Analysis of anti-platelet aggregation components of Rhizoma Zingiberis using chicken thrombocyte extract and high performance liquid chromatography

Background The conventional procedure for screening bioactive components from traditional Chinese medicine is time-consuming, expensive and low efficient. Therefore, some alternative strategies are needed urgently. A novel method for screening anti-platelet aggregation components from oleoresins was developed using chicken thrombocyte extract and high performance liquid chromatography. Methods The anti-platelet aggregation components of oleoresins were combined with receptors, channels and enzymes of chicken thrombocytes under physiological environment. Unbound substances were washed away and bound compounds were eluted using specific phosphate buffered solution （PBS）. Compounds released from target sites were collected and analyzed by high performance liquid chromatography and LC-MS. The activity of three compounds which were screened from this model was confirmed using platelet aggregation pharmacology in vivo. Results There were four typical compounds that bound to the thrombocytes： 6-gingerol, 8-gingerol, 6-shogaol and 10-gingerol, and all had shown anti-platelet aggregation activities. Eight-gingerol displayed the best anti-platelet aggregation effect. Conclusions Chicken thromobcyte extract can be used to isolate chemicals that are ligands of the receptor or other bio-targets on the platelet. This may therefore be a simple and efficient method to screen for anti-platelet aggregation compounds from traditional Chinese medicine.

Thrombocyte counts in mice after the administration of methanolic extract of Melastoma malabathricum

Objective:To investigate the effect of methanolic extract of Melastoma malabathricum(M.malabathricum)in thrombocyte counts in mice.Methods:Methanolic extract of M.malabathricum corresponding to 1.5 to 2 mg/10 g body weight in saline was administered to mices via oral route.Control group was given normal saline.Twenty five microlitres of blood were drawn at 0 h and thereafterat 1,2,4,8,12,20 h after dosing via tail bleeding technique.The thrombocytes were counted in the triple laminated middle 25 squares of the haemocytometer using light microscope.Results:The control group showed a moderate rise after the administration of saline whereas the M.malabathricum treated group shows significant rise within 2nd h in thrombocyte counts with an increment of 51.64%compared to baseline count.Conclusions:Based on the results,it can be concluded that M.malabathricum could be a potential remedy in treating thrombocytopenic condition.

Trombinol,a bioactive fraction of Psidium guajava,stimulates thrombopoietin expression in HepG2 cells

Objective:To study the regulation of trombinol on thrombopoietin,an essential regulator of thrombocyte production.Methods:Effect of trombinol on thrombopoietin regulation was evaluated at the m RNA and protein levels in human hepatoma Hep G2 cells.The m RNA expressions were revealed by PCR and real-time PCR,while the protein expressions were analyzed using western blotting and human ELISA kit.Statistical differences between the test were determined by student's t-test with P < 0.05 was considered statistically significant.Results:Trombinol significantly increased the expression of thrombopoietin at the level of m RNA and protein secretion in Hep G2 cell lines.Trombinol with the concentration of15 mg/m L,positively induces 2.5-fold of thrombopoietin expression.Up-regulation of GABP,a transcription factor of thrombopoietin,is suggested to be involved in cellular regulatory mechanisms of trombinol.Here,our result shows convincing evidence that trombinol affects the thrombopoietin productions in vitro.This molecular explanation of thrombopoietin's stimulating function is in line with the traditional use of Psidium guajava for treatment of diseases involving thrombocytopenia.Conclusions:Thrombopoietin stimulating function of trombinol could be potentially considered as one of alternative treatment for thrombocytopenia-related cases,including post chemotherapy shock,dengue fever and liver failure.

Mechanism of blood coagulation in common carp (Cyprinus carpio)

In vitro,carp blood was anticoagulated by using MgSO4 at a final concentration of 22.2 mmol L-1 and sodium citrate at a final concentration of 11.8 mmol L-1.The coagulation times for carp plasma diluted by ion-free water(1:1),and that of carp plasma to which thrombocytes and small lymphocytes were added,were measured at 23℃ using standard methods,and then contrasted with the coagulation times for plasma obtained from chickens and rabbits.The shapes of the thrombocytes and small lymphocytes,which were either wet mounted or stained with hematoxylin and eosin,were observed under a light microscope.We found that:(i)the coagulation reaction of carp blood was significantly(P<0.01)accelerated by the addition of ion-free water;(ii)the three types of blood cells(thrombocytes,small lymphocytes and red blood cells)promoted plasma coagulation to a similar extent(P>0.05);(iii)in carp Mg^(2+)plasma and K_(2)C_(2)O_(4) plasma,the thrombocytes were usually morphologically normal,but many small lymphocytes were destroyed and became aggregated;(iv)in the citrate plasma,thrombocytes were often aggregated,but the small lymphocytes were usually morphologically normal;and(v)the coagulation time for chicken and rabbit plasma was significantly extended by adding ion-free water.

Haematopoetic effect of methanol extract of Nigerian honey bee (Apis mellifera) propolis in mice

Objective:To investigate the haematopoetic effect of methanol extract of Nigerian honey bee propolis in mice.Methods:Fifteen white Albino mice were grouped into 3(A-C)of 5 animals each.Group A mice serve as control group,while groups B and C received 300 and 600 mL/kg of honey bee propolis respectively,for 21 days.The haematological parameters were determined using the automated haematologic analyzer Sysmex kx21,(product of Sysmex Corporation,Japan)using standard techniques.The data were analyzed using ANOVA and the level of significance was at P<0.05.Acute oral toxicity study was conducted to determine it's safety on acute exposure.Results;Results showed significant(P<0.05)dose dependent elevations in platelate count,mean platelate volume,plateletecrit,platelete distribution weight,white blood cells,granulocyte count,lymphocytes and mid cell total count.The extract however produce no significantly(P>0.05)alteration to the erythrocytic indices like red blood cells,haematocrite,haemoglobin,mean corpuscular haemoglobin,mean corpuscular volume and red cell distribution width,but increase mean corpuscular haemoglobin concentration in dose related fashion.Acute oral toxicity showed the extract to be relatively safe at a high dose on acute exposure.However,21-days of treatment with the extract do neither increase nor decrease the body weight of the mice.Conclusions:Administration of methanol extract of Nigerian honey bee propolis in mice at the doses investigated has brought about leucopoietic and thrombopoietic changes without any significant effect on red blood cells and factors that relate to it,except for the mean corpuscular haemoglobin concentration.