Effects of Cydonia oblonga Miller extracts on blood hemostasis, coagulation and fibrinolysis in mice, and experimental thrombosis in rats

Journal of Ethnopharmacology 154 (2014) 163–169

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Research Paper

Effects of Cydonia oblonga Miller extracts on blood hemostasis, coagulation and fibrinolysis in mice, and experimental thrombosis in rats Wenting Zhou a,1, Adil Abdurahman a,1, Anwar Umar a,b,n, Guldiyar Iskander a, Elzira Abdusalam a, Benedicte Berké b, Bernard Bégaud b, Nicholas Moore a,b,nn a b

Department of Pharmacology, Xinjiang Medical University, 830011 Urumqi, Xinjiang, People's Republic of China Department of Pharmacology, Université Bordeaux Segalen, F-33076 Bordeaux, France

art ic l e i nf o

a b s t r a c t

Article history: Received 4 February 2014 Received in revised form 18 March 2014 Accepted 23 March 2014 Available online 4 April 2014

Introduction: Cydonia oblonga Miller (COM) is traditionally used in Uyghur medicine for the prevention of cardiovascular disease. The present study is designed to explore the effects of COM extracts on models and markers of thrombosis and related biomarkers. Materials and methods: 20, 40, 80 mg/kg/day COM aqueous extracts and 5 mg/kg/day aspirin, orally for 14 days were compared to untreated controls in mice on bleeding and clotting times, using the tail cutting and glass slide methods and for death rates in collagen–epinephrine pulmonary thrombosis, thrombolysis in vitro and euglobulin lysis time (ELT). In rats, common carotid artery FeCl3-induced thrombus and inferior vena cava thrombosis occlusion time, plasma concentrations of thromboxane B2 (TXB2) and 6-keto-prostaglandine F1α (6-keto-PGF1α) were measured. Results and conclusion: Compared to controls, COM extracts dose-dependently prolonged bleeding by 2.17, 2.78 and 3.63 times, vs. aspirin 2.58, and the clotting time by 1.44, 2.47 and 2.48 times, vs. aspirin 1.91. COM reduced pulmonary embolus mortality by 27, 40 and 53%, vs. 47% for aspirin. COM dosedependently increased thrombolysis by 45, 55 and 63%, vs. 56% for aspirin, and shortened ELT to 71, 61 and 43%, vs. 43% for aspirin. In rats, venous occlusion time was prolonged. Arterial and venous thrombus weights were dose-dependently reduced in COM groups. TXB2 decreased and 6-keto-PGF1α increased with COM and aspirin, with an association between 6-keto-PGF1α/TXB2 and arterial or venous thrombus weight for all products, and for occlusion time with COM but not for aspirin. Conclusion: We confirm the experimental effects of COM on hemostasis and thrombosis. Further exploration of putative clinical effects appear justified. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cydonia oblonga Miller Thrombolysis Arterial thrombosis Venous occlusion 6-keto-PGF 1α Thromboxane

1. Introduction Cydonia oblonga Mill. (COM), the common Quince, belonging to the Rosaceae Family, Cydonia Genus (Sadik, 1993), has been used

Abbreviations: COM, Cydonia oblonga Mill.; ELT, euglobulin lysis time; TXB2, thromboxane B2; SPF, specific pathogen-free; RIA, radioimmunoassay; SD, standard deviation n Corresponding author at: Department of Pharmacology, Xinjiang Medical University, 830011 Urumqi, Xinjiang, People's Republic of China. Tel./fax: þ 86 991 43 62 421. nn Corresponding author at: Department of Pharmacology, University of Bordeaux Segalen, 33076 Bordeaux, France. Tel.: þ 33 557571560; fax: þ33 557574671. E-mail addresses: [email protected] (A. Umar), [email protected] (N. Moore). 1 Both are equal contributors to this work. http://dx.doi.org/10.1016/j.jep.2014.03.056 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

