Study On Preparation Technics Of Compound Blood-Activating And Analgesic Gel-Cream And Its Preliminary In Vivo And In Vitro Assessment

ObjectiveTo prepare the Compound Blood-activating and Analgesic gel-cream(CBAg)from the blood-circulation-promoting and blood-stasis-removing prescription recorded in the "Li Zhongyu Clinical Experience Collection",and optimize the extraction method of Panax notoginseng,Aconitum carmichaeli Debx and Aconitum kusnezoffii Reichb involved in the mentioned prescription,laying the foundation for preparing the anticipated CBAg;to screen out the best prescription and preparation technic for the CBAg,and select the optimum transdermal penetration enhancer;to assess the quality of the prepared CBAg;to provide accordance for the local skin in vivo pharmacokinetics study,this paper established an in vivo skin microdialysis analysis method;to validate the therapeutic efficacy of the CBAg.Methods1.Study on the extraction technics of the CBAgThe HPLC method for the determination of notoginsenoside R1 and aconitine was established to select the best extraction technics of Panax notoginseng,Aconitum carmichaeli Debx and A conitum kusnezoffii Reichb leveraging orthogonal test.2.Study on the preparation and in vitro transdermal release of the CBAgBased on the sensory evaluation,initial adhesion,and continuous adhesion as indices,the matrix composition of CBAg was optimized by orthogonal test on the basis of a single factor,and the transdermal penetration enhancer was optimized by Franz diffusion cell method regarding its type and amount,which is compared with the original prescriptions’ usage.3.Quality assessment of the CBAgAccording to the preparation quality standard of the Chinese Pharmacopoeia part Ⅳ(2015 edition),the traits,qualitative identification,shaping,heat resistance,cold resistance,initial adhesion,continuous adhesion,skin tracing-ability of the CBAg were tested.The drug content was also quantified.4.Establishment of the in vivo skin microdialysis method for CBAgThe UPLC analyzing method of dracorhodin was established.The effects of flow rate,concentration and dracorhodin concentration on the in vitro recovery of the probe were investigated by the incremental method and the reduction method and the stability of the in vivo recovery of the probe was investigated.5.Preliminary study on the pharmacodynamics of the CBAgThe preliminary study of the pharmacodynamics was carried out,setting weight and recovery of the injured muscle as indices and the impact damage model as a soft tissue injury model.Results1.Study on the extraction technics of the CBAgIt is preferred that the optimal extraction process of Panax notoginseng is 10 times 60%ethanol reflux extraction for 2 times,30 min per time;the optimal extraction process of A conitum carmichaeli Debx and Aconitum kusnezoffii Reichb is 8 times 70%ethanol reflux,extracted once and 30 min each time.2.Study on the preparation and in vitro transdermal release of the CBAgThe screened best prescription for CBAg is composed of NP700(4 g),glycerin(12 g),glycolic aluminum(0.08 g),CMC-Na(0.18 g),carbomer(0.1 g),tartaric acid(0.1 g),drug extract 3 mL,dracorhodin powder(1 g),and 5%transdermal penetration enhancer.The steady-state diffusion rates of dracorhodin and notoginsenoside R1 in CBAg were 0.2808 μg·cm-2·h-1 and 2.8611μg·cm-2·h-1 respectively,and the 12 h cumulative releasing amount is 3.1356μg·cm-2 and 23.8403 μg·cm-2.The steady-state diffusion rates of dracorhodin and notoginsenoside R1 in the original prescription were 0.1379 μg·cm-2·h-1 and 1.1733 μg·cm-2·h-1 respectively,and the cumulative transdermal volume in 12 hours reached 1.3374 μg·cm-2 and 13.6114 μg ·cm-2.3.Quality assessment of the CBAgThe preliminary quality evaluation results of CBAg displayed that the CBAg was a brown semi-solid preparation;notoginsenoside R1 and dracorhodin was identified,and aconitine was limitedly quantified;the test result of shaping,heat resistance,and cold resistance are in line with the requirements of the Chinese Pharmacopoeia(2015 edition);the three batches of CBAg were all able to adhere to the No.19 steel ball,and do not fall off on the test plate after more than 1 h;the average amount of three batches of CBAg was 26.42 g/100 cm2;every piece of CBAg contained 1.3026 mg of dracorhodin and 2.2775 g of notoginsenoside R1.4.Establishment of the in vivo skin microdialysis method for CBAgWith the increase of flow rate,the in vitro recovery of the dracorhodin probe gradually decreased,while the change of blood concentration and temperature had little effect on the recovery of the probe.At the respective flow rates of 1.0,1.5,2.0,and 2.5 μL/min,the recovery of the probes in the incremental method and the reduction method were(41.99±1.16)%and(39.05± 0.73)%,(34.56 ± 1.13)%and(31.26 ± 0.70)%,(29.99 ± 0.26)%and(29.59 ± 0.38)%,(16.88±0.19)%and(25.92±1.97%),respectively.In vivo recovery of the probes stayed stable relatively within 10 h.5.Preliminary study on the pharmacodynamics of the CBAgThe rats’weight of each group gradually increased with time,and there was a significant difference on the 3rd day and the 5th day compared with before those the administration(P<0.05).On the 3rd day,the rats’weight of each dose group showed a significant change compared with the blank matrix group(P<0.05),and the 5th day did not see such distinct change(P>0.05).With time flowing,the syndrome index assessment scores for soft tissue injury of each rats group declined step by step.On the 3rd day,a significant difference between each dose group and the blank matrix group(P<0.05),whereas the middle,high and low dose groups saw no significant change compared with the original prescription group(P>0.05).On the 5th day,a significant difference between the dose groups and the blank matrix group(P<0.05)still existed,and a significant difference between the low dose group and the original prescription group(P<0.05)was also seen,but the middle and high dose groups showed no significant difference from the original prescription groups(P>0.05).ConclusionThe blood-circulation-promoting and blood-stasis-removing prescription recorded in "Li Zhongyu Clinical Experience Collection" was converted into the CBAg.By optimizing the extraction process of the CBAg and the ratio of matrix prescription,the optimal transdermal enhancer was selected which brought a better penetration ability than the original prescription.The results of in vitro transdermal release test,quality assessment,and the preliminary pharmacodynamics test indicated that the preparation process of the CBAg is reasonable,feasible and stable,achieving the pre-anticipated research purpose.