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Chinese Journal of Chromatography
2026, Vol. 44, No. 8
Online: 08 August 2026

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Research methods and technological advancements in herbal material science LIU Yanfang, ZHOU Han, WANG Jixia, SHEN Aijin, LIU Dian, HAN Yang, YU Wenyi, YUAN Bin, WANG Zhen, ZENG Jie, XUE Xingya, FENG Jiatao, GUO Zhimou, LIANG Xinmiao 2026, 44 (8):  847-863.  DOI: 10.3724/SP.J.1123.2026.03008 Abstract ( 67 )   HTML ( 27 )   PDF (3704KB) ( 48 )   Traditional Chinese medicine （TCM）， as a complex system of substances， has long faced the challenges of an unclear material basis and obscure mechanisms of action， which has hindered its modernization and internationalization. To address these challenges， the “Herbalome” project was launched in 2007. This initiative aims to systematically reveal the material composition， structure， and biological functions of TCM through the integration of multidisciplinary technologies， thereby elucidating the synergistic mechanisms of multi-component and multi-target interactions. This paper reviews key methodological and technological advancements in the field of herbal material science. Specifically， regarding the composition and structural identification of herbal compounds， the integration of liquid chromatography-mass spectrometry with two-dimensional chromatography and intelligent data mining strategies has enabled high-throughput characterization of the complex chemical constituents of TCMs， as well as the discovery of novel compounds. Furthermore， advancements in multidimensional high-performance preparative chromatography and the development of novel multidimensional multi-channel separation and purification devices have overcome bottlenecks in the large-scale systematic preparation of TCM compounds. Additionally， nuclear magnetic resonance technology， enhanced by artificial intelligence techniques， such as intelligent heteronuclear single quantum coherence （HSQC） recognition and deep learning models， has significantly improved the efficiency and accuracy of structural identification. In the study of the biological effects of herbal substances， target-oriented and phenotype-oriented pharmacological technologies， such as cellular label-free integrative pharmacology， thermal proteome profiling， and affinity mass spectrometry， have facilitated the discovery of targets and the elucidation of mechanisms for bioactive components in TCM. Moreover， TCM databases and computational simulation techniques， including virtual screening， molecular dynamics， and artificial intelligence prediction models， have supported the construction and simulation of complex “multi-component and multi-target” interaction networks. Structural biology techniques， particularly cryo-electron microscopy， have provided atomic-level insights into the interactions between TCM bioactive components and target proteins， advancing our understanding of structure-activity relationships and multi-target mechanisms. In conclusion， the synergistic advancement of various techniques， including separation analysis， bioeffect evaluation， and computational modelling， is driving a paradigm shift in herbal material science from “experience-dependent” to “data-driven” approaches. The ongoing emergence and integration of novel technologies will continue to reveal unexplored areas within traditional Chinese medicine， systematically elucidate its multi-component， multi-target synergistic mechanisms， and enable the in-depth discovery of novel structures， targets， and biological effects. This progress will provide innovation insights to support the inheritance and innovation of traditional Chinese medicine as well as modern drug discovery.

