben had no anticoagulationeffect in vitro

Hemorheology is related to blood flow and pressureflow volume and resistance of blood vessels includingWBV PV ESR and PCV Hemorheology is commonly usedfor diagnosis and prevention of cardiovascular diseases inclinical practice [12ndash19] Red blood cells (RBCs) plateletsand plasmahave an influence onWBV becauseRBCs accountfor almost 50 of blood volume and constitute the majorityof the cellular content in blood In our research Ret Rea RbuPro Ali Pae and Pen groups could significantly decreaseWBV and PCV which suggested that the amelioration effectof compounds on WBV might be partly due to the PCVdecrease

PV plays an important role in WBV It showed thecoefficient between PV and ESR was positive in significantlevel [20] In our research Ret Rea Rbu Pro Ali Pae andPen groups could significantly decrease PV and ESR which

suggested that the amelioration effect of compounds on PVmight be partly due to the ESR decrease

Ret Rea Rbu Pro Ali Pae and Pen groups couldsignificantly extend TT APTT and PT and reduced thecontent of FIB in vivo which indicated the beneficial effectof them on intrinsic and extrinsic coagulation pathwaysand hinder fibrin formation However further study will berequired to clarify these additional mechanisms

TXA2 and PGI2 metabolites of arachidonic acid (AA)which are mutually antagonistic play an important role inthrombosis TXB2 and 6-Keto-PGF1120572 are the stable metabo-lites of TXA2 and PGI2 respectively [21] The dynamicequilibrium of TXB26-Keto-PGF1120572 is a vital factor for theregulation and control of vessel wall intensity and regionalflow [21 22]

Our study results suggested that Ret Rbu Ali andPen could significantly downregulate serum TXB2 level andupregulate serum 6-Keto-PGF1120572 level in blood stasis ratwhich indicated that the antithrombotic effects of themwere associated with the regulation of TXA2 and PGI2 Reacould significantly downregulate serum TXB2 level whichindicated that the antithrombotic effect of Rea was associatedwith the regulation of TXA2 Pro and Pen could significantlyupregulate serum 6-Keto-PGF1120572 level indicating that theantithrombotic action of Pro and Pen was associated with theregulation of PGI2

Endothelial dysfunction plays a pivotal role in throm-bosis [23 24] ET and NO a pair of mutually antagonisticbiological activity factor play an important role in regulatingcardiovascular system function and participate in a variety ofpathological processes of cardiovascular diseaseThe damageof vascular endothelial cells increases the release of ET andleads to vasoconstriction [25] NO synthase can reflect thechanges in endogenous NO a vasodilatory factor secretedby endothelial cells ENOS a constitutively present enzymein distinct subcellular locations within cardiomyocytes ismainly responsible for the endothelium-dependent vasodi-latation distributed in vascular endothelium And the activa-tion of eNOS promotes the continued release of NO [26 27]A research [28] reported that RPR could relaxe vascular

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 3

Con Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben Pro Con Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben Pro

Con Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben ProCon Brv Ret Rpe Rea Rbu Pae Ali Pen Oxy Ben Pro

0

10

20

30

40A

PTT

(s)

0

10

20

30

40

50

TT (s

)

PT (s

)FI

B (g

L)

0

2

4

6

8

10

0

2

4

6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 1 Anticoagulation activity of RPR extract and six compounds in vitro (119899 = 4 lowastlowastlowast119901 lt 0001 or 0001 lt lowastlowast119901 lt 001 or lowast119901 lt 005 versuscontrol group 119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus breviscapine)

3 Results

31 Effects on Plasma Coagulation Parameters In Vitro Asshown in Figure 1 compared with the control group ethanolextract of RPR (Ret) petroleum extract of RPR (Rpe) ethylacetate extract of RPR (Rea) n-butyl alcohol extract of RPR(Rbu) Pae Ali Pen Oxy and Pro could significantly prolongAPTT (001 lt 119901 lt 005 0001 lt 119901 lt 001 119901 lt 0001respectively) Above all there was no difference between Rbuand breviscapine (Brv) (119901 gt 005) In PT assay comparedwith the control group all of the extracts and six compoundscould significantly prolong PT (001 lt 119901 lt 005 0001 lt119901 lt 001 119901 lt 0001 respectively) Then the prolongingPT effects of Rpe Rea and Rbu were better than that of Brv(0001 lt 119901 lt 001 119901 lt 0001 resp) and Ret Pae Pen OxyBen and Pro had no difference with the Brv (119901 gt 005) InTT assay compared with the control group the Ret Rpe ReaRbu Pae Ali Pen and Pro could significantly prolong TT(119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005 resp) For FIBassay the Rpe Pae Ali Pen Oxy and Ben could significantlydecrease the content of FIB (119901 lt 0001) while the Ret ReaRbu and Pro could significantly increase the content of FIB(119901 lt 0001)

32 The Antithrombotic Effect of RPR Extract In Vivo

321 Effects on WBV PV ESR and PCV In Vivo The effectson WBV and PV were shown in Table 1 In model groupWBV and PV significantly increased at all shear rates in theblood stasis (119901 lt 0001) which indicated the blood stasisrat model was successfully establishedThe Ret Rea and Rbugroups could significantly decrease WBV and PV at all shear

rates (119901 lt 0001) and they decreased WBV and PV effects aswell as aspirin group (119901 gt 005)

The results of ESR and PCV were shown in Table 2ESR and PCV in the model group were higher than that ofcontrol group (119901 lt 0001) which indicated that the bloodstasis model was successfully established The Ret Rea andRbu groups could significantly decrease ESR and PCV (119901 lt0001) and they decreased ESR effects as well as aspirin group(119901 gt 005) The Rea and Rbu decreased PCV effects as well asaspirin group (119901 gt 005)

322 Effects on Plasma Coagulation Parameters To evaluatethe effect of the coagulation cascade on the antithromboticactivity of RPR extract the classical coagulation assays(APTT TT PT and FIB content) were determined in vivoCoagulation parameters were shown in Figure 2 APTTTT and PT were decreased and FIB content was increasedsignificantly in model group compared with control group(119901 lt 0001) RPR extracts could prolong PT APTT and TTand decrease FIB content significantly (119901 lt 0001)

323 TXB2 and 6-Keto-PGF1120572 Assay in Serum Results ofTXB2 and 6-Keto-PGF1120572 were shown in Table 3 and theplasma TXB2 level in model rats was prominently higherthan that of control group (119901 lt 0001) whereas the plasma6-Keto-PGF1120572 level in model rats was markedly lower thanthat of control group (119901 lt 005) Ret Rea and Rbu couldsignificantly counteract increase of TXB2 level (119901 lt 0001)and the effects of Ret and Rea were close to aspirin (119901 gt005) Ret and Rbu could significantly upregulate 6-Keto-PGF1120572 level in plasma compared with model rats (119901 lt 0001

4 BioMed Research International

Table 1 Effect on WBV (mPasdots) at various shear rates

Group WBV (mPasdots) PV (mPasdots)200S 30S 3S 30s

Con 434 plusmn 020 545 plusmn 033 988 plusmn 061 123 plusmn 003

Mod 544 plusmn 017lowastlowastlowast 680 plusmn 014lowastlowastlowast 1266 plusmn 025lowastlowastlowast 142 plusmn 003lowastlowastlowast

Asp 467 plusmn 047 575 plusmn 019 998 plusmn 036 127 plusmn 003

Ret 468 plusmn 017 604 plusmn 045 1039 plusmn 068 129 plusmn 001

Rea 467 plusmn 046 580 plusmn 056 1017 plusmn 041 129 plusmn 005

Rbu 464 plusmn 043 560 plusmn 061 1038 plusmn 054 126 plusmn 004

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 and 0001 lt 119901 lt 001 versus model group

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

Con Mod RetAsp Rea RbuCon Mod RetAsp Rea Rbu

0

10

20

30

40

50

APT

T (s

)

0

10

20

0 0

2

4

6

20

40

60

30

40

TT (s

)

PT (s

)FI

B (g

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779998779

998779998779

Figure 2 Anticoagulation activity of RPR extract in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group119901 lt 0001 versus aspirin 119901 lt 005 versus model group)

Table 2 Effect on ESR and PCV

Group Dose (gkg) ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 008 3715 plusmn 246

Mod mdash 1160 plusmn 114lowastlowastlowast 4683 plusmn 071lowastlowastlowast

Asp 01 740 plusmn 055 3892 plusmn 104

Ret 12 679 plusmn 084 4171 plusmn 130

Rea 12 780 plusmn 084 3973 plusmn 233

Rbu 12 680 plusmn 084 3973 plusmn 182

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 versus model group and 119901 lt 005 versus aspirin