in traditional medicine for a long time in Xinjiang. It is called “Biye” in Uygur, or Man Tan, and Quince in English. The chemical composition of COM is rich. Ripe fruits contain fructose, tannin, protopectin, organic acids, amino acids and other ingredients; pulp contain tetradecylic acid, vaccenic acid glyceride, volatile oil, flavonoids, etc.; pericarps contain heptyl ethyl ether and nonyl ethyl ether, etc. Seeds contain mostly mucilage, amygdalin, fatty oil, etc.; leaves contain alkaloids, glycosides, tannins, mucilage, ketose, aldose, lipids, vitamin C, cyanogenetic glycoside, etc. (Nanjing, 1997; Kurban, 2004). COM fruit are used to make jam and jelly in the western world. In China, the use of COM for medicine or food has not been developed. In traditional Uyghur medicine, COM leaves are used as a decoction to reinforce the individual's defenses, and prevent cardiovascular diseases among other virtues (Xakir et al., 2006). Interestingly, it is also commonly used in Turkey (Kultur, 2007) to treat or prevent diabetes and seems to have antidiabetic

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W. Zhou et al. / Journal of Ethnopharmacology 154 (2014) 163–169

and antioxidant effects (Aslan et al., 2010). In previous studies we found an effect of COM on lipid metabolism in hyperlipemic rats (Abliz et al., 2014), and on blood pressure in a renal hypertension model in rats (Zhou et al., 2014). Because of its traditional use in the prevention of cardiovascular disease, and the societal importance of such diseases, and having shown the effects of COM on the other main risk factors for cardiovascular diseases, lipids and hypertension we decided to test the effects of COM on coagulation and thrombosis, the endpoint of coronary disease. This was tested in vitro for coagulation and fibrinolysis in vivo, and on models of thrombosis and related biomarkers in mice and rats (Jiang et al., 2007), using common models (Umar et al., 2003b; Umar et al., 2004; Tohti et al., 2006), compared to the standard antiplatelet agent aspirin.

2. Materials and methods 2.1. Apparatus, materials, animals 2.1.1. Drugs and reagents Leaves of Cydonia oblonga Mill. were collected in October 2010 from the Kashgar area in Kargilik County, Xinjiang, PRC, dried and crushed then set aside. Aqueous extract of COM (Department of Pharmacology, Xinjiang Medical University, China) was obtained by steeping the dried and crushed fresh Cydonia oblonga Mill. leaves in water at 60 1C three times, once for two hours and twice one hour before first drying and then freeze-drying the extract thus obtained. One gram powder was equivalent to about 1.6 g crude leaves. Cydonia oblonga Mill. leaves were identified by Prof. Parida Abliz, and deposited in the herbarium of the Traditional Chinese Medicine Ethnical Herbs Specimen Museum of Xinjiang Medical University under number NO.TCMEHSM2013_100. Other drugs and reagents were Aspirin, collagen (Sigma Co, USA), Adrenaline Hydrochloride Injection (Xinzheng Pharmaceutical Co., Ltd., Tianjin, China), urethane, barbital (Miura Chemical Co., Ltd., Shanghai, China), sodium citrate (Beijing Chemical Plant, Beijing, China), 6-keto-PGF 1α RIA kit, and TXB2 RIA kit (Puerwei biotechnology Co., Ltd., Beijing, China). 2.1.2. Instruments RE-52 Rotary Evaporator (Yarong Biochemical Instrument Factory, Shanghai, China), DK-S24 digital electric thermostat water bath (Jinyun Medical Instrument Factory, Jiangsu, China), SK8210HP ultrasonator (Kedao Instrument Factory, Shanghai, China), BL1500 electronic balance (Sartorius Instrument Co., Ltd., Beijing, China), KH-120M desktop high-speed centrifuge (Anting Science Instrument Factory, Shanghai, China), analytical balance (Mettler – Toledo Instrument Factory, USA), and Automatic RIA analyzer (USTC affiliated Zhongjia Co., Ltd., Anhui, China). 2.1.3. Animals Male ICR mice weighing 18–22 g and male Wistar rats weighing 300–350 g, both specific pathogen free (SPF), were provided by the Experimental Animal Center of the Xinjiang Medical University (certificate number SYXK2003-0001). 2.2. Methods All animals received care in compliance with the Chinese Convention on Animal Care, and the study was approved by the Institutional Ethics Committee (No. IACUC – 20130129016).