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Development of high-performance chromatographic materials and their application in herbalome research since 2020 FENG Jing, XIA Donghai, SONG Chunying, QI Yi, JIN Gaowa, LIU Yanfang, LIANG Xinmiao, YU Dongping, GUO Zhimou 2026, 44 (8):  864-876.  DOI: 10.3724/SP.J.1123.2025.08025 Abstract ( 177 )   HTML ( 13 )   PDF (2230KB) ( 50 )   Traditional Chinese medicine （TCM）， a treasured cultural heritage of the Chinese nation， plays a significant role in treating various diseases. However， the extreme complexity of its chemical components makes the comprehensive elucidation of the material basis and pharmacological mechanisms a major challenge in modern TCM research. To deeply investigate the material basis of TCM and interpret its holistic regulation mechanism involving multiple components and targets， Liang Xinmiao and his colleagues proposed the concept of herbal materiomics， which focuses on two main research directions： the material basis （including qualitative/quantitative analysis and isolation of active compounds） and the functional mechanisms （in-depth study of biological effects and modes of action）. In herbalome research， chromatographic analysis and preparative purification are powerful tools for deciphering the composition of complex TCM systems， with the development of novel chromatographic separation materials being a key driver of progress. This review systematically summarizes recent advances in the development of novel high performance chromatographic separation materials since 2020 and their applications in herbalome research. It focuses on three chromatographic modes： reversed-phase liquid chromatography （RPLC）， hydrophilic interaction liquid chromatography （HILIC）， and supercritical fluid chromatography （SFC）. The structural and performance characteristics of these novel materials are highlighted， along with their applications in the analysis and preparative purification of key active components such as alkaloids， flavonoids， saponins and terpenoids. In RPLC， the separation selectivity and retention capacity of the stationary phases for compounds such as alkaloids and flavonoids have been effectively enhanced through two primary strategies： first， by incorporating polar groups such as fluorine atoms， amides， carboxyl groups into the traditional C18/C8/phenyl skeletons； second， by utilizing natural product derivatives like cardanol and rosin as functional ligands. Furthermore， this approach has led to the creation of novel materials possessing orthogonal separation characteristics. In HILIC， various functionalized stationary phases have been developed， including β-cyclodextrin， nitrogen-containing heterocycles， core-shell structured amides， and surface-polymerized phases. These phases significantly improve the retention and separation efficiency of strongly polar compounds （e.g.， saponins， oligosaccharides） by providing abundant hydrogen bonding， electrostatic， and π-π interactions. In SFC， the development of novel stationary phases extends from modified functional skeletons （e.g.， phenyl） to zwitterionic and nitrogen-containing heterocyclic hydrophilic stationary phases. Additionally， mixed-mode stationary phases have been developed to effectively ameliorate the peak tailing problems associated with basic compounds. Concurrently， the emergence of small-particle-size ordered mesoporous silica materials enables ultra-high column efficiency and rapid separation of components such as tanshinones. This review further illustrates the successful application of these novel materials in both high-performance analytical and preparative chromatography， using four important classes of TCM active ingredients—alkaloids， flavonoids， saponins， and terpenoids—as examples. These novel materials afford significant chromatographic improvements—notably in separation selectivity， peak symmetry， analysis speed， and the resolution of previously difficult-to-separate isomers. Critically， the integration of these advanced materials has facilitated the construction of highly orthogonal multidimensional chromatographic systems （e.g.， RPLC×RPLC， HILIC×RPLC）， thereby providing the capacity for in-depth profiling and identification of dozens to hundreds of components from complex TCM extracts. In the field of preparative chromatography， these materials enable efficient and large-scale purification of target monomers， including trace active ingredients， thereby significantly enriching compound libraries for herbalome research. This includes the implementation of sophisticated multi-dimensional and multi-mode preparative strategies， which have facilitated the discovery of novel compounds and unprecedented skeletons. Finally， the article concludes by outlining future development directions of chromatographic materials， emphasizing the need to enhance separation selectivity and orthogonality， improve separation efficiency and preparative loading capacity， and develop versatile stationary phases. These advancements are crucial to better support the in-depth development of herbalome research. This review aims to provide a material foundation and technical reference for resolving the material basis of complex TCM systems.

