| Acne is a chronic inflammatory disease of hair follicles and sebaceous glands.It is closely related to many factors,such as excessive sebum secretion,elevated androgen levels,abnormal keratinization of the sebaceous glands,bacterial infection,inflammation and immune response.There are many problems in the treatment of chemical drugs,however,the extracts of plant volatile oil or Chinese herbal medicine showed many excellent characteristics,and multiple factors can play a role in the pathogenesis of acne.Guixiong Quyu Cream includes Chinese angelica,chuanxiong,asarum and cassia twig of four drugs.The main reasons for the slow onset of traditional Chinese medicine are the complex composition,unknown effect components and low bioavailability.The solution to this problem is based on the theory of traditional Chinese medicine on the basis of traditional Chinese medicine preparation technology breakthrough.Part 1: Study on extraction process of the Gui Xiong Qu Yu CreamObjective: To select the appropriate extraction method and optimize extraction process.Method: compared the extraction rate of volatile oil by steam distillation and petroleum ether the percolation method and CO2 supercritical fluid extraction(SFE)to select the best extraction method.Using orthogonal test method,the extraction pressure,temperature and time of CO2 supercritical fluid extraction were investigated with the indexes of extracts and main components.Results and discussion: Ligustilide,cinnamaldehyde and eugenol were the main ingredient of Chuanxiong Quyu Cream.In order to determine their content in the samples,the HPLC detection methodswere established.Chromatographic condition: chromatographic column:Iinertsil ODS-3chromatographic column(4.6 mm×:150mm,5?m),Mobile phase: methanol-water(70?30),flow rate: 1.0 m L?min-1;test wavelength: 280 nm,Column temperature: room temperature,test volume: 20?L.The results showed that eugenol,ligustilide and cinnamaldehyde were in good linear relationshipin the scope of5.202~208.1?g?ml-1,3.797~759.4?g?m L-1,8.781?g?ml-1~439.0?g?ml-1respectively,with good precision,and were table in 12 h.The HPLC method can be used to determine the content of the samples of ligustilide and cinnamaldehyde and eugenol.Using the three methods,the extraction rate of Angelica sinensis was 0.5%,1.5%,5.2%respectively;Chuanxiong's was 0.7%,6.7% and 10.2%respectively;Cloves' was 1.8%,4.2% and 2.3%respectively;and Guizhi's was 0.8%,1.2% and 3.8%respectively.Comprehensive consideration,we selected CO2 SFE respectively.Angelica sinensis extract yield as index,extraction pressure,extraction temperature and extraction time were significantly affected(P? 0.05),and influencing factors of extraction time >extraction temperature >extraction pressure.To extract of Ligusticum lactone content as the index,extraction temperature and extraction time had significant influence too(P?0.05),and there are some effects of extraction pressure(P ? 0.10),and influencing factors of extraction time >extraction temperature >extraction pressure.According to the difference in the extract of Ligusticum lactone content analysis results,to determine the optimum extraction pressure is 35 MPa,extraction temperature is 45 ?,extraction time is 3h.The extraction rate was 5.04% and the content of ligustilide(100g material)689.95 mg.Chuan Xiong extract yield as index,extraction pressure,extraction temperature and extraction time were significantly affected(P? 0.05),and influencing factors of extraction temperature >extraction time>extraction pressure.To extract of ligusticum lactone content as the index,extraction temperature,extraction time and extraction pressure had significant influence too(P?0.05),and influencing factors of extraction temperature >extraction time>extraction pressure.According to the difference in the extract of Ligusticum lactone content analysis results,to determine the optimum extraction pressure is 25 MPa,extraction temperature is 40?,extraction time is 2.5h.The extraction rate was 5.04% and the content of ligustilide(100g material)4607.04 mg.Asarumextract yield as index,extraction time was significantly affected(P?0.05).there are some effects of extraction time(P ? 