The specific embodiment
Ginkgolide monomer compound of the present invention, all can obtain by buying commercially available prod, or by existing method separation and purification preparation.Through check, all monomeric compounds all conform to corresponding reference substance structure, and detect its purity all more than 98% through HPLC.
The two or more compositions of Semen Ginkgo diterpenes diterpenoids lactones of the present invention, can combine by corresponding monomeric compound.
The compositions that contains ginkgolectone AB C and bilobalide of the present invention, can directly buy commercially available bilobalide injection, perhaps the method by ZL200610103626.0 or ZL200610103625.6 makes, also can be by monomeric compound is combined.
Below illustrate beneficial effect of the present invention by test example.
The anti-platelet activity facilitation research of test example 1 bilobalide to ginkgolide monomer
1 materials and methods
1.1 laboratory animal
80 of Japan large ear rabbits, body weight (2.0 ± 0.2) kg, male and female half and half, provide [ the animal quality certification number: XCXK(Chongqing) 20020001 ] by Medical University Of Chongqing's Experimental Animal Center.
1.2 Experimental agents
(the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred provides ginkalide A, content: 5.0mg/ml), (Chengdu hundred is abundant to be provided ginkalide B, content: 5.0mg/ml), (Chengdu hundred is abundant to be provided ginkalide C, content: 5.0mg/ml), (Chengdu hundred is abundant to be provided the ginkalide A compositions, the part by weight of ginkalide A and bilobalide is 52:48, bilobalide content: 5.0mg/ml), (Chengdu hundred is abundant to be provided the ginkalide B compositions, the part by weight of ginkalide B and bilobalide is 52:48, bilobalide content: 5.0mg/ml), (Chengdu hundred is abundant to be provided the ginkalide C compositions, the part by weight of ginkalide C and bilobalide is 52:48, bilobalide content: 5.0mg/ml), (Chengdu hundred is abundant to be provided bilobalide, content: 96.0%).
1.3 reagent and instrument
Platelet activating factor (PAF) (cayman, lot number: 012328) be dissolved in PH be 7.6 containing in the Tris-NaCl solution of 0.25% bovin serum albumin, final concentration is 3.6nmol/L; Sodium citrate (Beijing biotech company of Zhong Shan Golden Bridge, lot number: 20130117) distilled water is made into 3.8% concentration; Rabbit β-thromboglobulin (β-TG) ELISA test kit (FOCUS, lot number: 20130224), and rabbit platelet factor 4(PF-4) ELISA test kit (FOCUS, lot number: 20130301).TYXN-96 multifunctional intellectual blood pool instrument (development of Shanghai GM technical research institute); Scanning electron microscope S-3000N(HIT); ELX-800 microplate reader (U.S. Bao Te company).
1.4 grouping and medication
80 of Japan large ear rabbits, be divided at random 8 groups, 10 every group: (1) normal saline group, (2) bilobalide group, (3) ginkalide A group, (4) ginkalide B group, (5) ginkalide C group, (6) ginkalide A compositions group, (7) ginkalide B compositions group, (8) ginkalide C compositions group.
The said medicine configured all is converted into to bilobalide or total lactone according to 1.00ml/kg(: dosage i.v. administration 5.0mg/kg), once a day, continuous 7d.
1.5 detection platelet aggregation rate
After administration 7d, every animal hearts is got blood 10.5ml, separate 1.5ml blood plasma and get serum, 3.8% sodium citrate 1:9 anticoagulant for all the other 9ml blood plasma, the centrifugal 10min of 800r/min, get supernatant and obtain platelet rich plasma (PRP), separate 100 μ lPRP for electron microscopic examination, all the other PRP are for the detection of platelet aggregation rate; The centrifugal 15min of remainder 3000r/min speed, get platelet poor plasma (PPP).Regulate PRP with PPP, make the PRP number of platelets 360 * 10
9/ L.Measure and be recorded in 10 μ lPAF and induce lower 1min, 5min and max platelet rate.
1.6 platelets is observed
Isolated 100 μ lPRP are placed in to silication EP pipe, the gathering that adds the PAF induced platelet of 1 μ l, after effect 15min, PRP is placed on the copper mesh sample carrier that is covered with the Formar film, hatch 10min for 37 ℃, ultra-pure water rinses, 3% glutaraldehyde is 5min fixedly, with ultra-pure water, rinse well again, after the specimen natural drying on copper mesh, at the golden film of its plated surface one deck 20nm, ultramicroscope S-3000N scanning cellular morphology, observe 100 platelet, calculate various platelet proportion.Under Electronic Speculum, the platelet typing comprises: (1) circle: rounded or oval.Volume is little, and central authorities are fine and close, and core is large, and peripheral zona pellucida is low narrow.(2) tree-like: send single or multiple podocytic processes from central dense area, elongated or lamellar, have branch sometimes.(3) flatten shape: there is dense-core in central authorities, and peripheral zona pellucida is wider, and periphery is smooth or little projection arranged.(4) assemble shape: often several, consist of to tens platelet, aggregation differs in size, and wherein visible platelet interconnects, complete being fused into one had, and peripheral part platelet podocytic process is obvious.