119901 lt 005) and there were no obvious differences with aspirin(119901 gt 005) Meanwhile Ret Rea and Rbu decreased theratio of TXB26-Keto-PGF1120572 compared with model group(119901 lt 0001) The ratios of TXB26-Keto-PGF1120572 in Ret waslower than that of aspirin (119901 lt 0001) and that of Rea wasclose to those of aspirin (119901 gt 005)

324 Effect of RPR on ET and eNOS In Figure 3 the plasmaET-1 level in blood stasis model rats (6590 plusmn 424 120583gL)was prominently higher than that of control group (4675 plusmn254 120583gL) (119901 lt 0001) whereas the plasma eNOS level in

BioMed Research International 5

Table 3 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose (gkg) 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 9179 plusmn 738 8336 plusmn 675 0911 plusmn 008

Mod mdash 8126 plusmn 205lowast 10878 plusmn 379lowastlowastlowast 134 plusmn 006lowastlowastlowast

Asp 01 9058 plusmn 456 8190 plusmn 438 0910 plusmn 007

Ret 12 9627 plusmn 813 8352 plusmn 374 0874 plusmn 010

Rea 12 8681 plusmn 610 8442 plusmn 796 0976 plusmn 011

Rbu 12 9124 plusmn 536 9924 plusmn 693 109 plusmn 009

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 or lowast119901 lt 005 versus control group 119901 lt 0001 or 001 lt 119901 lt 0001 or 119901 lt 005 versus model group119901 lt 0001 versus aspirin

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

ET-1

(120583g

L)

eNO

S (U

L)

0

20

40

60

80

100

0

5

10

15

20

25

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779

998779

Figure 3The effects of RPR treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus aspirin 119901 lt 005)

model rats (1896 plusmn 127UL) was markedly lower than thatof control group (2261 plusmn 073UL) (119901 lt 0001) Ret and Reacould effectively downregulate of ET-1 level and upregulateeNOS level in plasma compared with model rats (119901 lt 0001119901 lt 005 resp) and Rea downregulation of ET-1 level effectwas better than that of aspirin (119901 lt 0001) and the effect ofRet downregulation of ET-1 level had no obvious differencecompared to aspirin group (119901 gt 005) Ret Rea and Rbucould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 119901 lt 005 resp) and the effects of Rbuupregulation of eNOS level were better than that of aspirin(119901 lt 005) the effects of Rea upregulation of eNOS level hadno obvious difference compared to aspirin (119901 gt 005)

33 The Antithrombotic Effect of Active Compounds In Vivo

331 Effects onWBV PV ESR and PCV Theeffects onWBVand PV were shown in Table 4 In model group WBV andPV significantly increased at all shear rates in the blood stasis(119901 lt 0001) which indicated the blood stasis rat modelwas successfully established Pro Ali Pae and Pen couldsignificantly decrease WBV and PV at all shear rates (119901 lt0001 0001 lt 119901 lt 001 119901 lt 005) and they decreased WBVeffects as well as xiangdan injection (Xdi) (119901 gt 005) at lowshear rate

The results of ESR and PCV for each group are shown inTable 5 ESR and PCV in the model group were higher than

that of control group (119901 lt 0001) Pro Ali Pae and Pen couldsignificantly decrease ESR and PCV (119901 lt 0001 0001 lt 119901 lt001 119901 lt 005) Effects on ESR and PCV of Ali were equal toXdi (119901 gt 005) Effect on ESR of Pro was equal to Xdi (119901 gt005) The effect on PCV of Pae was equal to Xdi (119901 gt 005)

332 Effects on PlasmaCoagulation Parameters Coagulationparameters were shown in Figure 4 APTT TT and PT weredecreased and FIB content was increased very significantlyin model group compared with control group (119901 lt 0001)Pro Ali Pae and Pen could prolong PT APTT and TT anddecrease FIB content effectively (119901 lt 0001 119901 lt 005)

333 TXB2 and 6-Keto- PGF1120572 in Serum In Table 6 theplasma TXB2 level in blood stasis model was prominentlyhigher than that of control group (119901 lt 0001) whereas theplasma 6-Keto-PGF1120572 level in model group was markedlylower than that of control group (119901 lt 0001)

Ali and Pen could significantly counteract increase ofTXB2 level (119901 lt 0001) and they were close to those of Xdigroup (119901 gt 005) Pro Ali Pae and Pen could effectivelyupregulate 6-Keto-PGF1120572 level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and Pae andPen had no obvious difference compared to Xdi (119901 gt 005)Meanwhile Pro Ali Pae and Pen could decrease the ratioof TXB26-Keto-PGF1120572 compared with model group (119901 lt0001) Pro and Ali ratios of TXB26-Keto-PGF1120572 in the rat

6 BioMed Research International

Table 4 Effect on WBV (mPasdots) at various shear rates

Group Dose WBV (mPasdots) PV (mPasdots)200S 30S 3S 30S

Con mdash 396 plusmn 011 526 plusmn 009 1006 plusmn 021 127 plusmn 004

Mod mdash 529 plusmn 018lowastlowastlowast 660 plusmn 023lowastlowastlowast 1255 plusmn 043lowastlowastlowast 141 plusmn 003lowastlowastlowast

Xdi 36mLKg 414 plusmn 004 528 plusmn 019 1049 plusmn 086 127 plusmn 001

Pro 50mgKg 477 plusmn 009 582 plusmn 011 1110 plusmn 018 136 plusmn 003

Ali 50mgKg 437 plusmn 017 539 plusmn 026 1027 plusmn 047 133 plusmn 003

Pae 50mgKg 446 plusmn 011 558 plusmn 012 1062 plusmn 023 135 plusmn 003

Pen 50mgKg 463 plusmn 005 569 plusmn 099 1070 plusmn 039 134 plusmn 002

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 0001 lt 119901 lt 001 and 119901 lt 005 versus model group 119901 lt 00010001 lt 119901 lt 001 and 119901 lt 005 versus Xdi

0

20

40

60

APT

T (s

)

0

10

20

30

PT (s

)

0

20

40

60

TT (s

)

0

2

4

6

FIB

(gL

)

Con Mod Xdi Pro Ali Pae PenCon Mod Xdi Pro Ali Pae Pen

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779

998779

998779

998779998779

998779998779998779

Figure 4 Anticoagulation activity of active compounds in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or 119901 lt 005 versusmodel group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt 119901 lt 001)

were lower compared to the Xdi (119901 lt 0001) and those of thePae and Pen were close to those of Xdi (119901 gt 005)

334 Effect of Compounds on ET and eNOS As shown inFigure 5 the plasma ET-1 level in blood stasis model rats(9482 plusmn 419 120583gL) was prominently higher than that ofcontrol group (8708 plusmn 334 120583gL) (119901 lt 0001) whereasthe plasma eNOS level in model rats (2283 plusmn 145UL) wasmarkedly lower than that of control group (2677 plusmn 098UL)(119901 lt 0001) Ali and Pae could effectively downregulate ET-1 level and upregulate eNOS level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and the Pae group

downregulation of ET-1 level effect had no obvious differencecompared to Xdi group (119901 gt 005) Pro Ali Pae and Pencould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005) and Ali andPen upregulation of eNOS level effect was better than aspiringroup (119901 lt 0001 119901 lt 005)

4 Discussion

APTT PT TT and FIB are four indicators related toanticoagulant in clinical practice In our study Rpe PaeAli and Pen could significantly extend TT APTT and TT

BioMed Research International 7

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

ET-1

(ng

L)

eNO

S (U

L)

0

50

100

150

0

10

20

30

40

lowastlowast

lowastlowastlowast

998779998779998779

998779

998779

998779

Figure 5 The effects of active compounds treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 or 0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt lowastlowast119901 lt 001 versus control group)

Table 5 Effect on ESR and PCV

Group Dose ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 082 3604 plusmn 123

Mod mdash 1050 plusmn 058lowastlowastlowast 4580 plusmn 130lowastlowastlowast

Xdi 36mLKg 725 plusmn 050 3711 plusmn 173

Pro 5mgKg 850 plusmn 058 4073 plusmn 116

Ali 5mgKg 85 plusmn 129 3756 plusmn 237

Pae 5mgKg 875 plusmn 17 3868 plusmn 148

Pen 5mgKg 890 plusmn 084 3955 plusmn 148

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group and119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus model group and0001 lt 119901 lt 001 or 119901 lt 005 versus Xdi

and reduced the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways and hinder fibrin formation Ret Rea Rbu andProcould significantly extend TT APTT and PT but they couldnot reduce the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways Then the Oxy and Ben had no anticoagulationeffect in vitro

