Study On The Mechanism Of Jiufen San Transdermal Transport Based On HaCaT Cell Model

Jiufen San is from Qing Fei Shanshou’s《Emergency Remedy for First Aid》,which is included in the Chinese Pharmacopoeia.It is used to treat arthralgia and has remarkable curative effect.It can be taken internally or externally.Its prescription is composed of strychni semen,ephedra,frankincense,and myrrh,but due to the toxic traditional Chinese medicine of Strychnia semen in the prescription and can easily cause poisoning after long-term use,while external use can be directly used for local effect.At the same time,the volatile oil of frankincense and myrrh is rich in content,which can promote the transdermal absorption of the drug.Jiufen San prescription can be made into a suitable transdermal drug preparation,which will help to better exert the curative effect.In this dissertation,brucine and strychnine alkaloid in Strychnia semen and ephedrine hydrochloride alkaloids in ephedra are selected as the research objects.From the cellular and molecular point of view,the compatibility of frankincense myrrh volatile oil and three alkaloids on the skin its influence uptake viable epidermis cells,so as to provide a reference for the development of external preparations of nine dispersion.Meanwhile frankincense and myrrh volatile oil penetration enhancement mechanism was discussed,provide the basis for the use of frankincense and myrrh volatile oil.1.The toxic effect of the volatile oil of Frankincense and Myrrh in Jiufen San with brucine,strychnine and ephedrine hydrochloride on HaCaT cells.Using CCK-8 colorimetric method to study the single use of volatile oil of frankincense and myrrh and its drug pair,mixed volatile oil and three alkaloids of brucine,strychnine,and ephedrine hydrochloride（single alkaloid administered alone）and mixed alkaloids（three main alkaloids in co-administration）toxicity compatibility on HaCaT cells.The experimental results showed that frankincense volatile oil,myrrh volatile oil,drug pair volatile oil,mixed volatile oil,Azone,brucine,strychnine,and ephedrine hydrochloride all have obvious cytotoxic effects on HaCaT cells,and they are concentration-dependent.The cell non-toxic range of brucine is 0～18μg/m L;the cell non-toxic range of strychnine is 0～12μg/m L;the cell non-toxic range of ephedrine hydrochloride is 0～20μg/m L;the mixed alkaloid strychnine the non-toxic range of the cells is 0～12μg/m L.Under the safe concentration of volatile oil,when brucine is compatible with frankincense volatile oil,myrrh volatile oil,drug pair volatile oil,and mixed volatile oil,the cell non-toxic range of brucine is reduced to0～4.5μg/m L,0～4.5μg/m L,0～9μg/m L,0～9μg/m L;When strychnine is compatible with frankincense volatile oil,myrrh volatile oil,drug pair volatile oil,and mixed volatile oil,the cell non-toxic range of strychnine is reduced to 0～6μg/m L;When ephedrine hydrochloride is compatible with frankincense volatile oil,myrrh volatile oil,drug pair volatile oil,and mixed volatile oil,the cell non-toxic range of ephedrine hydrochloride is reduced to 0～10μg/m L,0～10μg/m L,0～10μg/m L,0～5μg/m L;When mixed alkaloids are compatible with frankincense volatile oil,myrrh volatile oil,drug pair volatile oil,and mixed volatile oil,the cell non-toxic range of strychnine in the mixed alkaloid is reduced to 0～6μg/m L.Preliminary consideration may be that under the safe concentrations of the three original alkaloids,the volatile oil of frankincense and myrrh promotes cellular uptake to some extent,which increases the concentration of alkaloids in the cells,which exceeds the original safe concentration,which leads to the three alkaloids nontoxic range decreased to some extent.2.The influence of the compatibility of Frankincense and Myrrh volatile oil in Jiufen San with brucine,strychnine and ephedrine hydrochloride on HaCaT cell intake.Establish LC-MS/MS methods for the determination of brucine,strychnine,and ephedrine hydrochloride.Set up different volatile oil groups,negative control solvent groups and positive control azone groups,and use cell uptake experiments to investigate the cellular uptake of brucine,strychnine,and ephedrine hydrochloride when administered alone（containing a single alkaloid）or co-administered（containing three alkaloids）under the action of volatile oil.The test results showed that when administered alone,the drug intake per unit protein of the brucine solvent group was 15.65±2.56ng/mg,and that of the frankincense group,the myrrh group,the drug pair group,the mixed group,and the azone group were respectively 40.69±1.79,42.41±3.66,42.07±2.19,43.37±3.36,16.25±1.83ng/mg,the enhancement ratio of each volatile oil group were 2.66,2.75,2.75,2.82,the azone group was 1.05,and all except the azone group were greater than2;The drug intake per unit protein of strychnine solvent group was 13.51±1.73ng/mg,and that of the frankincense group,the myrrh group,the drug pair group,the mixed group,and the azone group were respectively 31.64±2.84,34.94±3.16,38.69±3.06,42.20±4.38,13.85±1.80ng/mg,the enhancement ratio of each volatile oil group were 2.35,2.60,2.89,3.13,the azone group was 1.03,and all