| Jiufen San comes from《Facilities for the First Aid of the First Aid》by Shan Shou Fei,in the Qing Dynasty.And it has been included in the Chinese Pharmacopoeia,which has the functions of promoting blood circulation and removing blood stasis,reducing swelling and pain.It can be used for falls,bruises,swelling and pain.Clinically,it is mainly used for the treatment of rheumatoid arthritis.It can be taken orally or externally.Its prescription composition is strychni semen,ephedra,frankincense,and myrrh.Traditional Chinese medicine has a long history of transdermal administration.The aromas of aromatic medicines are commonly used.These medicines contain volatile components and can"lead Chinese medicine to the disease."It is speculated that frankincense and myrrh have similar effects in modern prescriptions as penetration enhancers in transdermal administration.In this thesis,we choose brucine,strychnine in strychni semen and ephedrine hydrochloride in ephedra as research objects,and explore the effect of frankincense myrrh essential oil on the transdermal absorption of these three alkaloids.So as to provide a reference for the development of Jiufen San external preparations.At the same time,the composition and mechanism of volatile oil of frankincense myrrh were discussed,which provided basis for the use of frankincense myrrh.1.GC-MS Comparative Analysis of Frankincense Myrrh on Volatile OilGC-MS technology was used to analyze the volatile oils of Frankincense,Myrrh,Drug Pair,and Mixed Group.Among them,26 components were identified in Frankincense volatile oil,accounting for 82.54% of the total volatile oil,of which the relative content was larger than 1%Octylacetate(27.91%),Nerolidol(25.84%),D-Limonene(4.48%),1-Octanol(2.70%),Linalool(1.29%),Dodecanoic acid(1.34%),Neocembren A(4.31%),D2-Carene(2.63%).These 8 components account for 70.5% of the total volatile oil;20 components were identified in the volatile oil of myrrh,which accounted for 79.28% of the total volatile oil.Among them,the relatively large content of more tha n 1%was 4-ethenyl-4-methyl-1-(propan-2-yl)-3-(prop-1-en-2-yl)cyclohexene(14.45%),Germacrene D(20.01%),α-Copaene(6.40%),α-Bourbonene(2.76%),β-ylangene(2.03%),(-)-g-Elemene(1.53%),β-Eudesmene(1.14%),Curzerene(5.59%),Naphthalene,1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-(1.09%),d-Cadinene(3.03%),α-Elemol(1.53%),β-Elemene(6CI)(10.39%),Chamigrene(2.20%),Elixene(2.75%).These 14 componen ts account for 74.9%of the total volatile oil;29 components were identified in the volatile oil of the drug pair,accounting for73.36%of the total volatile oil.Among them,the relatively large content of more th an 1%was Octylacetate(13.98%),Germacrene D(8.21%),and Nerolidol(10.96%),β-Elemene(6CI)(7.89%),α-Copaene(3.29%),α-Bourbonene(2.53%),4-ethenyl-4-methyl-1-(propan-2-yl)-3-(prop-1-en-2-yl)cyclohexene(5.54%),β-ylangene(1.43%),(-)-g-Elemene(2.54%),β-Eudesmene(1.01%),(-)-α-Selinene(2.96%),d-Cadinene(2.64%),α-Elemol(1.05%),D2-Carene(1.55%).These 14 components account for 65.58%of the total volati le oil;36 components were identified in the mixed volatile oil,accounting for 76.59%o f the total volatile oil.Among them,the relatively large content of more than 1%wa s Octylacetate(13.53%),Germacrene D(10.28%),and Nerolidol(12.46%),D-Limonene(1.80%),1-Octanol(1.11%),α-Copaene(3.14%),4-ethenyl-4-methyl-1-(propan-2-yl)-3-(pro p-1-en-2-yl)cyclohexene(6.18%),(-)-β-Bourbonene(1.44%),β-ylangene(1.08%),(-)-g-Elem ene(2.23%),Curzerene(3.38%),d-Cadinene(1.47%),β-Elemene(6CI)(3.85%),D2-Carene(1.08%),These 14 components account for 63.03%of the total volatile oil.To analyze the higher content of these 4 kinds of volatile oils.The two chemical components,Octylacetate and Nerolidol,which are higher in frankincense,have little change in the content of the volatile oils used as a drug pair or mixing.The highest content in myrrh is Germacrene D,which is slightly reduced in the drug pair and almost unchanged in the mixture.The contents and types of other ingredients have changed to some extent.The Principal Component Analysis method was used to analyze the chemical constituents of the volatile oils in the frankincense group,the drug pair group,and the mixed group.5 chemical components including Octylacetate,D-Limonene、1-Octanol、Linalool、Nerolidol were found to be the main components of frankincense in the drug pair and mixed group;To analysis the chemical constituents of volatile oil in myrrh group,drug pair group and mixed group,and Germacrene D、α-Cubebene、β-ylangene、opropa[1,2]benzene、Naphthalene,1,2,4a,5,6,8a-hexahydro-4,7-dimethyl-1-(1-methylethyl)-、α-Caryophyllene、β-Eudesmene、d-Cadinene、α-Elemol、β-Elemene(6CI)、α-Gurjunene、Curzerene are the 12 components that myrrh plays