Hemorheology is related to blood flow and pressureflow volume and resistance of blood vessels includingWBV PV ESR and PCV Hemorheology is commonly usedfor diagnosis and prevention of cardiovascular diseases inclinical practice [12ndash19] Red blood cells (RBCs) plateletsand plasmahave an influence onWBV becauseRBCs accountfor almost 50 of blood volume and constitute the majorityof the cellular content in blood In our research Ret Rea RbuPro Ali Pae and Pen groups could significantly decreaseWBV and PCV which suggested that the amelioration effectof compounds on WBV might be partly due to the PCVdecrease

PV plays an important role in WBV It showed thecoefficient between PV and ESR was positive in significantlevel [20] In our research Ret Rea Rbu Pro Ali Pae andPen groups could significantly decrease PV and ESR which

suggested that the amelioration effect of compounds on PVmight be partly due to the ESR decrease

Ret Rea Rbu Pro Ali Pae and Pen groups couldsignificantly extend TT APTT and PT and reduced thecontent of FIB in vivo which indicated the beneficial effectof them on intrinsic and extrinsic coagulation pathwaysand hinder fibrin formation However further study will berequired to clarify these additional mechanisms

TXA2 and PGI2 metabolites of arachidonic acid (AA)which are mutually antagonistic play an important role inthrombosis TXB2 and 6-Keto-PGF1120572 are the stable metabo-lites of TXA2 and PGI2 respectively [21] The dynamicequilibrium of TXB26-Keto-PGF1120572 is a vital factor for theregulation and control of vessel wall intensity and regionalflow [21 22]

Our study results suggested that Ret Rbu Ali andPen could significantly downregulate serum TXB2 level andupregulate serum 6-Keto-PGF1120572 level in blood stasis ratwhich indicated that the antithrombotic effects of themwere associated with the regulation of TXA2 and PGI2 Reacould significantly downregulate serum TXB2 level whichindicated that the antithrombotic effect of Rea was associatedwith the regulation of TXA2 Pro and Pen could significantlyupregulate serum 6-Keto-PGF1120572 level indicating that theantithrombotic action of Pro and Pen was associated with theregulation of PGI2

Endothelial dysfunction plays a pivotal role in throm-bosis [23 24] ET and NO a pair of mutually antagonisticbiological activity factor play an important role in regulatingcardiovascular system function and participate in a variety ofpathological processes of cardiovascular diseaseThe damageof vascular endothelial cells increases the release of ET andleads to vasoconstriction [25] NO synthase can reflect thechanges in endogenous NO a vasodilatory factor secretedby endothelial cells ENOS a constitutively present enzymein distinct subcellular locations within cardiomyocytes ismainly responsible for the endothelium-dependent vasodi-latation distributed in vascular endothelium And the activa-tion of eNOS promotes the continued release of NO [26 27]A research [28] reported that RPR could relaxe vascular

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

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ToxinsJournal of

VaccinesJournal of

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AntibioticsInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

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Pharmaceutics

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MEDIATORSINFLAMMATION

of

4 BioMed Research International

Table 1 Effect on WBV (mPasdots) at various shear rates

Group WBV (mPasdots) PV (mPasdots)200S 30S 3S 30s

Con 434 plusmn 020 545 plusmn 033 988 plusmn 061 123 plusmn 003

Mod 544 plusmn 017lowastlowastlowast 680 plusmn 014lowastlowastlowast 1266 plusmn 025lowastlowastlowast 142 plusmn 003lowastlowastlowast

Asp 467 plusmn 047 575 plusmn 019 998 plusmn 036 127 plusmn 003

Ret 468 plusmn 017 604 plusmn 045 1039 plusmn 068 129 plusmn 001

Rea 467 plusmn 046 580 plusmn 056 1017 plusmn 041 129 plusmn 005

Rbu 464 plusmn 043 560 plusmn 061 1038 plusmn 054 126 plusmn 004

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 and 0001 lt 119901 lt 001 versus model group

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

Con Mod RetAsp Rea RbuCon Mod RetAsp Rea Rbu

0

10

20

30

40

50

APT

T (s

)

0

10

20

0 0

2

4

6

20

40

60

30

40

TT (s

)

PT (s

)FI

B (g

L)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779

998779998779998779998779

998779998779

Figure 2 Anticoagulation activity of RPR extract in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group119901 lt 0001 versus aspirin 119901 lt 005 versus model group)

Table 2 Effect on ESR and PCV

Group Dose (gkg) ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 008 3715 plusmn 246

Mod mdash 1160 plusmn 114lowastlowastlowast 4683 plusmn 071lowastlowastlowast

Asp 01 740 plusmn 055 3892 plusmn 104

Ret 12 679 plusmn 084 4171 plusmn 130

Rea 12 780 plusmn 084 3973 plusmn 233

Rbu 12 680 plusmn 084 3973 plusmn 182

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 versus model group and 119901 lt 005 versus aspirin

119901 lt 005) and there were no obvious differences with aspirin(119901 gt 005) Meanwhile Ret Rea and Rbu decreased theratio of TXB26-Keto-PGF1120572 compared with model group(119901 lt 0001) The ratios of TXB26-Keto-PGF1120572 in Ret waslower than that of aspirin (119901 lt 0001) and that of Rea wasclose to those of aspirin (119901 gt 005)

324 Effect of RPR on ET and eNOS In Figure 3 the plasmaET-1 level in blood stasis model rats (6590 plusmn 424 120583gL)was prominently higher than that of control group (4675 plusmn254 120583gL) (119901 lt 0001) whereas the plasma eNOS level in

BioMed Research International 5

Table 3 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose (gkg) 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 9179 plusmn 738 8336 plusmn 675 0911 plusmn 008

Mod mdash 8126 plusmn 205lowast 10878 plusmn 379lowastlowastlowast 134 plusmn 006lowastlowastlowast

Asp 01 9058 plusmn 456 8190 plusmn 438 0910 plusmn 007

Ret 12 9627 plusmn 813 8352 plusmn 374 0874 plusmn 010

Rea 12 8681 plusmn 610 8442 plusmn 796 0976 plusmn 011

Rbu 12 9124 plusmn 536 9924 plusmn 693 109 plusmn 009

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 or lowast119901 lt 005 versus control group 119901 lt 0001 or 001 lt 119901 lt 0001 or 119901 lt 005 versus model group119901 lt 0001 versus aspirin

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

ET-1

(120583g

L)

eNO

S (U

L)

0

20

40

60

80

100

0

5

10

15

20

25

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779

998779

Figure 3The effects of RPR treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus aspirin 119901 lt 005)

model rats (1896 plusmn 127UL) was markedly lower than thatof control group (2261 plusmn 073UL) (119901 lt 0001) Ret and Reacould effectively downregulate of ET-1 level and upregulateeNOS level in plasma compared with model rats (119901 lt 0001119901 lt 005 resp) and Rea downregulation of ET-1 level effectwas better than that of aspirin (119901 lt 0001) and the effect ofRet downregulation of ET-1 level had no obvious differencecompared to aspirin group (119901 gt 005) Ret Rea and Rbucould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 119901 lt 005 resp) and the effects of Rbuupregulation of eNOS level were better than that of aspirin(119901 lt 005) the effects of Rea upregulation of eNOS level hadno obvious difference compared to aspirin (119901 gt 005)

33 The Antithrombotic Effect of Active Compounds In Vivo

331 Effects onWBV PV ESR and PCV Theeffects onWBVand PV were shown in Table 4 In model group WBV andPV significantly increased at all shear rates in the blood stasis(119901 lt 0001) which indicated the blood stasis rat modelwas successfully established Pro Ali Pae and Pen couldsignificantly decrease WBV and PV at all shear rates (119901 lt0001 0001 lt 119901 lt 001 119901 lt 005) and they decreased WBVeffects as well as xiangdan injection (Xdi) (119901 gt 005) at lowshear rate

The results of ESR and PCV for each group are shown inTable 5 ESR and PCV in the model group were higher than

that of control group (119901 lt 0001) Pro Ali Pae and Pen couldsignificantly decrease ESR and PCV (119901 lt 0001 0001 lt 119901 lt001 119901 lt 005) Effects on ESR and PCV of Ali were equal toXdi (119901 gt 005) Effect on ESR of Pro was equal to Xdi (119901 gt005) The effect on PCV of Pae was equal to Xdi (119901 gt 005)

