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Journal of Ethnopharmacology 116 (2008) 313–317

Selection of component drug in activating blood flow and removing bloodstasis of Chinese herbal medicinal formula for dairy cow mastitis

by hemorheological method

Yu Lu, Yuan-Liang Hu ∗, Xiang-Feng Kong, De-Yun WangInstitute of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine,

Nanjing Agricultural University, Nanjing 210095, PR China

Received 19 August 2007; received in revised form 1 October 2007; accepted 20 November 2007Available online 26 November 2007

bstract

bjective: In order to select the component drug in promoting blood circulation and removing blood stasis of Chinese herbal medicinal formulaor dairy cow mastitis.

ethods: 25 healthy rabbits were allocated randomly into five equal groups. The rabbits in four experimental groups were administered withecoctions of giant knotweed rhizome (GKR, rhizoma polygoni cuspidati), safflower (SF, flos carthami), red sage root (RSR, radix salviaeiltiorrhizae) and chuanxiong rhizome (CXR, rhizoma Chuanxiong) by gastrogavage, respectively, in control group, physiological saline, once aay for seven successive days. After the last administration, all rabbits were intravenously injected with 10% macromolecular dextran to inducelood stasis. The blood samples of all rabbits were collected before the first administration, at 2 h after the last administration and 1 h after injection

f dextran, respectively for determination of hemorheologic parameters by MVIS-2035 hemorheology auto-analyzing system.esults: The results showed that all of four kinds of herbs presented different degree of activating blood flow and removing blood stasis.onclusion: Red sage root was the best especially in resisting blood stasis induced by dextran, and would be selected as main component drug of

he prescription for dairy cow mastitis.2007 Elsevier Ireland Ltd. All rights reserved.

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eywords: Hemorheology; Chinese herbal medicinal formula for dairy cow ma

. Introduction

Mastitis is one of the most severe diseases in dairy cowespite the progress made in improving general udder healthn recent years. Epidemiological studies have revealed that the

ain pathogens are bacteria such as Staphylococcus aureus,taphylococcus epidermidis, Escherichia coli, Streptococcusysgalactiae and Streptococcus agalactiae and so on. Unfor-

Abbreviations: TCM, traditional Chinese medicine; GKR, giant knotweedhizome; SF, safflower; RSR, red sage root; CXR, chuanxiong rhizome; MW,olecular weight; ESR, erythrocyte sedimentation rate; PCV, packed cell vol-

me; WBV, whole blood viscosity; PV, plasma viscosity; HGB, hemoglobin;BC, red blood cell count; lWRV, whole blood reduced viscosity at low shear

ate; hWRV, whole blood reduced viscosity at high shear rate; EAI, erythrocyteccumulation index; K, K value for ESR equation; TRE, time for erythrocytelectrophoresis; ERC, erythrocyte rigid coefficient.∗ Corresponding author. Tel.: +86 25 84395203; fax: +86 25 84398669.

E-mail address: ylhu@njau.edu.cn (Y.-L. Hu).

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378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2007.11.031

; Component drug in promoting blood circulation and removing blood stasis

unately, treatment with antimicrobials was not successfulith bacteriological cure rates varying between 0% and 80%

Melchior et al., 2006).According to traditional Chinese medicinal theory (TCM the-

ry), the most cases of mastitis are caused by ‘blood stasis’ sohat suffered udder presents red, swelling, hot and painful andhe milk becomes brownish or yellowish purulent with flakes orlots. Therefore, the treatment principle of mastitis is not onlylearing away heat and toxic material, but also activating bloodow and removing blood stasis to alleviate tumefaction and painZhou et al., 2003). Many Chinese herbal formulas have beensed successfully to prevent and treat mastitis in China (Wangnd Zhang, 2004). The drugs with the action of promoting bloodirculation and removing blood stasis are necessary component

n these formulas.

Giant knotweed rhizome (GKR, rhizoma polygoni cuspidati),afflower (SF, flos carthami), red sage root (RSR, radix salviaeiltiorrhizae) and chuanxiong rhizome (CXR, rhizoma Chuanx-

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14 Y. Lu et al. / Journal of Ethnop

ong) were widely used to cure “blood stasis”. Resveratrol andolydatin purified from GKR (Xue, 2000), Safflor yellow fromF (Shi and Liu, 2006), tanshinone and tanshinol from RSRChen and Zhu, 2005) and Chuanxiongzine from CXR are effec-ive components and widely used for treating various kindsf chronic diseases, such as cardiovascular system diseases inhina, Japan, Korea, and Singapore, etc. (Liang et al., 2005).he four kinds of herbs also appeared in many prescriptions forairy cow mastitis (Wang and Zhang, 2004).

