Preparation And Interaction Mechanism Study Of Cyclodextrin Inclusion Complex With Mosla Chinensis “Jiangxiangru” Essential Oil

Objective:As a kind of natural cyclic oligosaccharide,cyclodextrin（CD）is often used in the solubilization research of insoluble substances due to the special structure of“hydrophobic inside and hydrophilic outside”and non-toxic and degradable properties.Essential oil（EO）has many biological properties and can be isolated and extracted from many medicinal plants.However,their application is limited owing to the great low water solubility and high volatility.In this study,the inclusion complexes（ICs）of Mosla Chinensis“Jiangxiangru”EO were prepared by different preparation methods,and the structure was characterized by several analysis methods.The dominant conformation of the IC was confirmed by the molecular simulation method,providing a theoretical reference and basis for further optimization of the preparation process of IC and the application of EO drugs.Method:Firstly,we have determined the physical and chemical properties of Mosla Chinensis“Jiangxiangru”EO from different batches.The chemical constituents of EO were analyzed by FI-IR and GC-MS.Then,the preparation conditions of three kinds of ICs were screened and optimized through experimental design.Finally,the relevant properties of the ICs prepared under the optimal ratio were investigated,and the inclusion mechanism was clarified by molecular simulation.The specific research contents are as follows:（1）A total of 8 batches of Mosla Chinensis“Jiangxiangru”EO were collected from Jiangzhong,Zhangshu Tianqitang,Sichuan New Lotus and Anhui Huifeng Traditional Chinese Medicine Pieces Co.,Ltd.The EO was extracted by steam distillation and the physical and chemical properties of EO were determined by the yield,color parameters,relative gravity,surface tension,rotation and acid value.Furthermore,the components and structure were qualitatively identified by FI-IR and GC-MS.The property differences between different batches of Mosla Chinensis“Jiangxiangru”EO were discussed by these results.（2）Theα,β,γ-CD IC were prepared by ultrasonic method to improve the stability and bio-availability of Mosla Chinensis“Jiangxiangru”EO.The six significant influencing factors were screened and optimized by Plackett-Burman design（PBD）and central composite design（CCD）,and the factors were CD/EO（A）,ultrasonic time（B）,ultrasonic power（C）,ultrasonic temperature（D）,water volume/CD（E）and EO/ethanol（F）,respectively.The encapsulation efficiency（Y₁）and loading capacity（Y₂）of theseα,β,γ-CD IC were determined by the ethanol extraction under ultraviolet spectrophotometer,and the encapsulation efficiency was used as the evaluation index.（3）The physical and chemical properties ofα,β,γ-CD IC were investigated by relevant morphological observation,SEM,FT-IR,TGA,in vitro dissolution（p H 1.2,6.8 and neutral）and thermal stability.The solubilization of EO withα,β,γ-CD was compared by the phase solubility.The inclusion mechanism and binding mode of carvacrol and cyclodextrin were elaborated through molecular docking and molecular dynamics.Result:（1）There was no significant difference between different batches of EO according to the physical and chemical properties results,and the color parameter showed the EO was transparent liquid with a normal yellow light,and the color get darker as the smaller the yield of EO at 100 g mass.The results of relative gravity showed that 8 batches of EO values were distributed from 0.9332 to 0.9471 m L/g.The results of surface tension and rotation indicated that the EO contained optically active chemicals,and the average surface tension（30.7 m N/m）was lower than the pure water（71.5 m N/m）.The average acid value of EO（2.61 mg/g）was lower than that of general vegetable oil,indicating that the Mosla Chinensis“Jiangxiangru”EO was not easily rancidity.The analysis of FI-IR showed that the components of 8batches of EO were similar.A total of 12 chemical components were identified by GC-MS with qualitative analysis.The main components were monoterpene dilute,monoterpene phenols and sesquiterpenes,among which the relatively high content group was divided into monoterpene phenols（60%）.