332 Effects on PlasmaCoagulation Parameters Coagulationparameters were shown in Figure 4 APTT TT and PT weredecreased and FIB content was increased very significantlyin model group compared with control group (119901 lt 0001)Pro Ali Pae and Pen could prolong PT APTT and TT anddecrease FIB content effectively (119901 lt 0001 119901 lt 005)

333 TXB2 and 6-Keto- PGF1120572 in Serum In Table 6 theplasma TXB2 level in blood stasis model was prominentlyhigher than that of control group (119901 lt 0001) whereas theplasma 6-Keto-PGF1120572 level in model group was markedlylower than that of control group (119901 lt 0001)

Ali and Pen could significantly counteract increase ofTXB2 level (119901 lt 0001) and they were close to those of Xdigroup (119901 gt 005) Pro Ali Pae and Pen could effectivelyupregulate 6-Keto-PGF1120572 level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and Pae andPen had no obvious difference compared to Xdi (119901 gt 005)Meanwhile Pro Ali Pae and Pen could decrease the ratioof TXB26-Keto-PGF1120572 compared with model group (119901 lt0001) Pro and Ali ratios of TXB26-Keto-PGF1120572 in the rat

6 BioMed Research International

Table 4 Effect on WBV (mPasdots) at various shear rates

Group Dose WBV (mPasdots) PV (mPasdots)200S 30S 3S 30S

Con mdash 396 plusmn 011 526 plusmn 009 1006 plusmn 021 127 plusmn 004

Mod mdash 529 plusmn 018lowastlowastlowast 660 plusmn 023lowastlowastlowast 1255 plusmn 043lowastlowastlowast 141 plusmn 003lowastlowastlowast

Xdi 36mLKg 414 plusmn 004 528 plusmn 019 1049 plusmn 086 127 plusmn 001

Pro 50mgKg 477 plusmn 009 582 plusmn 011 1110 plusmn 018 136 plusmn 003

Ali 50mgKg 437 plusmn 017 539 plusmn 026 1027 plusmn 047 133 plusmn 003

Pae 50mgKg 446 plusmn 011 558 plusmn 012 1062 plusmn 023 135 plusmn 003

Pen 50mgKg 463 plusmn 005 569 plusmn 099 1070 plusmn 039 134 plusmn 002

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 0001 lt 119901 lt 001 and 119901 lt 005 versus model group 119901 lt 00010001 lt 119901 lt 001 and 119901 lt 005 versus Xdi

0

20

40

60

APT

T (s

)

0

10

20

30

PT (s

)

0

20

40

60

TT (s

)

0

2

4

6

FIB

(gL

)

Con Mod Xdi Pro Ali Pae PenCon Mod Xdi Pro Ali Pae Pen

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779

998779

998779

998779998779

998779998779998779

Figure 4 Anticoagulation activity of active compounds in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or 119901 lt 005 versusmodel group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt 119901 lt 001)

were lower compared to the Xdi (119901 lt 0001) and those of thePae and Pen were close to those of Xdi (119901 gt 005)

334 Effect of Compounds on ET and eNOS As shown inFigure 5 the plasma ET-1 level in blood stasis model rats(9482 plusmn 419 120583gL) was prominently higher than that ofcontrol group (8708 plusmn 334 120583gL) (119901 lt 0001) whereasthe plasma eNOS level in model rats (2283 plusmn 145UL) wasmarkedly lower than that of control group (2677 plusmn 098UL)(119901 lt 0001) Ali and Pae could effectively downregulate ET-1 level and upregulate eNOS level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and the Pae group

downregulation of ET-1 level effect had no obvious differencecompared to Xdi group (119901 gt 005) Pro Ali Pae and Pencould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005) and Ali andPen upregulation of eNOS level effect was better than aspiringroup (119901 lt 0001 119901 lt 005)

4 Discussion

APTT PT TT and FIB are four indicators related toanticoagulant in clinical practice In our study Rpe PaeAli and Pen could significantly extend TT APTT and TT

BioMed Research International 7

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

ET-1

(ng

L)

eNO

S (U

L)

0

50

100

150

0

10

20

30

40

lowastlowast

lowastlowastlowast

998779998779998779

998779

998779

998779

Figure 5 The effects of active compounds treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 or 0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt lowastlowast119901 lt 001 versus control group)

Table 5 Effect on ESR and PCV

Group Dose ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 082 3604 plusmn 123

Mod mdash 1050 plusmn 058lowastlowastlowast 4580 plusmn 130lowastlowastlowast

Xdi 36mLKg 725 plusmn 050 3711 plusmn 173

Pro 5mgKg 850 plusmn 058 4073 plusmn 116

Ali 5mgKg 85 plusmn 129 3756 plusmn 237

Pae 5mgKg 875 plusmn 17 3868 plusmn 148

Pen 5mgKg 890 plusmn 084 3955 plusmn 148

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group and119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus model group and0001 lt 119901 lt 001 or 119901 lt 005 versus Xdi

and reduced the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways and hinder fibrin formation Ret Rea Rbu andProcould significantly extend TT APTT and PT but they couldnot reduce the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways Then the Oxy and Ben had no anticoagulationeffect in vitro

Hemorheology is related to blood flow and pressureflow volume and resistance of blood vessels includingWBV PV ESR and PCV Hemorheology is commonly usedfor diagnosis and prevention of cardiovascular diseases inclinical practice [12ndash19] Red blood cells (RBCs) plateletsand plasmahave an influence onWBV becauseRBCs accountfor almost 50 of blood volume and constitute the majorityof the cellular content in blood In our research Ret Rea RbuPro Ali Pae and Pen groups could significantly decreaseWBV and PCV which suggested that the amelioration effectof compounds on WBV might be partly due to the PCVdecrease

PV plays an important role in WBV It showed thecoefficient between PV and ESR was positive in significantlevel [20] In our research Ret Rea Rbu Pro Ali Pae andPen groups could significantly decrease PV and ESR which

suggested that the amelioration effect of compounds on PVmight be partly due to the ESR decrease

Ret Rea Rbu Pro Ali Pae and Pen groups couldsignificantly extend TT APTT and PT and reduced thecontent of FIB in vivo which indicated the beneficial effectof them on intrinsic and extrinsic coagulation pathwaysand hinder fibrin formation However further study will berequired to clarify these additional mechanisms

TXA2 and PGI2 metabolites of arachidonic acid (AA)which are mutually antagonistic play an important role inthrombosis TXB2 and 6-Keto-PGF1120572 are the stable metabo-lites of TXA2 and PGI2 respectively [21] The dynamicequilibrium of TXB26-Keto-PGF1120572 is a vital factor for theregulation and control of vessel wall intensity and regionalflow [21 22]

Our study results suggested that Ret Rbu Ali andPen could significantly downregulate serum TXB2 level andupregulate serum 6-Keto-PGF1120572 level in blood stasis ratwhich indicated that the antithrombotic effects of themwere associated with the regulation of TXA2 and PGI2 Reacould significantly downregulate serum TXB2 level whichindicated that the antithrombotic effect of Rea was associatedwith the regulation of TXA2 Pro and Pen could significantlyupregulate serum 6-Keto-PGF1120572 level indicating that theantithrombotic action of Pro and Pen was associated with theregulation of PGI2

Endothelial dysfunction plays a pivotal role in throm-bosis [23 24] ET and NO a pair of mutually antagonisticbiological activity factor play an important role in regulatingcardiovascular system function and participate in a variety ofpathological processes of cardiovascular diseaseThe damageof vascular endothelial cells increases the release of ET andleads to vasoconstriction [25] NO synthase can reflect thechanges in endogenous NO a vasodilatory factor secretedby endothelial cells ENOS a constitutively present enzymein distinct subcellular locations within cardiomyocytes ismainly responsible for the endothelium-dependent vasodi-latation distributed in vascular endothelium And the activa-tion of eNOS promotes the continued release of NO [26 27]A research [28] reported that RPR could relaxe vascular

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom

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ToxinsJournal of

VaccinesJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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Drug DeliveryJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

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Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

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Autoimmune Diseases

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Anesthesiology Research and Practice

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Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 5

Table 3 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose (gkg) 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 9179 plusmn 738 8336 plusmn 675 0911 plusmn 008

