| Objective: Compound Luotuopengzi ointment(CLO) is a traditional Uighur medicine treatment of Rheumatoid arthritis(RA). To improve its formulation defects and ease of use, a compound Luotuopengzi hydrogel patch(CLHP) based on the original formulation was developed. To confirm the pharmacodynamics of CLHP, compared the the effect of CLHP and CLO acute inflammation, chronic inflammation, pain,RA and skin irritation. Studied the pharmacokinetic of CLHP and CLO in rat base on Microdialysis technique, to acquire the process in vivo, transdermal properties and lay the foundation for further study. Methods:(1) Experiments of xylene induced ear edema in mice, carrageenan-induced rat paw edema and cotton-induced rat granuloma to study the CLO and CLHP anti-inflammatory effe; Through acetic acid writhing test in mice to observe the CLO and CLHP analgesia; Study the anti-rheumatoid arthritis of CLO and CLHP used complete Freund’s adjuvant-induced arthritis test in rats. The intact and damaged skins of New Zealand rabbits were used to carry out irritation trial of CLO and CLHP.(2) Use microdialysis technique and high performance liquid chromatography–fluorescence to study the pharmacokinetic of harmaline and harmine in vivo in rats. Homemade stable performance, flexible and can be used for linear microdialysis probe in vivo microdialysis study by microdialysis in vivo experiments to evaluate the perfusion rate, the impact of the drug concentration on the probe sample recovery. The probes were made using the skin of rats and blood microdialysis sampling synchronization, high performance liquid chromatography combined with the change in real-time monitoring of the skin and blood harmaline and harmine concentrations. The pharmacokinetics parameters were calculated by pharmacokinetics software DAS2.0 to investigate the influence. Results:(1) Compared with control group,the three dose group of CLHP and CLO could inhibit the acute inflammation of mouse ear swelling(P<0.05),the sub-acute inflammation of carrageenan induced rat foot swelling(P<0.05),the chronic inflammation of cotton-induced rat granuloma(P<0.05),the pain induced acetic acid(P<0.05).For th sub-acute inflammation and echronic inflammati, CLHP has a better effect than CLO(P<0.05).The CLHP and CLO have same effects on acute inflammation and analgesic.For complete Freund’s adjuvant-induced rat articular, the role of inflammation and pathological examination showed that the high and middle dose group of CLHP and the high dose group of CLO have a good effect. There is a slight skin irritation on New Zealand rabbits for CLO and CLHP.(2) High Performance Liquid Chromatography Fluorescence Determination of harmaline and harmine chromatographic conditions were as follows: acetonitrile-ammonium acetate buffer(3.4g ammonium acetate + 0.5m L of glacial acetic acid + 500 m L water) as the mobile phase = 18:82, column octadecyl silane bonded silica as a filler Shimadzu ODS- C18(5μm, 250 mm × 4.6mm), a flow rate of 1.0 m L/min; injection volume: 20 μL; column temperature: 30 ℃; harmaline excitation wavelength λex=375nm, emission wavelength λem=470nm; harmine excitation wavelength λex=320nm, emission wavelength λem=420nm. The valuation results shown that the linear ranges ofharmaline and hamine were 0.20~412.00ng/m L(r=0.9999), 0.89~453.13ng/m L(r=0.9999). Chromatography of specificity, precision, accuracy and stability are in line with the requirements of biological sample analysis. Determination of the increment and decrement technique probes in vitro method consistent recovery, the recovery of probe in vivo perfusion with increasing speed decreases, the recovery has nothing to do with the concentration of the drug probe, continuous sampling probe recovered within 20 h rate stability. After CLHP and CLO transdermal administration, harmaline and harmine in blood e were very lowwithin the sampling tim. Because of only individual points can be measured,so we can not express the blood after administration the kinetic characteristics of pharmacokinetics. The pharmacokinetic parameters of harmaline after the CLHP and CLO percutaneous administration: Cmax were 24.27 ± 1.98 and 8.27 ± 0.65(μg/L), Tmax were 13.00 and 13.67 ± 0.58(h), MRT were 11.58 ± 0.07 and 10.86 ± 0.11(h), AUC(0-t) were 151.25 ± 8.10 and 87.43 ± 1.77(μg·h/L), AUC(0-∞) were 164.26 ± 14.11 and 152.19 ± 6.57(ug·h/L).where Cmax, MRT, AUC(0-t) difference was statistically significant(P<0.05). The pharmacokinetic parameters of harmine after the CLHP and CLO percutaneous administration: Cmax were 80.25 ± 9.80 and 60.88 ± 3.68(μg/L), Tmax were 15.33 ± 0.58 and 15.00(h), MRT was 14.03 ± 0.17 and 13.07 ± 0.30(h), AUC(0-t) were 285.25 ± 31.65 and 148.13 ± 18.29(μg·h/L), AUC(0-∞) were 315.25 ± 37.27 and 150.21 ± 19.19(μg·h/L) where Cmax, MRT, AUC(0-t), AUC(0-∞) the difference was statistically significant(P<0.05).Conclusion: CLHP and CLO have good effects on anti-inflammatory,analgesic and anti-arthritic, but at the anti-chronic inflammation and ease of use in terms of CLHP than CLO. There is a slight skin irritation on New Zealand rabbits for CLO and CLHP. The homemade microdialysis probe have a stable performance.The result of Pharmacokinetic study suggest that the CLHP after administration changed the pharmacokinetic parameters of CLO.The CLHP have higher bioavailability and longer duration. It is expected to produce better efficacy. |
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