| BackgroundHemp,is an anual herb,its base is Cannabis sativa L..In China,hemp is a traditional economic crop and a medicinal plant with a long history.Its multiple tissues and organs can be used as medicine and have different therapeutic effects.Its seeds are used as medicine,called "hemp seed",which has the effect of laxative,promoting blood circulation and stranguria.The flower is used as medicine,called"Mabo",which is mainly used to treat aversion to wind,amenorrhea and forgetfulness.The husk and bracts are used as medicine,called "Maben",which has the effect of breaking the accumulation and dispersing pus,and can also be used to treat overwork injuries.The leaves can be used to formulate anesthetics because they contain anesthetic resins.Hemp contains terpene phenolic compounds and their derivatives,flavonoids and their glycosides,alkaloids,coumarins,terpenes and sterols,fatty acids and other chemical components.Terpene phenolic compounds are the main active substances of hemp,also known as cannabinoids.It is a natural product with a typical C21 terpene phenolic skeleton isolated and extracted from hemp,as well as compounds derived and transformed from it.Research purposesTaking the terpene phenolic components in hemp as the research object,the qualitative and quantitative analysis of the terpene phenolic components in hemp plants.It is proposed to study the metabolic differences of terpene phenolic compounds in different parts of hemp plants from the metabolic level,and lay a foundation for subsequent molecular research.Research results1.Collection and sorting of terpene phenolic components in hemp:a total of 121 terpene phenolic compounds that have been isolated and identified from hemp were collected,and the molecular formulas,accurate mass numbers and structural formulas of these compounds were sorted out.2.Establishment of a quantitative detection method for six main terpene phenolic components in hemp:using the Agilent Eclipse Plus C18(50 mm×2.1 mm,1.8μm)chromatographic column,the column temperature was 35℃,the mobile phases were 0.1%formic acid water(A)and methanol(B),and the flow rate was 0.3 mL/min.The gradient elution program is:2 min,5%～70%B;2～10min,70%～100%B;10～13min,100%B;13～14min,100%～5%B;14～15min,5%B.The mass spectrometer was in MRM mode with alternate scanning of positive and negative ions.The results show that the established method can achieve good separation of the six main terpene phenolic components in the sample;the six terpene phenolic components have a good linear relationship within the investigated range,with R2 ranging from 0.9991 to 1.000;the sample recovery rate is 99.36%～102.88%,the RSD of the sample recovery rate was less than 5%;the precision RSD was 0.53%-2.74%;the repeatability RSD was 0.29%-2.80%;the stability RSD was 2.58%-4.65%.The inspection results of the sample pretreatment method showed that the extraction efficiency of the six terpene phenolic components in hemp was the highest when the extraction solvent was 95%methanol and the solvent soaking time was 4℃ for 2 h.When the vacuum freeze-drying method was used,the properties of the six terpene phenolic compounds in the samples were relatively stable.3.The establishment of terpene phenolic spectrum in hemp:based on primary and.secondary mass spectrometry information of UPLC-QTF-MS,combined with compound fragmentation information in product ion mode of UPLC-QQQ-MS.Finally,76 terpene phenolic compounds were deduced in positive ion mode,and 28 terpene phenolic compounds were deduced in negative ion mode.4.Establishment of hemp-targeted metabolomics method:The mass spectrometry conditions of the 104 terpene phenolic compounds in hemp derived earlier were optimized.Q1 selects the parent ion information of each compound that has been determined in the MS SCAN mode of UPLC-QTOF-MS;selects two Q3 for each compound,one selects the characteristic ion fragmentation in the MS/MS mode of UPLC-QTOF-MS,and the other selects the characteristic ion fragmentation in the PI mode of UPLC-QQQ-MS;the DP value of the compound is uniformly set to 120V;using the MRM mode of UPLC-QQQ-MS to set isocratic collision energies for different ion pairs,select the collision energy value of the compound with the highest responsivity and the best peak shape as the optimal collision energy of the compound.Finally,established a stable and reliable targeted metabolomic method suitable for the analysis of cannabis terpenes and phenolic compounds.5.Spatial distribution of terpene and phenolic components in hemp plants:using the established targeted metabolomic method,104 terpene phenolic compounds in different parts(top,central,and bottom)of 3 tissues(flowers,bracts,leaves)of 5 hemp varieties were determined.The results showed that there were significant differences in the contents of terpene phenolic compounds in the top,central and bottom of hemp flowers,bracts and leaves.In the top and central hemp,28 differential compounds were screened from the flowers,17 from the bracts and 36 from the leaves.In the top and bottom hemp,25 differential compounds from the flowers,29 from the bracts and 38 from the leaves.In the central and bottom hemp,18 differential compounds were screened from flowers,23 differential compounds from bracts and 14 differential compounds from leaves.The metabolic trends of the differential compounds 3,36 and 68 in hemp flower were consistent,and the content gradually decreased from the top to the bottom.The metabolic trends of differential compounds 3,60 and 72 in hemp bracts were consistent,and their contents gradually decreased from the top to the bottom,but the metabolic trend of differential compound 49 was opposite to them.ConclusionIn this study,the 6 main terpene phenolic components in hemp(cannabidiol,tetrahydrocannabinol,cannabigerol,cannabidiolic acid,tetrahydrocannabinolic acid,cannabigerolic acid)quantitative detection method.The method has high sensitivity,good stability,simple and rapid operation,and is suitable for the determination of six terpene phenolic components in hemp.In this study,the qualitative study of terpene phenolic compounds in hemp was carried out based on LC-MS,and the hemp samples were analyzed by the product ion scanning technology of UPLC-QTOF-MS and UPLC-QQQ-MS.A total of 104 terpene phenolic compounds were identified by this method.At the same time,this study developed a targeted metabolomic method based on UPLC-QQQ-MS/MS technology for hemp terpene phenolic components.The method is stable and reliable,and is of great significance for the accurate detection of various terpene phenolic compounds in hemp.By analyzing the differential compounds in three tissues(flowers,bracts,and leaves)of 5 different varieties of hemp in different parts(top,central,and bottom),it was found that most of the differential compounds had the highest content in the top,but there were also some a few different compounds showed different trends in the three parts. |
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