| Background:Previous studies in the laboratory confirmed that the Z.bungeanum essential oil showed a good transdermal permeable effect on model series of drugs with different logP values and showed a concentration-dependent relationship and good clinical application prospects.However,the essential oil itself has the problems of volatility,instability and other issues.Besides,essential oil is not stable,and incompatibility with matrix in its transdermal formulations,affecting the transdermal drug delivery and clinical efficacy.In the early stage of the research group,an external analgesic for the treatment of osteoarthritis(composition of Chuanxiong,Zanthoxylum oil,and turpentine oil)was used as a model drug.The essential oil in the compound were used as the oil phase,and the extraction was the aqueous phase.The O/W-type microemulsion gel cream delivery system was developed and the relevant physical characterization of the system,the stability of volatile components in the system,in vitro release and transdermal,in vivo pharmacokinetics and pharmacodynamics were evaluated to verify the suitability of microemulsion gel cream delivery system and external compounds of Traditional Chinese Medicine.The results showed that the microemulsion gel cream system not only can improve the instability of essential components in the external compound of traditional Chinese medicine,but also can promote the transdermal absorption of the drug.At the same time,the components in prescription can be directly used as the oil phase and aqueous phase of the microemulsion,which can give full play to the application characteristics of Chinese medicine "drug-assisted combination",the proposed system provides theoretical and experimental basis for the improvement of Chinese herbal compound preparations containing volatile components.Therefore,it is necessary for the essential oil to adopt proper loading technology in the preparation of external preparations to solve the problems of instability of the essential oil in the external preparation and poor compatibility with the base material.The preparation of the microemulsion gel paste system is cumbersome and difficult to form;therefore,microemulsion gel system is easier to operate,simple to form,and industrially feasible.Objective:According to the statement on Li Yue Pian Wen(Published in Qing dynasty),an ancient classic literature on topical remedies in China,these herbs which are rich in essential oil,can effectively promote the percutaneous absorption of the active components in a prescription,and resemble the role of the transdermal penetration enhancers in modern transdermal delivery system.Microemulsion is a good bagging technology that can solve the problem of essential oil,but the effect of microemulsion on the penetration promoting effect is not clear,and the change of the component-enhancing effect of microemulsion containing essential oil is not clear.On this basis,this project will prepare a simpler microemulsion containing Z.bungeanum essential oil and its gel,examine the stability of Z.bungeanum oil in the preparation,and explore whether Z.bungeanum essential oil in the preparation of the traditional Chinese medicine to enhance the penetration of different components;and explore the mechanism of percutaneous promotion of Z.bungeanum essential oil preparations to provide the basis for the clinical application of volatile oil preparations.Methods:(1)The preparation and evaluation of microemulsion:①The first thing is selecting excipients with high solubility and compatibility for the model drug,and optimizing the proportion of suitable oil phase,surfactant and co-surfactant.The size,PDI and appearance quality were used as evaluation indicators and the simplex lattice method was used to optimize the formulation of microemulsion and the composition of the optimal blank prescription was determined.②The in vitro transdermal test was performed to determine the mode of adding the Z.bungeanum oil in the microemulsion(adding as a drug or the form of oil phase)to prepare the final drug-loaded microemulsion formulation for quality evaluation.③The in vitro transdermal test was performed to compare the presence or absence of competitive inhibition among the five model drugs in the drug-loaded microemulsion and to ensure the accuracy of the in vitro transdermal test results.