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Recent advances of affinity ultrafiltration-liquid chromatography-mass spectrometry in screening natural active substances XU Yongbing, NIU Yujia, GUO Mingquan 2026, 44 (8):  877-888.  DOI: 10.3724/SP.J.1123.2025.08020 Abstract ( 132 )   HTML ( 12 )   PDF (1252KB) ( 37 )   Medicinal plants serve as a valuable source of naturally occurring bioactive compounds， which have played a vital role in promoting human health， preventing and treating various diseases for thousands of years. However， the phytochemical profiles of medicinal plants， including traditional Chinese herbal medicines （TCHMs）， are notably complex and highly diverse， posing a tough challenge for the rapid screening and identification of their potential bioactive constituents. This challenge is central to pharmacological research and drug development efforts in medicinal plants including TCHMs. Therefore， the rapid and efficient screening of bioactive components from complex natural products has become a key research direction in academia. To address this issue， affinity ultrafiltration coupled with liquid chromatography-mass spectrometry （AUF-LC-MS） has emerged as an efficient technique that integrates affinity-based capture， ultrafiltration， and liquid chromatographic-mass spectrometric analysis to enable rapid screening and characterization of potential small-molecule bioactive compounds. Known for its low sample consumption， high specificity， exceptional sensitivity， operational simplicity and high efficiency， this approach leverages specific interactions between target molecules and chemical extracts to identify bioactive ligands from complex mixtures， making it particularly suitable for studying intricate matrices such as medicinal plant extracts and TCHM formulations. This study summarizes the principles， characteristics， and recent applications of AUF-LC-MS technology in the screening of potential active components in medicinal plants including TCHMs over the past decade （2015-2025）. Based on different levels of AUF-LC-MS screening， including compound level， single TCHM level， and TCHM formula level， a comprehensive summary is provided. AUF-LC-MS screening of single TCHM level is further analyzed and summarized based on the number of targets， including single target screening， dual-targets screening， and multi-targets screening. At the same time， comparisons were made between other different natural active substance screening approaches and the AUF-LC-MS method. In addition， it outlines prospective research directions for the further development of AUF-LC-MS with the aim of providing new insights for the study of the pharmacological substances of TCHMs and the development of new drugs.

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Research advances in the preparation and structural characterization of depolymerized oligosaccharides from traditional Chinese medicine LIU Junyi, ZHENG Yi, YAN Jingyu 2026, 44 (8):  889-899.  DOI: 10.3724/SP.J.1123.2025.08024 Abstract ( 99 )   HTML ( 18 )   PDF (632KB) ( 13 )   Polysaccharides derived from traditional Chinese medicine （TCM） have attracted widespread attention due to their excellent bioactivities， such as immunomodulatory， anti-inflammatory， and anti-tumor effects. However， these polysaccharides exhibit inherent characteristics including large molecular weights， complex monosaccharide compositions， and diverse branched structures. These features result in low bioavailability and difficulties in unraveling their structure-activity relationships （SARs）， thereby imposing significant limitations on their practical applications. In contrast， oligosaccharides possess distinct advantages： they have low molecular weights， relatively simple structures， and are easy to purify and characterize structurally. Owing to these properties， oligosaccharides serve as ideal models for deciphering the SARs of TCM polysaccharides. Consequently， the preparation， separation and purification， and structural characterization of TCM oligosaccharides have become the core research focuses in this field.This paper firstly reviews various degradation strategies from the perspective of degradation approaches. These strategies include chemical hydrolysis， oxidative degradation， enzymatic hydrolysis， and assisted degradation methods. For each method， a comparative analysis of their degradation mechanisms， product distribution characteristics， and applicable scopes is conducted. Meanwhile， it elaborates on the modification effects of different methods on the degree of polymerization （DP）， anomeric configuration， and branched structure of oligosaccharides， as well as their potential regulatory roles in the bioactivities of oligosaccharides. Subsequently， this paper carries out a systematic review on the chromatographic techniques commonly used for the separation and purification of degraded oligosaccharides， including gel filtration chromatography （GFC）， ion-exchange chromatography （IEC）， hydrophilic interaction chromatography （HILIC）， and porous graphitized carbon chromatography （PGC）. For each chromatographic technique， this paper provides a detailed account of its separation principle， applicable scenarios， and specific applications in the separation of degraded oligosaccharides. Finally， regarding the oligosaccharide structural characterization techniques-such as monosaccharide composition analysis， methylation analysis， and nuclear magnetic resonance （NMR） spectroscopy-the paper clarifies the complementarity between different techniques and their respective limitations.In summary， this paper focuses on the application of combined strategies-such as chemical hydrolysis， enzymatic hydrolysis， and physicochemical hybrid methods—in the preparation of degraded oligosaccharides derived from TCM， as well as the roles and key points of separation techniques and characterization techniques in structural elucidation. Looking ahead， the preparation， separation and purification， and structural analysis of TCM oligosaccharides require the further establishment of standardized protocols. Additionally， the integrated application of artificial intelligence and multi-omics technologies should be actively promoted in this research field. It is believed that with the continuous in-depth exploration of degraded oligosaccharides， their SARs will become increasingly explicit. It is anticipated that progress in the purification and structural elucidation of TCM-degraded oligosaccharides， together with the convergence of artificial intelligence and multi-omics approaches， will bring clarity to their structure-activity relationships. This will， in turn， enable their more precise and impactful application in functional food design and pharmaceutical development.

