Mass spectrometry-based profiling and imaging strategy, a fit-for-purpose tool for unveiling the transformations of ginsenosides in Panax notoginseng during processing.

BACKGROUND: Panax notoginseng, a valuable medicinal plant, is traditionally used to treat trauma, body pain, and cardiovascular diseases in two clinical forms including raw (crude) and processed form. Processing-triggered compound transformation is responsible for the distinct bioactivity between raw and processed Panax notoginseng. Nevertheless, investigating the chemical diversity and dynamic transformation pattern of processed Panax notoginseng is challenging. METHODS: A new approach, which integrates multi-components characterization, processing trajectory depiction, discovery of differential markers, transformation mechanism of metabolites, in situ spatial distribution and transformation of metabolites, was established to elucidate the role of processing on the holistic chemical transformations of Panax notoginseng (PN). RESULTS: In this study, 136 ginsenosides (mainly rare ginsenosides) were identified or tentatively characterized and the temperature-dependent chemical variation trajectory was depicted via principal component analysis (PCA). Nineteen processing-associated markers were confirmed by orthogonal partial least squares-discriminant analysis (OPLS-DA). For the first time, the transformation pathway of ginsenosides during processing were elucidated by integrating the precursor ion scan (PIS) and mimic processing strategy that involves with deglycosylation, dehydration, hydration, acetylation, and isomerization. Results of mass spectrometry imaging (MSI) revealed the major ginsenosides M-Rb1, R1, Rg1, Rb1, Rd, and Re exhibited distinct spatial distribution pattern that are highly abundant in the xylem and showed a downward trend during processing. We firstly depicted the spatial distribution of processing-triggered rare ginsenosides (Rg3, Rk1, Rg5, etc.), and in situ transformation of ginsenosides was discovered in the process of steaming. Additionally, this variation trend was consistent with untargeted metabolomics results. CONCLUSION: This study comprehensively revealed chemical diversity and dynamic transformation pattern and depicted the spatial distribution of ginsenosides of PN during processing. It could provide a clue for the distinct bioactivities between raw and processed PN and elucidate the role of processing on the holistic chemical transformations of natural products, more importantly, the proposed strategy is valuable for the quality evaluation and control of the processing of natural product.

Characterizing the influence of different drying methods on chemical components of Panax notoginseng leaves by heart-cutting two-dimensional liquid chromatography coupled to orbitrap high-resolution mass spectrometry.

Panax notoginseng leaves (PNL) was considered as a promising functional food ingredient with abundant protopanaxdiol ginsenosides. In this study, the influence of different drying methods on chemical components in PNL was characterized by a newly developed heart-cutting 2D-LC-HRMS. Our data indicates that vigorous ginsenoside transformation occurs in PNL processed by sun-air drying and hot-air drying (HAD) at 50 °C, but not shade-air drying (SAD), HAD at 25 °C and steaming prior to drying (SD). Specifically, the main components of PNL, ginsenosides Rb3, Rc, Rb2, Rb1 and Rd, can be transformed into notoginsenosides Fd and Fe, ginsenoside Rd2, Gypenoside XVII and ginsenoside F2, respectively, by highly selective cleavage of ß-1,2-glucosidic linkage at the C-3 position. Only SD can inactivate the proteins that mediate this transformation. Different drying methods also greatly affect the quality of PNL products extracted by the conventional decoction method. These findings offer the scientific basis to design industrial drying methods for ensuring the quality of PNL.

Quantitative Characterization of Ginsenoside Biotransformation in Panax notoginseng Inflorescences and Leaves by Online Two-Dimensional Liquid Chromatography Coupled to Mass Spectrometry.

Panax notoginseng inflorescences (PNI) and leaves (PNL) are commonly used as folk medicine and food supplements. In this study, an online two-dimensional hydrophilic interaction × reversed-phase liquid chromatography coupled to linear trap quadropole mass spectrometry method was developed to determine 24 ginsenosides, including two novel compounds, in PNI and PNL extracted by water and methanol. Our data demonstrated that ginsenosides Rd, Rc, Rb2, Rb3, Rb1, Ra2, Ra1, and Ra3 in both PNI and PNL extracted by water rather than methanol can be transformed to ginsenoside F2, notoginsenoside Fe, ginsenoside Rd2, notoginsenoside Fd, gypenoside XVII, PN02, PN01, and PN03, respectively, by selectively cleaving the ß-(1→2)-glucosidic linkage at the C-3 position. Ginsenoside transformation was further verified to be mediated by the proteins isolated from samples. Additionally, the two newly discovered transformed products, namely, PN02 and PN03, were prepared and identified as novel compounds by nuclear magnetic resonance. Our findings provide new insight into the importance of extraction solvents on the component profile of natural products.

Remarkable Impact of Acidic Ginsenosides and Organic Acids on Ginsenoside Transformation from Fresh Ginseng to Red Ginseng.

Panax ginseng contains many chemical components, including acidic ginsenosides and organic acids. However, whether these acidic substances play a role in ginsenoside transformation during steaming treatment has not yet been explored. In this paper, the content of neutral ginsenosides, acidic ginsenosides, and their degradation products in unsteamed and steamed P. ginseng were simultaneously quantified by high-performance liquid chromatography. We observed that neutral ginsenosides were converted to rare ginsenosides during the root steaming but not during the individual ginsenoside steaming. In contrast, acidic malonyl ginsenosides released malonic acid and acetic acid through demalonylation, decarboxylation, deacetylation reactions during the steaming at 120 °C. These malonyl ginsenosides not only were converted to rare ginsenosides but also promoted the degradation of neutral ginsenosides. Further studies indicated that a low concentration of organic acid was the determining factor for the ginsenoside conversion. The related mechanisms were deduced to be mainly acidic hydrolysis and dehydration. In summary, acidic ginsenosides and organic acids remarkably affected ginsenoside transformation during the steaming process. Our results provide useful information for precisely understanding the ginsenoside conversion pathways and mechanisms underlying the steaming process.