Mod mdash 8126 plusmn 205lowast 10878 plusmn 379lowastlowastlowast 134 plusmn 006lowastlowastlowast

Asp 01 9058 plusmn 456 8190 plusmn 438 0910 plusmn 007

Ret 12 9627 plusmn 813 8352 plusmn 374 0874 plusmn 010

Rea 12 8681 plusmn 610 8442 plusmn 796 0976 plusmn 011

Rbu 12 9124 plusmn 536 9924 plusmn 693 109 plusmn 009

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 or lowast119901 lt 005 versus control group 119901 lt 0001 or 001 lt 119901 lt 0001 or 119901 lt 005 versus model group119901 lt 0001 versus aspirin

Con Mod RetAsp Rea Rbu Con Mod RetAsp Rea Rbu

ET-1

(120583g

L)

eNO

S (U

L)

0

20

40

60

80

100

0

5

10

15

20

25

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779

998779

Figure 3The effects of RPR treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus aspirin 119901 lt 005)

model rats (1896 plusmn 127UL) was markedly lower than thatof control group (2261 plusmn 073UL) (119901 lt 0001) Ret and Reacould effectively downregulate of ET-1 level and upregulateeNOS level in plasma compared with model rats (119901 lt 0001119901 lt 005 resp) and Rea downregulation of ET-1 level effectwas better than that of aspirin (119901 lt 0001) and the effect ofRet downregulation of ET-1 level had no obvious differencecompared to aspirin group (119901 gt 005) Ret Rea and Rbucould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 119901 lt 005 resp) and the effects of Rbuupregulation of eNOS level were better than that of aspirin(119901 lt 005) the effects of Rea upregulation of eNOS level hadno obvious difference compared to aspirin (119901 gt 005)

33 The Antithrombotic Effect of Active Compounds In Vivo

331 Effects onWBV PV ESR and PCV Theeffects onWBVand PV were shown in Table 4 In model group WBV andPV significantly increased at all shear rates in the blood stasis(119901 lt 0001) which indicated the blood stasis rat modelwas successfully established Pro Ali Pae and Pen couldsignificantly decrease WBV and PV at all shear rates (119901 lt0001 0001 lt 119901 lt 001 119901 lt 005) and they decreased WBVeffects as well as xiangdan injection (Xdi) (119901 gt 005) at lowshear rate

The results of ESR and PCV for each group are shown inTable 5 ESR and PCV in the model group were higher than

that of control group (119901 lt 0001) Pro Ali Pae and Pen couldsignificantly decrease ESR and PCV (119901 lt 0001 0001 lt 119901 lt001 119901 lt 005) Effects on ESR and PCV of Ali were equal toXdi (119901 gt 005) Effect on ESR of Pro was equal to Xdi (119901 gt005) The effect on PCV of Pae was equal to Xdi (119901 gt 005)

332 Effects on PlasmaCoagulation Parameters Coagulationparameters were shown in Figure 4 APTT TT and PT weredecreased and FIB content was increased very significantlyin model group compared with control group (119901 lt 0001)Pro Ali Pae and Pen could prolong PT APTT and TT anddecrease FIB content effectively (119901 lt 0001 119901 lt 005)

333 TXB2 and 6-Keto- PGF1120572 in Serum In Table 6 theplasma TXB2 level in blood stasis model was prominentlyhigher than that of control group (119901 lt 0001) whereas theplasma 6-Keto-PGF1120572 level in model group was markedlylower than that of control group (119901 lt 0001)

Ali and Pen could significantly counteract increase ofTXB2 level (119901 lt 0001) and they were close to those of Xdigroup (119901 gt 005) Pro Ali Pae and Pen could effectivelyupregulate 6-Keto-PGF1120572 level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and Pae andPen had no obvious difference compared to Xdi (119901 gt 005)Meanwhile Pro Ali Pae and Pen could decrease the ratioof TXB26-Keto-PGF1120572 compared with model group (119901 lt0001) Pro and Ali ratios of TXB26-Keto-PGF1120572 in the rat

6 BioMed Research International

Table 4 Effect on WBV (mPasdots) at various shear rates

Group Dose WBV (mPasdots) PV (mPasdots)200S 30S 3S 30S

Con mdash 396 plusmn 011 526 plusmn 009 1006 plusmn 021 127 plusmn 004

Mod mdash 529 plusmn 018lowastlowastlowast 660 plusmn 023lowastlowastlowast 1255 plusmn 043lowastlowastlowast 141 plusmn 003lowastlowastlowast

Xdi 36mLKg 414 plusmn 004 528 plusmn 019 1049 plusmn 086 127 plusmn 001

Pro 50mgKg 477 plusmn 009 582 plusmn 011 1110 plusmn 018 136 plusmn 003

Ali 50mgKg 437 plusmn 017 539 plusmn 026 1027 plusmn 047 133 plusmn 003

Pae 50mgKg 446 plusmn 011 558 plusmn 012 1062 plusmn 023 135 plusmn 003

Pen 50mgKg 463 plusmn 005 569 plusmn 099 1070 plusmn 039 134 plusmn 002

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 0001 lt 119901 lt 001 and 119901 lt 005 versus model group 119901 lt 00010001 lt 119901 lt 001 and 119901 lt 005 versus Xdi

0

20

40

60

APT

T (s

)

0

10

20

30

PT (s

)

0

20

40

60

TT (s

)

0

2

4

6

FIB

(gL

)

Con Mod Xdi Pro Ali Pae PenCon Mod Xdi Pro Ali Pae Pen

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779

998779

998779

998779998779

998779998779998779

Figure 4 Anticoagulation activity of active compounds in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or 119901 lt 005 versusmodel group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt 119901 lt 001)

were lower compared to the Xdi (119901 lt 0001) and those of thePae and Pen were close to those of Xdi (119901 gt 005)

334 Effect of Compounds on ET and eNOS As shown inFigure 5 the plasma ET-1 level in blood stasis model rats(9482 plusmn 419 120583gL) was prominently higher than that ofcontrol group (8708 plusmn 334 120583gL) (119901 lt 0001) whereasthe plasma eNOS level in model rats (2283 plusmn 145UL) wasmarkedly lower than that of control group (2677 plusmn 098UL)(119901 lt 0001) Ali and Pae could effectively downregulate ET-1 level and upregulate eNOS level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and the Pae group

downregulation of ET-1 level effect had no obvious differencecompared to Xdi group (119901 gt 005) Pro Ali Pae and Pencould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005) and Ali andPen upregulation of eNOS level effect was better than aspiringroup (119901 lt 0001 119901 lt 005)

4 Discussion

APTT PT TT and FIB are four indicators related toanticoagulant in clinical practice In our study Rpe PaeAli and Pen could significantly extend TT APTT and TT

BioMed Research International 7

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

ET-1

(ng

L)

eNO

S (U

L)

0

50

100

150

0

10

20

30

40

lowastlowast

lowastlowastlowast

998779998779998779

998779

998779

998779

Figure 5 The effects of active compounds treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 or 0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt lowastlowast119901 lt 001 versus control group)

Table 5 Effect on ESR and PCV

Group Dose ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 082 3604 plusmn 123

Mod mdash 1050 plusmn 058lowastlowastlowast 4580 plusmn 130lowastlowastlowast

Xdi 36mLKg 725 plusmn 050 3711 plusmn 173

Pro 5mgKg 850 plusmn 058 4073 plusmn 116

Ali 5mgKg 85 plusmn 129 3756 plusmn 237

Pae 5mgKg 875 plusmn 17 3868 plusmn 148

Pen 5mgKg 890 plusmn 084 3955 plusmn 148

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group and119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus model group and0001 lt 119901 lt 001 or 119901 lt 005 versus Xdi

and reduced the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways and hinder fibrin formation Ret Rea Rbu andProcould significantly extend TT APTT and PT but they couldnot reduce the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways Then the Oxy and Ben had no anticoagulationeffect in vitro

Hemorheology is related to blood flow and pressureflow volume and resistance of blood vessels includingWBV PV ESR and PCV Hemorheology is commonly usedfor diagnosis and prevention of cardiovascular diseases inclinical practice [12ndash19] Red blood cells (RBCs) plateletsand plasmahave an influence onWBV becauseRBCs accountfor almost 50 of blood volume and constitute the majorityof the cellular content in blood In our research Ret Rea RbuPro Ali Pae and Pen groups could significantly decreaseWBV and PCV which suggested that the amelioration effectof compounds on WBV might be partly due to the PCVdecrease

