Volatile Terpenoid Compounds In Rhododendron Species From Baili Rhododendron Forest Region In Guizhou Province

Allelochemicals are widely distributed in the tissues and organs of plants,which can be released through natural pathways(leaching,volatilization,decomposition and root exudation).Different plants release allelochemicals into the environment in different ways,the volatilization is one of the major routes.The dispersal of a large amount of volatile allelopathic substances will cause a high degree of canopy closure in the forest,leading to the natural regeneration from seeds of Rhododendron was difficult to complete.Rhododendron is one of the most important forest resources of Baili Rhododendron National Forest Park,and has important economic,environmental,and health values.Aiming at solving the ecological problems,that the hierarchical structure of Rhododendron community was simple in the Baili Rhododendron forest area,and the difficulty in regenerating the seeds under the forest.Besides,in order to clearly identify the chemical factors that affect the natural regeneration of the Rhododendron community,the 6 main Rhododendron species(Rhododendron stamineum,Rhododendron decorum,Rhododendron delavayi,Rhododendron agastum,Rhododendron irroratum and Rhododendron annae)in the Rhododendron National Forest Park was selected.The analysis method of HS-SPME and comprehensive two-dimensional gas chromatography combined with the quadrupole time-of-flight mass spectrometry and hydrogen flame ionization detector(GC × GC-QTOFMS / FID)to analyze volatile terpenoids of the Rhododendron was developed.The research explored the natural regeneration barrier factors of the Rhododendron community for future research,providing data support and theoretical basis for the sustainable development of Rhododendron communities in Baili Azalea National Forest Park.The main results are as follows:1.This study adopted headspace solid-phase microextraction as an extraction method for volatile terpenoids.We collected six Rhododendron varieties(Rhododendron stamineum,R.decorum,R.delavayi,R.agastum,R.irroratum and R.annae),including their leaves(fresh leaves and litters),stems,roots and flowers.The obtained samples were freeze-dried in a vacuum freeze dryer at-70 ° C for one week,and then ground into powders to obtain a mixture of rhododendron tissues as materials to establish experimental method.Five HS-SPME parameters that affected extraction efficiency of volatile terpenoids were optimized,they are solid-phase microextraction coating fiber,sample weight,equilibrium temperature,equilibrium time and extraction time,respectively.The HS-SPME method optimization results indicated that the best extraction efficiency can be obtained when using PDMS/DVB/CAR fiber equilibrated at 70 ° C for 20 minutes,then extracted for 15 min.To minimize analytical errors,the stability and repeatability under the optimized SPME conditions were evaluated.Intra-day and inter-day accuracy are expressed as relative standard deviation(RSD %).The results show that the RSD of almost all test compounds are below 20 %,proving the stability and repeatability of the established method conditions.In addition,the automated SPME injection also improves the great reproducibility.The relative standard deviations of the one-dimensional and two-dimensional retention times of the terpenoid compounds in the Rhododendron samples are less than 1%.These RSD values in the acceptance range fully prove that the data obtained by GC × GC-QTOFMS method has sufficient stability and accuracy.2.Quantitative and qualitative analysis methods of volatile terpenoid compounds were established.The paper compared the separation ability of one-dimensional gas chromatography and comprehensive two-dimensional gas chromatography in detecting volatile terpenoid compounds.The results confirmed that comprehensive two dimension gas chromatography can detect 35 volatile terpenoids,while one-dimensional gas chromatography can only separate 18 terpenoids.Subsequently,GC × GC-QTOFMS was used to identify and analyze all the rhododendron samples.Through NIST mass spectrometry matching,combined with accurate molecular mass and retention index(RI),the compounds could be more accurately identified.The QTOFMS spectrum was compared with a standard library(NIST 17)compounds were firstly selected with a minimum match factor of 800.Subsequently,compounds with precise molecular mass deviations within 5 ppm were retained.Finally,the retention index information was used for further compound identification.The acceptable RI difference between the experimental value(RIexp)and the literature value(RIlit)was 0–40(| RIexp-RIlit |).Based on the above three criteria,all terpenoid compounds were identified with high similarity.The average forward match factor was 862 and the reverse match factor was 891.Finally,a total of 35 volatile terpenoid compounds in six Rhododendrons were identified.At the same time,quantitative analysis of the compound content was with the internal standard 1,4-cineole.The results show that monoterpenes and their oxygen-containing derivatives are the main components of all Rhododendron varieties,among which d-limonene,p-cymene,?-pinene and ?-terpineol have higher content,and these chemicals also have allelochemical properties.3.Due to the types and contents of volatile terpenoid compounds in different tissues of Rhododendron may range in each season,the established GC × GC-QTOFMS / FID method was applied to research terpenoid compounds in different types of Rhododendron samples.The results showed that:(1)The content of monoterpene compounds in each Rhododendron are more abundant than sesquiterpenes compounds;(2)The contents of terpenoid compounds in different tissues of each Rhododendron sample indicated that more volatile terpenoid compounds can be detected the litters and roots,which suggested that terpenoids compounds are mostly released into the environment through litter decay or root secretion pathways;(3)From the release of total amount of terpenoid compounds in four seasons,spring and autumn can release more substitute compounds compare to summer and winter;(4)The PCA method was used to study the differences in the concentration of terpenoid compounds in different Rhododendron varieties.The PCA model divides the different tissues of plant into three well-defined clusters,explaining the internal connections of the various parts between the Rhododendron varieties.