Stiff silkworm polypeptide with antiplatelet aggregation activity as well as preparation method and application of stiff silkworm polypeptide
An anti-platelet aggregation and silkworm technology is applied in the application of anti-thrombotic drugs. The preparation of the polypeptide, in the field of anti-platelet aggregation polypeptide, can solve problems such as neutropenia and achieve good protective effect.
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Embodiment 1
[0021] Embodiment 1: Preparation of BB octapeptide
[0022] (1) Take white silkworm, soak and wash it, pulverize and homogenate, and add 60% ethanol according to the ratio of W / V 1:6. Reflux 3 times, the time is 1h, 40min, 20min respectively, and combine the reflux liquid for 3 times, and the reflux temperature is 80°C.
[0023] (2) Use a microporous membrane to remove particles from the reflux liquid, concentrate the obtained filtrate under reduced pressure, then centrifuge at 10000×g for 30 min, and take the supernatant. The supernatant was concentrated under reduced pressure and lyophilized.
[0024] (3) Dissolve the lyophilized Bombyx mori extract powder in a small amount of 20mM PBS pH7.6, and perform gel filtration chromatography on Sephadex G-50. The eluent is 20mM PBS pH7.6, the flow rate is 0.3ml / min, and the collection unit is 3ml. 4 peaks were obtained, the third peak was collected, peak B, lyophilized as figure 1 shown.
[0025] (4) Peak B was dissolved in a s...
Embodiment 2
[0028] Example 2: In vitro anti-platelet aggregation activity of BB octapeptide
[0029] (1) male rabbit is anesthetized, common carotid artery blood is taken and placed in the 15ml centrifuge tube containing 3.2% sodium citrate of 1ml, (every tube final volume is about 10ml, guarantees that rabbit blood and anticoagulant volume ratio are 9:1), centrifuged at 120×g for 10 min at 20° C. to obtain the supernatant, namely platelet-rich plasma. The remaining blood samples were centrifuged at 1600×g for 10 min to obtain platelet-poor plasma.
[0030] (2) Take 300 μl of platelet-poor plasma and place it in the test area of the platelet aggregation meter to adjust to zero, then draw 270 μl of platelet-rich plasma and place it in the preheating tank, add 30 μl of BB octapeptide with different concentration gradients prepared in normal saline (100, 300, 600, 900, 1000 μg / ml) and test beads, preheated at 37°C, placed in the test area after 1 min, added 30 μl of inducer collagen (fina...
Embodiment 3
[0035] Example 3: The protective effect of BB octapeptide on the acute pulmonary thromboembolism model in mice
[0036] (1) Take 36 healthy ICR mice, weighing 20-25 g, and divide them into 4 groups with 9 mice in each group. BB octapeptide was diluted with normal saline into different dose gradients of 10, 30 and 50 mg / kg, and the negative control was normal saline. All the above groups were given tail vein administration, once a day, for 4 consecutive days, and the administration volume was 10ml / kg.
[0037] (2) 15 minutes after the last administration, a mixed inducer of collagen 12 mg / kg and adrenaline 1 mg / kg was injected into the tail vein to form acute pulmonary thromboembolism, and the death or hemiplegia of each group within 15 minutes after the injection of the mixed inducer was recorded (loss of integrity within 30 s) reflex) the number of mice. The protection rate of acute pulmonary thromboembolism was calculated by the following formula: [1-(X / Y)]×100%. X repres...
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