Study On Systematic Profiling Of Alkaloids In Multiple Botanical Origins Of Uncariae Rammulus Cum Uncis Based On Liquid Chromatography-High Resolution Mass Spectrometry

Uncariae Ramulus Cum Uncis（Gou-Teng）with alkaloids as the main active constituents was a common TCM used to extinguish wind and settle convulsion.It has been applied in clinics to treat the cardiovascular and central nervous system diseases.In Chinese Pharmacopoeia（2015 edition）,the stems and hooks of five species from the Rubiaceae family,including Unacria rhynchophylla（Miq.）Miq.Ex Havil.,U.macrophylla Wall.,U.hirsuta Havil.,U.sinensis（Oliv.）Havil.,and U.sessilifructus Roxb.,are officially recorded as the botanical origins of Gou-Teng.While differences of the active alkaloids between five botanical origins of Gou-Teng remained to be unclear,which lacked systematic chemical profiling and comparison.As the vital quality control markers,it has been difficult to prepare oxindole alkaloids.Effective methods to recognize botanical origins of commercial Gou-Teng were missing.All of these have hindered the effective quality control of Gou-Teng.In view of the above problems,we proposed a strategy for systematic research of“active alkaloid group”based on liquid chromatography tandem high-resolution mass spectrometry（LC-HRMS）analytical platform,developed sensitive and efficient targeted profiling approaches to uncover the chemical material basis of Gou-Teng with multiple botanical origins,and provided a scientific basis for the quality control.1.Study on alkaloids in five speciesA sample chamber was constructed,and 150 herbal samples from five Uncaria species were collected and authenticated.An in-house structure library including 143alkaloids from the Uncaria genus was constructed based on UNIFI software.Chemical profiles of five botanical origins of Gou-Teng were analyzed in the positive mode using UHPLC/LTQ-Orbitrap MS,and alkaloids were targeted by searching in-house library and comparing MSⁿ data.The LC-MS guided studies on chemical constituents of U.macrophylla,U.hirsuta,U.sinensis and U.rhynchophylla were performed.As a result,38 alkaloids,including 9 new compounds,were isolated by dereplication and unambiguously identified.2.Study on preparation of unstable reference standardsTaking the isolation of four oxindole alkaloids in U.macrophylla as an example,this research developed an approach for preparing unstable alkaloid reference standards.Supercritical fluid chromatography（SFC）with CO₂ as mobile phase was used.And acetonitrile,in which four compounds were stable,was selected as the modifier of mobile phase.After further optimizations of stationary phases,additives,column temperatures,ABPR pressures and flow rates,two ultra-performance SFC methods with Torus 1-AA and Torus Diol columns were developed.Accordingly,the four alkaloids with the purities higher than 95%were finally prepared on a Viridis Prep Silica 2-EP OBD column using preparative SFC.3.Systematic profiling and comparison of alkaloids in five speciesConsidering that the differences of the alkaloids between five species remained unclear,we proposed multiple novel LC-MS analysis strategies to systematically profile and compare alkaloids in five species,by the fragmentation rules of reference standards,two dimensional liquid chromatography（2D LC）and target profiling methods.（1）In the positive mode,the MSⁿ fragmentation behaviors of 48 alkaloid standards were systematically studied using UHPLC/LTQ-Orbitrap MS.Accordingly,fragmentation rules of different subtype alkaloids were summarized.Fragmentation behaviors of alkaloids in CID（collision-induced dissociation）and HCD（high-energy C-trap dissociation）modes were compared,and a method for fragment acquisition named MS²/CID-MS³/HCD was proposed.（2）Methods for targeted profiling of alkaloids were investigated using UHPLC/LTQ-Orbitrap MS,and thus three methods based on mass defect filter（MDF）were established.Method one:a method combined classical rectangular MDF and neutral loss filter（NLF,162.05 Da）was developed to separately screen free alkaloids and alkaloid glucosides.Ultimately,60 free alkaloids and 32 alkaloid glucosides were characterized from the extract of U.rhynchophylla.Method two:a polygonal MDF algorithm was constructed to screen potential alkaloids.Then,a precursor ion list（PIL）was applied to selectively trigger MSⁿ fragmentation.Using this method,198alkaloids were finally characterized from the stems,leaves and flowers of U.sinensis.Method three:a PIL based on-line raster MDF approach was developed.By integrating the step-wise PIL and optimal parent mass width（±55 mDa）,the raster MDF could be achieved during the data acquisition,and fragmentations of targeted alkaloids were sensitively triggered.（3）By combining the orthogonal separations of the positively charged reversed phase column and traditional reversed phase column in different pH conditions,a 2D LC system was constructed with XCharge C18 in acid and Kinetex EVO C18 in base separately as the first and second dimensional columns.By integrating 2D LC system and the step-wise PIL based raster MDF scan,alkaloids in five botanical origins of Gou-Teng were systematically characterized.Finally,1227 alkaloids,including 657 from U.rhynchophylla,361 from U.hirsuta,407 from U.macrophylla,410 from U.sinensis and 248 from U.sessilifructus,were characterized.After systematic comparison of alkaloids in five species,characteristic alkaloid group of each specie and distribution characters of alkaloid subtypes were obtained.4.Differentiation of five species and recognization of botanical origins of commercial Gou-Teng samplesMulti-batch samples of five species were systematically compared using the plant metabolomic method.By unsupervised principle component analysis（PCA）,it was demonstrated that good differentiations between different species,except that between U.hirsuta and U.sinensis,could be achieved.Differences within one species were much smaller than that between different species.In addition,a support vector machine model with characteristic alkaloids as variables was developed and applied to distinguish and identify five species and to recognize botanical origins of commercial Gou-Teng.The results suggested that the commercial Gou-Teng samples were mainly botanically originated from U.rhynchophylla.By the researches mentioned above,1)the methods for analysis and preparation of unstable oxindole alkaloids were developed,which provided a solution to prepare TCM reference standards;2)the efficient and sensitive targeted profiling strategies for alkaloids were proposed,which provided a reference for the selective characterization of target components in the TCM complex systems;3)a support vector machine model was developed to distinguish five species and recognize botanical origins of commercial Gou-Teng,which supplied a reference approach for the analysis of TCM botanical origins;4)alkaloids in five Uncaria species were systematically characterized and compared to illuminate the differences,which laid a foundation for further evaluating the rationality of the concurrent use of five species and provided the scientific basis for constructing scientific and reasonable quality control standard of Gou-Teng.