2.2.1. Experiments in mice 2.2.1.1. Treatment groups. In each of the following experiments, male ICR mice, weighing 18–22 g were randomly divided into groups of 10–15 animals each, treated as follows: One group of mice received only neutral saline preparation (control group). Three groups received COM extract at the dose of respectively 20, 40 and 80 mg/kg (low, medium, high doses). One group received aspirin 5 mg/kg. All drugs were given orally once daily for 14 days, dissolved in water. After the last administration, mice were anesthetized with urethane.

2.2.1.2. Bleeding time. One hour after the last administration, the tail was cut 5 mm from the end. Timing was started when the blood flowed. The tail end was blotted every 15 s until the end of bleeding (natural stop of blood flow) which was the bleeding time (McKay et al., 1971; Li, 2004; Xu et al., 2005; Furie and Furie, 2008; Stoll et al., 2008). 2.2.1.3. Clotting time in vitro. One hour after last treatment administration, an eye was rapidly removed with ophthalmic elbow tweezers, two drops of blood were dropped at the end of a slide and timing immediately started with a stopwatch. One of the drops of blood was pricked lightly with a pin every 30 s. When a blood streak appeared, timer was stopped to record the whole blood clotting time. The other drop of blood was used for retesting (Li, 1991). 2.2.1.4. Collagen–epinephrine induced pulmonary thrombosis. Thirty minutes after the last drug administration, acute pulmonary thromboembolism was induced by 0.2 ml of collagen and epinephrine 10:1 mg/kg injected into the tail vein of the mice (Umetsu and Sanai, 1978; DiMinno and Silver, 1983; Yu and Hu, 1997; Zhou et al., 2006). The number of the deaths was counted within 15 min. The inhibitory rate was computed using the following formula: Inhibition¼((number of deaths in treated number of deaths in controls)/number of deaths in controls)  100, eventually adjusted for different number of animals per groups. 2.2.1.5. Thrombolysis in vitro. One hour after last treatment administration, an eye was rapidly removed with ophthalmic elbow tweezers, the blood was dropped in a plastic tube with a precisely weighed 3 cm-long No. 4 surgical silk thread, left standing for 10 min, then the blood was coagulated. The silk thread was then pulled out with the thrombus sticking on gently, cut into 2 cm sections and placed in a tube, 2 ml of a solution of normal saline, containing COM 4 g/L, 2 g/L, or 1 g/L, aspirin 0.25 g/L, or Ginkgo biloba 2 g/L, were added and left standing for 24 h at 37 1C, before measuring the thrombolytic rate (Pang et al., 1996). 2.2.1.6. Euglobulin lysis time (ELT). One hour after the last drug administration, 1 ml blood was drawn from the retro-orbital venous plexus. 3.8% sodium citrate was added in the test tube and the ratio of anticoagulant and blood was strictly controlled to 1:9. The blood was centrifuged at 3000 rpm for 10 min, then 0.25 ml plasma was separated, 4.5 ml distilled water and 0.05 ml 1% acetic acid were added in turn, then adjusted to pH ¼9. After it was fully mixed, the mixture was placed in a refrigerator at 4 1C for 10 min. The mixture was centrifuged at 3000 rpm for 10 min and the euglobulin was precipitated. The supernatant was removed and the tube placed upside down on filter paper to absorb