(2)Preparation and evaluation of microemulsion gel and stability of essential oil;①The main factors influencing the preparation of microemulsion gels are the preparation of microemulsions and the preparation of gel backbones.On the basis of the preparation of the drug-loaded microemulsion in the early stage,the following focus on the preparation is the gel skeleton.The microemulsion gel method:add the unswollen gel skeleton into the microemulsion,overnight,and stir it evenly after it is fully swelled.The appearance of the gel matrix(clear clarity,presence or absence of precipitation,matte),formability,and viscosity are used as evaluation indexes to screen the gel matrix and its amount,and finally prepare the microemulsion gel for quality evaluation.The in vitro transdermal test was conducted to examine the manner of the drug added to the microemulsion gel(the drug was added to the microemulsion or added to the gel matrix).②Based on the final microemulsion gel formulation,using fingerprint similarity evaluation,the stability of Zanthoxylum oil in the formulation was investigated.(3)In vitro evaluation of microemulsion gel preparation:①According to the above preparation method,drug-loaded microemulsions and microemulsion gels were prepared and evaluated in vitro.Using the cumulative release rate(Q24%)for 24 hours as an indicator,the in vitro release behavior of the five model drugs in the microemulsion gel was examined using a modified Franz diffusion cell method.②The transdermal behavior of microemulsion and microemulsion gels were examined using the improved Franz diffusion cell method with the cumulative permeation volume(Q24),steady-state skin penetration rate(Jss),and ER(enhanced penetration factor)of 5 model drugs.(4)Study on the mechanism of promoting penetration of microemulsion gel preparation:①Fourier transform infrared spectroscopy(ATR-FTIR),differential scanning calorimetry(DSC)and HE staining were used to investigate the effects of Z.bungeanum oil on the microscopic and molecular structure of the stratum corneum.②The microemulsion containing fluorescent probe P4(provided by Wu Wei of Fudan University)was prepared,and C6 was used as a model drug to explore the fate after microemulsion administration based on laser confocal technology,which is to explore the passage of microemulsion through the skin after administration(complete microemulsion structure or microemulsion rupture release).Results:(1)The preparation and evaluation of microemulsion:①The optimal blank prescription is:Labrasol 29.336%,propylene glycol 7.334%,Oleic acid 5.305%,Water 58.025%.②The transdermal flow rate and the cumulative transmutation for 24h(Q24)of five model drugs were compared by in vitro transdermal test to determine where the oil phase is added in the microemulsion.It was confirmed that adding Z.bungeanum oil as oil phase not only facilitates the percutaneous penetration of the model drug,but also embodies the feature of"drug-assisted combination".③In vitro transdermal tests showed that there was no competitive inhibition of the five model drugs in drug-loaded microemulsions,which ensured the accuracy of in vitro transdermal test results.Finally,the drug-loading microemulsion was prepared by the following methods:29.336%with Labrasol as surfactant,7.334%with propylene glycol as cosurfactant and 5.305%with Z.bungeanum oil(extracted based on laboratory original technology)as oil phase,58.025%with water as the aqueous phase,weighed accurately according to the prescription,and after the magnetic stirrer was uniformly mixed,then according to the drug loading to add 5 model drugs:OT 18 mg·g-1,TMP 23 mg·g-1 and FA 20 mg·g-1,PR 30 mg·g-1,GP 31 mg·g-1,magnetic stirring until completely dissolved.The formed microemulsion had clear and transparent appearance,good fluidity,pale yellow,average particle size of(118.77±3.37)nm,PDI of(0.282±0.02),Zeta of(0.346±0.05)mV,and good stability.(2)Preparation and evaluation of microemulsion gel and stability of essential oil:①Preparation method of microemulsion gel:preparing microemulsion according to the above microemulsion prescription,adding 1%carbomer,overnight,fully swell,add appropriate amount of triethanolamine,stir while adding.②The fingerprint similarity evaluation was used to compare the stability of Z.bungeanum oil in microemulsion gels and ordinary hydrogels.The results showed that Z.bungeanum oil has better stability in microemulsion gels.(3)In vitro evaluation of microemulsion gel preparation:①Q24%of osthole in microemulsion gel was(1.28±0.16)%,Q24%of tetramethylpyrazine was(85.90±4.57)%,Q24%of ferulic acid was(43.82±5.34)%,and Q24 of puerarin was(46.87±5.26)%and Q24 of geniposide was(88.60±5.46)%.Except osthole,the release of the other four model drugs was in line with the first-order kinetic equation,and the release mechanism was drug diffusion and the dual mechanism of the skeleton.The in vitro release of ostholes complies with the zero-order kinetic equation.