（2）A two-step experimental design（PBD and CCD）was used to select and optimize the preparation conditions of EO being encapsulated byα,β,γ-CD.The results showed that three factors,α-CD/EO（A）,water volume/α-CD（E）and EO/ethanol（F）had an impact on the formation ofα-CD IC,and when A=9.22（g:g）,E=8（m L:g）,F=1.28（g:g）,encapsulation efficiency（Y₁）and loading capacity（Y₂）were 48.91%and 4.89%,respectively.Three factors,β-CD/EO（A）,ultrasonic power（C）and EO/ethanol（F）,had an impact on the formation ofβ-CD IC,and when A=8.73（g:g）,C=213.56（W）,F=0.5（g:g）,encapsulation efficiency（Y₁）and loading capacity（Y₂）were 85.52%and 9.03%,respectively.These two factors of ultrasonic time（B）and water volume/γ-CD（E）had an impact on the formation ofγ-CD IC,and when B=41.39（min）,D=48.31（^oC）,E=13.89（m L/g）,the encapsulation efficiency（Y₁）and loading capacity（Y₂）reached 74.41%and 8.32%,respectively.（3）Phase solubility results showed that cyclodextrin could increase the solubility of EO in water,and the inclusion constant ofβ-CD and EO was up to 100.69 L/g when the concentration ofβ-CD was 0-6 mmol/L and the temperature was 328 K,indicating that theβ-CD IC was more stable thanα-CD IC andγ-CD IC.The results of morphology observation and FI-IR analysis showed that the formation of new substances may indicate the successful formation of ICs,showing that the inclusion reaction could occur between cyclodextrin molecules and hydrophobic substances and be stable in the cyclodextrin with non-covalent action.TGA and thermal stability results showed that the EO encapsulated by cyclodextrin could improve thermal stability.Similarly,FWO models showed that theβ-CD IC was more stable thanα-CD IC andγ-CD IC.The average E_a of the thermodynamic parameters ofβ-CD IC was161.48 k J/mol,which was larger than that ofβ-CD PM（147.32 k J/mol）.The average E_a ofα-CD IC was 153.53 k J/mol lower thanα-CD PM（171.63 k J/mol）.The average E_a of the thermodynamic parameter values ofγ-CD IC was 150.13 k J/mol smaller thanγ-CD PM（169.00 k J/mol）.The in vitro dissolution of EO and its ICs and PMs at different p H values showed thatβ-CD IC was released more slowly thanα-CD IC andγ-CD IC in a short time,showing sustained release characteristics.Under acidic conditions,the maximum cumulative release ofα-CD IC andβ-CD IC were 79.60%and 63.79%,respectively.When p H was neutral,the maximum cumulative release ofγ-CD IC was 79.39%.Meanwhile,molecular docking and dynamic simulation showed that the binding molar ratio of carvacrol toα,βandγ-CD was 1:1,indicating a dynamic equilibrium.Conclusion:1.Physical and chemical parameters show that the Mosla Chinensis“Jiangxiangru”EO obtained from the same place has no obvious difference,and the EOs are yellow and transparent.Most of the chemical components of EO are dextral substances and the average acid value is 2.61 mg/g with non-rancidity.In qualitative analysis,the highest content with EO more than 60%is phenolic.Thus,the EO is easily oxidized resulting lose the pharmacological activity.2.The three ICs（α、β、γ-CD IC）with the encapsulation efficiency as the evaluation index,various factors affect the preparation of ICs with different degrees under the same preparation method and guest molecules.Theα-CD/EO,water volume/α-CD and EO/ethanol have an impact on the preparation ofα-CD IC.The effects ofβ-CD/EO,ultrasonic power and EO/ethanol on the preparation ofβ-CD IC are significant.The effect of ultrasonic time and water amount/γ-CD on the preparation ofγ-CD IC is significant,which indicated the two-step experimental design（PBD and CCD）can effectively screen the preparation conditions and reduce the manpower and material resources.At the same time,the preparation of the IC is affected by the type and preparation conditions of cyclodextrin,andβ-CD is more suitable for the encapsulation of EO of Mosla Chinensis“Jiangxiangru”.3.In a certain concentration range,cyclodextrin can effectively increase the solubility of EO in water.The inclusion type ofα-CD and EO is A_L type,whichα-CD IC is forming with a 1:1 molar ratio.In the range of 0 to 6 mmol/L,the inclusion type ofβ-CD and EO is A_N type,which could formβ-CD IC with a 1:1 or 1:2 molar ratio.The thermal stability of EO can be increased by encapsulating with it three cyclodextrins.After the release of high-energy binding water within cyclodextrin,the IC is stabilized by non-covalent bonding.Compared withα-CD IC andγ-CD IC,β-CD IC shows slower release behavior under acidic conditions with the maximum release（63.79%）.In addition,the formation of ICs is not simply adsorbed,but stabilized by non-covalent bonding（hydrogen bonding or hydrophobic interaction）.The binding ratio of carvacrol to cyclodextrin was 1:1,andβ-CD is the most stable with binding energy（-4.1 kcal/mol）.