1.7 the mensuration of PF-4 and β-TG contents level in serum
To the PAF that adds 1 μ l in 1.5ml blood plasma, the release of inducing PF-4 and β-TG, blood plasma, in 4 ℃ of standing 4h, is got 200 μ l serum, is detected.Concrete operations are carried out according to the test kit description, microplate reader reading result data.
1.8 statistical procedures is mean ± standard deviation (x ± s) expression for experimental result, carry out statistical analysis with SPSS18.0 software, adopt the t check of two sample mean comparisons to carry out statistical analysis to each group platelet aggregation rate, platelet form percent and PF-4 and β-TG content.There is statistical significance P<0.05 for difference.
2 results
2.1 platelet aggregation test result
Under PAF induces, the max platelet rate of ginkalide A compositions group, ginkalide B compositions group, ginkalide C compositions group is compared with the normal saline group, significant difference (p<0.01) all occurs, and each is organized platelet aggregation inhibition rate and is significantly increased; The max platelet rate of ginkalide A group, ginkalide B group, ginkalide C group is compared with the normal saline group, all variant variations (p<0.05), and each is organized platelet aggregation inhibition rate and is improved; And the bilobalide group is compared with the normal saline group, the little or there was no significant difference of difference.Illustrate that the platelet aggregation that ginkalide A, ginkalide B, ginkalide C, ginkalide A compositions group, ginkalide B compositions group, ginkalide C compositions group can be induced PAF produces inhibitory action, but the suppression ratio that can find out the compositions group from experimental data is higher than the suppression ratio of set of monomers, and the platelet aggregation that the bilobalide group is induced PAF does not produce inhibitory action or inhibitory action is very weak.
Table 1, bilobalide compositions platelet aggregation test result
Group |
1min |
5min |
MAX |
Platelet aggregation inhibition rate (%) |
The ginkalide A group |
30.28±5.64* |
40.56±3.70* |
52.02±4.53* |
25.92% |
The ginkalide B group |
34.01±4.25* |
38.97±6.54* |
55.32±4.76* |
21.22% |
The ginkalide C group |
34.56±5.10* |
39.23±4.65* |
56.89±5.84* |
18.98% |
Ginkalide A compositions group |
25.10±5.85** |
36.66±4.86** |
46.96±4.23** |
33.12% |
Ginkalide B compositions group |
31.32±4.34** |
34.78±5.43** |
48.34±5.56** |
31.16% |
Ginkalide C compositions group |
29.25±3.28** |
35.86±5.37** |
45.50±6.83** |
35.20% |
The bilobalide group |
34.34±6.59 |
58.79±8.32 |
59.84±8.92 |
6.73% |
The normal saline group |
36.85±6.14 |
68.32±9.17 |
70.22±11.40 |
0.00% |
Compare * * p<0.01, * p<0.05 with the normal saline group.
2.2 platelets testing result
Mainly observe four kinds of platelet forms under the scanning electron microscope of 2000 times: circular, tree-like, flatten shape and assemble shape.Under PAF induces, the strong activation occurs in normal saline group platelet, and platelet adhesion power strengthens, and can see red blood cell adhesion on platelet.The platelet form is irregular, volume increases, stretch out and be the outstanding pseudopodium of spore shape, assemble the shape platelet counts and increase.Under PAF induces, Ginkgolide A. B. C group and each compositions group Platelet Size are more consistent, smooth surface, and the accumulation type platelet is more rare, but more rare than monomer bilobalide group of the aggregation platelet of bilobalide compositions group.
Table 2, bilobalide compositions are respectively organized the platelet form relatively after adding PAF
Compare * * p<0.01, * p<0.05 with the normal saline group.
2.3 platelet PF-4 and β-TG testing result
With the normal saline group, compare, the PF-4 level of ginkalide A compositions group, ginkalide B compositions group, ginkalide C compositions group significantly reduces (p<0.01); β-TG level also significantly reduces (p<0.01); PF-4 and β-TG level reduces, and illustrates that platelet release function is suppressed; The PF-4 of ginkalide A group, ginkalide B group, ginkalide C group and β-TG level changes (p<0.05), but change, is not so good as the obvious of compositions group; And the bilobalide group does not have significant change.