W. Zhou et al. / Journal of Ethnopharmacology 154 (2014) 163–169

remnant liquid (Li, 1991; Xu et al., 2005; Zhou et al., 2006). Then 0.25 ml borate buffer (pH ¼9.0) was added to the test tube, stirred with a fine glass rod carefully for about 1 min to dissolve it and the tube was placed in water bath at 37 1C for 2 min. 0.25 ml 0.025% calcium chloride was added to solidify the euglobulin. The time until the clot was completely dissolved was recorded (ELT). 2.2.2. Experiments in rats 2.2.2.1. Common carotid artery thrombosis. Forty-two male wistar rats, weighing 300–350 g, were randomly divided into groups of seven rats as follows: sham group, model group, COM extracts low 20 mg/kg, medium 40 mg/kg, high 80 mg/kg dose, and aspirin 5 mg/kg. Treatments were given orally once daily for 14 days, dissolved in water. Rats were anesthetized with 3% barbital (0.5 ml/100 g i.p.) 1 h after the last administration. Under sterile conditions, the rats were fixed on anatomical plane in supine position, the hairs on the throat were sheared and the skin was disinfected with iodine and draped. An incision was made of about 3 cm in the midline on the throat (Kurz et al., 1990; Broersma et al., 1991; Imura et al., 1995; Liu and Xu, 1995; Zhang et al., 1998; Dieude et al., 2009; Eckly et al., 2011). The left common carotid artery was isolated for 2 cm in length carefully and a plastic sheet (3 cm  1.5 cm) was placed under the vessel to separate it from the surrounding tissue. The surface of carotid artery was covered with a piece of filter paper (1 cm  1 cm) saturated with 40% FeCl3 solution (normal saline in sham group) (Reyers et al., 1980; Johnstone et al., 2001; Liang et al., 2006; Jiang et al., 2007; Nakata et al., 2008; Zhou et al., 2009). The temperature of the distal arterial surface was monitored by a thermometer. The time from when the filter paper was placed to a sudden drop in the temperature was recorded as thrombosis occlusion time (OT). An injured carotid artery segment (0.6 cm) was then cut off and placed on the filter paper to dry and was then weighed. The rate of thrombosis inhibition was calculated as: thrombosis inhibition (%) ¼(A A1)/A  100%, where A was the wet weight of the thrombus in the model group and A1 was that in agentstreated groups. 2.2.2.2. Inferior vena cava thrombosis. Forty-two male wistar rats, weighing 300–350 g, were also divided into 6 groups of seven rats, as described in Section 2.2.2.1. Under sterile conditions, the rats were fixed on anatomical planes in supine position, the hairs on the abdomen were sheared and the skin was disinfected with iodine and draped. An abdominal incision was made along the medio-ventral line. Inferior vena cava was isolated and ligated with silk thread below the left renal vein branch. The abdominal walls were subsequently closed. 4 h later the abdomen was reopened, the inferior vena cava was clamped about 2 cm below the ligature and other branches were ligated. The inferior cava vein was opened lengthwise, the thrombus was removed and placed on the filter paper to dry, then was weighed (Rhodes et al., 2000; Johnstone et al., 2001). Thrombosis inhibition was calculated with the same equation described in Section 2.2.1. 2.2.2.3. Measurement of the plasma concentration of TXB2 and 6-keto-PGF1α. Forty-two male wistar rats, weighing 300–350 g, were divided into groups and treated as above in the arterial thrombosis model. Under sterile conditions, 90 min after surgery, the abdominal aorta was isolated, and 3 ml blood were collected by abdominal aortic puncture and immediately added with 0.2 ml Indomethacin–EDTA  Na2 in 1 ml syringe and mixed. The blood samples were centrifuged at 3000 r/min and 4 1C for 15 min. The plasma was separated and stored at 20 1C. The plasma concentrations of TXB2 and 6-keto-PGF1α were measured by

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radioimmunoassay (RIA) (Shan et al., 1998; Xu et al., 2005; Liang et al., 2008; Liu et al., 2011). 2.3. Statistical analysis All the results are given as mean values 7standard deviation (mean7SD). Differences between the control and treatment groups were tested using Student's t-test. All analyses were done with the Statistical Package for Social Sciences (SPSS, version 16.0).

