molecules Article A New Method for Simultaneous Determination of Phenolic Acids, Alkaloids and Limonoids in Phellodendri Amurensis Cortex Yao Chen 1, Zhao Zhang 1,*, Yang Zhang 1, Xiaomei Zhang 2, Zhipeng Zhang 1, Yonghong Liao 1 and Bengang Zhang 1 1 Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences and Peking Union Medical College, Beijing 100193, China; [email protected] (Y.C.); [email protected] (Y.Z.); [email protected] (Z.Z.); [email protected] (Y.L.); [email protected] (B.Z.) 2 China-ASEAN Traditional Medicine Exchange and Cooperation Center, Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, China; [email protected] * Correspondence: [email protected]; Tel.: +08-601-0628-99773 Academic Editor: Nicola Marchetti Received: 17 December 2018; Accepted: 13 February 2019; Published: 15 February 2019 Abstract: Phellodendri Amurensis Cortex (PAC) is a well-known herbal medicine in China with complex components, but the previous research has mostly focused on its alkaloids analysis. For the first time, a simpler and more efficient method was proposed in this paper to simultaneously determine the content of three different kinds of compounds—phenolic acids, alkaloids and limonoids—in PAC. The phenolic acids included 3-O-feruloylquinic acid, 4-O-feruloylquinic acid and syringin. The alkaloids include magnoflorine, phellodendrine, jatrorrhizine, palmatine and berberine, while the limonoids include obaculactone and obacunone. An approach combining multi-wavelength and HPLC-DAD was used in this study due to the great difference in maximum absorption wavelength of the various components. Four wavelengths at 215, 275, 280 and 310 nm, respectively, were chosen for monitoring. It has been indicated through appropriate tests that this approach is of high accuracy, good repeatability and stability and provides a scientific basis for the quality assessment of PAC and associated derivatives. In addition, the chromatographic fingerprints method combined with multivariate statistical analysis chosen in this study was proved to be effective and reasonable for an accurate classification of 33 batches of samples collected from different locations. Keywords: phellodendri amurensis cortex; phenolic acids; alkaloids; limonoids; multi-wavelength; quality evaluation 1. Introduction Phellodendri Amurensis Cortex (PAC), known as “Guan Huang Bai” in China, is derived from the dried bark of Phellodendron amurense Rupr. and mainly distributed in the northeastern region of China [1]. In recent years, in addition to the traditional efficacy of clearing heat and eliminating dampness, detoxifying and eliminating inflammation; further biological activity has been discovered in modern pharmacological studies of PAC, including antioxidant, hypoglycemic, antitumor activity, induced specific immune tolerance, neuroprotection and so on [2–7]. For centuries, PAC has been utilized solely or in combination with other medicines for the clinical treatment of various diseases, and used to produce drugs such as Huangbai capsule, Zhi-bai-di-huang pills, Huang-lian-jie-du decoction, Er-miao pills, Er-xian decoction, Si-miao pills, etc. [8–11]. Phytochemical studies in the literature show that PAC was known to contain a complex mixture of ingredients, including alkaloids, phenolics, limonoids and so on [12–14]. Most studies have reported Molecules 2019, 24, 709; doi:10.3390/molecules24040709 www.mdpi.com/journal/molecules Molecules 2019, 23, x FOR PEER REVIEW 2 of 15 Molecules 2019, 24, 709 2 of 15 Phytochemical studies in the literature show that PAC was known to contain a complex mixture of ingredients, including alkaloids, phenolics, limonoids and so on [12–14]. Most studies have on the biological activities of the alkaloids derived from PAC, including their antimicrobial activity [15], reported on the biological activities of the alkaloids derived from PAC, including their antimicrobial anti-inflammation activity [16], and neuroprotective activity [5,17]. Phenolic acids are also a widely activity [15], anti-inflammation activity [16], and neuroprotective activity [5,17]. Phenolic acids are distributed group of secondary metabolites that possess strong anti-oxidative activities, and they also a widely distributed group of secondary metabolites that possess strong anti-oxidative activities, areand generally they are added generally into added medicinew into medicinew to prevent to cancer prevent and cancer bacterial and cellbacterial growth cell [ 18growth,19]. Limonoids [18,19]. showLimonoids protective show effects protective against effects glutamate-induced against glutamat neurotoxicitye-induced neurotoxicity in primary in culturesprimary ofcultures rat cortical of cellsrat [cortical15,20]. Thuscells [15,20]. it can be Thus seen it PAC can isbe a seen medicinal PAC is plant a medicinal with high plant medicinal with high value, medicinal showing value, strong andshowing far-ranging strong biological and far-ranging and pharmacological biological and pharmacological activity, which activity, makes is which worthy makes of further is worthy research of andfurther development. research and development. ConsideringConsidering the the complexity complexity ofof herbalherbal medicines, slight slight differences differences in in various various components components may may greatlygreatly affect affect the the therapeutic therapeutic effects effects [21,22 [21,22].]