Table 3, bilobalide compositions platelet PF-4 and β-TG testing result
Group |
PF-4(μg/ml) |
β-TG(μg/ml) |
The ginkalide A group |
1.482±0.165* |
1.452±0.254* |
The ginkalide B group |
1.403±0.187* |
1.413±0.172* |
The ginkalide C group |
1.454±0.145* |
1.430±0.146* |
Ginkalide A compositions group |
1.350±0.125** |
1.426±0.158** |
Ginkalide B compositions group |
1.332±0.162** |
1.405±0.165** |
Ginkalide C compositions group |
1.339±0.197** |
1.430±0.201** |
The bilobalide group |
1.583±0.245 |
1.604±0.244 |
Normal saline |
1.733±0.294 |
1.740±0.215 |
Compare * * p<0.01, * p<0.05 with the normal saline group.
Brief summary:
From above-mentioned result of the test, bilobalide is used and there is no anti-platelet activity separately, and this result with bibliographical information is consistent.After bilobalide and Ginkgolides a and B or C are used in combination, although bilobalide dosage only has single compound half,, the drug activity of compositions but significantly is better than single compound, this just shows, bilobalide has the effect that promotes the bilobalide anti-platelet activity.
The anti-platelet activity facilitation research of test example 2 bilobalide to the bilobalide compositions
1 materials and methods
1.1 laboratory animal
80 of Japan large ear rabbits, body weight (2.0 ± 0.2) kg, male and female half and half, provide [ the animal quality certification number: XCXK(Chongqing) 20020001 ] by Medical University Of Chongqing's Experimental Animal Center.
1.2 Experimental agents
(the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred provides ginkgolectone AB, content: 5.0mg/ml, ginkalide A: B=20:30), ginkalide B C(Chengdu hundred abundant providing, content: 5.0mg/ml, ginkalide B: C=30:10), ginkalide A C(Chengdu hundred abundant providing, content: 5.0mg/ml, ginkalide A: C=20:10), (Chengdu hundred is abundant to be provided ginkgolectone AB+bilobalide group, the part by weight of ginkgolectone AB and bilobalide is 52:48, bilobalide content: 5.0mg/ml), (Chengdu hundred is abundant to be provided ginkalide B C+ bilobalide group, the part by weight of ginkalide B C and bilobalide is 52:48, bilobalide content: 5.0mg/ml), (Chengdu hundred is abundant to be provided ginkalide A C+ bilobalide group, the part by weight of ginkalide A C and bilobalide is 52:48, bilobalide content: 5.0mg/ml), (Chengdu hundred is abundant to be provided bilobalide, content: 96.0%).
1.3 reagent and instrument
Platelet activating factor (PAF) (cayman, lot number: 012328) be dissolved in PH be 7.6 containing in the Tris-NaCl solution of 0.25% bovin serum albumin, final concentration is 3.6nmol/L; Sodium citrate (Beijing biotech company of Zhong Shan Golden Bridge, lot number: 20130117) distilled water is made into 3.8% concentration; Rabbit β-thromboglobulin (β-TG) ELISA test kit (FOCUS, lot number: 20130224), and rabbit platelet factor 4(PF-4) ELISA test kit (FOCUS, lot number: 20130301).TYXN-96 multifunctional intellectual blood pool instrument (development of Shanghai GM technical research institute); Scanning electron microscope S-3000N(HIT); ELX-800 microplate reader (U.S. Bao Te company).
1.4 grouping and medication
80 of Japan large ear rabbits, be divided at random 8 groups, 10 every group: (1) normal saline group, (2) bilobalide group, (3) ginkgolectone AB group, (4) ginkalide B C group, (5) ginkalide A C group, (6) ginkgolectone AB+bilobalide compositions group, (7) ginkalide B C+ bilobalide compositions group, (8) ginkalide A C+ bilobalide compositions group.
The said medicine configured all is converted into to Ginkgo total lactones according to 1.00ml/kg(: dosage i.v. administration 5.0mg/kg), once a day, continuous 7d.
1.5 detection platelet aggregation rate
After administration 7d, every animal hearts is got blood 10.5ml, separate 1.5ml blood plasma and get serum, 3.8% sodium citrate 1:9 anticoagulant for all the other 9ml blood plasma, the centrifugal 10min of 800r/min, get supernatant and obtain platelet rich plasma (PRP), separate 100 μ lPRP for electron microscopic examination, all the other PRP are for the detection of platelet aggregation rate; The centrifugal 15min of remainder 3000r/min speed, get platelet poor plasma (PPP).Regulate PRP with PPP, make the PRP number of platelets 360 * 10
9/ L.Measure and be recorded in 10 μ lPAF and induce lower 1min, 5min and max platelet rate.
