Characteristics Research Of Percutaneous Penetration Specific Chromatogram Of Rhei Radix Et Rhizoma,Cortex Phellodendri Amurensis,Lycopi Herba,Cacumen Platycladi And Shuangbai Spray Release Agent

Shuangbai powder is the experience side of Guangzhou University of Traditional Chinese Medicine. The side which is made of Rhei Radix Et Rhizoma, Cortex Phell odendri Amurensis, Lycopi Herba, Cacumen Platycladi and mint, with blood stasis, clearing heat and detoxification, swelling and pain, have been widely used in the treatment of acute soft tissue injury, sores, bed sores, fractures, phlebitis and other diseases. Aiming at the deficiencies of the traditional formulations of Shuangbai powder, the study group has developed Shuangbai spray release agent, which contain dual advantages of spray and film former. This thesis on the basis of preliminary study of percutaneous penetration of Shuangbai powder and its component herbs, has furtherly optimizaed the method of HPLC specific chromatogram and parameter of percutaneous penetration, and studied specific chromatogram of percutaneous penetration of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi and Shuangbai Spray release agent with exploring characteristics of different constituent groups of Chinese herbal medicine.Objective1. To establish HPLC specific chromatogram analysis method of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi and provide analytical tools for quality control and percutaneous penetration test; To study the correlation of specific chromatogram between four of herbs and their extracts.2. To establish specific chromatogram analysis method of Shuangbai spray release agent and study out specific chromatogram of different constituent groups, and provide analytical tools for quality control and percutaneous penetration test.3. With traditional Chinese medicine specific chromatogram technology as well as percutaneous penetration test combinated, to establish percutaneous penetration specific chromatogram of ethanol extracts and decoctums of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi and explore characteristics of different constituent groups of Chinese herbal medicine, and compare transdermal promotion of menthol.4. To establish percutaneous penetration specific chromatogram of Shuangbai Spray release agent and explore characteristics of different constituent groups of Chinese herbal medicine; To clarify advantages of Shuangbai spray release agent percutaneous penetration and technical rationality through comparison between Shuangbai spray release agent and its single herbs.Methods1. The HPLC specific chromatogram analysis method of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi and their purification position was established and correlations of them were researched with traditional Chinese medicine HPLC specific chromatogram technology as well as specific chromatogram similarity evaluation system software writed by the State Pharmacopoeia Commission Specific chromatogram similarity evaluation system software (2004A version) adopted.2. The HPLC specific chromatogram analysis method of Shuangbai spray release agent was established with two detection wavelengths. Component of anthraquinones were analyzed at 430 nm while component of phenolic acids, alkaloids, flavones and other types were analyzed at 280 nm.3. The percutaneous penetration research of ethanol extracts and decoctum of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi and Shuangbai spray release agent were carried out with percutaneous penetration test adopted. Franze diffusion cell method was used with saline as receiving liquid, abdominal skin of nude mice as a transdermal barrier, collection period of receiving liquid as 2,4,6,8,10,12 hours; According to the clinical use usually, test temperatures were adjusted with ethanol extracts as (32 ± 0.5) ℃ and decoctums as (37 ± 0.5) ℃.With analysis methods of HPLC specific chromatogram established adopted, specific chromatogram of various samples before and after percutaneous permeation were analyzed; With correlated coefficient method of SPSS software used, similarities of specific chromatogram of various samples before and after percutaneous permeation were analyzed.4. With traditional Chinese medicine specific chromatogram technology as well as percutaneous penetration test combinated and principal components of percutaneous permeation parameters calculated refered, and characteristic peak areas of various samples before and after percutaneous permeation refered, parameters of percutaneous permeation specific chromatogram were studied out: (1) Converted peaks area of cumulative penetration of characteristic peaks (QA); (2) Cumulative percentageS of characteristic peaks (QA%); (3) Zero-order equation of characteristic peaks cumulative percentages of transmission-time (QA%=Ja*t+B), Transdermal rate constant of characteristic peaks (JA, h-1) and Correlated parameters (r1; (4) First-order equation of characteristic peaks cumulative percentages of transmission-time (QA%= JB*t1/2+C and Correlated parameters (r2).Results1. The HPLC specific chromatogram analysis methods of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi were optimizaed and correlations of four herbs and their extracts were analyzed.