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Paper text:
, there has been an increased resistance to antiplatelet drugs widely used in the treatment of vascular diseases .
it has been documented that some natural compounds in the diet can inhibit the platelet activation .
alpha - lipoic acid ( -la ) is a naturally occurring compound , synthesized in small amounts by plants and animals , including humans .
currently , -la is used as a dietary supplement for preventing and treating vascular , neurodegenerative , and endocrine diseases .
studies have reported that -la inhibit platelet aggregation in vitro and in type i diabetic patients .
dihydroquercetin ( dhq ) is a flavonoid founded in larix dahurica , larix sibirica , and pseudotsuga taxifolia .
despite the fact that dihydroquercetin possesses antioxidative , anti - inflammatory , hepatoprotective , and anticancer activities , it is not widely used .
thus , the objective of the study was to investigate the antiplatelet activity of -la and dhq and their combined use .
the rats were kept in the vivarium ( at temperature 22  2c , relative humidity of 55  15% , 12/12 h light - dark cycle ) .
food for conventional laboratory small rodents ( cjsc  assortment - agro  , russia ) and water was freely available .
the food contained antioxidants : vitamins a ( 30,000 iu / kg ) , e ( 130,000 iu / kg ) , c ( 78.7 mg / kg ) , and selenium ( 0.07 mg / kg ) .
rats were fasted for 12 hours preceding the experiment , but had access to water .
permission for experiments was received from the local ethics committee of the e.d.goldberg institute of pharmacology , siberian branch , russian academy of medical sciences ( tomsk , russia ) .
all experimental procedures with animals were performed in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ( march 18 , 1986 ; strasburg ; ets 123 ) and the requirement of public health ministry of russian federation order no .
diethyl ether (  kuzbassorgchem  ltd , russia ) , dhq (  ametis  ltd , russia ) , -la (  marbiofarm  ltd , russia ) , dimethylsulfoxide (  marbiofarm  ltd , russia ) , na3c6h5o7 (  sigma  co ltd , the usa ) , adenosine diphosphate ( adp ;  sigma  co ltd , the usa ) were used .
wistar rats were anesthetized with diethyl ether , and blood was collected from the common carotid artery .
blood was anticoagulated with sodium citrate ( 3.8% ; 1:9 , v / v ) and centrifuged at 400 g for 10 min at room temperature , platelet - rich plasma ( prp ) was obtained from the resulting supernatant .
the residue was centrifuged at 1,500 g for another 15 min to produce platelet - poor plasma ( ppp ) .
prp was diluted with ppp to a platelet concentration of 4  10 cells / ml before using in the aggregation assays .
-la and dhq were dissolved in dimethylsulfoxide ( dmso ) and added to platelet suspension correspondingly to the final concentration of 0.3 , 0.6 , 1.2 , and 0.6 , 2.5 , 5.0 mm 2 min before addition of adp .
equivolume quantity of dmso ( the final concentration of 0.6 mm ) was added in the control samples .
aggregation was stimulated by adp at a final concentration of 4  10 m and measured with a platelet aggregometer ( at-2 ,  npf  ltd , russia ) .
data were expressed as the means  standard error of the mean ( sem ) .
the rats were kept in the vivarium ( at temperature 22  2c , relative humidity of 55  15% , 12/12 h light - dark cycle ) .
food for conventional laboratory small rodents ( cjsc  assortment - agro  , russia ) and water was freely available .
the food contained antioxidants : vitamins a ( 30,000 iu / kg ) , e ( 130,000 iu / kg ) , c ( 78.7 mg / kg ) , and selenium ( 0.07 mg / kg ) .
rats were fasted for 12 hours preceding the experiment , but had access to water .
permission for experiments was received from the local ethics committee of the e.d.goldberg institute of pharmacology , siberian branch , russian academy of medical sciences ( tomsk , russia ) .
all experimental procedures with animals were performed in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ( march 18 , 1986 ; strasburg ; ets 123 ) and the requirement of public health ministry of russian federation order no .