1.6 platelets is observed
Isolated 100 μ lPRP are placed in to silication EP pipe, the gathering that adds the PAF induced platelet of 1 μ l, after effect 15min, PRP is placed on the copper mesh sample carrier that is covered with the Formar film, hatch 10min for 37 ℃, ultra-pure water rinses, 3% glutaraldehyde is 5min fixedly, with ultra-pure water, rinse well again, after the specimen natural drying on copper mesh, at the golden film of its plated surface one deck 20nm, ultramicroscope S-3000N scanning cellular morphology, observe 100 platelet, calculate various platelet proportion.Under Electronic Speculum, the platelet typing comprises: (1) circle: rounded or oval.Volume is little, and central authorities are fine and close, and core is large, and peripheral zona pellucida is low narrow.(2) tree-like: send single or multiple podocytic processes from central dense area, elongated or lamellar, have branch sometimes.(3) flatten shape: there is dense-core in central authorities, and peripheral zona pellucida is wider, and periphery is smooth or little projection arranged.(4) assemble shape: often several, consist of to tens platelet, aggregation differs in size, and wherein visible platelet interconnects, complete being fused into one had, and peripheral part platelet podocytic process is obvious.
1.7 the mensuration of PF-4 and β-TG contents level in serum
To the PAF that adds 1 μ l in 1.5ml blood plasma, the release of inducing PF-4 and β-TG, blood plasma, in the standing 4h of 4oC, is got 200 μ l serum, is detected.Concrete operations are carried out according to the test kit description, microplate reader reading result data.
1.8 statistical procedures is mean ± standard deviation for experimental result
mean, with SPSS18.0 software, carry out statistical analysis, adopt the t check of two sample mean comparisons to carry out statistical analysis to each group platelet aggregation rate, platelet form percent and PF-4 and β-TG content.There is statistical significance P<0.05 for difference.
2 results
2.1 platelet aggregation test result
Under PAF induces, the max platelet rate of ginkalide A C compositions group, ginkgolectone AB compositions group, ginkalide B C compositions group is compared with the normal saline group, significant difference (p<0.01) all occurs, and each is organized platelet aggregation inhibition rate and is significantly increased; The max platelet rate of ginkalide A C group, ginkgolectone AB group, ginkalide B C group is compared with the normal saline group, and variant (p<0.05), but suppression ratio is not so good as the obvious of compositions group; And the bilobalide group is compared with the normal saline group, the little or there was no significant difference of difference.
Table 4, bilobalide compositions platelet aggregation test result
Group |
1min |
5min |
MAX |
Platelet aggregation inhibition rate (%) |
The ginkgolectone AB group |
29.82±5.45* |
39.54±5.32* |
50.67±4.42* |
27.84% |
Ginkalide B C group |
30.56±4.87* |
39.95±5.64* |
53.46±3.76* |
23.87% |
Ginkalide A C group |
30.64±4.69* |
40.23±3.34* |
52.59±6.45* |
25.11% |
Ginkgolectone AB+bilobalide group |
24.26±6.48** |
34.46±3.49** |
44.48±4.57** |
36.66% |
Ginkalide B C+ bilobalide group |
25.51±5.64** |
36.38±5.76** |
45.39±5.77** |
35.36% |
Ginkalide A C+ bilobalide group |
26.67±5.13** |
37.68±5.46** |
44.61±6.42** |
36.47% |
The bilobalide group |
34.34±6.59 |
58.79±8.32 |
59.84±8.92 |
6.73% |
The normal saline group |
36.85±6.14 |
68.32±9.17 |
70.22±11.40 |
0.00% |
Compare * * p<0.01, * p<0.05 with the normal saline group.
2.2 platelets testing result
Mainly observe four kinds of platelet forms under the scanning electron microscope of 2000 times: circular, tree-like, flatten shape and assemble shape.Under PAF induces, the strong activation occurs in normal saline group platelet, and platelet adhesion power strengthens, and can see red blood cell adhesion on platelet.The platelet form is irregular, volume increases, stretch out and be the outstanding pseudopodium of spore shape, assemble the shape platelet counts and increase.Under PAF induces, ginkalide A C group, ginkgolectone AB group, ginkalide B C and bilobalide compositions group Platelet Size are more consistent, smooth surface, the accumulation type platelet is more rare, but the accumulation type platelet of compositions group that has added bilobalide is than not adding accumulation type in each group of bilobalide still less.
Table 5, bilobalide compositions are respectively organized the platelet form relatively after adding PAF
Compare * * p<0.01, * p<0.05 with the normal saline group.
2.3 platelet PF-4 and β-TG testing result
With the normal saline group, compare, the PF-4 level of ginkalide A C group, ginkgolectone AB group, ginkalide B C group, each group of bilobalide compositions significantly reduces (p<0.01); β-TG level also significantly reduces (p<0.01); PF-4 and β-TG level reduces, and illustrates that platelet release function is suppressed;
And the PF-4 of bilobalide group and β-TG level does not all have significant change.