To prepare the new formula for the treatment of cow masti-is, based on our previous investigations (Lu et al., 2005), weave selected Huangbo (cortex phellodendri) and RSR among4 kinds of herbs using bacteriostasis test in vitro on the majorathogenic bacteria causing cow mastitis. In order to select theain drug in promoting blood circulation and removing blood

tasis of the formula for dairy cow mastitis, this paper comparedhe effect of the four kinds of herbs on hemorheology, taking theigh molecular weight dextran-induced blood stasis in rabbitss model.

. Preparation of plant materials

.1. Preparation of drug solution reagents

GKR, SF, RSR and CXR were purchased from Dahua tra-itional Chinese medicine company, Nanjing, Jiangsu provincend authenticated by the Institute of Traditional Chinese Vet-rinary Medicine of Nanjing Agricultural University accordingo Veterinary Pharmacopoeia of the People’s Republic ofhina (Veterinary Pharmacopoeia commission of the People’sepublic of China, 2005). Five hundred grams of each drug wasxtracted with 4.0 L water decocting twice, once a hour, andondensed into 1.0 g of crude drug per milliliter. The extrac-ive rates (w/w) of GKR, SF, RSR and CXR were 18.95%,3.26%, 40.27% and 24.91%, respectively. The decoctions wereterilized by pasteurization and stored at 4 ◦C for the test.

.2. Reagents and instruments

High molecular weight dextran (MW 500,000) was pur-hased from Amersham Biosciences (Sweden) and dilutedo 10% with physiologic saline before injection. MVIS-2035emorheology auto-analyzing system was made in Chongqingianhai Medical Equipment Co. Ltd., China; Automaticrythrocyte sedimentation rate analyzer was made in Electaab SRI, Italy.

.3. Animals and treatment

Fifty conventional New Zealand rabbits (2.20 ± 0.30 kg)ere purchased from Qinglongshan Experimental Animal Farm,anjing, Jiangsu province (No. SCXK (Su) 2002-0027). Theyere maintained in an air-conditioned room with light from

7:00 h to 19:00 h. The room temperature (25 ± 1 ◦C) andumidity were controlled automatically. They were fed waternd food ad libitum. After 1 week of acclimation, 50 rabbitsere randomly divided into five equal groups. The rabbits in

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acology 116 (2008) 313–317

our experimental groups were administered with decoctions ofKR, SF, RSR and CXR by gastrogavage, respectively, in con-

rol group, physiological saline, 1.0 mL/kg, once a day for sevenuccessive days. After the last administration, all rabbits werenjected with 10% macromolecular dextran via ear marginalein, 10 mL/kg and finished within 3 min to induce blood sta-is (Yun et al., 2001; Kong et al., 2006a; Kong et al., 2006b).he blood samples of all rabbits were collected before the firstdministration, at 2 h after the last administration and 1 h afternjection of dextran, respectively for determination of hemorhe-logic parameters. The experiments followed the guidelines ofhe regional Animal Ethics Committee.

.4. Determination of hemorheological parameters

The 5.0 mL of fresh blood sample each rabbit was put intolastic test tube containing heparin (0.2 mg/mL) as an anticoag-lant, of which 1.2 mL blood was used for test of erythrocyteedimentation rate (ESR) by automatic erythrocyte sedimenta-ion rate analyzer, 1.0 mL blood for test of packed cell volumePCV) by Wintrobe test and the rest for test of whole blood vis-osity (WBV) at the shear rates of 200 s−1, 100 s−1, 30 s−1 ands−1, plasma viscosity (PV) and other hemorheological param-ters by MVIS-2035 hemorheology auto-analyzing system.

.5. Statistical analysis

Data are expressed as the mean ± S.D. Statistical analysisf data were performed with the SPSS package (Version 11.5,PSS Inc., US) and was performed using a one-way analysisf variance (ANOVA) followed by Duncan’s post hoc test toompare the experimental groups and control group. A value of< 0.05 was selected as indicating statistical significance.