0.10),The effects of the extraction pressure was not significant(P > 0.10).The influencing factors was extraction temperature >extraction time >extractionpressure.To extract of eugenol as the index,extraction temperature,extraction time and extraction pressure had significant influence too(P?0.05),and influencing factors of extraction pressure>extraction time>extraction temperature.According to the difference in the extract of Asarum content analysis results,to determine the optimum extraction pressure is 25 MPa,extraction temperature is 40?,extraction time is 2.5h.The extraction rate was 4.03% and the content of ligustilide(100g material)268.39 mg.Cassia twigextract yield as index,extraction temperature was significantly affected(P?0.05),The effects of the extraction pressure and the extractiontime were not significant,and influencing factors of extraction time>extraction pressureextraction temperature >.To extract of cinnamaldehydeas the index,extraction temperature,extraction time and extraction pressure had significant influence(P?0.05),and influencing factors of extraction time = extraction pressure>extraction temperature.According to the difference in the extract of Asarum content analysis results,to determine the optimum extraction pressure is 20 MPa,extraction temperature is 40?,extraction time is 2h.The extraction rate was 3.85% and the content of ligustilide(100g material)592.83 mg.Part 2: Research on molding process of Gui Xiong Qu Yu CreamObjective: To study the preparation technology of the Gui Xiong Qu Yu Cream.Method: The dosage of emulsifier in cream were study on by physical property and the emulsification conditions were investigated by orthogonal experiment.Results and discussion: Using Labrasol and TEFOSE63 as emulsifier,when the ratio was 2:1,the consistency of the cream was moderate.Using the reflect-line orthogonal simplex method,investigating the extension of cream,combined with the stability test results,the formula would be optimized.The results showed that the matrix formula was liquid paraffin-emulsifier(1:1).The temperature,the dosage of emulsifier,the stirring speed and the emulsification time of the main medicine with matrix were taken as the variable factors,and each factor was divided into three levels(the orthogonal table of four factors and three levels),and the ligustilide,cinnamaldehyde and eugenol detection rate were taken as the quantitative indexes.The results showed that theoptimum temperature of the emulsification conditions was 65 ?,theoptimumdosage of emulsifier was 10%,theoptimumof stirring speed was 300r/min,and theoptimum of emulsification time was 30 min.The pilot test results showed that the preparation process was reproducible and the method was feasible.Part 3: Study on the quality standard of the Guixiong Qu Yu CreamObjective: To protocol the quality standard draft of the Guixiong Quyu Cream Method and Results: The results showed that the cream samples and identification of eugenol and cinnamaldehyde control using HPLC assay had the same position of the chromatographic peaks.And the method can be used for qualitative detection.Particle size,volume,microbial limits were in line with the requirements of the Chinese Pharmacopoeia 2010 Edition.Ligustilide(C21H25NO4)is the main active constituents of volatile oil from the principal drug-angelica and Guixiong.HPLC method was used to determine the content in the cream.The method was reliable and reproducible,and the content of component was more than 0.130 mg.The 24 month of 3 batches of Gui Xiong Qu Yu Cream samples were classified on survey,the results show that the Gui Xiong Qu Yu Cream quality is relatively stable,no significant changes in the indicators,could comply with the quality standards.Therefore,the effective period of the provisional of the Gui Xiong Qu Yu Cream is 24 months.It shows that the product has good long-term stability.This period can meet the requirements of the quality control of the drug,and can meet the needs of applications.Based on the above studies,we have formulated the quality standard of the preparation.Experiment 4: Study on the transdermal kinetics of the Gui Xiong Qu Yu CreamObjective: To investigate the transdermal absorption of the Gui Xiong Qu Yu Cream.Methods: Isolated Ba Ma incense porket skin was placed in Franz diffusion pool,with 30% ethanol saline solution as absorption solution,constant temperature water bath at 37 C,constant speed stirring.Theconcentration of 1%,2.5% or 