(1) The study of HPLC specific chromatogram of components of anthraquinones and other types of Rhei Radix Et Rhizoma were carried out with detection wavelength in 430 nm and 280 nm optimizaed.14 characteristic peaks of anthraquinones were marked with 5 Free anthraquinone (aloe emodin, rhein, emodin, chrysophanol and emodin monomethyl ether) identified in 430 nm; 10 characteristic peaks were marked with 2 specific compounds (gallic acid and catechins) identified in 280 nm. Similarities of Rhei Radix Et Rhizoma and its ethanol extract in the same batches were during 0.992-0.999 and 0.987-0.999 in 430 nm and 280 nm, prompting that routine alcohol extraction process can preserve characteristic components of Rhei Radix Et Rhizoma.(2) Cortex Phellodendri Amurensis has marked 17 characteristic peaks and its ethanol extracts has marked 15～17 characteristic peaks with 4 specific compounds (magnoflorine, jatrorrhizine, palmatine, berberine) identified. Similarities of Cortex Phellodendri Amurensis and its ethanol extract in the same batches were during 0.926～0.999, prompting that routine alcohol extraction and macroporous resin purification process can preserve characteristic components of Cortex Phellodendri Amurensis.(3) Lycopi Herba has marked 20 characteristic peaks with 1 specific compounds (caffeic acid) identified. Similarities of Lycopi Herba and its ethanol extract in the same batches were during 0.988-0.998, prompting that routine alcohol extraction and macroporous resin purification process can preserve characteristic components of Lycopi Herba.(4) Cacumen Platycladi has marked 17 characteristic peaks with 2 specific compounds (quercitrin and quercetin) identified. Similarities of Cacumen Platycladi and its extract in the same batches were during 0.779-0.985, prompting that routine decoctum process can preserve characteristic components of Cacumen Platycladi.2. The percutaneous penetration specific chromatogram of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi and their ethanol extracts, ethanol extracts containing 0.1% menthol and decoctums were established. The percutaneous permeation characteristics of main component groups of four herbs were gained.(1) In 430 nm wavelength, component groups of anthraquinones of Rhei Radix Et Rhizoma ethanol extracts and decoctums could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a large gap, while similarities of them were only during 0.394-0.568 and similarities of receiving liquid during different time periods were mostly over 0.84.The transmittance constant (JA) of 10 characteristic peaks of anthraquinones components in three concentrations of ethanol extracts were during 0.003～0.183, whose differences were 61 times. JA of peak 5, peak 7 (rhein) and peak 6 (aloe rhubarb prime) were during 0.069-0.183,0.036-0.118 and 0.021-0.034, and the other were below 0.029; JA of peak 8 (emodin), peak 9 (chrysophanol) and peak 10 (physcion) were during 0.003-0.012, significantly smaller than the other characteristic peaks. After adding 0.1% mint, JA of characteristic peaks of Rhei Radix Et Rhizoma anthraquinones components increased from 0.02 to 0.90 times; Percutaneous permeation properties of decoctums of Rhei Radix Et Rhizoma anthraquinones components were better than ethanol extracts.In 280 nm wavelength, components group of phenolic acids and other types of Rhei Radix Et Rhizoma ethanol extracts and decoctums could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a large gap, while similarities of them were below 0.766 and similarities of receiving liquid during different time periods were over 0.49. JA of 10 characteristic peaks of phenolic acids and other types in three concentrations of ethanol extracts were during 0.023-1.199, and JA of peak l-peak 2 were during 0.036-0.238.JA of peak 9, were larger than 0.36, significantly larger than the other characteristic peaks. After adding 0.1% mint,JA of characteristic peaks of Rhei Radix Et Rhizoma anthraquinones components increased from 0.02 to 0.37 times; Percutaneous permeation properties of decoctums of Rhei Radix Et Rhizoma phenolic acids and other types components were similar to ethanol extracts.