. Results

.1. Effects of four drugs on normal hemorheologicalarameter

After the last administration, as compared with the beforedministraion, the whole blood viscosities at low shear ratelWRV) and erythrocyte rigid coefficient (ERC) in GKR, SFnd RSR groups were markedly smaller (Table 3, p < 0.05);GB contents in GKR and SF groups were significantly larger

Table 2, p < 0.05); the rest hemorheological parameters had noignificant change among experimental groups and the controlroup (Tables 1–3, p > 0.05).

.2. Effects of four drugs on stasis-inducedemorheological parameter

After injection of dextran, all hemorheological parameteralues in control group were significantly larger than those of

efore injection except HGB and RBC (Tables 1–3, p < 0.05).he most hemorheological parameter values in four experimen-

al groups had increase in different degree incomparison withefore injection.

Y. Lu et al. / Journal of Ethnopharmacology 116 (2008) 313–317 315

Table 1Changes of whole blood viscosity and plasma viscosity (mPa s)

Group Whole blood viscosity PV

200 s−1 100 s−1 30 s−1 3 s−1 100 s−1

Before administration 4.64 ± 0.25 c 5.01 ± 0.27 e 5.73 ± 0.32 c 7.42 ± 0.45 c 1.41 ± 0.03 c

After administrationGKR 4.70 ± 0.43 c 5.08 ± 0.47 e 5.81 ± 0.54 c 7.53 ± 0.69 c 1.43 ± 0.03 cSF 4.56 ± 0.41 c 4.93 ± 0.45 e 5.64 ± 0.52 c 7.30 ± 0.69 c 1.42 ± 0.02 cRSR 4.44 ± 0.33 c 4.80 ± 0.36 e 5.48 ± 0.43 c 7.07 ± 0.57 c 1.40 ± 0.02 cCXR 4.62 ± 0.18 c 4.99 ± 0.20 e 5.70 ± 0.23 c 7.36 ± 0.31 c 1.40 ± 0.04 cControl 4.51 ± 0.25 c 4.87 ± 0.28 e 5.56 ± 0.34 c 7.87 ± 0.48 c 1.44 ± 0.05 c

After stasis-inducedGKR 6.66 ± 0.09 a 7.23 ± 0.12 ab 8.34 ± 0.17 a 10.96 ± 0.30 ab 1.95 ± 0.01 aSF 6.36 ± 1.01 a 6.90 ± 1.14 bc 7.95 ± 1.38 ab 10.42 ± 2.00 ab 1.88 ± 0.13 bRSR 5.73 ± 0.83 b 6.20 ± 0.91 cd 7.09 ± 1.08 b 9.18 ± 1.46 b 1.87 ± 0.15 bCXR 6.00 ± 0.86 a 6.48 ± 0.94 d 7.42 ± 1.09 ab 9.60 ± 1.47 b 1.94 ± 0.06 aControl 6.85 ± 0.74 a 7.44 ± 0.81 a 8.57 ± 0.97 a 11.25 ± 1.35 a 1.88 ± 0.20 b

a–e: Column data marked without the same superscripts differ significantly (p < 0.05).

Table 2Changes of hemorheological parameters correlative to red blood cell

Group PCV HGB (g/L) RBC(1012/L) ESR (mm/h) TRE (S)

Before administration 0.40 ± 0.03 ab 125.24 ± 8.08 b 4.51 ± 0.29 ab 1.40 ± 0.55 c 14.93 ± 0.81 e

After administrationGKR 0.43 ± 0.03 a 131.75 ± 10.28 a 4.74 ± 0.37 a 1.50 ± 0.58 c 15.27 ± 1.40 eSF 0.42 ± 0.03 ab 130.20 ± 8.77 a 4.69 ± 0.32 a 1.75 ± 0.50 c 14.83 ± 1.33 eRSR 0.42 ± 0.04 ab 128.65 ± 12.53 ab 4.63 ± 0.45 a 1.00 ± 0.00 c 14.44 ± 1.08 eCXR 0.40 ± 0.04 ab 122.45 ± 12.01 ab 4.41 ± 0.43 ab 1.50 ± 0.58 c 15.01 ± 0.57 eControl 0.40 ± 0.06 ab 124.00 ± 17.35 ab 4.47 ± 0.63 ab 2.00 ± 0.00 c 14.67 ± 0.83 e

