Study On Quantitative Detection Technology Of Numb-Taste Substances In Zanthoxylum Bungeanum Based On High Performance Liquid Chromatography

Zanthoxylum bungeanum belongs to the Zanthoxylum L.plant of the Rutaceae.Its fruit is one of the traditional"eight seasonings"in China,and it is also a traditional Chinese medicinal material.In recent years,with the adjustment of the agricultural industry structure and the implementation of returning farmland to forests,Z.bungeanum has been planted on a large scale as an economically preferred tree species,and its planting area has increased at a rate of 20%to 30%every year.However,with the rapid development of Z.bungeanum industry and the upgrading of intensive processing industries,the phenomenon of mixed harvest of different varieties of Z.bungeanum and shoddy products has gradually appeared in the market,resulting in uneven quality of commercially available Z.bungeanum and their intensive processing products.Therefore,the quality rating of Z.bungeanum and its intensively processed products has become the industry’s need.Numb-taste,as the unique flavor of Z.bungeanum,is one of the most important indicators for evaluating the quality of Z.bungeanum and its intensively processed products.However,in most of the current quality evaluation standards of Z.bungeanum,numb-taste substances are judged only by the sense of smell,which is highly subjective and poor in accuracy.Although some standards include the content of numb-taste substances into the scope of Z.bungeanum quality inspection and use high performance liquid chromatography（HPLC）to determine the content of Z.bungeanum numb-taste substance,there are still problems,such as imperfect pre-processing methods of samples,inaccurate selection of detection wavelength,low standard variety and supply and high prices of standard products.Thus,those standards cannot achieve accurate quantitative detection of numb-taste substances in different types of Z.bungeanum and its intensively processed products,and it is also impossible to evaluate its quality evaluation.Therefore,improving the application of HPLC in the quantitative detection of numb-taste substances in Z.bungeanum,developing a method that can accurately quantitatively detect numb-taste substances in Z.bungeanum have become an urgent problem,which is of great significance to scientifically evaluate the quality of different types of Z.bungeanum and its intensively processed products.This study intends to take 31 kinds of Z.bungeanum（20 kinds of Z.bungeanum Maxim,11 kinds of Z schinifolium sieb.et zucc）and 8 kinds of intensively processed products of Z.bungeanum（3 kinds of Z.bungeanum flavoring oil,3 kinds of hotpot condiment and2 kinds of Z.bungeanum oleoresin）as the main research objects.Through exploring the UV spectrum characteristics of the numb-taste substances,optimizing the conditions of HPLC（mobile phase composition,column temperature,flow rate）and pre-treatment conditions with ultrasonic-assisted solvent extraction（extraction solvent,Z.bungeanum particlesize,material-to-liquid ratio,ultrasonic power,ultrasonic temperature,single ultrasonic time,repeated extraction times）,investigating the methodological characteristics;the HPLC quantitative detection method of numb-taste substances was perfected and established.Moreover,on this basis,a new quantitative detection method-Quantitative Analysis of Multi-components by Single marker（QAMS）was introduced.Then,a QAMS of numb-taste substances in Z.bungeanum was established by determining the internal reference material,investigating the durability under different conditions（chromatographic system,chromatographic column,detection wavelength,column temperature,flow rate,injection volume,internal reference concentration）,determining the relative correction factor（f）value,checking reproducibility and verifying feasibility.In addtion,the established QAMS was used to quantitatively analyze39 Z.bungeanum samples,so as to scientifically evaluate the quality of Z.bungeanum from different origins and different varieties and its refined products,improving the quantitative detection technique and quality evaluation system for the numb-taste substances in Z.bungeanum.The main findings are as follows:（1）Research on HPLC-based quantitative detection technology of numb-taste substances in Z.bungeanum.By studying the UV spectrum characteristics of the numb-taste substances,optimizing the conditions of HPLC and pre-treatment conditions in Z.bungeanum samples and its intensively processed products,and comparing the extraction effect of optimized pre-treatment method with traditional extraction methods and local standard method,conducting methodological investigations.Solving the problem of inaccurate selection of detection wavelength and imperfect sample preparation,the HPLC-based quantitative detection technology of numb-taste substances in Z.bungeanum.was established.The results are as follows:1)The results of ultraviolet spectroscopy showed that one of the four absorption peaks was sharp and stable at about 270 nm in the UV full-band scanning spectrum of all samples,and the other three absorption peaks are weak or unstable.The HPLC response value of all Z.bungeanum samples was the highest at 270 nm,and the diode array detector（DAD）scan spectrum showed that Theλ_max of Hydroxy-ε-sanshool（HεSS）,Hydroxy-α-sanshool（HαSS）,Hydroxy-β-sanshool（HβSS）,Hydroxy-γ-sanshool（HγSS）and Hydroxy-γ-isosanshool（HγISS）is about 270 nm,and theλ_max of HαSS,which accounts for the largest proportion of numb-taste substances,was also 270 nm.Therefore,270 nm is the best detection wavelength for numb-taste substances.2)After multiple experiments,the final HPLC conditions was as follows:acetonitrile-water（45%:55%,V/V）,isocratic elution,detection wavelength was 270 nm,flow rate was 0.8m L/min,column