PV plays an important role in WBV It showed thecoefficient between PV and ESR was positive in significantlevel [20] In our research Ret Rea Rbu Pro Ali Pae andPen groups could significantly decrease PV and ESR which

suggested that the amelioration effect of compounds on PVmight be partly due to the ESR decrease

Ret Rea Rbu Pro Ali Pae and Pen groups couldsignificantly extend TT APTT and PT and reduced thecontent of FIB in vivo which indicated the beneficial effectof them on intrinsic and extrinsic coagulation pathwaysand hinder fibrin formation However further study will berequired to clarify these additional mechanisms

TXA2 and PGI2 metabolites of arachidonic acid (AA)which are mutually antagonistic play an important role inthrombosis TXB2 and 6-Keto-PGF1120572 are the stable metabo-lites of TXA2 and PGI2 respectively [21] The dynamicequilibrium of TXB26-Keto-PGF1120572 is a vital factor for theregulation and control of vessel wall intensity and regionalflow [21 22]

Our study results suggested that Ret Rbu Ali andPen could significantly downregulate serum TXB2 level andupregulate serum 6-Keto-PGF1120572 level in blood stasis ratwhich indicated that the antithrombotic effects of themwere associated with the regulation of TXA2 and PGI2 Reacould significantly downregulate serum TXB2 level whichindicated that the antithrombotic effect of Rea was associatedwith the regulation of TXA2 Pro and Pen could significantlyupregulate serum 6-Keto-PGF1120572 level indicating that theantithrombotic action of Pro and Pen was associated with theregulation of PGI2

Endothelial dysfunction plays a pivotal role in throm-bosis [23 24] ET and NO a pair of mutually antagonisticbiological activity factor play an important role in regulatingcardiovascular system function and participate in a variety ofpathological processes of cardiovascular diseaseThe damageof vascular endothelial cells increases the release of ET andleads to vasoconstriction [25] NO synthase can reflect thechanges in endogenous NO a vasodilatory factor secretedby endothelial cells ENOS a constitutively present enzymein distinct subcellular locations within cardiomyocytes ismainly responsible for the endothelium-dependent vasodi-latation distributed in vascular endothelium And the activa-tion of eNOS promotes the continued release of NO [26 27]A research [28] reported that RPR could relaxe vascular

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

6 BioMed Research International

Table 4 Effect on WBV (mPasdots) at various shear rates

Group Dose WBV (mPasdots) PV (mPasdots)200S 30S 3S 30S

Con mdash 396 plusmn 011 526 plusmn 009 1006 plusmn 021 127 plusmn 004

Mod mdash 529 plusmn 018lowastlowastlowast 660 plusmn 023lowastlowastlowast 1255 plusmn 043lowastlowastlowast 141 plusmn 003lowastlowastlowast

Xdi 36mLKg 414 plusmn 004 528 plusmn 019 1049 plusmn 086 127 plusmn 001

Pro 50mgKg 477 plusmn 009 582 plusmn 011 1110 plusmn 018 136 plusmn 003

Ali 50mgKg 437 plusmn 017 539 plusmn 026 1027 plusmn 047 133 plusmn 003

Pae 50mgKg 446 plusmn 011 558 plusmn 012 1062 plusmn 023 135 plusmn 003

Pen 50mgKg 463 plusmn 005 569 plusmn 099 1070 plusmn 039 134 plusmn 002

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 0001 lt 119901 lt 001 and 119901 lt 005 versus model group 119901 lt 00010001 lt 119901 lt 001 and 119901 lt 005 versus Xdi

0

20

40

60

APT

T (s

)

0

10

20

30

PT (s

)

0

20

40

60

TT (s

)

0

2

4

6

FIB

(gL

)

Con Mod Xdi Pro Ali Pae PenCon Mod Xdi Pro Ali Pae Pen

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

998779998779998779

998779998779998779

998779998779998779

998779

998779

998779

998779998779

998779998779998779

Figure 4 Anticoagulation activity of active compounds in vivo (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 or 119901 lt 005 versusmodel group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt 119901 lt 001)

were lower compared to the Xdi (119901 lt 0001) and those of thePae and Pen were close to those of Xdi (119901 gt 005)

334 Effect of Compounds on ET and eNOS As shown inFigure 5 the plasma ET-1 level in blood stasis model rats(9482 plusmn 419 120583gL) was prominently higher than that ofcontrol group (8708 plusmn 334 120583gL) (119901 lt 0001) whereasthe plasma eNOS level in model rats (2283 plusmn 145UL) wasmarkedly lower than that of control group (2677 plusmn 098UL)(119901 lt 0001) Ali and Pae could effectively downregulate ET-1 level and upregulate eNOS level in plasma compared withmodel rats (119901 lt 0001 0001 lt 119901 lt 001) and the Pae group

downregulation of ET-1 level effect had no obvious differencecompared to Xdi group (119901 gt 005) Pro Ali Pae and Pencould upregulate eNOS level in plasma compared withmodelrats (119901 lt 0001 0001 lt 119901 lt 001 119901 lt 005) and Ali andPen upregulation of eNOS level effect was better than aspiringroup (119901 lt 0001 119901 lt 005)

4 Discussion

APTT PT TT and FIB are four indicators related toanticoagulant in clinical practice In our study Rpe PaeAli and Pen could significantly extend TT APTT and TT

BioMed Research International 7

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

ET-1

(ng

L)

eNO

S (U

L)

0

50

100

150

0

10

20

30

40

lowastlowast

lowastlowastlowast

998779998779998779

998779

998779

998779

Figure 5 The effects of active compounds treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 or 0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt lowastlowast119901 lt 001 versus control group)

Table 5 Effect on ESR and PCV

Group Dose ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 082 3604 plusmn 123

Mod mdash 1050 plusmn 058lowastlowastlowast 4580 plusmn 130lowastlowastlowast

Xdi 36mLKg 725 plusmn 050 3711 plusmn 173

Pro 5mgKg 850 plusmn 058 4073 plusmn 116

Ali 5mgKg 85 plusmn 129 3756 plusmn 237

Pae 5mgKg 875 plusmn 17 3868 plusmn 148

Pen 5mgKg 890 plusmn 084 3955 plusmn 148

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group and119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus model group and0001 lt 119901 lt 001 or 119901 lt 005 versus Xdi

and reduced the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways and hinder fibrin formation Ret Rea Rbu andProcould significantly extend TT APTT and PT but they couldnot reduce the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways Then the Oxy and Ben had no anticoagulationeffect in vitro

Hemorheology is related to blood flow and pressureflow volume and resistance of blood vessels includingWBV PV ESR and PCV Hemorheology is commonly usedfor diagnosis and prevention of cardiovascular diseases inclinical practice [12ndash19] Red blood cells (RBCs) plateletsand plasmahave an influence onWBV becauseRBCs accountfor almost 50 of blood volume and constitute the majorityof the cellular content in blood In our research Ret Rea RbuPro Ali Pae and Pen groups could significantly decreaseWBV and PCV which suggested that the amelioration effectof compounds on WBV might be partly due to the PCVdecrease

PV plays an important role in WBV It showed thecoefficient between PV and ESR was positive in significantlevel [20] In our research Ret Rea Rbu Pro Ali Pae andPen groups could significantly decrease PV and ESR which

suggested that the amelioration effect of compounds on PVmight be partly due to the ESR decrease

Ret Rea Rbu Pro Ali Pae and Pen groups couldsignificantly extend TT APTT and PT and reduced thecontent of FIB in vivo which indicated the beneficial effectof them on intrinsic and extrinsic coagulation pathwaysand hinder fibrin formation However further study will berequired to clarify these additional mechanisms

TXA2 and PGI2 metabolites of arachidonic acid (AA)which are mutually antagonistic play an important role inthrombosis TXB2 and 6-Keto-PGF1120572 are the stable metabo-lites of TXA2 and PGI2 respectively [21] The dynamicequilibrium of TXB26-Keto-PGF1120572 is a vital factor for theregulation and control of vessel wall intensity and regionalflow [21 22]

Our study results suggested that Ret Rbu Ali andPen could significantly downregulate serum TXB2 level andupregulate serum 6-Keto-PGF1120572 level in blood stasis ratwhich indicated that the antithrombotic effects of themwere associated with the regulation of TXA2 and PGI2 Reacould significantly downregulate serum TXB2 level whichindicated that the antithrombotic effect of Rea was associatedwith the regulation of TXA2 Pro and Pen could significantlyupregulate serum 6-Keto-PGF1120572 level indicating that theantithrombotic action of Pro and Pen was associated with theregulation of PGI2