. In accordance In accordance with thewith Chinese the Chinese Pharmacopoeia, Pharmacopoeia, palmatine andpalmatine berberine and were berberine used as were the chemicalused as the indicators chemical of indicators PAC when of thePAC pharmacodynamics when the pharmacodynamics function was notfunction yet clear. was The not current yet clear. quality The controlcurrent of quality PAC is control insufficient, of PAC as is most insufficient, of the reports as most merely of the focus reports on the qualitativemerely focus and quantitativeon the qualitative analysis and of quantitative its alkaloids analysis [23–26]. of Although its alkaloids several [23–26]. analytical Although methods several based onanalytical HPLC, 1 H-NMR,methods HPLC-DAD-MSbased on HPLC, have1H-NMR, been HPLC-DAD-MS employed to quantify have been chemical employed markers, to quantify they have simultaneouslychemical markers, determined they have only simultaneously same type of determined compound [only23–26 same]. Few type studies of compound so far have [23–26]. reported Few on thestudies simultaneous so far have determination reported on of the contentsimultaneous of the determination multiple bioactive of the components content of ofthe PAC, multiple making a rapidbioactive and components validated multicomponentof PAC, making a rapid analytical and validated method multicomponent still highly desirable analytical for themethod systematic still highly desirable for the systematic evaluation of its quality. HPLC-DAD-ESI-MS/MS has been used evaluation of its quality. HPLC-DAD-ESI-MS/MS has been used for the systematic characterization for the systematic characterization and identification of chemical constituents, and multi-component and identification of chemical constituents, and multi-component quantification. Therefore, in present quantification. Therefore, in present study, we attempted to establish a comprehensive and study, we attempted to establish a comprehensive and systematical method that combines qualitative systematical method that combines qualitative assessment and quantitative analysis of phenolic assessment and quantitative analysis of phenolic acids, alkaloids and limonoids for quality control of acids, alkaloids and limonoids for quality control of PAC. The objectives of this study were thus to PAC.develop The objectivesan improved of this studyHPLC-DAD-ESI-MS/MS were thus to develop method an improved for the HPLC-DAD-ESI-MS/MS simultaneous separation, method foridentification the simultaneous and quantitation separation, of identification ten phenolic ac andids, quantitation alkaloids and of limonoids ten phenolic in PAC acids, extract alkaloids (Figure and limonoids1). in PAC extract (Figure1). O O OH OH HO HO O HO HO OH OCH3 HO O O OH H3CO OH O 3-O-feruloylquinic acid 4-O-feruloylquinic acid (Phenolic acid) (Phenolic acid) H CO 3 OH O O HO OH HO OH OH Syringin (Phenolic acid) H3CO H3CO HO + N N+ HO HO N+ H H3CO HO OCH3 OCH H CO 3 OCH 3 OH 3 H Magnoflorine Phellodendrine Jatrorrhizine (Alkaloid) (Alkaloid) (Alkaloid) Figure 1. Cont. Molecules 2019, 23, x FOR PEER REVIEW 3 of 15 Molecules 2019, 24, 709 3 of 15 Figure 1. Cont. H3CO O N+ N+ H3CO O OCH3 OCH3 OCH OCH3 3 Palmatine Berberine (Alkaloid) (Alkaloid) O O O O O O O O H O O H O O O O O H H Obaculactone Obacunone (Limonoid) (Limonoid) FigureFigure 1. Structures 1. Structures of of the the 10 10 constituents constituents identified identified in in Phellodendri Phellodendri AmurensisAmurensis Cortex Cortex (PAC). (PAC). 2. Results2. Results and and Discussion Discussion 2.1.2.1. Identification Identification of Constituentsof Constituents TheThe identity identity of theof the various various chromatographic chromatographic peaks peaks was was identifiedidentified by comparing their their retention retention time,time, accurate accurate mass mass and and MS/MS MS/MS fragmentation fragmentation patternspatterns withwith the data reported reported in in the the literature literature or or commercialcommercial standards. standards. Considering Considering the the complexity complexity ofof thethe chemicalchemical constituents