the main role in the drug pair and the mixed group.2.Study on the effect of volatile oils of frankincense myrrh in Jiufen San on percutaneous penetration of alkaloids in vitroEstablished HPLC method for the determination of brucine,strychnine,and ephedrine hydrochloride.Different volatile oil groups were set up,a solvent group as a negative control and an azone group as a positive control.Franz diffusion cell method was used to investigate the effects of volatile oil alone.Transdermal absorption of brucine,strychnine,and ephedrine hydrochloride when administered and co-administered(co-administration of three alkaloids).The experimental results show that the transdermal absorption of brucine in accordance with the Higuchi equation when administered alone.The cumulative transdermal volume of brucine in the solvent group for 12h is 56.72±0.58μg/cm~2.The frankincense group,myrrh group,drug pair group,mixed group and azone group were 114.85±7.19,113.40±27.58,120.69±21.44,97.71±6.98,135.06±4.39μg/cm~2,and the enhancement ratio of the volatile oil in each group were 2.13,1.94,2.14,and 1.68.The azone group was 2.87,all greater than 1.It shows that frankincense and myrrh volatile oil alone,compatible and mixed have a certain penetration promoting effect on brucine,and there is no significant difference,similar to the positive control group.The transdermal absorption of strychnine was consistent with the Higuchi equation when administered alone.The cumulative transdermal volume of strychnine in the solvent group for 12h was 186.88±19.82μg/cm~2.The frankincense group,myrrh group,drug pair group,mixed group and the azone group was 291.09±11.37,288.42±9.47,268.78±30.27,249.90±10.08,236.14±12.41μg/cm~2,and the enhancement ratio of the volatile oil in each group were 1.58,1.61,1.55 and 1.51,and the azone group was 1.34,all Greater than 1.It shows that frankincense and myrrh volatile oil alone,compatible and mixed have a certain penetration promoting effect on strychnine,and there is no significant difference,similar to the positive control group.The transdermal absorption of ephedrine hydrochloride in accordance with the Higuchi equation when administered alone.The cumulative transdermal volume of ephedrine hydrochloride in the solvent group for 12h was 26.33±3.68μg/cm~2.The frankincense group,myrrh group,drug pair group,mixed group and the azone group was 114.22±7.99,105.49±12.02,112.55±4.86,96.18±5.18,110.80±1.70μg/cm~2,the enhancement ratio of the volatile oil in each group were 3.42,3.29,3.37 and 3.18,and the azone group was 3.59,all Greater than 1.It shows that frankincense and myrrh volatile oil alone,compatible and mixed can promote the penetration of ephedrine hydrochloride,and there is no significant difference,similar to the positive control group.The experimental results show that the transdermal absorption of brucine conforms to the Higuchi equation when co-administered.The cumulative transdermal volume of brucine in the solvent group for 12h is 48.75±14.63μg/cm~2.The frankincense group,myrrh group,drug pair group,the mixed group and the azone group were 73.08±12.40,63.02±7.24,82.77±14.21,85.60±15.16,73.80±2.08μg/cm~2,and the enhancement ratio of the volatile oils in each group were 1.44,1.29,1.57,1.71 and the azone group is 1.63,all are greater than 1.It shows that frankincense and myrrh volatile oil alone,compatible and mixed have a certain penetration promoting effect on brucine,and there is no significant difference,similar to the positive control group.Compared with the single administration,the enhancement ratio of the frankincense group decreased significantly.It is considered that the percutaneous penetration of brucine is affected by the other two alkaloids.When co-administration,the transdermal absorption of strychnine was in accordance with the Higuchi equation.The cumulative transdermal volume of strychnine in the solvent group for 12h was 137.71±20.71μg/cm~2.The frankincense group,myrrh group,drug pair group,mixed group,and the azone group was 209.00±17.39,177.14±9.46,206.46±16.29,192.94±16.09,211.86±11.54μg/cm~2,and the enhancement ratio of the volatile oils in each group were 1.46,1.28,1.37 and 1.39,and the azone group was 1.61.Greater than 1.It shows that frankincense and myrrh volatile oil alone,compatible and mixed have a certain promoting effect on strychnine and there is no significant difference,similar to the positive control Azone group.Compared with the single administration,the enhancement ratio of myrrh group significantly decreased,and the transdermal penetration of strychnine was initially considered to be affected by the other two alkaloids.The transdermal absorption of ephedrine hydrochloride in co-administration conforms to the