②In vitro transdermal experiments were used to compare the percutaneous penetration of five model drugs in microemulsions and microemulsion gel.The experimental results showed that,except geniposide,the effect of different preparations of Z.bungeanum oil on the penetration of the other model drugs was:5%prickly ash oil>microemulsion>microemulsion gel>blank microemulsion>blank solution;the effect on the penetration of geniposide was:5%prickly ash oil>microemulsion gel>microemulsion>blank microemulsion>blank solution.(4)Study on the mechanism of promoting penetration of microemulsion gel preparation:①ATF-FTIR and differential scanning calorimetry(DSC)were used to determine the effect of Z.bungeanum essential oil on the molecular structure of the stratum corneum skin.HE staining was used to visually observe the changes of the cuticle on the skin.The experimental results show that the direct application of Zanthoxylum oil,drug-loaded microemulsion,and the microemulsion gel all can shift the symmetrical vibration absorption peak(vs)and asymmetric vibration peak(vas)of lipid CH toward higher wavelengths,indicating that they are all have a certain influence on the order of lipids of the stratum corneum.The ratio of the amide Ⅰ band to the amide Ⅱ band area suggests changes in the water content of the stratum corneum.The ratio of amide Ⅰ band to amide Ⅱ band area of 5%Z.bungeanum essential oil,drug-loaded microemulsion,and microemulsion gel was significantly different,and the change in hydration intensity was 5%Z.bungeanum essential oil>drug-loaded microemulsion>microemulsion gel.Differential scanning calorimetry(DSC)results showed that the peaks of the phase transition peaks of the cuticular oil layer treated with Zanthoxylum oil,microemulsion,and gel were migrated to the low temperature region,and the peak value of the corresponding phase change peak was reduced,and the peak area also reduced or even disappeared significantly.The results showed that the Z.bungeanum essential oil,microemulsion,and gel all destroyed the tight structure of the lipid layer of the stratum corneum,improved its lipid fluidity,and changed the conformation of the protein,thus exerting its promoting effect.The results of HE staining showed that the skin structure of the saline-treated group was intact,the covered squamous epithelium was intact,the stratum corneum was intact,and the collagen fiber bundles were tightly arranged;the skin keratotic layer structure of the Z.bungeanum essential oil,microemulsion and microemulsion gel groups was incomplete.The arrangement of epidermal cells was disordered,the intercellular space was enlarged,and the boundaries between the layers of the skin were blurred.Among them,the effect of Z.bungeanum essential oil was stronger,followed by microemulsion,and the microemulsion gel was weaker.It can be seen that the microemulsion and gel skin are less irritating and safe to use.②The particle size of the microemulsion encapsulated with the fluorescent probe was(87.59±1.21)nm,and the PDI was(0.355±0.03).The observation by the laser confocal microscope showed that the microemulsion could partially penetrate through the microemulsion after being administered.Through the stratum corneum of the skin,it can reach the dermis;at the same time,the hair follicles in the skin are also important channels for microemulsion to enter the skin.After 24 hours of administration,the fluorescence intensity was decreased because the transdermal microemulsion was absorbed or degraded.Conclusion:In summary,this topic is based on the pre-laboratory study that Z.bungeanum essential oil showed a good transdermal permeable effect on model series of drugs with different logP values.To solve the solution of the problem in practical application of essential oil,such as instability,the drug-loaded microemulsion and microemulsion gel with Zanthoxylum oil as the oil phase were prepared to enhance the stability of Zanthoxylum oil,and the microemulsion and microemulsion gel were investigated.The transdermal permeation characteristics of different constituents were also investigated.ATR-FTIR,DSC,etc.were used to preliminarily elucidate the percutaneous transport mechanism of microemulsion and microemulsion gels.Finally,microemulsion was explored using laser confocal technology to investigate digestive kinetics in the skin after administration.The research of this topic can guide the rational preparation and application of the essential oil in the compound preparations for external use. |
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