A robust platform based on ultra-high performance liquid chromatography Quadrupole time of flight tandem mass spectrometry with a two-step data mining strategy in the investigation, classification, and identification of chlorogenic acids in Ainsliaea fragrans Champ.

The chlorogenic acids are the major bioactive constituents of the whole plant of Ainsliaea fragrans Champ. (Xingxiang Tuerfeng). These compounds are usually present as isomeric forms in Xingxiang Tuerfeng. Therefore, an efficient approach should be developed for the rapid discovery and identification of chlorogenic acids isomers through the fragmentation pathway and rules. In this study, the collision induced dissociation tandem mass spectrometry (CID-MS/MS) fragmentation routes of chlorogenic acids were systematically investigated by UHPLC-QTOF-MS/MS in the negative ion mode using eight chlorogenic acids standards. As a result, diagnostic product ions for rapid discovery and classification of chlorogenic acids isomers were determined according to their MS/MS fragmentation patterns and intensity analysis. Based on these findings, a novel two-step data mining strategy was established. The first key step was to screen different kinds of substitution and the skeleton of the quinic acid using the characteristic product ions and neutral losses. The second key step was to screen and classify different types of chlorogenic acids using their diagnostic product ions. It was apply to the rapid investigation, classification, and identification of chlorogenic acids. And the same carbon skeletons from a complex extract of Ainsliaea fragrans Champ. were effectively identified. 88 constituents, including 14 chlorogenic acids types, were rapidly discovered and identified, and in particular, 12 types of chlorogenic acids, including p-CoQC, FQA, BQC, CQA-Glu, CFQA, p-Co-CQC, di-p-CoQC, BCQA, di-CQA-Glu, PCQA, tri-QCA, and P-di-CQA, were first discovered in Ainsliaea fragrans Champ. In conclusion, UHPLC-QTOF-MS/MS method together with a systematic two-step data mining strategy was established as a feasible, effective, and rational technique for analyzing chlorogenic acids. Additionally, this study laid a foundation for the study of the active substances and quality control of Ainsliaea fragrans Champ.

A strategy for comprehensive identification of sequential constituents using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer, application study on chlorogenic acids in Flos Lonicerae Japonicae.

The analytical methodologies for evaluation of multi-component system in traditional Chinese medicines (TCMs) have been inadequate or unacceptable. As a result, the unclarity of multi-component hinders the sufficient interpretation of their bioactivities. In this paper, an ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap (UPLC-LTQ-Orbitrap)-based strategy focused on the comprehensive identification of TCM sequential constituents was developed. The strategy was characterized by molecular design, multiple ion monitoring (MIM), targeted database hits and mass spectral trees similarity filter (MTSF), and even more isomerism discrimination. It was successfully applied in the HRMS data-acquisition and processing of chlorogenic acids (CGAs) in Flos Lonicerae Japonicae (FLJ), and a total of 115 chromatographic peaks attributed to 18 categories were characterized, allowing a comprehensive revelation of CGAs in FLJ for the first time. This demonstrated that MIM based on molecular design could improve the efficiency to trigger MS/MS fragmentation reactions. Targeted database hits and MTSF searching greatly facilitated the processing of extremely large information data. Besides, the introduction of diagnostic product ions (DPIs) discrimination, ClogP analysis, and molecular simulation, raised the efficiency and accuracy to characterize sequential constituents especially position and geometric isomers. In conclusion, the results expanded our understanding on CGAs in FLJ, and the strategy could be exemplary for future research on the comprehensive identification of sequential constituents in TCMs. Meanwhile, it may propose a novel idea for analyzing sequential constituents, and is promising for quality control and evaluation of TCMs.

Identification and characterization of five new classes of chlorogenic acids in burdock (Arctium lappa L.) roots by liquid chromatography/tandem mass spectrometry.