Table 6, bilobalide compositions platelet PF-4 and β-TG testing result
Group |
PF-4(μg/ml) |
β-TG(μg/ml) |
The ginkgolectone AB group |
1.282±0.148** |
1.407±0.134** |
Ginkalide B C group |
1.278±0.156** |
1.398±0.167** |
Ginkalide A C group |
1.280±0.145** |
1.387±0.196** |
Ginkgolectone AB compositions group |
1.248±0.138** |
1.239±0.148** |
Ginkalide B C compositions group |
1.242±0.125** |
1.231±0.162** |
Ginkalide A C compositions group |
1.239±0.177** |
1.229±0.195** |
The bilobalide group |
1.583±0.245 |
1.604±0.244 |
Normal saline |
1.733±0.294 |
1.740±0.215 |
Compare * * p<0.01 with the normal saline group.
Brief summary:
From above-mentioned result of the test, bilobalide is used and there is no anti-platelet activity separately, and this result with bibliographical information is consistent.By the combination of two thing of Ginkgolide A. B. C with after bilobalide is combined use, although the bilobalide accumulated dose reduces by half, but, drug activity after coupling but significantly is better than bilobalide combination of two thing, this just shows, bilobalide has the effect that promotes bilobalide compositions anti-platelet activity.
The anti-platelet activity facilitation research of test example 3 bilobalide to the bilobalide compositions
1 materials and methods
1.1 laboratory animal
50 of Japan large ear rabbits, body weight (2.0 ± 0.2) kg, male and female half and half, provide [ the animal quality certification number: XCXK(Chongqing) 20020001 ] by Medical University Of Chongqing's Experimental Animal Center.
1.2 Experimental agents
Bilobalide injection (the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred, lot number: 20120303, method according to the embodiment bis-in granted patent ZL200610103625.6 makes, wherein, ginkalide A: ginkalide B: ginkalide C=20:30:10, the part by weight of Ginkgolide A. B. C total amount and bilobalide is 52:48, bilobalide content: 4.91mg/ml), the abundant scientific and technological pharmaceutical Co. Ltd in ginkgolectone AB C(Chengdu hundred provides, content: 97.5%).
1.3 reagent and instrument
Platelet activating factor (PAF) (cayman, lot number: 012328) be dissolved in PH be 7.6 containing in the Tris-NaCl solution of 0.25% bovin serum albumin, final concentration is 3.6nmol/L; Sodium citrate (Beijing biotech company of Zhong Shan Golden Bridge, lot number: 20130117) distilled water is made into 3.8% concentration; Rabbit β-thromboglobulin (β-TG) ELISA test kit (FOCUS, lot number: 20130224), and rabbit platelet factor 4(PF-4) ELISA test kit (FOCUS, lot number: 20130301).TYXN-96 multifunctional intellectual blood pool instrument (development of Shanghai GM technical research institute); Scanning electron microscope S-3000N(HIT); ELX-800 microplate reader (U.S. Bao Te company).
1.4 grouping and medication
50 of Japan large ear rabbits, be divided at random 5 groups, 10 every group: dosage group, (5) hundred abundant bilobalide injection low dose group in (1) normal saline group, (2) ginkgolectone AB C group, (3) hundred abundant bilobalide injection high dose group, (4) hundred abundant bilobalide injections.
Hundred high, medium and low group of abundant bilobalide injectors (can be called for short hundred abundant high, medium and low) are amounted to Ginkgo total lactones (comprising ginkgolectone AB C+ bilobalide) according to 1.02ml/kg, 0.51ml/kg and 0.255ml/kg(respectively and are respectively: 5.0mg/kg, 2.5mg/kg, 1.25mg/kg) dosage i.v. administration.Ginkgolectone AB C5.13mg/kg(amounts to ginkgolectone AB C5.0mg/kg) by the dilution process dilution the i.v. administration that provide.Once a day, continuous 7d.
1.5 detection platelet aggregation rate
After administration 7d, every animal hearts is got blood 10.5ml, separate 1.5ml blood plasma and get serum, 3.8% sodium citrate 1:9 anticoagulant for all the other 9ml blood plasma, the centrifugal 10min of 800r/min, get supernatant and obtain platelet rich plasma (PRP), separate 100 μ lPRP for electron microscopic examination, all the other PRP are for the detection of platelet aggregation rate; The centrifugal 15min of remainder 3000r/min speed, get platelet poor plasma (PPP).Regulate PRP with PPP, make the PRP number of platelets 360 * 10
9/ L.Measure and be recorded in 10 μ lPAF and induce lower 1min, 5min and max platelet rate.