3. Results 3.1. Hemostasis parameters in mice 3.1.1. Bleeding time As shown in Table 1, all the doses of water extract of COM significantly prolonged the bleeding time compared with the saline control group (P o0.05). Compared with the aspirin group the high dose of water extract of COM 80 mg/kg significantly prolonged the bleeding time (P o0.05). Bleeding time appeared to increase with dose of COM 3.1.2. Clotting time As shown in Table 1, middle and high dose 40, 80 mg/kg of water extract of COM significantly prolonged the clotting time compared with the saline control group P o0.05. Compared with the aspirin group, high dose of water extract of COM 80 mg/kg significantly prolonged the clotting time P o0.05. There was increased effect of 40 and 80 mg/kg compared to 20 mg/kg, but no difference between 40 and 80 mg/kg, probably indicating maximal effect has been reached. 3.1.3. Collagen–epinephrine induced pulmonary thrombosis As shown in Table 2, the inhibitory rate on pulmonary thrombosis mortality of low, middle and high dose 20, 40, 80 mg/kg of water extract of COM were 26.7%, 46.7% and 53.3% respectively. Compared Table 1 Effect of COM leaf extracts, aspirin on bleeding time and clotting time, (mean 7 SD) after 14 days of oral treatment in mice. Group

n

Dose (mg/kg)

Bleeding time (min)

Clotting time (min)

Normal Aspirin COM-low dose COM-medium dose COM-high dose

8 9 10 10 10

0 5 20 40 80

3.017 0.45 7.78 7 2.45n 6.53 7 2.08n 8.377 2.24n 10.94 7 2.84n#▲

139.8 7 51.0 268.27 32.2n 201.9 7 78.4n 346.2 7 58.1n 347.17 61.7n#▲

n

# ▲

P o 0.05 vs. saline control group. Po 0.05 vs. aspirin group. P o0.05 vs. Ginkgo biloba group.

Table 2 Effects of COM leaf extracts, aspirin on collagen–epinephrine induced pulmonary thrombosis (mean7 SD) after 14 days of oral treatment in mice. Group

N

Dose (mg/kg)

No. Dead

Survival rate (%)

Saline control Aspirin COM-low dose COM-medium dose COM-high dose

15 15 15 15 15

5 20 40 80

15/15 8/15 11/15 9/15 7/15

– 46.7n 26.7 40.0 53.3n

n

P o 0.05 vs. control group.

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with the aspirin group, high dose of water extract of COM 80 mg/kg significantly increased the inhibitory rate (Po0.05). 3.1.4. in vitro thrombolysis As shown in Table 3, compared with the saline control group, all doses of water extract of COM significantly reduced the weight of thrombus in vitro both initially and 24 h later P o0.01, and the thrombolysis rates were 45.4%, 54.8% and 63.3%. Compared with the aspirin group, high dose of water extract of COM 80 mg/kg significantly reduced the weight of thrombus in vitro both initially and 24 h later (P o0.01). 3.1.5. Euglobulin lysis time (ELT) As shown in Table 3, all the doses of water extract of COM significantly and dose-dependently shortened the euglobulin lysis time (ELT) compared with the saline control group (P o0.01). Compared with the aspirin group, low dose of water extract of COM 20 mg/kg had significantly longer ELT (P o0.05).

or venous thrombus weight (Fig. 1), and a slightly more complex relationship between the 6-keto-PGF1α to TXB2 ratio and arterial occlusion time: though the value for the highest dose of COM was in line with control and aspirin values, the values for low and medium doses of COM were clearly above that line (Fig. 2). Table 4 Effect of COM extracts on FeCl3-induced common carotid artery thrombosis in rats (mean7 SD, n¼ 7 per group). Group

N

Dose (mg/kg)

OT (min)

Weight of thrombus (mg)

Inhibitory rate (%)

Model

7 7 7 7 7

– 20 5 20 40

8.71 71.20 15.78 74.07n 19.5 72.52n 27.0 73.85n 33.375.98n

15.157 0.59 7.147 1.23n 6.40 7 1.53n 9.93 7 1.72n 6.96 7 1.62n

– 52.9 57.8 34.4n 54.0n

7

80

Ginkgo Aspirin COM-L COMM COM-H

40.7 75.80n#▲

5.377 1.48n#▲

64.5n#▲

OT: thrombosis Occlusion Time.