After stasis-inducedGKR 0.33 ± 0.04 cd 102.30 ± 11.18 ab 3.68 ± 0.40 d 85.33 ± 7.02 b 21.64 ± 0.29 abSF 0.29 ± 0.02 d 90.93 ± 7.16 ab 3.27 ± 0.26 e 94.67 ± 16.26 a 20.67 ± 3.28 bcRSR 0.31 ± 0.03 cd 97.13 ± 7.80 ab 3.50 ± 0.28 de 95.33 ± 10.79 a 18.63 ± 2.71 dCXR 0.34 ± 0.06 bcd 106.43 ± 17.63 ab 3.83 ± 0.64 cd 93.67 ± 14.57 a 19.49 ± 2.79 cdControl 0.37 ± 0.04 bc 114.70 ± 13.15 ab 4.13 ± 0.47 bc 84.00 ± 19.80 b 22.26 ± 2.39 a

a–e: Column data marked without the same superscripts differ significantly (p < 0.05).

Table 3Changes of other hemorheological parameters (mPa s)

Group hWRV lWRV ERC EAI K

Before administration 5.65 ± 0.23 de 10.51 ± 0.39 f 0.94 ± 0.02 e 5.25 ± 0.39 cd 4.56 ± 2.00 c

After administrationGKR 5.35 ± 0.26 e 9.99 ± 0.35 g 0.89 ± 0.03 f 5.25 ± 0.42 cd 5.62 ± 2.82 cSF 5.32 ± 0.38 e 9.94 ± 0.60 g 0.89 ± 0.04 f 5.18 ± 0.50 cd 6.23 ± 2.23 cRSR 5.26 ± 0.07 e 9.81 ± 0.15 g 0.89 ± 0.05 f 5.07 ± 0.36 d 3.45 ± 0.66 cCXR 5.83 ± 0.45 de 10.81 ± 0.84 f 0.97 ± 0.07 e 5.25 ± 0.31 cd 4.48 ± 1.10 cControl 5.26 ± 0.71 e 10.44 ± 0.87 f 0.90 ± 0.08 ef 4.97 ± 0.40 d 6.50 ± 1.55 c

After stasis-inducedGKR 7.38 ± 0.78 b 14.13 ± 1.62 b 1.19 ± 0.13 b 5.63 ± 0.19 b 194.67 ± 17.66 bSF 8.07 ± 1.01 a 15.34 ± 1.86 a 1.31 ± 0.09 a 5.51 ± 0.75 bc 180.65 ± 15.05 bRSR 6.54 ± 0.10 c 12.38 ± 0.20 d 1.15 ± 0.04 c 4.88 ± 0.37 d 200.37 ± 6.63 bCXR 6.14 ± 0.91 cd 11.60 ± 1.70 e 1.07 ± 0.15 d 4.95 ± 0.62 d 232.07 ± 71.39 aControl 7.20 ± 0.86 b 13.57 ± 1.82 c 1.10 ± 0.13 c 5.98 ± 0.10 a 238.49 ± 100.90 a

a–f: Column data marked without the same superscripts differ significantly (p < 0.05).

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However, compared with control group, the values of RBC,AI and K in GKR group; WBV of 100 s−1, PCV, RBC, TRE,AI and K in SF group; WBV of 100 s−1 and 3 s−1, RBC, TRE,WRV, lWRV, EAI and K in RSR group; WBV of 100 s−1 ands−1, TRE, hWRV, lWRV, ERC and EAI, are markedly smaller

Tables 1–3, p < 0.05). Furthermore, the data of whole bloodiscosity at all shear rates, plasma viscosity, time for erythro-yte electrophoresis and erythrocyte accumulation index in RSRroup are the smallest among four experiment groups.

. Discussion and conclusion

Hemorheology is the study of the relation of blood flow toressure, flow volume, and resistance of blood vessel includinglood viscosity, erythrocyte deformability, erythrocyte aggre-ability and blood platelet aggregation, which reflects theemocircular status of the whole body and the developing pro-ess of diseases. Therefore, hemorheology is commonly used foriagnosis and prevention of disease and evaluation of the clinicalherapeutic effect of drugs (Qin, 1999). Whole blood viscosity,rythrocyte sedimentation rate, whole blood reduced viscos-ty and other indexes selected in this experiment are the basicemorheological parameters and can comprehensively evaluatehe therapeutic efficacy of drugs in promoting blood circula-ion and removing blood stasis. In the present study, changesf the parameters between before and after administration weres follows: in the RSR and SF groups, whole blood reducediscosity at low shear rate and erythrocyte rigid coefficient offter administration were significantly smaller than those ofefore. It is generally known that reduction of blood viscos-ty could decrease the risk of the formation of blood clots (Hout al., 2007) and reduction of erythrocyte rigid coefficient couldncrease the erythrocyte deformability. So RSR and SF had thectivity of activating blood flow on normal hemorheology; com-aring with before administration, in the GKR group only wholelood reduced viscosity at low shear rate after administrationas markedly decreased, and in the CXR group whole bloodiscosity, HGB, RBC and K of after administration were quietlyecreased. So GKR and CXP had the lower activity of activatinglood flow on normal hemorheology.