1.6 platelets is observed
Isolated 100 μ lPRP are placed in to silication EP pipe, the gathering that adds the PAF induced platelet of 1 μ l, after effect 15min, PRP is placed on the copper mesh sample carrier that is covered with the Formar film, hatch 10min for 37 ℃, ultra-pure water rinses, 3% glutaraldehyde is 5min fixedly, with ultra-pure water, rinse well again, after the specimen natural drying on copper mesh, at the golden film of its plated surface one deck 20nm, ultramicroscope S-3000N scanning cellular morphology, observe 100 platelet, calculate various platelet proportion.Under Electronic Speculum, the platelet typing comprises: (1) circle: rounded or oval.Volume is little, and central authorities are fine and close, and core is large, and peripheral zona pellucida is low narrow.(2) tree-like: send single or multiple podocytic processes from central dense area, elongated or lamellar, have branch sometimes.(3) flatten shape: there is dense-core in central authorities, and peripheral zona pellucida is wider, and periphery is smooth or little projection arranged.(4) assemble shape: often several, consist of to tens platelet, aggregation differs in size, and wherein visible platelet interconnects, complete being fused into one had, and peripheral part platelet podocytic process is obvious.(result is referring to Fig. 1~5)
1.7 the mensuration of PF-4 and β-TG contents level in serum
To the PAF that adds 1 μ l in 1.5ml blood plasma, the release of inducing PF-4 and β-TG, blood plasma, in 4 ℃ of standing 4h, is got 200 μ l serum, is detected.Concrete operations are carried out according to the test kit description, microplate reader reading result data.
1.8 statistical procedures is mean ± standard deviation for experimental result
mean, with SPSS18.0 software, carry out statistical analysis, adopt the t check of two sample mean comparisons to carry out statistical analysis to each group platelet aggregation rate, platelet form percent and PF-4 and β-TG content.There is statistical significance P<0.05 for difference.
2 results
2.1 platelet aggregation test result
Under PAF induces, ginkgolectone AB C group, and the max platelet rate of bilobalide injection senior middle school low dose group all with the normal saline group, compare, significant difference (p<0.01) occurs, the above platelet aggregation inhibition rate of respectively organizing is significantly increased.Ginkgolectone AB C is described, reaches the platelet aggregation generation inhibitory action that hundred abundant each groups can be induced PAF.
Table 7, hundred abundant bilobalide injection platelet aggregation test results
Group |
1min |
5min |
MAX |
Platelet aggregation inhibition rate (%) |
Ginkgolectone AB C |
26.37±5.89** |
40.67±8.48** |
45.43±7.49** |
35.30% |
Normal saline |
36.85±6.14 |
68.32±9.17 |
70.22±11.40 |
0.00% |
Hundred is abundant low |
28.16±5.42** |
34.01±7.50** |
42.69±7.26** |
39.21% |
Hundred abundant in |
25.09±5.26** |
34.95±7.63** |
42.71±7.25** |
39.18% |
Hundred abundant height |
24.42±5.04** |
30.84±7.22** |
39.49±7.38** |
43.76% |
Compare * * p<0.01 with the normal saline group
2.2 platelets testing result
Mainly observe four kinds of platelet forms under the scanning electron microscope of 2000 times: circular, tree-like, flatten shape and assemble shape.Under PAF induces, the strong activation occurs in normal saline group platelet, and platelet adhesion power strengthens, and can see red blood cell adhesion on platelet.The platelet form is irregular, volume increases, stretch out and be the outstanding pseudopodium of spore shape, assemble the shape platelet counts and increase.Under PAF induces, ginkgolectone AB C group and bilobalide injection be high, normal, basic, and respectively to organize Platelet Size more consistent, smooth surface, and the accumulation type platelet is more rare.
Table 8, hundred abundant bilobalide injections are respectively organized the platelet form relatively after adding PAF
Compare * * p<0.01, * p<0.05 with the normal saline group
2.3 platelet PF-4 and β-TG testing result
With the normal saline group, compare, ginkgolectone AB C and bilobalide injection are respectively organized the PF-4 level and are significantly reduced (p<0.01); β-TG level also significantly reduces (p<0.01); PF-4 and β-TG level reduces, and illustrates that platelet release function is suppressed.
Table 9, hundred abundant bilobalide injection platelet PF-4 and β-TG testing result
Compare * * p<0.01 with the normal saline group
3. discuss
In bilobalide injection, Ginkgo total lactones content is up to 99%, and the bilobalide by 48% and 52% ginkgolectone AB C form.This test ginkgolectone AB C consumption (5.0mg/kg) is identical with Ginkgo total lactones in the bilobalide injection high dose group, and the bilobalide in Ginkgo total lactones does not possess the anti-platelet aggregation effect or effect is very weak, ginkgolectone AB C is generally acknowledged platelet aggregation antagonist, and its action pathway is the activation of antagonism platelet activating factor (PAF).This result of the test shows: adding of bilobalide can significantly improve the effect of bilobalide compositions anti-platelet aggregation.