3.2. Effect on thrombosis in rats

n

3.2.1. FeCl3-induced common carotid artery thrombosis As shown in Table 4, all doses of COM extracts significantly and dose-dependently prolonged thrombosis occlusion time, reduced the weight of thrombus and increased the inhibitory rate, compared with the model group (P o0.01). Aspirin 5 mg/kg had about the same effect as the medium doses of COM (40 mg/kg) for inhibition of thrombus weight, but less effect on occlusion time. 3.2.2. Inferior vena cava thrombosis As shown in Table 5, COM extracts significantly and dose -dependently reduced the weight of thrombus, increasing inhibitory rate compared with the model group (Po0.01). Again the effects of aspirin were not very different from that of the medium dose of COM.

# ▲

Table 5 Effect of COM extracts on inferior vena cava thrombosis in rats (x7 s, n ¼7). Group

N

Dose (mg/kg)

Weight of thrombus (mg)

Inhibitory rate (%)

Model

7 7 7 7 7 7

– 20 5 20 40 80

19.4 79.5 7.8 72.4n 6.5 71.8n 10.9 74.0n 7.5 72.2n 5.0 72.1n#▲

– 59.9n 66.3n 43.7n 61.4n 74.4n#▲

Ginkgo Aspirin COM-L COM-M COM-H n

#

3.2.3. Plasma concentrations of TXB2 and 6-keto-PGF1α As shown in Table 6, compared with the normal group, the arterial plasma concentration of 6-keto-PGF1α was significantly decreased (P o0.05) and that of TXB2 was significantly increased (P o0.05) in the model group. Compared with this model group, in all the COM extracts groups, the plasma concentration of TXB2 was decreased significantly and dose-dependently (P o0.05) and that of 6-keto-PGF1α was increased significantly but with an inversed dose-dependence (P o0.05). Aspirin clearly inhibited the secretion of both 6-keto-PGF1α and TXB2 as expected from its pharmacological properties. The 6-keto-PGF1α to TXB2 ratio went from 2.5 in normal controls to 0.37 in model animals (indicating strong platelet activation). With aspirin it was 2.19. With increasing doses of COM it went from 0.94 at the lowest dose to 1.75 at 40 mg, and 6.10 at the highest dose (Figs. 1 and 2). There was an asymptotic relationship between this 6-keto-PGF1α to TXB2 ratio and arterial

P o0.01 vs. model group. Po 0.05 vs. aspirin group. P o0.05 vs. Ginkgo group.

▲

P o0.01 vs. model group. Po 0.05 vs. aspirin group. P o0.05 vs. Ginkgo group.

Table 6 Effect of COM extracts on the plasma concentrations of TXB2 and 6-keto-PGF1α in rats (x7 s, n¼7). Group

n

Dose (mg/kg)

6-keto-PGF1α (pg/ml)

TXB2 (pg/ml)

Normal Model

7 7 7 7 7 7 7

– – 20 5 20 40 80

593.96 7 357.89 267.22 7 255.05 498.807 30.98n 170.607 103.77 558.52 7 314.22n 334.777 156.02n 384.42 7 334.22n#

242.207 117.40 717.32 7 299.21 255.25 7 199.68n 78.217 42.47n 411.017 225.02n 191.55 7 154.36n 91.59 7 62.04n▲

Ginkgo Aspirin COM-L COM-M COM-H n

# ▲

P o0.01 vs. model group. Po 0.05 vs. aspirin group. P o0.05 vs. Ginkgo group.

Table 3 Effects of COM leaf extracts or aspirin on thrombolysis and on euglobulin Lysis time (mean 7SD). Group

n

Dose (mg/kg)

Weight of thrombus at 0 h (mg)

Weight of thrombus at 24 h (mg)

Thrombolysis (%)

Euglobulin lysis time (min)

Saline control Aspirin COM-low COM-middle COM-high

10 10 10 10 10

0 5 20 40 80

197.17 49.5 104.37 25.9n 121.7 7 65.3n 104.87 50.2n 104.07 49.2n#▲

169.1 751.5 55.0 712.0n 72.2 754.8n 42.8 742.6n 37.1 722.5n#▲

13.0 7 8.23 46.4 7 9.07n 45.4 7 15.3n 54.8 7 14.1n 63.3 7 13.6n#▲

257.8 7 52.2 112.3 7 57.7n 183.0 7 49.3n# 157.5 7 11.2n 111.7 7 37.9n▲

n

# ▲

Po 0.01 vs. saline control group. Po 0.05 vs. aspirin group. Po 0.05 vs. Ginkgo biloba group.