Endothelial dysfunction plays a pivotal role in throm-bosis [23 24] ET and NO a pair of mutually antagonisticbiological activity factor play an important role in regulatingcardiovascular system function and participate in a variety ofpathological processes of cardiovascular diseaseThe damageof vascular endothelial cells increases the release of ET andleads to vasoconstriction [25] NO synthase can reflect thechanges in endogenous NO a vasodilatory factor secretedby endothelial cells ENOS a constitutively present enzymein distinct subcellular locations within cardiomyocytes ismainly responsible for the endothelium-dependent vasodi-latation distributed in vascular endothelium And the activa-tion of eNOS promotes the continued release of NO [26 27]A research [28] reported that RPR could relaxe vascular

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 7

Con Mod Xdi Pro Ali Pae Pen Con Mod Xdi Pro Ali Pae Pen

ET-1

(ng

L)

eNO

S (U

L)

0

50

100

150

0

10

20

30

40

lowastlowast

lowastlowastlowast

998779998779998779

998779

998779

998779

Figure 5 The effects of active compounds treatment on eNOS and ET-1 level in thrombosis rats (119899 = 8 lowastlowastlowast119901 lt 0001 versus control group119901 lt 0001 or 0001 lt 119901 lt 001 versus model group 119901 lt 0001 or 119901 lt 005 versus Xdi 0001 lt lowastlowast119901 lt 001 versus control group)

Table 5 Effect on ESR and PCV

Group Dose ESR (mmsdothminus1) PCV ()Con mdash 300 plusmn 082 3604 plusmn 123

Mod mdash 1050 plusmn 058lowastlowastlowast 4580 plusmn 130lowastlowastlowast

Xdi 36mLKg 725 plusmn 050 3711 plusmn 173

Pro 5mgKg 850 plusmn 058 4073 plusmn 116

Ali 5mgKg 85 plusmn 129 3756 plusmn 237

Pae 5mgKg 875 plusmn 17 3868 plusmn 148

Pen 5mgKg 890 plusmn 084 3955 plusmn 148

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group and119901 lt 0001 or 0001 lt 119901 lt 001 or 119901 lt 005 versus model group and0001 lt 119901 lt 001 or 119901 lt 005 versus Xdi

and reduced the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways and hinder fibrin formation Ret Rea Rbu andProcould significantly extend TT APTT and PT but they couldnot reduce the content of FIB in vitro which indicated thebeneficial effect of themon intrinsic and extrinsic coagulationpathways Then the Oxy and Ben had no anticoagulationeffect in vitro

Hemorheology is related to blood flow and pressureflow volume and resistance of blood vessels includingWBV PV ESR and PCV Hemorheology is commonly usedfor diagnosis and prevention of cardiovascular diseases inclinical practice [12ndash19] Red blood cells (RBCs) plateletsand plasmahave an influence onWBV becauseRBCs accountfor almost 50 of blood volume and constitute the majorityof the cellular content in blood In our research Ret Rea RbuPro Ali Pae and Pen groups could significantly decreaseWBV and PCV which suggested that the amelioration effectof compounds on WBV might be partly due to the PCVdecrease

PV plays an important role in WBV It showed thecoefficient between PV and ESR was positive in significantlevel [20] In our research Ret Rea Rbu Pro Ali Pae andPen groups could significantly decrease PV and ESR which

suggested that the amelioration effect of compounds on PVmight be partly due to the ESR decrease

Ret Rea Rbu Pro Ali Pae and Pen groups couldsignificantly extend TT APTT and PT and reduced thecontent of FIB in vivo which indicated the beneficial effectof them on intrinsic and extrinsic coagulation pathwaysand hinder fibrin formation However further study will berequired to clarify these additional mechanisms

TXA2 and PGI2 metabolites of arachidonic acid (AA)which are mutually antagonistic play an important role inthrombosis TXB2 and 6-Keto-PGF1120572 are the stable metabo-lites of TXA2 and PGI2 respectively [21] The dynamicequilibrium of TXB26-Keto-PGF1120572 is a vital factor for theregulation and control of vessel wall intensity and regionalflow [21 22]

Our study results suggested that Ret Rbu Ali andPen could significantly downregulate serum TXB2 level andupregulate serum 6-Keto-PGF1120572 level in blood stasis ratwhich indicated that the antithrombotic effects of themwere associated with the regulation of TXA2 and PGI2 Reacould significantly downregulate serum TXB2 level whichindicated that the antithrombotic effect of Rea was associatedwith the regulation of TXA2 Pro and Pen could significantlyupregulate serum 6-Keto-PGF1120572 level indicating that theantithrombotic action of Pro and Pen was associated with theregulation of PGI2

Endothelial dysfunction plays a pivotal role in throm-bosis [23 24] ET and NO a pair of mutually antagonisticbiological activity factor play an important role in regulatingcardiovascular system function and participate in a variety ofpathological processes of cardiovascular diseaseThe damageof vascular endothelial cells increases the release of ET andleads to vasoconstriction [25] NO synthase can reflect thechanges in endogenous NO a vasodilatory factor secretedby endothelial cells ENOS a constitutively present enzymein distinct subcellular locations within cardiomyocytes ismainly responsible for the endothelium-dependent vasodi-latation distributed in vascular endothelium And the activa-tion of eNOS promotes the continued release of NO [26 27]A research [28] reported that RPR could relaxe vascular

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

8 BioMed Research International

Table 6 Effect on TXB2 and 6-Keto-PGF1120572 in serum

Group Dose 6-Keto-PGF1120572 (ngL) TXB2 TXB26-Keto-PGF1120572Con mdash 1358 plusmn 011 10074 plusmn 258 1358 plusmn 011

Mod mdash 1872 plusmn 010lowastlowastlowast 10963 plusmn 100lowastlowastlowast 1872 plusmn 010lowastlowastlowast

Xdi 36mLKg 1178 plusmn 015 10024 plusmn 316 1178 plusmn 015

Pro 5mgKg 1479 plusmn 004 10625 plusmn 358 1479 plusmn 004

Ali 5mgKg 1406 plusmn 011 10036 plusmn 105 1406 plusmn 011

Pae 5mgKg 1208 plusmn 008 10642 plusmn 232 1208 plusmn 008

Pen 5mgKg 1198 plusmn 007 10123 plusmn 235 1198 plusmn 007

Data represent mean plusmn SD 119899 = 8 lowastlowastlowast119901 lt 0001 versus control group 119901 lt 0001 versus model group 119901 lt 0001 or 001 lt 119901 lt 0001 versus Xdi

smooth muscle via endothelium-dependent and Akt- andSOCE-eNOS-cGMP-mediated pathways through activationof both KCa and KATP channels and inhibition of L-typeCa2+ channels In our research Rbu Pro and Pen couldsignificantly increase eNOS level Then Ret Rea Ali and Paecould significantly decrease ET level and markedly increaseeNOS level These results indicated that Ret Rea Ali andPae had a protective effect on vascular endothelium andcould regulate the tension of vascular smooth muscle bydownregulating ET level and upregulating eNOS level

In addition Wang et al [29] investigated water extract ofRPR against myocardial ischemia in mice induced by isopro-terenol and found that RPR has the cardioprotective activitieson isoproterenol-induced myocardial ischemia Myocardialischemia is associated withmyocardial infarction and suddendeath hence antithrombotic effects of RPRmay contribute tothe improvement of myocardial ischemia The research [30]reported that Pae has many pharmacological activities suchas protection function of the blood brain barrier cerebralblood flow brain edema after cerebral ischemia antitumoureffect immune regulation and hypoglycemic effect Anotherresearch [31] showed that Pae and Ali could increase thenumbers of white blood cells and reversed the atrophy ofthymus However cardiovascular research on Pen and Prowas little In our study Pro Ali Pae and Pen were fourantithrombotic components of RPR

5 Conclusion

The present study suggested that the RPR extract and thePro Ali Pae and Pen possessed remarkable antithromboticproperty in blood stasis model rats induced by ice waterbath and subcutaneous injection of norepinephrine Thisproperty could be associated with activating blood flowanticoagulation activity the regulation of active substances invascular endothelium and maintaining the balance of TXA2and PGI2 In the end the Pro Ali Pae and Pen may be themain antithrombotic ingredient of RPR

Ethical Approval

The authors declare that the ethical background to this studywas approved by national ethical committee