The anti-platelet activity facilitation research of test example 4 bilobalide to the bilobalide compositions
1 materials and methods
1.1 laboratory animal
50 of Japan large ear rabbits, body weight (2.0 ± 0.2) kg, male and female half and half, provide [ the animal quality certification number: XCXK(Chongqing) 20020001 ] by Medical University Of Chongqing's Experimental Animal Center.
1.2 Experimental agents
The abundant scientific and technological pharmaceutical Co. Ltd in bilobalide compositions 1(Chengdu hundred provides, method according to embodiment tri-in granted patent L200610103625.6 makes, wherein, ginkalide A: ginkalide B: ginkalide C=20:30:10, ginkalide A, B, the part by weight of C total amount and bilobalide is 5:65, bilobalide total content: 4.0mg/ml), the abundant scientific and technological pharmaceutical Co. Ltd in bilobalide compositions 2(Chengdu hundred provides, method according to embodiment tri-in granted patent ZL200610103625.6 makes, wherein, ginkalide A: ginkalide B: ginkalide C=20:30:10, ginkalide A, B, the part by weight of C total amount and bilobalide is 60:5, bilobalide total content: 5.0mg/ml), the abundant scientific and technological pharmaceutical Co. Ltd in ginkgolectone AB C(Chengdu hundred provides, content: 97.5%), (the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred provides bilobalide, content: 96.0%).
1.3 reagent and instrument
Platelet activating factor (PAF) (cayman, lot number: 012328) be dissolved in PH be 7.6 containing in the Tris-NaCl solution of 0.25% bovin serum albumin, final concentration is 3.6nmol/L; Sodium citrate (Beijing biotech company of Zhong Shan Golden Bridge, lot number: 20130117) distilled water is made into 3.8% concentration; Rabbit β-thromboglobulin (β-TG) ELISA test kit (FOCUS, lot number: 20130224), and rabbit platelet factor 4(PF-4) ELISA test kit (FOCUS, lot number: 20130301).TYXN-96 multifunctional intellectual blood pool instrument (development of Shanghai GM technical research institute); Scanning electron microscope S-3000N(HIT); ELX-800 microplate reader (U.S. Bao Te company).
1.4 grouping and medication
50 of Japan large ear rabbits, be divided into 5 groups at random, 10 every group: (1) normal saline group, (2) bilobalide group, (3) ginkgolectone AB C group, 1 group of (4) bilobalide compositions, 2 groups of (5) bilobalide compositionss.
1 group of bilobalide compositions is amounted to Ginkgo total lactones (comprising ginkgolectone AB C+ bilobalide 5:65) according to 1.25ml/kg(and is: dosage i.v. administration 5.0mg/kg), 2 groups of bilobalide compositionss are amounted to Ginkgo total lactones (comprising ginkgolectone AB C+ bilobalide 60:5) according to 1.00ml/kg(and are: dosage i.v. administration 5.0mg/kg), the amount i.v. administration of after the bilobalide dilution, pressing 5.0mg/kg, ginkgolectone AB C5.13mg/kg(amounts to ginkgolectone AB C5.0mg/kg) by the dilution process dilution the i.v. administration that provide.Once a day, continuous 7d.
1.5 detection platelet aggregation rate
After administration 7d, every animal hearts is got blood 10.5ml, separate 1.5ml blood plasma and get serum, 3.8% sodium citrate 1:9 anticoagulant for all the other 9ml blood plasma, the centrifugal 10min of 800r/min, get supernatant and obtain platelet rich plasma (PRP), separate 100 μ lPRP for electron microscopic examination, all the other PRP are for the detection of platelet aggregation rate; The centrifugal 15min of remainder 3000r/min speed, get platelet poor plasma (PPP).Regulate PRP with PPP, make the PRP number of platelets 360 * 10
9/ L.Measure and be recorded in 10 μ lPAF and induce lower 1min, 5min and max platelet rate.
1.6 platelets is observed
Isolated 100 μ lPRP are placed in to silication EP pipe, the gathering that adds the PAF induced platelet of 1 μ l, after effect 15min, PRP is placed on the copper mesh sample carrier that is covered with the Formar film, hatch 10min for 37 ℃, ultra-pure water rinses, 3% glutaraldehyde is 5min fixedly, with ultra-pure water, rinse well again, after the specimen natural drying on copper mesh, at the golden film of its plated surface one deck 20nm, ultramicroscope S-3000N scanning cellular morphology, observe 100 platelet, calculate various platelet proportion.Under Electronic Speculum, the platelet typing comprises: (1) circle: rounded or oval.Volume is little, and central authorities are fine and close, and core is large, and peripheral zona pellucida is low narrow.(2) tree-like: send single or multiple podocytic processes from central dense area, elongated or lamellar, have branch sometimes.(3) flatten shape: there is dense-core in central authorities, and peripheral zona pellucida is wider, and periphery is smooth or little projection arranged.(4) assemble shape: often several, consist of to tens platelet, aggregation differs in size, and wherein visible platelet interconnects, complete being fused into one had, and peripheral part platelet podocytic process is obvious.