diethyl ether (  kuzbassorgchem  ltd , russia ) , dhq (  ametis  ltd , russia ) , -la (  marbiofarm  ltd , russia ) , dimethylsulfoxide (  marbiofarm  ltd , russia ) , na3c6h5o7 (  sigma  co ltd , the usa ) , adenosine diphosphate ( adp ;  sigma  co ltd , the usa ) were used .
wistar rats were anesthetized with diethyl ether , and blood was collected from the common carotid artery .
blood was anticoagulated with sodium citrate ( 3.8% ; 1:9 , v / v ) and centrifuged at 400 g for 10 min at room temperature , platelet - rich plasma ( prp ) was obtained from the resulting supernatant .
the residue was centrifuged at 1,500 g for another 15 min to produce platelet - poor plasma ( ppp ) .
prp was diluted with ppp to a platelet concentration of 4  10 cells / ml before using in the aggregation assays .
-la and dhq were dissolved in dimethylsulfoxide ( dmso ) and added to platelet suspension correspondingly to the final concentration of 0.3 , 0.6 , 1.2 , and 0.6 , 2.5 , 5.0 mm 2 min before addition of adp .
equivolume quantity of dmso ( the final concentration of 0.6 mm ) was added in the control samples .
aggregation was stimulated by adp at a final concentration of 4  10 m and measured with a platelet aggregometer ( at-2 ,  npf  ltd , russia ) .
data were expressed as the means  standard error of the mean ( sem ) .
the rats were kept in the vivarium ( at temperature 22  2c , relative humidity of 55  15% , 12/12 h light - dark cycle ) .
food for conventional laboratory small rodents ( cjsc  assortment - agro  , russia ) and water was freely available .
the food contained antioxidants : vitamins a ( 30,000 iu / kg ) , e ( 130,000 iu / kg ) , c ( 78.7 mg / kg ) , and selenium ( 0.07 mg / kg ) .
rats were fasted for 12 hours preceding the experiment , but had access to water .
permission for experiments was received from the local ethics committee of the e.d.goldberg institute of pharmacology , siberian branch , russian academy of medical sciences ( tomsk , russia ) .
all experimental procedures with animals were performed in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes ( march 18 , 1986 ; strasburg ; ets 123 ) and the requirement of public health ministry of russian federation order no .
diethyl ether (  kuzbassorgchem  ltd , russia ) , dhq (  ametis  ltd , russia ) , -la (  marbiofarm  ltd , russia ) , dimethylsulfoxide (  marbiofarm  ltd , russia ) , na3c6h5o7 (  sigma  co ltd , the usa ) , adenosine diphosphate ( adp ;  sigma  co ltd , the usa ) were used .
wistar rats were anesthetized with diethyl ether , and blood was collected from the common carotid artery .
blood was anticoagulated with sodium citrate ( 3.8% ; 1:9 , v / v ) and centrifuged at 400 g for 10 min at room temperature , platelet - rich plasma ( prp ) was obtained from the resulting supernatant .
the residue was centrifuged at 1,500 g for another 15 min to produce platelet - poor plasma ( ppp ) .
prp was diluted with ppp to a platelet concentration of 4  10 cells / ml before using in the aggregation assays .
-la and dhq were dissolved in dimethylsulfoxide ( dmso ) and added to platelet suspension correspondingly to the final concentration of 0.3 , 0.6 , 1.2 , and 0.6 , 2.5 , 5.0 mm 2 min before addition of adp .
equivolume quantity of dmso ( the final concentration of 0.6 mm ) was added in the control samples .
aggregation was stimulated by adp at a final concentration of 4  10 m and measured with a platelet aggregometer ( at-2 ,  npf  ltd , russia ) .
data were expressed as the means  standard error of the mean ( sem ) .