Competing Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contributions

Pingyao Xie Lili Cui and Wen-yi Kang conceived anddesigned the experiments Lili Cui Pingyao Xie and YuanShan performed the experiments Lili Cui and Pingyao Xiewrote the first draft of the manuscript Wen-yi Kang revisedthe draft and approved the version submitted Pingyao XieLili Cui and Wen-yi Kang contributed equally to this workPingyao Xie and Lili Cui are listed as co-first authors

Acknowledgments

This work was supported by Henan Province UniversityScience and Technology Innovation Team (16IRTSTHN019)Science and Technology Innovation Team of Kaifeng City(2016-124) Kaifeng City Science and Technology InnovationTalent (1509010) and Key Project in Science and TechnologyAgency of Kaifeng (1603111)

References

[1] C N Berry D Girard S Lochot and C Lecoffre ldquoAntithrom-botic actions of argatroban in rat models of venous lsquomixedrsquoand arterial thrombosis and its effects on the tail transectionbleeding timerdquo British Journal of Pharmacology vol 113 no 4pp 1209ndash1214 1994

[2] Q Q Bao L Zhang X Q Li Z L Li and Y M LiuldquoClinical significance of acute infection in elderly patients withthrombotic diseaserdquo Chinese Journal of Gerontology vol 29 no17 pp 2253ndash2255 2009

[3] F R Rosendaal ldquoRisk factors for venous thrombotic diseaserdquoThrombosis and Haemostasis vol 82 no 2 pp 610ndash619 1999

[4] A-P Lu H-W Jia C Xiao and Q-P Lu ldquoTheory of traditionalchinese medicine and therapeutic method of diseasesrdquo WorldJournal of Gastroenterology vol 10 no 13 pp 1854ndash1856 2004

[5] S Xu L Yang R Tian et al ldquoSpecies differentiation and qualityassessment of Radix Paeoniae Rubra (Chi-shao) by means ofhigh-performance liquid chromatographic fingerprintrdquo Journalof Chromatography A vol 1216 no 11 pp 2163ndash2168 2009

[6] J Su P Zhang J-J Zhang X-M Qi Y-G Wu and J-J ShenldquoEffects of total glucosides of paeony on oxidative stress in the

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

BioMed Research International 9

kidney from diabetic ratsrdquo Phytomedicine vol 17 no 3-4 pp254ndash260 2010

[7] P Zhang J-J Zhang J Su X-M Qi Y-G Wu and J-JShen ldquoEffect of total glucosides of paeony on the expressionof nephrin in the kidneys from diabetic ratsrdquo The AmericanJournal of Chinese Medicine vol 37 no 2 pp 295ndash307 2009

[8] H-X Li S-Y Han X-W Wang et al ldquoEffect of the carthaminsyellow from Carthamus tinctorius L on hemorheological disor-ders of blood stasis in ratsrdquo Food and Chemical Toxicology vol47 no 8 pp 1797ndash1802 2009

[9] L Liu J-A Duan Y Tang et al ldquoTaoren-Honghua herb pairand its main components promoting blood circulation throughinfluencing on hemorheology plasma coagulation and plateletaggregationrdquo Journal of Ethnopharmacology vol 139 no 2 pp381ndash387 2012

[10] A Gromotowicz J Szemraj A Stankiewicz et al ldquoStudy ofthe mechanisms of aldosterone prothrombotic effect in ratsrdquoJRAASmdashJournal of the Renin-Angiotensin-Aldosterone Systemvol 12 no 4 pp 430ndash439 2011

[11] H Chen M Jin Y-F Wang et al ldquoEffect of Toona microcarpaharms leaf extract on the coagulation systemrdquo BioMed ResearchInternational vol 2014 Article ID 615363 7 pages 2014

[12] T Gomi T Ikeda and F Ikegami ldquoBeneficial effect of 120572-blockeron hemorheology in patients with essential hypertensionrdquoBiorheology vol 10 no 8 pp 886ndash892 1997

[13] R Koppensteiner E Minar and H Ehringer ldquoEffect oflovastatin on hemorheology in type II hyperlipoproteinemiardquoAtherosclerosis vol 83 no 1 pp 53ndash58 1990

[14] C-L Hsieh Y-C Lin G-C Yen and H-Y Chen ldquoPreventiveeffects of guava (Psidium guajava L) leaves and its activecompounds against 120572-dicarbonyl compounds-induced bloodcoagulationrdquo Food Chemistry vol 103 no 2 pp 528ndash535 2007

[15] O Elwan S Al-Ashmawy S El-Karaksy and A A H HassanldquoHemorheology stroke and the elderlyrdquo Journal of the Neurolog-ical Sciences vol 101 no 2 pp 157ndash162 1991

[16] J Dormandy E Ernst A Matrai and P T Flute ldquoHemorrheo-logic changes following acute myocardial infarctionrdquo AmericanHeart Journal vol 104 no 6 pp 1364ndash1367 1982

[17] P Gorog C D Ridler G M Rees and I B Kovacs ldquoEvidenceagainst hypercoagulability in coronary artery diseaserdquo Throm-bosis Research vol 79 no 4 pp 377ndash385 1995

[18] J R Wang ldquoHemorrheology often relationship betweenmeasuring index and application valuerdquo Chinese Journal ofHemorheology vol 15 no 2 pp 304ndash305 2005

[19] A R Folsom K K Wu W D Rosamond A R Sharrett andL E Chambless ldquoProspective study of hemostatic factors andincidence of coronary heart disease the Atherosclerosis Riskin Communities (ARIC) Studyrdquo Circulation vol 96 no 4 pp1102ndash1108 1997

[20] Y G Zhao Y Xu and L Wang ldquoResearch related to Blood sed-imentation and plasma viscosityrdquo Chinese Journal of Hemorhe-ology vol 7 no 3 pp 19ndash20 1997

[21] F Homo-Delarche J F Bach and M Dardenne ldquoIn vitroinhibition of prostaglandin production by azathioprine and 6-merchaptopurinerdquo Prostaglandins vol 35 no 4 pp 479ndash4911988

[22] N Furuhashi M Tsujiei H Kimura A Yajima H Nagae andC Kimura ldquoMaternal and fetal atrial natriuretic peptide levelsmaternal plasma renin activity angiotensin II prostacyclin andthromboxane A2 levels in normal and preeclamptic pregnan-ciesrdquo Tohoku Journal of Experimental Medicine vol 165 no 2pp 79ndash86 1991

[23] H Cai and D G Harrison ldquoEndothelial dysfunction in car-diovascular diseases the role of oxidant stressrdquo CirculationResearch vol 87 no 10 pp 840ndash844 2000

[24] D S Celermajer K E Sorensen V M Gooch et al ldquoNon-invasive detection of endothelial dysfunction in children andadults at risk of atherosclerosisrdquoThe Lancet vol 340 no 8828pp 1111ndash1115 1992

[25] W G Haynes and D J Webb ldquoContribution of endogenousgeneration of endothelin-1 to basal vascular tonerdquo The Lancetvol 344 no 8926 pp 852ndash854 1994

[26] U Forstermann and T Munzel ldquoEndothelial nitric oxide syn-thase in vascular disease from marvel to menacerdquo Circulationvol 113 no 13 pp 1708ndash1714 2006

[27] P L Huang Z Huang H Mashimo et al ldquoHypertension inmice lacking the gene for endothelial nitric oxide synthaserdquoNature vol 377 no 6546 pp 239ndash242 1995

[28] S N Jin J F Wen T T Wang D G Kang H S Lee and K WCho ldquoVasodilatory effects of ethanol extract of Radix PaeoniaeRubra and its mechanism of action in the rat aortardquo Journal ofEthnopharmacology vol 142 no 1 pp 188ndash193 2012

[29] Z BWang H Yang and C Y Guo ldquoProtection of water extractfrom paeoniae radix rubra against myocardial ischemia in miceinduced by isoproterenolrdquo Chinese Herbal Medicines vol 5 no4 pp 256ndash259 2013

[30] S C Zheng X Y Li Y B Ou and R Sun ldquoResearch develop-ment on pharmacological of paeoniflorinrdquo Chinese Journal ofPharmacovigilance vol 9 no 2 pp 100ndash103 2012

[31] Y Zhu L Wang Z Yang et al ldquoHematopoietic effects ofpaeoniflorin and albiflorin on radiotherapy-inducedmyelosup-pression micerdquo Evidence-Based Complementary and AlternativeMedicine vol 2016 Article ID 5789381 8 pages 2016

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Submit your manuscripts athttpswwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in Pharmacological Sciences

Tropical MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AddictionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Autoimmune Diseases

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Pharmaceutics

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of