W. Zhou et al. / Journal of Ethnopharmacology 154 (2014) 163–169

Fig. 1. Relation between the 6-keto-PGF1α to TXB2 ratio and the arterial (AT) or venous (VT) weight (g).

Fig. 2. Relationship between the 6-keto-PGF1α to TXB2 ratio and the arterial occlusion time.

4. Discussion COM leaf decoctions are traditionally used in Uyghur medicine in Xinjiang for cardiovascular prevention. In addition to Xinjiang, COM leaves are also used traditionally in Turkey, which is culturally and ethnically related to Uyghurs, to treat diabetes. It was found to have antihyperglycaemic properties (Aslan et al., 2010) and antioxidant properties more powerful than those of green tea (Costa et al., 2009), perhaps because of its richness in polyphenols (Oliveira et al., 2007), similar to those found in other antithrombotic compounds we have tested (Umar et al., 2003a, 2003b, 2005; Tohti et al., 2006). We show here that there may be some biological legitimacy to this traditional use, based on a biological effect on hemostasis and thrombolysis that is comparable to the effects of aspirin. Drugs most commonly used for cardiovascular prevention act either on the risk factors for atheroma such as blood pressure or dyslipidemia, or on the thrombotic events themselves. The prevention of the actual thrombotic events is mostly done with antiplatelet agents such as aspirin, or with anticoagulants, or with thrombolytic agents when vascular obstruction has already occurred. Therefore the first point to look at in claims of potential cardiovascular prevention would be hemostasis and coagulation and its corollary, thrombolysis (DiMinno and Silver, 1983). The duration of bleeding time is mainly affected by the quantity and function of the platelets (Gu and Men, 1991; Li, 1991; Xu et al., 2005; Hirsh et al., 2008; Weitz et al., 2008). As shown in our experiment, compared with the saline control group, all the doses of water extract of COM significantly prolonged the bleeding time in mice tails (Po0.05), to a degree similar to that of aspirin with the intermediate dose, to an even greater degree with the higher dose.