After blood stasis was induced by high molecular weightextran, compared with control group, whole blood viscosity atll rates, whole blood reduced viscosity at low and high shearates, erythrocyte accumulation index, PCV and RBC in RSRroup significantly decreased, while ESR markedly increased,hich demonstrated that RSR possessed strong antithrombotic

ctivity. Hou et al. (2007) also proved that RSR could improvebnormal hemorheological parameters in aging guinea pigs byecreasing blood viscosity and viscoelasticity, reducing ery-hrocyte membrane malondialdehyde levels, and increasing thexygen transport efficiency of whole blood. 100 s−1 of WBV,CV, RBC, TRE, EAI and K in SF group; 100 s−1 and 3 s−1

f WBV, TRE, hWRV, lWRV, ERC and EAI in CXR group;

nd RBC and K in GKR group significantly decreased, whichhowed that SF, CXP and GKP possessed different degree ofntithrombotic activity, consistent with those of many studiesLiao, 2002; Xiao et al., 1996; Yan et al., 2003). Jared Clarke

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acology 116 (2008) 313–317

t al. (2005) reported that safflower oil (main component of SF)ould increase blood flow in the aftermath of trauma, reducelood viscosity and decrease several factors of the intrinsiclotting pathway. To make a comparison of four experimentroups, it could be seen that WBV at 200 s−1, TRE, hWRV,ERV, EAI and ERC in RSR group were significantly lowerhan those in SF group, WBV at 200 s−1, PV and K, thanXR group, WBV, TRE, hWRV, lERV, EAI and ERC, thanKR group, which indicated that RSR had the best anti-stasis

ffect.Blood stasis is a secondary pathogenic factor of mastitis.

n the one hand, it causes slowness of blood flow, dilation oflood vessel, increase of vascular permeability and inflamma-ory medium in blood. Macrophages are activated and releaseytokines which allures the aggregation of more leukocytes andymphocytes to exacerbate inflammatory response and tissuenjury. The exudates containing coagulation factors coagulateduickly in the udder to induce dysgalactia. On the other hand,t causes swelling of mammary gland and decrease of lacticxcretion. Bacteria and their toxic product cannot be eliminatedollowing milk in time and destroy mammary gland cell. Thewelling of mammary gland also inhibit milk secretion as a reflexGruet et al., 2001; Max et al., 2002; Zhang, 1993). Therefore,t is most important for treatment of mastitis to remove bloodtasis, thus ameliorating pathway of blood and milk, eliminat-ng pathogen and lessening inflammatory symptom. This muste realized by activating blood flow of whole body or local.

It was reported that high molecular weight dextran couldnduce blood stasis by promoting platelet aggregation andormation of microthrombi and increasing fibrin degradationroducts (Yun et al., 2001). This experiment results showed thathe hemorheologic changes in blood stasis model induced byigh molecular weight dextran are similar to those in mastitis.herefore this blood stasis model can be used for the selection ofomponent drug with the action of promoting blood circulationnd removing blood stasis of Chinese herbal medicinal formulaor mastitis.

To sum up, all of four kinds of herbs presented differentegree of activating blood flow and removing blood stasis, inhich RSR had the best efficacy. Furthermore, RSR had bet-

er bacteriostatic effect (Lu et al., 2005), anti-endotoxic actionJennifer Man-Fan Wan et al., 2006). RSR also could inhibitontraction of smooth muscle (Kong et al., 2006a; Kong et al.,006b) and activities of cytokines and inflammatory mediumChen et al., 2001; Yu and Xu, 1994). Therefore red sage rootould be selected as main component drug of Chinese herbaledicinal prescription for dairy cow mastitis.

cknowledgements

We thank Department of Hematology Nanjing General Hos-

ital of PLA for hematological measurements, and all othertaff in the Institute of Traditional Chinese Veterinary Medicinef Nanjing Agricultural University for their assistances in thexperiments.

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