The Interactional Characterization of Lentil Protein Isolate (LPI) with Cyanidin-3-O-Glucoside (C3G) and Their Effect on the Stability and Antioxidant Activity of C3G
The interaction between lentil protein isolate (LPI) and cyanidin-3-O-glucoside (C3G) was investigated via with UV–vis spectroscopy, circular dichroism, and fluorescence spectroscopy and the stability of anthocyanin was also evaluated. After LPI mixed with C3G, the turbidity and foaming capacity increased and the particle size and surface charge did not change significantly, while the surface hydrophobicity decreased significantly (p < 0.05). The fluorescence results indicated that C3G quenched the intrinsic of LPI by static quenching and LPI bound with C3G via hydrophobic effects with Ka of 3.24 × 106 M−1 at 298 K. The addition of LPI significantly (p < 0.05) slightly decreased the thermal and oxidation degradation of C3G by up to 90.23% and 54.20%, respectively, while their antioxidant activity was inhibited upon mixing. These alterations of physicochemical properties might be attributed to their structural changes during the interaction. The obtained results would be of help in stabilizing bioactive compounds and the development of functional foods.
2. Materials and Methods
2.1. Materials and Reagents
Cyanidin-3-O-Glucoside（C3G ）(98% purity) was obtained from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd. (Chengdu, China).
A novel ion mobility separation-enabled and precursor ions list-included high-definition data-dependent acquisition (HDDDA) approach: Method development and its application to the comprehensive multicomponent characterization of Fangji Huangqi Decoction
To elucidate the chemical composition of a traditional Chinese medicine (TCM) formula necessitates the development of more potent analytical strategies, because of the complexity as a result of the superposition of multiple drugs. Fangji Huangqi Decoction (FHD) is a four-component TCM formula composed by complicated chemical constituents. By using the VionTM ion mobility quadrupole time-of-flight (IM-QTOF) mass spectrometer, we present a novel IM separation-enabled and precursor ions list (PIL)-included high-definition data-dependent acquisition (HDDDA) approach, and apply it to the multicomponent characterization of FHD by coupling to ultra-high performance liquid chromatography. Chromatographic separation was conducted on a CORTECS® UPLC® T3 column, while HDDDA was employed for MS2 data acquisition in both the negative and positive electrospray-ionization (ESI) modes. The PILs of FHD in two ESI modes were created based on the phytochemical knowledge of four component drugs and mass defect filtering, which ultimately could obtain 316 and 258 targeted masses, respectively, from the full-scan MS1 data. Interestingly, the additional inclusion of PILs in HDDDA could improve the coverage on the target components by 12% (ESI–) and 48% (ESI + ). Structural elucidation was performed by in-house database-driven automatic peak annotation using UNIFITM. We could identify or tentatively characterize 203 components from FHD, involving 25 alkaloids, 86 flavonoids, 48 triterpenoids (saponins), 16 lactones, and 28 others). It is the first report regarding the method development of HDDDA that targets the global TCM components characterization, and the findings obtained would benefit the quality control and the secondary development of FHD.
2. Materials and methods
2.1. Reagents and chemicals
In total, 62 compounds (the chemical structures are given in Fig. 1 and the detainled information offered in Table S1, as Supplementary Materials) involving 16 flavonoids (numberred as 1–16), 15 alkaloids (17–31), 7 saponins (32–38), and 24 others (39–62), purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China), were used as the reference compounds in this work.
Fig. 1. Chemical structures of the 62 reference compounds.
