Anti-Thrombotic Effect of Carthamus tinctorius Linn Extracts in Rats

Purpose: To explore the effects of Carthamus tinctorius L. (CTL) extracts on thrombosis in rats. Methods: CTL extract was obtained in hot water (60 o C), dried in a hot air oven and then freeze-dried. The rats were divided into 6 groups: normal group, control group, reference group (aspirin 5 mg/kg) as well as groups that received 20, 40 and 80 mg/kg doses of CTL, respectively. For each group, treatment was given orally once daily for 14 days. Common carotid artery FeCl 3 -induced thrombus and inferior vena cava thrombosis occlusion time, as well as plasma concentrations of thromboxane B2 (TXB2) and 6-keto-prostaglandine F1 α (6-keto-PGF1 α ) were measured in rats. Results: Compared with the control group, all doses of CTL extracts significantly and dose-dependently prolonged thrombosis occlusion time, reduced the weight of thrombus and increased inhibition rate (p < 0.01). Plasma TXB2 concentration of all CTL extracts groups decreased dose-dependently (p < 0.05) while that of 6-keto-PGF1 α was increased (p < 0.05). There was association between 6-keto-PGF1 α /TXB2 and arterial or venous thrombus weight for all treatments, and also with occlusion time for CTL treatment but not for aspirin. Conclusion: CTL has a significant effect on thrombosis in rats. However, further studies are required to determine its clinical potentials.


INTRODUCTION
Carthamus tinctorius L. or safflower, commonly called Honghua in Chinese, is an annual or biennial herbal plant in the family of Compositae. The red tubular flowers without ovary are usually picked in the summer when the color of flowers changes from yellow into red, and then dried in shady and well-ventilated places for the clinical usage [1]. With the increasingly extensiveness of studies on the chemical constituents of Chinese Material Medica, phytochemical investigations have also been conducted on safflower. Currently, over 104 compounds from this plant have been isolated and identified, and they include quinochalones, flavonoids, alkaloids, polyacetylene, aromatic glucosides, organic acids, etc [2].

Animals
SPF Male Wistar hypertensive rats weighing 300-350 g were provided by the Experimental Animal Center of Zhengjiang Province (Certificate no. SYXK 2004-0002).The animals had free access to food and water, and were allowed to acclimatize for at least one week before use. The rat experiment was approved by the Animal Care and Use Committee of Wenzhou Medical University (approval ref no. 20131013) and was carried out in compliance with the Directive 2010/63/EU on the handling of animals used for scientific purposes [10].

Animal group
The rats were randomly divided into 6 groups of eight rats: normal group, control group, reference group (aspirin 5 mg/kg) as well as CTL extract groups, namely, 20, 40 and 80 mg/kg doses. Treatments were given orally once daily for 14 days, dissolved in water.

Common carotid artery thrombosis study
After the last administration, rats were anesthetized with 3 % barbital sodium (0.5 ml/100 g i.p.). 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. An incision was made of about 3 cm in the midline on the throat [11][12][13][14]. 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 % FeCl 3 solution (normal saline in sham group) [15,16]. 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 (Ti) was computed as in Eq 1.
where A and A1 are the wet weight of the thrombus in the control group and aspirin-or extract-treated groups.

Inferior vena cava thrombosis study
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 [17,18]. Thrombosis inhibition was calculated as in Eq 1 above.

Measurement of the plasma concentration of 6-keto-PGF1α and TXB2
Under sterile conditions, 90 min after surgery, the abdominal aorta was isolated and punctured for collecting 3 ml blood. The plasma was separated and stored at -20 o C. The plasma concentrations of 6-keto-PGF1α and TXB22 were measured by radioimmunoassay (RIA) [19,20].

Statistical analysis
Values are expressed as mean ± SD. Significant differences between the groups were analyzed using one-way analysis of variance (ANOVA) followed by two-paired Student's t-test. P < 0.05 was considered statistically significant.

FeCl 3 -induced common carotid artery thrombosis
As shown in Table 1, compared with the control l group, all doses of CTL extracts significantly and dose-dependently prolonged thrombosis occlusion time, reduced the weight of thrombus and increased inhibition rate (p < 0.01). Aspirin (5 mg/kg) had the same effect as CTL (30 mg/kg) for inhibition of thrombus weight, but less effect on occlusion time.

Inferior vena cava thrombosis
As shown in Table 2, compared with the control group, CTL extracts significantly and dosedependently reduced the weight of thrombus, increasing inhibitory rate (p < 0.01). The effects of aspirin were similar to that of the medium dose of CTL.

Plasma TXB2 and 6-keto-PGF1α
As shown in Table 3, compared with the normal group, arterial plasma 6-keto-PGF1α concentration was decreased (p < 0.05) and TXB2 concentration was increased (p < 0.05) in the control group. Compared with the control group, the plasma TXB2 concentration of all CTL extracts groups decreased dose-dependently (p < 0.05) while that of 6-keto-PGF1α increased dose-dependently (p < 0.05). Aspirin inhibited the secretion of both 6-keto-PGF1α and TXB2 significantly (p < 0.05).
6-Keto-PGF1α to TXB2 ratio went from 2.5 in normal control to 0.35 in control animals indicating strong platelet activation. It went from 1.0 at the lowest dose to 1.92 at 30 mg, and 6.28 at the highest dose with increasing doses of CTL. And it was 2.24 with aspirin (Figs 1 and 2). There was an asymptotic relationship between this 6keto-PGF1α to TXB2 ratio and arterial 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 CTL was in line with control and aspirin values, the values for low and medium doses of CTL were clearly above that line (Fig 2).

DISCUSSION
CTL is traditionally used in Chinese medicine for cardiovascular prevention in China. The effects of the CTL extracts on hemostasis were confirmed in experimental thrombosis rats, both venous and arterial. Injury of blood vessel endothelium by FeCl 3 can induce platelet adhesion and aggregation, which leads to thrombosis [21]. Ligation of veins causes focal blood stasis, injury of vascular endothelial cell and hypoxia [22]. In both cases, before activation of the intrinsic coagulation system, blood coagulation factors and thrombin are activated locally. Endothelial cell degeneration and necrosis then leads to exposure of subendothelial 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 decreased and plasma TXA2 increased [23], further promoting platelet adhesion and aggregation and imbalance of TXA2/PGI2, which leads to vasoconstriction, platelet aggregation, and thrombosis [24]. The biological half-life of serum TXA2 is only 30 s, and TXA2 is rapidly transformed to TXB2. Therefore, TXB2 was measured in this experiment.
Compared with the model group, CTL extracts dose-dependently prolonged OT, reduced the weight of arterial and venous thrombosis. The extracts also decreased the plasma TXB2 concentrations and increased 6-keto-PGF1α, thereby increasing the 6-keto-PGF1α to TXB2 ratio. The anti-thrombotic effect of CTL extracts was probably mediated by acting 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 was linearly related to the ratio for control, aspirin and CTL-H (r 2 = 0.999) but CTL-M and CTL-L appear not to lie on the same line (Fig 2). Furthermore, CTL perhaps had some additional effect 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 CTL doses. It is uncertain whether this is related to a differential effect on platelets rather than to actual thrombosis, and therefore needs to be further investigated [25,26].