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Collagen and epinephrine are inducers of platelet aggregation. When both are injected together, even at very low doses as used here, the synergism creates venous thrombi that result in fatal pulmonary embolism (Umetsu and Sanai, 1978; DiMinno and Silver, 1983; Yu and Hu, 1997). Aspirin inhibits mostly collageninduced platelet aggregation, and can at the dose used reduce by half the fatality rate in mice injected with epinephrine and collagen (Table 2). COM also inhibits collagen–epinephrin-induced mortality in a dose-dependent manner, the middle dose having about the same effect as aspirin, the higher dose being slightly more potent, as it was also for prolongation of bleeding time. This antiplatelet action of COM is consistent with an inhibitory effect of COM on thromboxane formation, as we have found elsewhere. The second element in the prevention of thrombosis is inhibition of coagulation, which is induced by platelet activation, or by other factors (Chen, 1990; Wang and Zhou, 2001). The process of thrombosis includes platelet adhesion, aggregation and release, the formation of white thrombus, through endogenous and exogenous coagulation system, activation of factor and the formation of thrombin leading to blood coagulation, the red and white blood cells flowing in the fibrin network ultimately form the clots (Zhang and Shi, 2000). Both endogenous and exogenous coagulation pathway participates in blood coagulation. By interfering with the activity of the extrinsic coagulation factors, in vitro experiments explore how the drug can inhibit the formation of fibrin. Clotting time is the time from when the blood is isolated from the body to coagulation, related to the extrinsic pathway. The duration of the process is related to the content and functions of various coagulation factors (Li, 1991; Xu et al., 2005). In our experiment, the results showed that low, middle and high dose (20, 40, or 80 mg/kg) COM prolonged the clotting time compared with the saline control group (Po 0.05). The middle and high doses had about the same effect, which was greater than that of aspirin. That the plateau in the dose response curve was obtained from the middle dose, which was not the case for the antiplatelet effects described above, suggests that the effect on coagulation may be mediated through a different mechanisms from that of the antiplatelet effect. The final process in thrombosis is that of fibrinolysis, which restores vascular lumen patency. The process of fibrinolysis can be divided into two stages (Li, 1991; Xu et al., 2005; Zhou et al., 2006). In the first stage plasminogen is activated into plasmin by its activator; in the second stage fibrinogen is cleaved by plasmin into fibrin degradation products (FDP), then the fragments are dissolved. In our thrombolytic experiment in vitro, we found a dosedependent effect of COM on thrombolysis, the effect of the lower dose being similar to that of aspirin. Again this suggests that COM may also have a thrombolytic activity that is superior to that of aspirin (Jin et al., 1996; Zhang, 1997). Fibrinolysis is mostly related to the activation of plasminogen. Euglobulin lysis time removes the effects of natural antiplasmin and explores the effects of plasmin and plasminogen activation (Xu et al., 2005). Again we found that COM dose-dependently shortened euglobulin lysis time, indicating plasmin activation: in this model the effect of aspirin was about the same as that of the highest dose of COM. The real impact of the effects of these COM extracts on hemostasis was then confirmed in experimental thrombosis, both venous and arterial, using standard experimental models in rats. Injury of blood vessel endothelium by FeCl3 can induce plate let adhesion and aggregation, which leads to thrombosis (Du et al., 2007). Ligation of vein causes focal blood stasis, injury of vascular endothelial cell and hypoxia (Eckly et al., 2011). In both cases, blood coagulation factors and thrombin are activated locally, before activation of the intrinsic coagulation system. Endothelial cell degeneration and necrosis then leads to exposure

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of sub-endothelial collagen, which activates the extrinsic coagulation system. The activated intrinsic and extrinsic coagulation systems activate thrombin and the blood coagulation factors. Due to injury of vascular endothelial cell, synthesis of PGI2 is reduced and TXA2 in plasma is increased (Bult et al., 1988), further promoting platelet adhesion and aggregation and imbalance of TXA2/PGI2, which leads to vasoconstriction, platelet aggregation, and thrombosis (Fuchs et al., 2010), as we found in the model group. Compared with this model group, COM extracts dose-dependently prolonged OT, reduced the weight of arterial and venous thrombosis. They decreased the plasma concentrations of TXB2 and increased that of 6-keto-PGF1α, thereby increasing the 6keto-PGF1α to TXB2 ratio. The COM extracts' anti-thrombotic effect was probably mediated by the effect on the prostacyclin/thromboxane balance, acting on both sides of the ratio, resulting in a ratio that was dependently related to thrombus weight (Fig. 1). Arterial occlusion time (Fig. 2) was linearly related to the ratio for control, aspirin and COM-H (r2 ¼0.999) but COM-M and COM-L appear not to lie on the same line. There may be some additional effect in COM that prolongs occlusion time beyond what would be expected from the effect on cyclo-oxygenase, or more generally on the synthesis of thromboxane and PGI2, especially for the lower COM doses. Whether this is related to a differential effect on platelets rather than to actual thrombosis is uncertain (Baxi et al., 2008; Kaptanoglu et al., 2008).

5. Conclusion In conclusion, the traditional use of COM leaves in the prevention of vascular disease in Xinjiang may be supported by experimentally demonstrable anti-thrombotic activity, probably at least in part related to an antithromboxane effect. Clinical exploration of this effect might be justified, to confirm these effects in man, test other putative effects on diabetes and lipid metabolism that might also impact on cardiovascular risk, and verify the absence of the gastro-intestinal harm that is associated with aspirin.

Role of the funding source The funding source had no input on the study protocol, methodology or analysis, in the interpretation of results or in the content of the present paper or the decision to submit the manuscript.

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