5% Gui Xiong Qu Yu Creamwas taken toevenly on the 0.5g on blood stasis for drug pool skin,respectively in 1H,3h,5h,7h,9h,12 h,1m L for receiving liquid precision draw samples,receiving liquid content determination of ligustilide,cinnamaldehyde and eugenol by using HPLC method respectively.The Higuchi equation was calculated respectively,and the penetration rate constant(Jss)was gotten.Results and Conclusions: The r value of ligustilide Higuchi equation of theconcentration of 1%,2.5% or 5% Gui Xiong Qu Yu Cream is 0.9538,0.8805,0.9193 respectively,and the Jss value is 0.7995?g·cm-2·h-1,1.094?g·cm-2·h-1,2.313?g·cm-2·h-1respectively.The r value of cinnamaldehyde Higuchi equation is 0.9762,0.9221,0.9527,respectively,and the Jss value is 27.85?g·cm-2·h-1,47.16?g·cm-2·h-1,110.2?g·cm-2·h-1respectively.The r value of eugenol Higuchi equation is 0.9513,0.9014,0.9500 respectively,and the Jss value is 1.239?g·cm-2·h-1,1.804?g·cm-2·h-1,3.355?g·cm-2·h-1respectively.The results showed that the cumulative permeation quantity(Qn)showed a good linear relationship with the time(t),the greater content of transdermal drug,ligustilide,cinnamaldehyde and eugenol absorbed more quickly.Experiment5: Pharmacodynamics study of the Gui Xiong Qu Yu Cream Objective:To study the effect of the Gui Xiong Qu Yu Cream on sebaceous glands of golden hamsters Method:48 male golden hamsters were randomly divided into 6 groups,8 rats in each group.It was isovitamin A acid cream group,Bing Huang cream group,1% Gui Xiong Qu Yu cream group,2.5% Gui Xiong Qu Yu cream group,5% Gui Xiong Qu Yu cream group and blank emulsion grouprespectively.Back hair was shaved4cm×6cm to expose the sebaceous glands of the back.The corresponding drugs were applied in the experiment,each time 0.5g,3 times per day,andcontinuous administration for 30 days.Each group of golden hamsters were weighed,and each group randomly selected two that weretaked two photos,and the reaction of different groups of golden hamsters was recorded within the dosing process every 15 daysbefore administration and twentieth days,thirtieth days after administrationrespectively.The hamster's behavior,signs,medication skin reaction,feeding and excretion were monitored every day.After 30 days,each group selects onegolden hamster with sebaceous gland spot was sacrificed.The upper left,upper right,lower left,lower right skin were cut respectively,and some of the skin for the typical sebaceous gland spotswas taked.The skin which was cut was put into 4% paraformaldehyde mixed with 0.04% glutaraldehyde and fixed 24 h and marked subsequently.Results and conclusions: There was no significant change in the skin of the blank emulsion group,and there were obvious sebaceous glands.Sixth days after administration,the coated skin area of the hamsters of Bing Huang cream group began to appear a large number of exfoliate,and the surface of the skin become rough,the color of the sebaceous glands was lighter,and the number was reduced,and touch the skin feeled thick hard.The change of skin in the isovitamin A acid cream group was the most obvious.An average of 6 days later,there were different degrees of skin lesions in the coated area;average 10 days later,scab formed;the average 16 days off the scab;the average 20 days in the drug area of the second damage;an average of 23 days the second scab formed;and an average of 30 days the second times to remove scab.After using different concentrations of Gui Xiong Qu Yu cream,the sebaceous glands of the skin were improved to varying degrees,the skin became smooth,the number of sebaceous glands decreased and the color became shallow.5% concentration was the best,followed by the concentration of 2.5%,better than the concentration of 1%.The tissue sections showed that the isovitamin A acid cream leaded to inflammatory cell infiltration,necrosis and edema of the back skin tissue of the hamster dorsals.There wasn't treatment effect on the back of golden hamster sebaceous gland hyperplasia with Bing Huang cream.There was no obvious abnormality in the skin of the matrix group and the blank group,and a small amount of inflammatory cell infiltration in the superficial,middle and deep dermis.Compared with the blank group,the skin was significantly improved with Gui Xiong Qu Yu cream. |
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