except the azone group were greater than 2;The unit protein drug intake of the ephedrine hydrochloride solvent group was 11.08±1.15ng/mg,and that of the frankincense group,the myrrh group,the drug pair group,the mixed group,and the azone group were respectively27.82±1.85,25.91±1.99,24.37±2.21,30.90±2.13,8.45±1.40ng/mg,the enhancement ratio of each volatile oil group were 2.52,2.35,2.21,2.80,the azone group was 0.76,and all except the azone group were greater than 2.When co-administered,the drug intake per unit protein of the brucine solvent group was 13.58±1.80ng/mg,and that of the frankincense group,the myrrh group,the drug pair group,the mixed group,and the azone group were respectively 20.75±3.11,22.56±3.28,20.48±2.13,21.64±2.40,11.68±1.34ng/mg,the enhancement ratio of each volatile oil group were 1.52,1.66,1.51,1.60,the azone group was 0.86,and all except the azone group were greater than 1;The drug intake per unit protein of Strychnine solvent group was 15.44±2.01ng/mg,and that of the frankincense group,the myrrh group,the drug pair group,the mixed group,and the azone group were respectively 24.92±3.07,29.24±2.13,26.95±2.74,28.45±2.08,12.47±2.56ng/mg,the enhancement ratio of each volatile oil group were 1.62,1.91,1.75,1.86,the azone group was 0.80,and all except the azone group were greater than1;The unit protein drug intake of the ephedrine hydrochloride solvent group was12.06±0.67ng/mg,and that of the frankincense group,the myrrh group,the drug pair group,the mixed group,and the azone group were respectively 17.95±1.71,19.09±1.46,19.71±2.55,19.49±1.48,7.92±1.77ng/mg,the enhancement ratio of each volatile oil group were 1.49,1.58,1.63,1.61,the azone group was 0.65,and all except the azone group were greater than 1.Compared with the solvent group,the volatile oil of Frankincense and Myrrh,both single-use and compatible volatile oil groups,have certain penetration promoting effects on brucine,strychnine,and ephedrine hydrochloride,and they have different penetration promoting effects on different drugs.The effect specifically shows that the volatile oil of frankincense and myrrh has better effects on fat-soluble brucine and strychnine than the water-soluble ephedrine hydrochloride.Considering the volatile oil of frankincense and myrrh as amphiphilic natural penetration enhancers.The penetration rate of the azone group is lower than that of the solvent group.It may be considered that the amount of azone added is the same as the essential oil of frankincense and myrrh,but it exceeds the safe concentration of azone itself,causing cell death and entering the cell.There are fewer drugs than the solvent group.At the same time,when comparing the penetration enhancement multiples of the volatile oil group administered alone and co-administered according to the prescription,it was found that the enhancement ratio of each volatile oil group of brucine,strychnine,and ephedrine hydrochloride were at the time of co-administer significantly decline,and it is speculated that these three alkaloids may interfere with each other during the common transdermal penetration enhancement process.However,the total uptake of the three alkaloids was greater than that of single administration,indicating that the volatile oil of Frankincense and Myrrh still had a significant effect on promoting penetration.3.Study on the effect of volatile oil of Frankincense and Myrrh on HaCaT cell fluidity and membrane potential.The fluorescence polarization method was used to study the influence of the volatile oil of Frankincense and Myrrh on the fluidity of HaCaT cell membranes with the fluorescence polarization degree P and microviscosityηas indicators.The experimental results showed that compared with the blank group,the P andηvalues of the negative control group did not change significantly（P>0.05）,and the P andηvalues of the low and medium-dose azone group were slightly lower than those of the blank group（P>0.05）,but with the increase of the concentration,the P andηvalues of the cell membrane decrease,that is,the high-dose azone group had a significant increase in the fluidity of the cell membrane（P<0.05）.The P andηvalues of the cell membrane in the middle-dose group of frankincense and the drug pair were significantly reduced（P<0.05）,and the P andηvalues of the high-dose group were significantly reduced（P<0.01）;the cell membrane of the myrrh,mixed medium and high-dose groups P andηvalues were significantly reduced（P<0.01）.It shows that different concentrations of frankincense,myrrh,drug pair,and mixed volatile oil groups can also significantly increase cell membrane fluidity and present a concentration-dependent relationship,showing similar action characteristics to the chemical permeability enhancer azone.Using Flow cytometry,the lipophilic anionic fluorescent probe Di BAC4（3）was used to study the effects of the volatile oil of frankincense and myrrh on the membrane potential of HaCaT cells.The experimental results showed that,compared with the blank group,the fluorescence intensity value of the negative control group did not show a significant difference（P>0.05）,and the low-dose frankincense group had no significant