(2) The components group of alkaloids of Cortex Phellodendri Amurensis ethanol extracts and decoctums could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a small gap, while similarities of them were during 0.881-0.969 and similarities of receiving liquid during different time periods were during 0.841-0.999.JA of 13 characteristic peaks of alkaloids in three concentrations of ethanol extracts were during 0.041-1.651, and JA of peaks 4 (magnoflorine), peak 10 (jatrorrhizine) and peak 11 (palmatine) and peak 12 (berberine) were larger than 0.06,0.05,0.06 and 0.07, and JA of peak 13, were larger than 0.94. After adding 0.1% mint, JA of characteristic peaks of Cortex Phellodendri Amurensis alkaloids components increased from 0.01 to 1.08 times; Percutaneous permeation properties of decoctums of Cortex Phellodendri Amurensis alkaloids components were similar to ethanol extracts, but JA were smaller than ethanol extracts, prompting that percutaneous permeation of them were weaker.(3) The components group of phenolic acids and other types of Lycopi Herba ethanol extracts and decoctums could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a small gap, while similarities of them were only during 0.465-0.915 and similarities of receiving liquid during different time periods were during 0.564-0.999. JA of 12 characteristic peaks of phenolic acids and other types in ethanol extracts, ethanol extracts containing 0.1% menthol and decoctums of Lycopi Herba were during 0.006-0.329, and JA of peaks 2 (caffeic acid) were during 0.097-0.235. After adding 0.1% mint, excepting peak 1,JA of characteristic peaks of Lycopi Herba phenolic acids and other types components increased from 0.07 to 1.82 times; Percutaneous permeation properties of decoctums of Lycopi Herba phenolic acids and other types components were similar to ethanol extracts, but JA were larger than ethanol extracts, prompting that percutaneous permeation of them were stronger.(4) The component groups of flavones and other types of Cacumen Platycladi ethanol extracts and decoctums could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a small gap, while similarities of them were only during 0.158-0.918 and similarities of receiving liquid during different time periods were during 0.634-0.992. JA of 12 characteristic peaks of flavones and other types in ethanol extracts, ethanol extracts containing 0.1% menthol and decoctums of Cacumen Platycladi were during 0.006-0.346, and JA of peaks 11 (quercitrin) were during 0.006-0.053. After adding 0.1% mint, JA of characteristic peaks of Lycopi Herba phenolic acids and other types components increased from 0.26 to 6.06 times, while peaks 11 (quercitrin) and peaks 13(quercetin) increased 3.55 and 2.37 times; Percutaneous permeation properties of decoctums of Cacumen Platycladi flavones and other types components were similar to ethanol extracts, but JA were larger than ethanol extracts, prompting that percutaneous permeation of them were stronger.3. The percutaneous penetration specific chromatogram of Shuangbai Spray release agent were established. The percutaneous permeation characteristics of main component groups of Shuangbai Spray release agent were gained.(1) In 430 nm wavelength, components group of anthraquinones of Shuangbai Spray release agent could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a large gap, while similarities of them were only 0.793 and similarities of receiving liquid during different time periods were during 0.988-0.998. The transmittance constant (JA) of 10 characteristic peaks of anthraquinones components were during 0.012～0.696, and JA of peak 1-peak 7 were during 0.130-0.696. JA of peak 5, peak 6 (aloe rhubarb prime) and peak 7 (rhein) were larger than 0.366,0.391 and 0.696; JA of peak 8 (emodin), peak 9 (chrysophanol) and peak 10 (physcion) were 0.028,0.048 and 0.012, significantly smaller than the other characteristic peaks.(2) In 280 nm wavelength, component groups of phenolic acids, alkaloids, flavones and other types of Shuangbai Spray release agent could percutaneous penetration basically, but the relative ratio of characteristic peaks of specific chromatogram before and after percutaneous permeation made a large gap, while similarities of them were only 0.409 and similarities of receiving liquid during different time periods were during 0.986-0.999. The transmittance constant (JA) of 18 characteristic peaks of phenolic acids, alkaloids, flavones and other types were during 0.050-1.939, and JA of peak 1 (gallic acid), peak 4(caffeic acid), peak 7 (magnoflorine), peak 17 (berberine) and peak 18 (palmatine) were 0.212,0.354,0.163,0.184 and 0.157.4. The characteristic analysis of four herbs and Shuangbai Spray release agent were carried out, while the percutaneous permeation characteristics of different component groups of Chinese medicine were gained preliminarily.