temperature was 40℃.Under this condition,the separation of numb-taste substances was high,and the detection limits of HεSS,HαSS,HβSS,HγSS and HγISS were 0.0542,0.0127,0.0116,0.0406 and 0.0138μg/m L respectively.In addition,the relative standard deviation（RSD）of the methodological investigation results were lower than 3.0%,indicating good precision of instrument and high method repeatability.3)The pretreatment conditions of ultrasonic-assisted solvent extraction of numbing substances in different Z.bungeanum and its intensively processed products was optimized,and the extract effect were compared between traditional methods and local standard methods.The results showed that the optimal extraction solvents（analytical pure methanol）,ultrasonic power（360W）,and repeated extraction times（2 times）of Z.bungeanum oleoresin,Z.bungeanum powder（375μm）,Z.bungeanum seasoning oil,and hot pot bottom material were consistent.However,there are differences in the optimization results of other conditions.The best material-to-liquid ratios were 4 mg/m L,20,20,and 15 m L/g,and the best ultrasonic temperatures were 40,45,40,and 45℃,respectively.The best single ultrasound times were 20,40,20,30 min.Under these conditions,the extraction volume were increased by 21.79,11.40,29.36,5.79%compared with the traditional extraction method,and 9.23,7.60,2.79,10.91%higher than the local standard method,indicating that the ultrasonic-assisted solvent extraction method had high extraction volume,and was time-saving and high-efficiency.（2）Research on quantitative detection technology of numb-taste substances in Z.bungeanum based on QAMSOn the basis of perfecting and establishing the HPLC quantitative detection of numb-taste substances,a new quantitative detection method（QAMS）is introduced.The accuracy,durability and reproducibility of this method were investigated by selecting internal reference materials,determining relative correction factors,and a QAMS-based quantitative detection method for numb-taste substances in Z.bungeanum was established,that solving the problem of low supply and high price of the standard,effectively reducing testing costs and improving detection efficiency;Meanwhile,the established QAMS was used to quantitatively analyze 39 Z.bungeanum samples,so as to scientifically evaluate Z.bungeanum from different origins and varieties.and to further improve the quantitative detection technology and quality evaluation system of numb-taste substances.The results are as follows:1)Used HεSS,HαSS,HβSS,HγSS and HγISS as internal reference materials to calculate the RSDs of the relative correction factor（f）,and the RSD values of each component were less than 5.0%,which means all five numb-taste substances can be used as internal reference materials for quantitative analysis.Among them,the RSD value of HαSS was smallest,but the accuracy was highest.In addition,it has the advantages of simple structure,easy availability,low price,so it is more in line with the selection criteria of internal reference materials.To sum up,HαSS was selected as the internal reference material for the establishment of QAMS.2)HαSS was used as the internal reference material,the durability inspection results showed that different conditions had little effect on the f of HεSS,HβSS,HγSS,and HγISS,with the RSD value of the f is less than 1.0%,and the accuracy is high.The average f values of HεSS,HβSS,HγSS and HγISS wre 1.8479,1.0197,1.9639,0.3975 respectively.The RSD values of durability and reproducibility under different conditions were all small.And there were no significant difference of quantitative results between QAMS and external standard method（ESM）,indicating that QAMS can replace ESM to quantitatively analyze numb-taste substances in Z.bungeanum.In other words,only HαSS standard is required to realize the simultaneous determination of HεSS,HβSS,HγSS and HγISS.3)Useing QAMS to quantitatively analyze the numb-taste substances in 31 kinds of Z.bungeanum samples and 8 kinds of Z.bungeanum intensively processed products.In terms of perspective of the total content of numb-taste substances,Z.bungeanum oleoresin was the highest,Z.bungeanum seasoning oil and hot pot bottom were relatively low,and the overall content of Z.bungeanum Maxim is higher than that of Z schinifolium sieb.et zucc.In addtion,HαSS accounted for the largest proportion of all samples,,and the content of HγSS in Z.bungeanum Maxim is significantly higher than that of Z schinifolium sieb.et zucc.So,it can realize the identification of different Z.bungeanum varieties by analyzing the content level of HγSS.In summary,by solving the problems of inaccurate selection of detection wavelength and imperfect sample pretreatment in the content determination of the numb-taste substances,the HPLC-based quantitative detection method for the numb-taste substances was established to improve the quantitative detection system of the numb-taste substances;On this basis,using HαSS as an internal reference,a QAMS-based quantitative detection method for numb-taste substances was established,which further solved the key problems of restricting the application of HPLC on numb-taste substances.The problems are the low variety and supply,and high price of numb-taste substance standards,which due to difficulties in preparation and unstable nature of the standards.In addition,the method developed reducing the test costs and improving the detection efficiency.The solution of which have better perfected the research on the quantitative detection technology of numb-taste substances in Z.bungeanum,and provided corresponding theoretical basis and feasible methods for the quality evaluation of Z bungeanum,so as to promote the development of deep processing and comprehensive utilization technology of Z.bungeanum and expand the application scope of Z.bungeanum products.