effect on the membrane potential of HaCaT cells（P>0.05）,but with the concentration increases,fluorescence intensity gradually increased membrane potential and exhibit a significant difference（P<0.01）,indicating that Azone volatile oil and frankincense HaCaT cells under the action of membrane potential depolarization occurs,and the higher the concentration,the depolarization phenomenon is more obvious,the lower the membrane potential.Myrrh,drug pair,and mixed low,medium and high dose groups can significantly reduce the membrane potential of HaCaT cells（P<0.01）.This indicates that the volatile oil of frankincense and myrrh can promote the absorption of drugs by increasing the fluidity of the HaCaT cell membrane and reducing the membrane potential.It is inferred that it may be one of the mechanisms by which the volatile oil of frankincense and myrrh has the effect of promoting penetration.4.Study on the effect of volatile oil of frankincense and myrrh on the balance of Ca²⁺in HaCaT cells.Using Flow cytometry,Fluo-3-AM were used as fluorescent probes to study the effects of the volatile oil of Frankincense and Myrrh on the concentration of Ca²⁺in HaCaT cells.The experimental results showed that compared with the blank group,the intracellular Ca²⁺concentration of the negative control group had no significant change（P>0.05）.The low-dose azone group had no significant effect on the intracellular Ca²⁺concentration（P>0.05）,but with the concentration Increased,the fluorescence intensity of intracellular Ca²⁺concentration increased significantly（P<0.01）,indicating that azone can promote the intracellular Ca²⁺concentration of HaCaT cells,and can increase the intracellular Ca²⁺concentration of HaCaT cells.Frankincense,myrrh,drug pair,mixed medium and high dose groups can significantly increase the intracellular Ca²⁺concentration in HaCaT cells（P<0.01）,and are concentration-dependent,suggesting that frankincense and myrrh volatile oil can also increase HaCaT cells the role of Ca²⁺concentration.The ultra-trace Ca²⁺-ATPase assay kit was used to determine the effect of Frankincense and Myrrh volatile oil on the Ca²⁺-ATPase activity in HaCaT cells.The experimental results showed that compared with the blank group,the intracellular Ca²⁺-ATPase activity of the negative control group was slightly reduced,but it was not statistically significant（P>0.05）.Azone,frankincense,myrrh,drug pair,and mixed were low,medium and high dose group can significantly reduce Ca²⁺-ATPase activity（P<0.01）,and exhibits a concentration-dependent manner,indicating that frankincense and myrrh volatile oil can significantly reduce the activity of Ca²⁺-ATPase.It indicates that the volatile oil of Frankincense and Myrrh can reduce the Ca²⁺-ATPase activity in HaCaT cells to a certain extent and increase the concentration of Ca²⁺in HaCaT cells,which may change the balance of Ca²⁺in HaCaT cells.It is inferred that it may be Frankincense and Myrrh volatile oil has a permeation promotion One of the mechanisms of action.That is to say,the volatile oil of Frankincense and Myrrh affects the concentration of Ca²⁺in HaCaT cells,thereby changing the membrane potential and membrane fluidity of active HaCaT cells to increase the permeability of the drug.5.Study on the effect of volatile oil of Frankincense and Myrrh on the signal pathway of inositol phospholipid in HaCaT cells.Auto Dock＿Vina software was used for molecular docking,virtual screening and prediction of the interaction of the active ingredients of Frankincense and Myrrh volatile oil with PLC,PKC and G protein.The experimental results show that the main active components of Frankincense volatile oil,Octylacetate,Nerolidol,and the main active components of Myrrh volatile oil,α-Copaene,Germacrene D,can interact with PLC and PKC and the combine energy is less than-5.0.The ELISA kit was used to study the effects of the volatile oil of Frankincense and Myrrh on PLC activity and PKC activity in HaCaT cells,and to verify the molecular docking results.The experimental results showed that Frankincense,Myrrh,drug pair,and the mixed middle and high dose groups were significantly increase PLC and PKC activities（P<0.01）,which was consistent with the predicted results of molecular docking.It is inferred that it may be the volatile oil of Frankincense and Myrrh one of the mechanisms of action of penetration-promoting,the increased activity of PLC and PKC can change the balance of Ca²⁺in HaCaT cells,change the membrane potential and the membrane fluidity of HaCaT cells,and increase the amount of drug penetration.In summary,the essential oil of Frankincense and Myrrh in Jiufen San has a good penetration enhancing effect on the three alkaloids of brucine,strychnine and ephedrine hydrochloride.Considering that the essential oil of Frankincense and Myrrh are amphiphilic natural penetration enhancers,and the volatile oil of Frankincense and Myrrh may increase the fluidity of the HaCaT cell membrane,decrease the membrane potential,increase the intracellular Ca²⁺concentration,decrease the intracellular Ca²⁺-ATPase activity,increase the PLC and PKC activities to achieve the permeabilization effect.