(1) The correlated parameters r1 of zero-order equation of characteristic peaks cumulative percentages of transmission-time of different samples mostly over r2, suggesting that in the circumstances of that the activity of test skins were better, the percutaneous permeation of Chinese medicine contained uniform state.(2) Percutaneous permeation of anthraquinones components of Shuangbai Spray release agent were stronger than Rhei Radix Et Rhizoma ethanol extracts and Rhei Radix Et Rhizoma ethanol extracts containing 0.1% menthol, suggesting that transdermal penetration of Shuangbai Spray release agent towards them were excellent; In the Shuangbai Spray release agent, characteristic peaks of component groups of phenolic acids, alkaloids, flavones and other types, Percutaneous permeation of peak 1(gallic acid), peak 4 (caffeic acid), peak 7(magnoflorine), peak 14 (quercitrin), peak 17 (berberine) and peak 18 (palmatine) were stronger than Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi ethanol extracts and their ethanol extracts containing 0.1% menthol, suggesting that transdermal penetration of Shuangbai Spray release agent towards them were excellent, and clarifying that process is reasonable and meet the percutaneous penetration need of drugs for external use.(3) In the ethanol extracts of four herbs, JA of Rhei Radix Et Rhizoma phenolic acids and other types components were 0.057-0.304, and peak 1 (gallic acid) was 0.238, while percutaneous permeation of them were basically more excellent than Rhei Radix Et Rhizoma anthraquinones components and other three herbs; JA of Cortex Phellodendri Amurensis alkaloids components were 0.080-0.176, and peak 4(magnoflorine), peak 11 (palmatine) and peak 12(berberine) were 0.139,0.147 and 0.176, while percutaneous permeation of them were more excellent than Cacumen Platycladi and Lycopi Herba; Jh of Cacumen Platycladi peak 1～7 were 0.015-0.086, while percutaneous permeation of them were slightly more excellent Lycopi Herba, but peak 2(caffeic acid) was 0.137 and percutaneous permeation of it was similar to Cortex Phellodendri Amurensis.(4) In the decoctums of four herbs, percutaneous permeation of Rhei Radix Et Rhizoma phenolic acids and other types components were basically more excellent than anthraquinones components; In the decoctums of Cacumen Platycladi, flavones and other types components were similar to Rhei Radix Et Rhizoma phenolic acids and other types components, and JA of peak 1 were 0.545 which was larger than the other peaks; Percutaneous permeation of Lycopi Herba decoctum was slightly weaker than Cacumen Platycladi; In the decoctum of Cortex Phellodendri Amurensis JA of main characteristic peaks were smaller than 0.080, while percutaneous permeation of them were basically weaker than the other three herbs.Conelus ion1. The HPLC specific chromatogram analysis methods of Rhei Radix Et Rhizoma, Cortex Phellodendri Amurensis, Lycopi Herba, Cacumen Platycladi optimizaed are feasible, which could be used for quality control of herbs and reference of technology; The HPLC specific chromatogram analysis methods of Shuangbai spray release agent is feasible, which could take into account analysis of the different types of ingredients group, providing a method for the preparation of quality standards. The percutaneous penetration properties of different component groups of Shuangbai spray release agent were better than single herb, suggesting that the process of it is reasonable.2. The different components of Chinese medicine could percutaneous penetration basically, but the performance of them had a big gap, suggesting that they had a certain percutaneous permeation characteristics. Affected by the analytical conditions, most of the characteristic components in Chinese herbal formula had problems of low contentration, less penetration and difficult to detect. We could gain more percutaneous penetration information of characteristic components with a single herb percutaneous penetration test of suitable concentrations.3. Because components of Chinese medicines are very complex. When we could not get enough reference substance, we could characterizate percutaneous penetration of characterization of the different characteristic peaks performance effectively and solve the lack of percutaneous penetration research of only using the individual component as index through establishments of zero-order equation of characteristic peaks cumulative percentages of transmission-time with transmittance constant (JA, h-1) as the main indicators.4. The research methods of Chinese medicine percutaneous penetration based on specific chromatogram technology are feasible, which could be used for a new method and thinking of traditional Chinese medicine percutaneous penetration research.5. The research could provide a new research idea of material basis of drugs for external use.