-la and dhq inhibited platelet aggregation induced by adp in a concentration - dependent manner in vitro [ table 1 ] .
however , incubation of prp with -la ( 0.3 mm ) and dhq ( 0.6 mm ) did not change the aggregation amplitude and was comparable to control groups .
aggregation amplitudes in -la group ( 0.6 mm ) and dhq group ( 2.5 mm ) were less than control group by 1.6 and 1.5 times , respectively .
the antiplatelet activity of -la at 1.2 mm was more significant ( p < 0.05 ) and pronounced than that of dhq at 5.0 mm [ table 1 ] . while combination of -la and dhq in equal concentration of 0.6 mm reduced the aggregation amplitude as compared to the control group by 2.2 times ( p < 0.05 ) .
adp contributes to platelet activation occurring both during protective hemostasis ( formation of the initial platelet monolayer ) and during formation of occlusive platelet - rich thrombi .
two g protein - coupled receptors are known to be largely responsible for platelet responses to adp : p2y1 , which activates phospholipase c and mobilization of ca , and p2y12 , which suppresses camp formation so that activation can occur .
-la ( 0.3 - 0.8 mm ) dose - dependently inhibited platelet aggregation induced by collagen , adp , or arachidonic acid .
investigators link the antiplatelet activity of -la with an elevation of cyclic adenosine monophosphate ( camp ) formation , involving the subsequent inhibition of thromboxane a2 ( txa2 ) , cyclooxygenase-1 , protein kinase c-mediated pathways , peroxisome proliferator - activated receptors ( ppar ) , ca mobilization and inhibition of reactive oxygen species formation , and the increase of platelet membrane fluidity .
dhq ( 1 - 5 mm ) is able to inhibit platelet aggregation induced by adp and thrombin .
kubatiev et al . , showed that dhq produced the decrease in the levels of both base and adp- or thrombin - induced cytoplasmic ca levels and elevated camp and cyclic guanosine monophosphate ( cgmp ) contents in native and thrombin - activated platelets .
the present study demonstrates the inhibition of platelet aggregation by combined use of -la and dhq in vitro .
our study showed that -la and dhq possess antiplatelet activity in the concentration range reported by other authors .
from the data presented in the table 1 , ic50 values of 0.69  0.03 and 3.76  0.42 mm for -la and dhq were calculated .
secondly , dhq also increased the antiplatelet activity of -la by 1.4 times [ table 1 ] .
however , the mechanisms underlying dhq - mediated potentiation of platelet aggregation needs further research . on the basis of the facts mentioned above , -la and dhq have some common mechanisms of antiplatelet activity ( via camp , and its influence on ca mobilization ) .
moreover , antiplatelet effect of combination of the effect being observed from -la and dhq use was greater than the sum of -la and dhq effects then used separately .
enhanced antiplatelet activity of -la and dhq simultaneous use may be determined by the ability of dhq to raise the level of cgmp and as a result increase phosphodiesterase type 3 ( pde3 ) activity .
pde3 has a relatively high affinity for both camp and cgmp , but a much lower efficacy of hydrolysis for cgmp , behaving essentially as a pure camp pde ; it has also been referred to as cgmp - inhibited camp pde because cgmp may competitively inhibit amp hydrolysis . in conclusion , our study demonstrated that -la and dhq possessed the high antiplatelet activity and dhq increased the activity of -la .
objectives : to investigate the antiplatelet activity of alpha - lipoic acid ( -la ) and dihydroquercetin ( dhq).materials andmethods : antiplatelet activity of the -la and dhq was evaluated in rich platelet plasma of rat . 
 the platelet aggregation was induced by adenosine diphosphate ( adp ) in concentration of 4  10 - 5 m.results:-la and dhq inhibited platelet aggregation in concentration - dependent manner . 
 the antiplatelet activity of -la was more pronounced than dhq . 
 dhq also increased the antiplatelet activity of -la by 1.4 times.conclusion:combined simultaneous use of -la and dhq possessed the high antiplatelet activity , and dhq potentiated the activity of -la .