1.7 the mensuration of PF-4 and β-TG contents level in serum
To the PAF that adds 1 μ l in 1.5ml blood plasma, the release of inducing PF-4 and β-TG, blood plasma, in 4 ℃ of standing 4h, is got 200 μ l serum, is detected.Concrete operations are carried out according to the test kit description, microplate reader reading result data.
1.8 statistical procedures is mean ± standard deviation for experimental result
mean, with SPSS18.0 software, carry out statistical analysis, adopt the t check of two sample mean comparisons to carry out statistical analysis to each group platelet aggregation rate, platelet form percent and PF-4 and β-TG content.There is statistical significance P<0.05 for difference.
2 results
2.1 platelet aggregation test result
Under PAF induces, the max platelet rate of ginkgolectone AB C group and bilobalide compositions group is compared with the normal saline group, and significant difference (p<0.01) all occurs, and each is organized platelet aggregation inhibition rate and is significantly increased; And the bilobalide group is compared with the normal saline group, the little or there was no significant difference of difference.Illustrate that the platelet aggregation that ginkgolectone AB C, bilobalide compositions group can be induced PAF produces inhibitory action, and the platelet aggregation that the bilobalide group is induced PAF does not produce inhibitory action or inhibitory action is very weak.
Table 10, bilobalide compositions platelet aggregation test result
Group |
1min |
5min |
MAX |
Platelet aggregation inhibition rate (%) |
Ginkgolectone AB C group |
26.37±5.89** |
40.67±8.48** |
45.43±7.49** |
35.30% |
1 group of bilobalide compositions |
29.21±5.75** |
41.52±7.32** |
46.29±7.58** |
34.82% |
2 groups of bilobalide compositionss |
24.93±3.21** |
35.79±6.98** |
42.38±8.67** |
38.84% |
The bilobalide group |
34.34±6.59 |
58.79±8.32 |
59.84±8.92 |
6.73% |
The normal saline group |
36.85±6.14 |
68.32±9.17 |
70.22±11.40 |
0.00% |
Compare * * p<0.01 with the normal saline group
2.2 platelets testing result
Mainly observe four kinds of platelet forms under the scanning electron microscope of 2000 times: circular, tree-like, flatten shape and assemble shape.Under PAF induces, the strong activation occurs in normal saline group platelet, and platelet adhesion power strengthens, and can see red blood cell adhesion on platelet.The platelet form is irregular, volume increases, stretch out and be the outstanding pseudopodium of spore shape, assemble the shape platelet counts and increase.Under PAF induces, ginkgolectone AB C group and bilobalide compositions group Platelet Size are more consistent, smooth surface, and the accumulation type platelet is more rare.
Table 11, bilobalide compositions are respectively organized the platelet form relatively after adding PAF
Compare * * p<0.01, * p<0.05 with the normal saline group.
2.3 platelet PF-4 and β-TG testing result
With the normal saline group, compare, the PF-4 level of ginkgolectone AB C, bilobalide compositions group significantly reduces (p<0.01); β-TG level also significantly reduces (p<0.01); PF-4 and β-TG level reduces, and illustrates that platelet release function is suppressed; And the PF-4 of bilobalide group and β-TG level does not have significant change.
Table 12, bilobalide compositions platelet PF-4 and β-TG testing result
Group |
PF-4(μg/ml) |
β-TG(μg/ml) |
Ginkgolectone AB C |
1.270±0.186** |
1.394±0.176** |
1 group of bilobalide compositions |
1.279±0.403** |
1.305±0.268** |
2 groups of bilobalide compositionss |
1.245±0.139** |
1.236±0.170** |
The bilobalide group |
1.583±0.245 |
1.604±0.244 |
Normal saline |
1.733±0.294 |
1.740±0.215 |
Compare * * p<0.01 with the normal saline group.
Brief summary:
From above-mentioned result of the test, bilobalide can promote the anti-platelet activity of ginkgolectone AB C, and in ginkgolectone AB C total amount: bilobalide=5~60:5~65 this in a big way in, all can bring into play facilitation preferably.
Sum up:
Although bilobalide does not have obvious anti-platelet aggregation activity, but, the present invention studies discovery, bilobalide not only can obviously promote the anti-platelet activity of different Semen Ginkgo diterpenes diterpenoids lactones monomers, can also produce remarkable facilitation to the anti-platelet activity of the different proportion compositions of two or more Semen Ginkgo diterpenes diterpenoids lactones.
By above-mentioned result of the test, can be drawn the following conclusions: bilobalide can promote the anti-platelet activity of Semen Ginkgo diterpenes diterpenoids lactones, and no matter is that monomer or compositions all have remarkable facilitation.
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