Burdock (Arcticum lappa L.) roots are used in folk medicine and also as a vegetable in Asian countries especially Japan, Korea, and Thailand. We have used LC-MS(n) (n = 2-4) to detect and characterize in burdock roots 15 quantitatively minor fumaric, succinic, and malic acid-containing chlorogenic acids, 11 of them not previously reported in nature. These comprise 3-succinoyl-4,5-dicaffeoyl or 1-succinoyl-3,4-dicaffeoylquinic acid, 1,5-dicaffeoyl-3-succinoylquinic acid, 1,5-dicaffeoyl-4-succinoylquinic acid, and 3,4-dicaffeoyl-5-succinoylquinic acid (M(r) 616); 1,3-dicaffeoyl-5-fumaroylquinic acid and 1,5-dicaffeoyl-4-fumaroylquinic acid (M(r) 614); 1,5-dicaffeoyl-3-maloylquinic acid, 1,4-dicaffeoyl-3-maloylquinic acid, and 1,5-dicaffeoyl-4-maloylquinic acid (M(r) 632); 1,3,5-tricaffeoyl-4-succinoylquinic acid (M(r) 778); 1,5-dicaffeoyl-3,4-disuccinoylquinic acid (M(r) 716); 1,5-dicaffeoyl-3-fumaroyl-4-succinoylquinic acid and 1-fumaroyl-3,5-dicaffeoyl-4-succinoylquinic acid (M(r) 714); dicaffeoyl-dimaloylquinic acid (M(r) 748); and 1,5-dicaffeoyl-3-succinoyl-4-dimaloylquinic acid (M(r) 732). All the structures have been assigned on the basis of LC-MS(n) patterns of fragmentation, relative hydrophobicity, and analogy of fragmentation patterns if compared to caffeoylquinic acids.

Hypoglycemic activity of leaf organic extracts from Smallanthus sonchifolius: Constituents of the most active fractions.

The aim of the present study was to determine the in vivo hypoglycemic activity of five organic extracts and enhydrin obtained from yacon leaves. The main constituents of the most active fraction were identified. Five organic extracts and pure crystalline enhydrin were administered to normoglycemic, transiently hyperglycemic and streptozotocin (STZ)-diabetic rats. The fasting and post-prandial blood glucose, and serum insulin levels were estimated and an oral glucose tolerance test (OGTT) was performed for the evaluation of hypoglycemic activity and dose optimization of each extract. We found that the methanol, butanol and chloroform extracts showed effective hypoglycemic activity at minimum doses of 50, 10 and 20mg/kg body weight, respectively, and were selected for further experiments. Oral administration of a single-dose of each extract produced a slight lowering effect in the fasting blood glucose level of normal healthy rats, whereas each extract tempered significantly the hyperglycemic peak after food ingestion. Daily administration of each extract for 8 weeks produced an effective glycemic control in diabetic animals with an increase in the plasma insulin level. Phytochemical analysis of the most active fraction, the butanol extract, showed that caffeic, chlorogenic and three dicaffeoilquinic acids were significant components. Additionally, enhydrin, the major sesquiterpene lactone of yacon leaves, was also effective to reduce post-prandial glucose and useful in the treatment of diabetic animals (minimum dose: 0.8mg/kg body weight). The results presented here strongly support the notion that the phenolic compounds above as well as enhydrin are important hypoglycemic principles of yacon leaves that could ameliorate the diabetic state.

Evaluation of the immunosensitizing potential of chlorogenic acid using a popliteal lymph node assay in BALB/c mice.

It has yet to be established whether chlorogenic acid (CGA), a common xenobiotic with potential exposure risk to humans, is associated with immune-mediated hypersensitivity reactions (HRs). The primary limitation in evaluating this potential relationship is the lack of an effective animal model for use in predicting the immunosensitizing potential of low molecular weight compounds (LMWCs). Currently, the popliteal lymph node assay (PLNA) is considered a very promising tool for assessing immunosensitizing potential of LMWCs. To determine whether CGA may possess an intrinsic capacity to stimulate or dysregulate immune responses, and if so, what mechanisms may be involved, we characterized the popliteal lymph node reaction induced by CGA in naive female BALB/c mice using both a direct PLNA (d-PLNA) and a reporter antigen PLNA (RA-PLNA) method. Our results show that CGA failed to induce immunoreactivity following a single subcutaneous injection either alone or when combined with TNP-OVA or TNP-Ficoll. These results indicated that CGA lacks the intrinsic capacity to sensitize or stimulate immune responses in BALB/c mice. Moreover, these results suggest that exposure to CGA may not represent a safety concern for humans and that removal of CGA from Traditional Chinese Medicine Injections may not significantly decrease the prevalence of HRs.