Anti-thrombolytic effect of Salvia miltiorrhiza Bge extract in rats

Purpose: To study the effects of Salvia miltiorrhiza Bge. extract (SMBE) on thrombosis in rats. Methods: SMBE was obtained in water at 60 C in an oven and then freeze-drying. Rats were divided into 6 groups of ten rats each: normal group, control group, reference group (aspirin 5 mg/kg) as well as three groups of SMBE groups (25, 50 and 100 mg/kg doses). Treatments were given orally once daily for 14 days. Common carotid artery FeCl3-induced thrombus and inferior vena cava thrombosis occlusion time, plasma concentrations of thromboxane B2 (TXB2) and 6-keto-prostaglandine F1α (6keto-PGF1α) were measured in the rats. Results: Compared with control group, all doses of SMBE significantly and dose-dependently prolonged thrombosis occlusion time, reduced the weight of thrombus and increased the inhibition rate of thrombus (p < 0.01). Plasma TXB2 concentration of all SMBE groups decreased dose-dependently (p < 0.05) while that of 6-keto-PGF1α increased with decrease in extract dose (p < 0.05). There was association between 6-keto-PGF1α/TXB2 and arterial or venous thrombus weight for all treatments, and also with occlusion time for SMBE treatment, but not for aspirin. Conclusion: The results demonstrate the anti-thrombosis effect of SMBE in rats. This finding suggests that the plant is a potential therapy for thrombosis.


INTRODUCTION
Danshen, the dry root and rhizome of Salvia miltiorrhiza Bge (Labiatae), is one of the popular herbs used in China and the neighboring countries.This herb is widely applied in traditional Chinese medicine for promotion of blood flow to overcome blood stasis and to resolve abscesses [1].Moreover, the extracts from the roots of this plant effectively prevented development of bone loss and increase blood estrogen level in a rat model of osteoporosis [2].Several preparations that contain its major bioactive ingredients are also effective for microcirculation and coronary vasodilation and prevent inflammatory factors, and are therefore used against hypertension and inflammatory diseases [3].Furthermore, a purified extract of this plant containing its major constituents, cryptotanshinone, tanshinone I, and tanshinone IIA can protect against liver toxicity in vivo and in vitro [4].It has been reported that cryptotanshinone, tanshinones I and IIA, and salvianolic acid B are the major bioactive hydrophilic constituents present in roots of Salvia miltiorrhiza Bge.collected from different areas of China.In addition, Salvia Miltiorrhiza Bge. has been shown to possess unique efficacy in treating thromboangiitis obliterans [5].
Sequel to the use of Salvia Miltiorrhiza Bge. in the prevention of cardiovascular disease [6], this study was performed to study the effect of SMBE on models of thrombosis in rats [7].[8].The rats were randomly divided into 6 groups of ten rats: normal group, control group, reference group (aspirin 5 mg/kg) as well as SMBE groups, namely, 25, 50 and 100 mg/kg doses.Treatments were given orally once daily for 14 days.

Studies on common carotid artery thrombosis
After the last administration, rats were anesthetized with 3 % barbital sodium (0.5 ml/100 g i.p.).Under sterile conditions of class SPF animal room, 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 [9][10][11][12].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) [13,14].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. Ti (%) = {(A -A1)/A}100 ………………….. (1) where A and A1 are the wet weight of the thrombus in the control l group and aspirin-or extract-treated groups, respectively.

Studies on inferior vena cava thrombosis
Under sterile conditions of class SPF animal room, the rats were fixed on anatomical planes in supine position, the hairs of the abdomen were scraped off 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 [15,16].Thrombosis inhibition was calculated as in Eq 1 above.

Measurement of plasma concentration of 6keto-PGF1α and TXB2
One and half hours after surgery, the abdominal aorta was isolated and punctured for collecting 3 mL blood.Plasma was separated and stored at -20 o C. The plasma concentrations of 6-keto-PGF1α and TXB2 2 were measured by radioimmunoassay (RIA) [17,18].

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

FeCl 3 -induced common carotid artery thrombosis
Compared with the control group, SMBE 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 SMBE (50 mg/kg) for inhibition of thrombus weight, but less effect on occlusion time (Table 1).

Inferior vena cava thrombosis
Compared with the control group, SMBE significantly and dose-dependently reduced the weight of thrombus, increasing inhibitory rate (P < 0.01).The effects of aspirin were similar to that of the medium dose of SMBE (Table 2).
Compared with the control group, the plasma TXB2 concentration of all SMBE groups was decreased dose-dependently (p < 0.05) while that of 6-keto-PGF1α was increased but with a reversed dose-dependence (p < 0.05).
Aspirin inhibited the secretion of both 6-keto-PGF1α and TXB2 significantly.

DISCUSSION
Many clinical studies showed that Danshen and its preparations are effective for the treatment of coronary artery diseases, angina pectoris, myocardial infarction, cerebrovascular diseases, various types of hepatitis and chronic renal failure [19].Ligation of veins causes focal blood stasis, injury of vascular endothelial cell and hypoxia [20].Danshen and its medicinal products are widely used in Asian area for supporting cardiovascular function; evaluation of the active constituents in this herb is essential to ensure the efficiency of medication.Studies showed that this herb contains several pharmacologically active compounds, especially the diterpene diketones known as tanshinones.Due to injury of vascular endothelial cell, synthesis of PGI 2 decrease and plasma TXA 2 increased [21], further promoting platelet adhesion and aggregation and imbalance of TXA 2 /PGI 2 , which leads to vasoconstriction, platelet aggregation, and thrombosis [22].The biological half-life of serum TXA2 is only 30 seconds, and TXA2 transformed to TXB2 quickly.Therefore, we measured TXB2 in our experiment.
Compared with the model group, SMBE dosedependently prolonged OT, reduced the weight of arterial and venous thrombosis.The extracts also decrease plasma TXB2 concentrations and increased 6-keto-PGF1α, thereby increasing the 6-keto-PGF1α to TXB2 ratio.The anti-thrombotic effect of SMBE was probably mediated by regulating the prostacyclin/thromboxane balance and the ratio, resulting in a ratio that was dependently related to thrombus weight.Arterial occlusion time was linearly related to the ratio for control, aspirin and SMBE -H (r 2 = 0.999) but SMBE -M and SMBE -L appear not to lie on the same line.Furthermore, SMBE perhaps had some additional effect that prolongs occlusion time beyond what would be expected from the effect on cycloxygenase, or more generally on the synthesis of thromboxane and PGI2, especially for the lower SMBE 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 [23,24].

CONCLUSION
The findings of this study indicate the antithrombosis effect of SMBE in Further studies are, however, required to ascertain its therapeutic significance in the treatment of thrombosis in humans.

DECLARATIONS
Salvia miltiorrhiza Bge. were collected from Shiyan City, Hubei Province in China in May 2015.Taxonomic identification of the plant was performed by Professor Ping He of College of Pharmacy of Southern Medical University in China.A voucher specimen of herbarium (no.

Table 3 :
Effect of SMBE on the plasma concentrations of 6-keto-PGF1α and TXB2 in rats