Configuration of the ion exchange chromatography, hydrophilic interaction chromatography, and reversed-phase chromatography as off-line three-dimensional chromatography coupled with high-resolution quadrupole-Orbitrap mass spectrometry for the multicomponent characterization of Uncaria sessilifructus
Herbs represent complex chemical systems involving various primary and secondary metabolites that are featured by large spans of acid-base property, polarity, molecular mass, and content, etc., which thus poses great challenges to characterize the metabolites contained. Here, the combination of multiple-mechanism chromatography coupled with improved data-dependent-MS2 acquisition (DDA-MS2) is presented as a strategy to support the deep metabolites characterization. Targeting Uncaria sessilifructus, a reputable medicinal herb containing alkaloids and triterpenic acids (TAs) as the main pharmacologically bioactive ingredients, a three-dimensional liquid chromatography (3D-LC) system was established by integrating ion exchange chromatography, hydrophilic interaction chromatography, and reversed-phase chromatography (IEC-HILIC-RPC). The first-dimensional chromatography, configuring a PhenoSphere SCX column eluted by methanol/20 mM ammonium acetate-0.05% formic acid in water, could well fractionate the total extract into two fractions (unretained ingredients and alkaloids). The subsequent HILIC using an XAmide column and RPC by a CSH Phenyl-Hexyl column achieved the sufficient resolution of the total TAs and total alkaloids, respectively. A polarity-switching precursor ions list-including DDA approach by Q-Orbitrap-MS enabled the high-efficiency, coverage-enhanced identification of alkaloids and TAs. This 3D-LC/Q-Orbitrap-MS system was validated as precise (RSD < 5% for intra-day/inter-day precision), Up to 308 components were separated from U. sessilifructus, and 128 thereof (including 85 alkaloids, 29 TAs, and 14 others) were identified or tentatively characterized, exhibiting superiority over the conventional one-dimensional LC/MS. Conclusively, 3D-LC/MS in an off-line mode can facilitate the flexible configuration of multiple chromatography to accomplish the fit-for-purpose characterization of the metabolites from an herbal extract or a biosample.
Fig. 1. A flflow chart for the off-line 3D-LC/Q-Orbitrap-MS system used for the characterization of the multiple ingredients from U. sessilifructus
2.1. Chemicals and reagents
A total of 15 compounds (HPLC-UV purity>98%; Fig. S1 and Table S1) purchased either from Chengdu Desite Biotech.Co. , Ltd.(Chengdu, China), were used as the reference compounds in this work.
Dimension-Enhanced Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry Combined with Intelligent Peak Annotation for the Rapid Characterization of the Multiple Components from Seeds of Descurainia sophia
The complex composition of herbal metabolites necessitates the development of powerful analytical techniques aimed to identify the bioactive components. The seeds of Descurainia sophia (SDS) are utilized in China as a cough and asthma relieving agent. Herein, a dimension-enhanced integral approach, by combining ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) and intelligent peak annotation, was developed to rapidly characterize the multicomponents from SDS. Good chromatographic separation was achieved within 38 min on a UPLC CSH C18 (2.1 × 100 mm, 1.7 μm) column which was eluted by 0.1% formic acid in water (water phase) and acetonitrile (organic phase). Collision-induced dissociation-MS2 data were acquired by the data-independent high-definition MSE (HDMSE) in both the negative and positive electrospray ionization modes. A major components knockout strategy was applied to improve the characterization of those minor ingredients by enhancing the injection volume. Moreover, a self-built chemistry library was established, which could be matched by the UNIFI software enabling automatic peak annotation of the obtained HDMSE data. As a result of applying the intelligent peak annotation workflows and further confirmation process, a total of 53 compounds were identified or tentatively characterized from the SDS, including 29 flavonoids, one uridine derivative, four glucosides, one lignin, one phenolic compound, and 17 others. Notably, four-dimensional information related to the structure (e.g., retention time, collision cross section, MS1 and MS2 data) was obtained for each component by the developed integral approach, and the results would greatly benefit the quality control of SDS.
