| Objective:To establish a detection method of liquid chromatography-tandem mass spectrometry(LC-MS)for the determination of vitamin K2 in rat plasma,simultaneously to analyze the bioequivalence of the three preparations of vitamin K2 and to compare the effects and the mechanisms of the above three vitamin K2 preparations in osteoporotic rats.Methods1.The rats were intragastrically given a single time and single dose of the test drugs or reference preparationof vitamin K2,and LC-MS method was used to measure the drug concentration in the plasma at different time points after administration,and to evaluate the bioequivalence of the above three preparations of vitaminK2 through calculating the main pharmacokinetic parameters.2.The model of osteoporosis in female SD rats was duplicated by ovariectomy.The wet,dry and gray weight of the right femur and the third lumbar vertebra were determined and the content of bone calcium was determined by flame atomic absorption spectrometry.The dual energy X-ray absorption method was used to test BMD and BMC of the left femur and the fourth lumbar spine.The left femur and the fourth lumbar vertebrae were biomechanically measured by bone strength meter.The bone microstructure of the left femur was observed by hematoxylin-eosin.3.RT Q-PCR technology was used to detect the mRNA expression level of Runx2 in the bone tissue.Results1.Pharmacokinetics:Compared with reference preparation,in the group of vitamin K2 powder,Cmax was lower than reference preparation(P<0.05),but AUC(0-72 h)had no statistically significant;compared with reference preparation,Cmax and AUC(0-72 h)had no statistical significance in the group of vitamin K2 oil(P>0.05).The values of Tmax in the above three groups were 6,4 and 4 h,respectively.2.Anti-osteoporotic effects(pharmacodynamics):(1)Bone weight and bone calcium content test results showed:compared with the model group,the three vitamin K2 preparations significantly increased bone calcium content of femur and vertebrae,and increased wet,dry and gray weight(P<0.05).Compared with the reference preparation,vitamin K2 powder and oil preparations had no obvious difference on the above indexes of vertebrae.(2)Bone mineral density results showed:Compared with the model group,the three vitamin K2 preparations significantly increased BMD and BMC of femur and vertebra(P<0.05).Compared with the reference preparation,vitamin K2 powder and oil preparations had the similar effect on BMD and BMC of femur and vertebrae.(3)Bone biomechanics results show that compared with the model group,vitamin K2 powder could increase the femoral load(P<0.05),and had no significant effect on vertebral load;vitamin K2 oil and reference preparations significantly increased the maximum load of the femur and lumbar spine(P<0.05).Compared with the reference preparation,the effect of vitamin K2 powder and oil on femoral load and vertebral load was not significantly different.(4)Bone morphometrics results show:compared with the model group,vitamin K2 powder had no significant effect on trabecular bone spacing,increased trabecular bone count,trabecular bone width,and trabecular bone surface area percentage(P<0.05);vitamin K2 oil and reference preparations could increase trabecular bone count,trabecular bone surface area percentage and trabecular bone width,reduce trabecular bone space(P<0.05).Compared with the reference preparation,vitamin K2 powder and oil had no obvious difference on the above indicators.3.RT Q-PCR results showed that compared with the model group,the three vitamin K2 preparations significantly increased 2-??Ct of Runx2(P<0.01).Compared with the reference preparation,the effect of powder and oil had no obvious difference.Conclusions1.The powder and oil preparations of vitamin K2 are bioequivalent to the reference vitamin K2 oil.2.All the three vitamin K2 preparations can improve osteoporosis.The effect of vitamin K2 oil is comparable to that of reference vitamin K2 oil,and vitamin K2 powder slightly lower than that of reference vitamin K2 oil.3.Upregulation of Runx2 mRNA expression may be the action mechanism of the above three vitamin K2 preparations,and vitamin K2 oils and powders are equivalent to reference vitamin K2 oils. |
PDF Full Text Request