Higuchi equation.The cumulative transdermal volume of ephedrine hydrochloride in the solvent group for 12 h is 72.82±18.30μg/cm~2.The frankincense group,myrrh group,drug pair group,mixed group,and the azone group was 114.40±15.54,88.68±10.34,96.64±15.71,111.22±16.73,98.82±5.5μg/cm~2,and the enhancement ratio of the volatile oils in each group were 1.51,1.23,1.24,and 1.51,and the azone group was 1.45,all were Greater than 1.It shows that frankincense and myrrh volatile oil alone,compatible and mixed can promote the penetration of ephedrine hydrochloride,and there is no significant difference,similar to the positive control group.Compared with the single administration,the enhancement ratio of each volatile oil group decreased significantly.It is considered that the transdermal penetration of ephedrine hydrochloride is affected by the other two alkaloids.3.In Vivo Microdialysis Penetration of Frankincense and Myrrh Volatile Oil in Jiufen San to AlkaloidsLC-MS/MS quantitative analysis method was established and methodological investigation was conducted.The microdialysis technique was used to investigate the effect of volatile oil on percutaneous permeability of brucine,strychnine and ephedrine hydrochloride in vivo.The results showed that the LC-MS/MS quantitative analysis method met the requirements.Using 20%ethanol-saline as the perfusate,a flow rate of 1μl/min,and a sampling interval of 0.5h,the recovery and transfer rate of the probe in vitro are almost the same.Therefore,the transfer rate of the probe can be used instead of the recovery rate to correct the obtained data.The results showed that the enhancement ratio of frankincense,myrrh,drug pair and mixed group to brucine were 35.86,77.59,56.98 and 41.79,respectively,which were greater than 3.34 of the azone group;The enhancement ratio of frankincense,myrrh,drug pair and mixed group to Strychnine were 26.80,49.85,44.81 and28.57,all of which were greater than the enhancement ratio of 5.77 in the Azone group;The enhancement ratio of frankincense,myrrh,drug pair and mixed group to ephedrine hydrochloride were 88.83,111.50,86.25 and 63.33,all of which are greater than the enhancement ratio of 19.75 in the Azone group;and for the fat-soluble alkaloids brucine,strychnine and the water-soluble alkaloid ephedrine hydrochloride,The cumulative transdermal volume and steady-state flow rate of each volatile oil group were significantly different from those of the negative control solvent group(**p<0.01),which indicates that each volatile oil group has a good penetration promoting effect on brucine,strychnine and ephedrine hydrochloride.Frankincense and myrrh essential oil are amphiphilic penetration enhancers.4.Effects of Volatile Oil from Frankincense and Myrrh in Jiufen San on SC and Blood Flow of Rat SkinTransmission electron microscopy(TEM)was used to directly observe the micromorphological effects of frankincense and myrrh essential oil on the stratum corneum structure of the skin.Transmission electron microscopy results showed that the number of stratum corneum in the skin of rats treated with each volatile oil group decreased,and the distance between the layers increased.This shows that frankincense and myrrh essential oil can achieve the effect of promoting penetration by changing the tightness and order of the microstructure of the stratum corneume of the skin.The laser Doppler blood flow monitor(LDF)was used to exclude the effect of anesthesia on the microcirculation blood flow in the skin of rats and examine the effect of volatile oil on the microcirculation blood flow perfusion in the skin of rats.From the perspective of skin microcirculation blood flow,To explore the mechanism of traditional Chinese medicine volatile oil in promoting the transdermal absorption of drugs.The results showed that the ratio of skin blood flow to basal blood flow in the ears of rats was about 1 at 10,20,30,40,50,and 60 minutes after the application of the drug-free solvent base and drug-containing solvent.This shows that neither the drug-free solution nor the drug-containing solution has any effect on the blood flow of the skin in the ears of rats.At 10,20,30,40,50,and 60 minutes after the administration of the volatile oil-containing drug solution,the blood flow of the ear skin of the rats in each volatile oil group increased significantly.The ratio is basically around 2.The effect of Azone group on accelerating skin blood flow is weak,and the value of skin blood flow change is stable at about 1.3.This suggests that accelerating blood flow to the skin may be one of the mechanisms by which the volatile oil of traditional Chinese medicine promotes percutaneous penetration. |
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