2 Materials and Methods
Twenty compounds (Fig. 2) were used as reference compounds in this work. These compounds were isoquercitrin (1, C21H20O12), quercetin-7-O-β-D-glucopyranoside (2, C21H20O12), kaempferol-3-O-neohesperidoside (3, C27H30O15), kaempferol-3-O-rutinoside (4, C27H30O15), kaempferol (5, C15H10O6),luteolin (6, C15H10O6), quercetin (7, C15H10O7), apigenin (8, C15H10O5), isorhamnetin-3-O-glucoside (9,C22H22O12), kaempferol-7-O-glucoside (10, 21H20O11), kaempferol-3-O-β-D-glucuronide (11,C21H18O12), apigenin-7-O-β-D-glucuronide (12, C21H20O10), rutin (13, C27H30O16), leutherodide A (14,C33H40O20), β-sitosterol (15, C35H60O6), glucosinalbin (16, C29H50O), scopolin (17, C14H19NO10S),psoralen (18, C16H18O9), sinapic acid (19, C11H6O3), and vanillic acid (20, C8H8O4). They were purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China)
Holistic quality evaluation of Saposhnikoviae Radix (Saposhnikovia divaricata) by reversed-phase ultra-high performance liquid chromatography and hydrophilic interaction chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry-based untargeted metabolomics
Untargeted metabolomics more suits the quality evaluation of TCM because of its holistic property. To assess the holistic quality difference of Saposhnikoviae Radix (the roots of Saposhnikovia divaricata), we integrate ultra-high-performance liquid chromatography coupled with ion mobility/quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS)-based untargeted metabolomics and quantitative assay. A BEH C18 column in the reversed-phase mode and a BEH Amide column in Hydrophilic Interaction Chromatography (HILIC) mode were utilized for metabolites profiling, which enabled high coverage of the non-polar to polar components in Saposhnikoviae Radix. Moreover, the application of major components knockout strategy enlarged the exposure of those minor components. Integrated use of high-definition MSE (HDMSE) and data-dependent acquisition (DDA) could enhance the metabolites characterization by providing reliable fragmentation information and collision cross section values. Computational in-house library-driven automated peak annotation of the HDMSE and DDA data assisted to characterize 104 components from Saposhnikoviae Radix. Chemometric analyses of the commercial Saposhnikoviae Radix samples (64 batches collected from 11 cultivars aging from 1 to 4 years), based on the positive MSE data, in general could indicate large discrimination between Guan-Fang-Feng (from Heilongjiang) and the others, but negligible difference among Saposhnikoviae Radix from the other ten provinces of China and with different ages. Quantitative assays of prim-O-glucosylcimifugin and 4′-O-β-D-glucosyl-5-O-methylvisamminol, by a rapid and fully validated UHPLC-UV method, could primarily deduce that Guan-Fang-Feng aging 2 and 3 years exhibited better quality. The methods established can holistically assess the quality of TCM with wide spans of plant metabolites covered.
2. Experimental section
2.1. Materials and reagents
Twenty-four compounds (Fig. 1 and Table S2), involving psoralen (1), 5-methoxypsoralen (2), methoxsalen (3), isopimpinellin (4), imperatorin (5), phellopterin (6), isoimperatorin (7), (+)-marmesin (8), nodakenin (9), umbelliferone (10), 7-hydroxy-6,8-dimethoxychromen-2-one (11), scopoletin (12), decursin (13), 5-methoxyfuro[2,3-h]chromen-2-one (14), 4′-O-β-D-glucosyl-5-O-methylvisamminol (15; GMV), cimifugin (16), prim-O-glucosylcimifugin (17; PGC), sec-O-glucosylhamaudol (18), tectochrysin (19), quercetin (20), rutin (21), wogonin (22), trans-ferulic acid (23), and adenosine (24), purchased from Chengdu Desite Biotechnology Co., Ltd. (Chengdu, China)
基于 UPLC 指纹图谱的防己及其混伪品鉴别研究
目的:通过UPLC指纹图谱比较正品防己及其混伪品广防己的差异,并结合化学计量学方法为防己药材的鉴别提供依据。方法:选用ACQUITY UPLC HSS T3（100 mm×2.1 mm,1.8μm）色谱柱,以0.2%磷酸溶液-乙腈为流动相,梯度洗脱;流速为0.2 mL/min;检测波长为210 nm。结果:建立的指纹图谱共标定了18个共有峰,通过与对照品比对,指认了5个成分,分别为木兰箭毒碱、波尔定碱、木兰花碱、防己诺林碱、粉防己碱;22批正品防己的相似度均在0.940以上,混伪品的相似度均≤0.239。聚类分析、主成分分析、正交偏最小二乘判别分析的结果一致,26批防己、广防己样品聚为2类,并筛选出3个差异标志性成分。结论:该方法能有效区分防己及其混伪品,可为控制防己药材的质量提供参考。
材料 木兰箭毒碱(批号: DST200426-014)、波尔定碱(批号: DST200320-092)、木兰花碱(批号:DST190625-004)、防己诺林碱(批号: DST190701-005)、粉防已碱(批号:DST200801-004)对照品均购自成都德思特生物技术有限公司；
DOI:10. 13863 / j. issn1001-4454. 2022. 11. 024