皮肤冻存对肉桂挥发性成分体外透皮特性的影响

The effect of transdermal properties in vitro on the volatile components in Cinnamomum cassia by cryopreservation of skin

关键词:  肉桂油  体外透皮  冻存  皮肤活力  CCK8  MTT

Key words:  cinnamon oil  in vitro transdermal  cryopreservation  skin vitalty  CCK8  MTT

摘要:

目的 建立同时测定肉桂挥发油中香豆素、肉桂醇、肉桂酸、肉桂醛和邻甲氧基肉桂醛含量的HPLC方法，并比较离体皮肤冻存对这些成分体外透皮特性的影响。方法 采用Franz扩散池法对肉桂油进行体外透皮吸收实验，以冻存前后的小鼠离体皮肤做渗透屏障，同步用MTT法和CCK8法监测皮肤活力。采用Agilent C18色谱柱(4.6 mm ×250 mm，5 μm)，乙腈-0.2%磷酸(30：70)为流动相，流速1mL·min-1，检测波长为250 nm和278 nm，同时测定透皮过程中肉桂这5个挥发性成分的含量变化，统计分析其28天内多点下的24h渗透规律。结果 香豆素、肉桂醇、肉桂酸、肉桂醛和邻甲氧基肉桂醛等成分在该色谱条件下分离度好，进样量分别在0.0013 ~ 0.7414、0.0043 ~ 2.3799、0.0105 ~ 5.7974、0.0389 ~ 44.5830、0.0027 ~ 1.5224 μg范围内线性关系良好(r＞0.999)。离体皮肤在常用冻存液中保存28天，5个成分24h的渗透速率变化范围分别是1.64 ~ 6.43、4.88 ~ 22.84、3.76 ~ 60.40、197.53 ~ 274.39、8.20 ~ 12.60 μg·cm-2·h-1；冻存皮肤的活力前7天下降明显，之后仍继续缓慢下降。结论 所建立的HPLC方法可简便、快捷地用于肉桂油中5个挥发性成分的含量测定，可为相关药物的研发提供参考。离体皮肤冻存28天，期间其活力会持续下降，5个成分24h的渗透速率变化规律不同，香豆素和肉桂酸的会增大，肉桂醇的会减小，而肉桂醛和邻甲氧基肉桂醛的变化不大。用CCK8法和MTT法监测皮肤活力变化的趋势相近。

Abstract:

OBJECTIVE To develop an HPLC determination method of coumarin，cinnamyl alcohol，cinnamic acid，cinnamaldehyde and 2-methoxy cinnamaldehyde in cinnamon oil，and to evaluate the in vitro transdermal properties of these components utilizing cryopreserved skin. METHODS The transdermal absorption of cinnamon oil was examined using Franz diffusion cells with cryopreserved mouse skin as the permeation barrier. Simultaneously，skin vitality was monitored using MTT and CCK8 assays. An Agilent C18 chromatographic column(4.6 mm × 250 mm，5 μm) with acetonitrile-0.2% phosphoric acid(30：70) as the mobile phase at a flow rate of 1 mL·min-1 was employed，Detection was performed at 250 nm and 278 nm to simultaneously monitor the content changes of these 5 volatile components，the 24-hour permeation regularities were analyzed over a period of 28 days. RESULTS Excellent separation of coumarin，cinnamyl alcohol，cinnamic acid，cinnamaldehyde and 2-methoxy cinnamaldehyde was achieved under the chromatographic conditions，good linear relationships(r＞0.999) were observed within the concentration ranges of 0.0013 ~ 0.7414、0.0043 ~ 2.3799、0.0105 ~ 5.7974、0.0389 ~ 44.5830、0.0027 ~ 1.5224 μg respectively.The range of 24-hour permeation rate changes of the above components is as follows:1.64 ~ 6.43、4.88 ~ 22.84、3.76 ~ 60.40、197.53 ~ 274.39、8.20 ~ 12.60 μg·cm-2·h-1.The vitality of cryopreservation skin decreased rapidly within the first 7 days then more gradually thereafter. CONCLUSION The proposed HPLC method offers a rapid and convenient means for determining the 5 volatile components in cinnamomum cassia oil，providing valuable insights for drug development.The vitality of cryopreservation skin in vitro continued to decline over 28 days.The variation patterns of 24-hour permeation rates of these components are different. Specifically，coumarin and cinnamic acid showed increased permeation，cinnamyl alcohol decreased，while cinnamaldehyde and 2-methoxy cinnamaldehyde remained relatively unchanged.Both CCK8 and MTT assays demonstrated similar trends in monitoring skin vitality.