Preparation And Pharmacokinetics Study Of Cyclodextrin Inclusion Complex Of Chuanxiong Rhizoma Volatile Oil

Chuanxiong is derived from the dried rhizome of umbelliferae Ligusticum chuanxiong Hort.,with bitter taste and warm nature,which has the effect of promoting Qi activating blood and dispelling wind to relieving pain.Chuanxiong is also praised as"blood gas medicine".It is a commonly used Chinese medicine in prescriptions,used for chest pain,swelling and pain,irregular menstruation,headache,etc.Chuanxiong Rhizoma volatile oil（Chuanxiong oil）is one of the effective components of Chuanxiong,its main component is Ligustilide,showing a strong antispasmodic,antiasthmatic,sedative and other effects,which is clinically effective in treating migraine and ischemic cerebrovascular disease.However,the chemical properties of Ligustilide are unstable and prone to various isomerization reactions such as dehydrogenation,oxidation,hydrolysis,and degradation.In addition,Chuanxiong oil has low water solubility and poor oral bioavailability,which greatly limits its clinical application.In order to solve the above problems,Chuanxiong oilβ-cyclodextrin（β-CD）and hydroxypropyl-β-cyclodextrin（HP-β-CD）inclusion complex were prepared.We study its solubility,stability and pharmacokinetic characteristics.The main research contents are as follows:1 Determination of the inclusion rate of Chuanxiong oil inclusion complexThe sum of the inclusion rate of Ligustilide and Senkyunolide A in Chuanxiong oil was used as the evaluation index.Using HPLC method,two solvents were selected for ultrasonic extraction to measure the total amount of inclusion complex and the amount of uninclusion complex.Choosing polar ethanol or methanol as the extraction solvent,investigating respectively about the extraction effects of 70%ethanol,80%ethanol,95%ethanol,absolute ethanol and methanol on the Chuanxiong oilβ-CD and HP-β-CD inclusion complex.Choosing low polarity’s petroleum ether as the elution solvent,at the same time investigated the number of elutions.The final determination method of inclusion rate was:The total amount of inclusion complex was measured by ultrasonic extraction with 70%ethanol,and the amount of uninclusion complex was measured by ultrasonic washing twice with petroleum ether.The total amount of inclusion complex subtracted the amount of uninclusion complex and divided the actual input amount to obtain the inclusion rate.2 Study on the inclusion process of Chuanxiong oil cyclodextrinChuanxiong oilβ-CD inclusion complex was prepared by ultrasonic stirring method,taking the comprehensive score of the inclusion rate and the yield as the evaluation index,the orthogonal test method optimized the best inclusion process,and the final preparation method was determined as follows:According to the mass ratio of Chuanxiong oil andβ-CD as 1:10,weighβ-CD and dissolved it in 20 times distilled water to prepare a saturated solution.Under the condition of ultrasonic and stirring,slowly added Chuanxiong oil（diluted with equal volume of ethanol）,mixed well,and stired magnetically for 30 min（temperature 55℃,speed 750 rpm）,cooled to room temperature,refrigerated at 4℃for 24 h,centrifuged（8000 rpm,10 min,room temperature）,the precipitate was dried under reduced pressure at 70℃,ground,and passed through an 80-mesh sieve.According to the preferred inclusion process,the average inclusion rate of the 3 batches of inclusion complex was 87.57%;the average yield was 65.41%;the comprehensive score was 80.92.Chuanxiong oil HP-β-CD inclusion complex was prepared by the single-phase method,taking the inclusion rate as the evaluation index.The orthogonal test method optimized the best inclusion process.The final preparation method was determined as follows:According to the mass ratio of Chuanxiong oil and HP-β-CD as 1:10,weighing HP-β-CD and dissolving it in 2 times 95%ethanol.Adding Chuanxiong oil dropwise to HP-β-CD ethylene-ethanol solution,mix well,recover ethanol under reduced pressure,dry under reduced pressure at 70°C,grind,and pass through an80-mesh sieve to obtain it.According to the preferred inclusion process,the average inclusion rate of 3 batches of inclusion complex was 92.34%.3 Study on the solubility of inclusion complexTaking phase solubility method to investigate the solubilization effects ofβ-CD and HP-β-CD on Chuanxiong oil.Results:In the range of 0～15 mmol/L,the concentration of Ligustilide and Senkyunolide A showed a non-linear increase with the increase ofβ-CD concentration,the inclusion type was A_Ntype,which could form a insoluble inclusion complex with the moore ratio was 1:1 or 2:1,the linear regression and K value calculation cannot been performed.The maximum solubilization multiples of Ligustilide and Senkyunolide A were 2.95 and 2.36;In the range of 0～60 mmol/L,the concentration of Ligustilide and Senkyunolide A increased linearly with the increase of the concentration of HP-β-CD.The inclusion type is A_Ltype,which could form a soluble inclusion complex with the moore ratio of 1:1,the regression equations were respectively Y=0.1378X+0.5311（R2=0.9971）,Y=0.0787X+0.5195（R2=0.9943）.The equilibrium constants K of Ligustilide and Senkyunolide A were 300.93 and 164.42 respectively,indicating that the formed inclusion complex was relatively stable,HP-β-CD had a good inclusion effect on Chuanxiong oil,the maximum solubilization factor of Ligustilide and Senkyunolide A could reach 15.57 and 10.80.4 Study on the stability of the inclusion complexThe stability of Chuanxiong oil,Chuanxiong oilβ-CD physical mixture,Chuanxiong oil HP-β-CD physical mixture,Chuanxiong oilβ-CD inclusion complex and Chuanxiong oil HP-β-CD inclusion complex were compared and studied.Weighing an appropriate amount of each sample into a closed clean weighing bottle,spread it into a thin layer≤5 mm thick,and seal it.Place in a 60℃constant temperature incubator,inspect for 10 days,take samples on day 0,day 5,and day 10respectively,and calculate the relative retention rate by dividing the measured content by the content of the sample on day 0.After 10 days,the relative retention rates of Ligustilide in each group were:80.99%,41.56%,43.03%,98.66%,90.36%,and the relative retention rates of Senkyunolide A were:64.95%,62.60%,62.69%,100%,96.59%.The results showed that theβ-CD and HP-β-CD inclusion complex can significantly improve the stability of Chuanxiong oil.5 Study on the pharmacokinetics of the inclusion complexHPLC method was used to determine the concentration of Ligustilide in plasma after oral administration of Chuanxiong oil and its cyclodextrin inclusion with osthol as the internal standard.Using DAS3.2 software to analyze the data to obtain related drugs kinetic parameters.The AUC_0-∞of Chuanxiong oil,β-CD inclusion complex and HP-β-CD inclusion complex were（2.16±0.51）,（1.99±0.75）,（2.56±0.83）mg/L·h respectively;MRT_0-∞were（5.99±2.77）,（6.68±3.18）,（5.57±1.23）h respectively;t_1/2were（3.86±1.84）,（4.53±3.36）,（4.46±0.96）h respectively.Taking Chuanxiong oil as the control group,the data of each group was subjected to independent sample t test.The results showed that there was no significant difference in AUC_0-∞,MRT_0-∞,t_1/2of each group,but there was a significant difference in the C_maxand T_maxof HP-β-CD inclusion complex group.It showed thatβ-CD and HP-β-CD inclusion complexs could not significantly improve the bioavailability of Ligustilide in Chuanxiong oil,in addition,Chuanxiong oil HP-β-CD inclusion complex may had quick-acting effects.