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Recent advances on bioaffinity fishing technology for discovery of active components from natural products QU Qingli, WANG Xiaofang, DI Duolong, PEI Dong 2026, 44 (8):  900-910.  DOI: 10.3724/SP.J.1123.2025.08021 Abstract ( 197 )   HTML ( 15 )   PDF (953KB) ( 62 )   Natural products have consistently served as an invaluable source for novel drug development， and the efficient and targeted screening of bioactive constituents from these complex mixtures is crucial for advancing drug discovery. Bioaffinity fishing techniques exploit the highly specific interactions between ligands and their biological targets—such as antigen-antibody， enzyme-substrate， and hormone-receptor interactions—to simultaneously separate and identify active compounds from complex natural product extracts. When coupled with liquid chromatography-mass spectrometry， bioaffinity fishing enables the integrated separation， screening， and analysis of bioactive molecules in a single step. This comprehensive review focuses specifically on bioaffinity fishing materials， which are fundamental to the effectiveness and reproducibility of the fishing process. The review systematically explores recent advances over the past three years， categorizing the discussion along two primary dimensions： carrier materials and immobilization methods. In terms of carrier materials， the article provides a comprehensive review of both carrier-free and carrier-based systems. Carrier-free approaches， including affinity ultrafiltration and size exclusion chromatography， utilize free targets in solution， while carrier-based systems immobilize the target onto a solid support. The review details a wide spectrum of innovative supports such as magnetic beads， which facilitate easy separation； metal-organic frameworks， known for their high surface area and tunable porosity； silica-based materials （e.g.， mesoporous silica and silica chromatographic stationary phases）， prized for their stability and versatility； gel-based materials like agarose and synthetic polymers， which offer superior biocompatibility； microchannel-based systems， including capillary columns and microfluidic chips that enable high-throughput screening with minimal sample consumption； fluorescent materials that integrate fishing with real-time activity monitoring and imaging； and other emerging materials such as graphene， carbon nanotubes， and paper-based substrates. Each material system is evaluated regarding its preparation， advantages， limitations， and representative applications in fishing active ingredients from natural products. Regarding immobilization strategies， the review compares three principal methodologies： physical adsorption， which is simple but often prone to leakage and low efficiency； physical encapsulation， which protects the target yet may incur mass transfer limitations and potential activity loss； and chemical cross-linking， which enhances stability but risks altering the target’s conformation and activity. A significant portion of the discussion is devoted to advanced directional immobilization techniques designed to preserve biological activity and improve efficiency. These include various bioorthogonal reactions such as Halo-tag/alkyl chloride， His-tag/Ni-NTA， SpyTag/SpyCatcher， SNAP-tag/benzylguanine， and the use of unnatural amino acid incorporation combined with click chemistry （e.g.， azide-alkyne cycloaddition， thiol-ene addition）. These strategies allow for precise orientation and covalent attachment of proteins， leading to improved stability， reduced nonspecific binding， enhanced chromatographic performance， and higher binding affinity. Notably， these methods enable the one-step immobilization of even low-abundance or endogenous targets without the need for prior purification， greatly expanding the technique’s applicability. Finally， the review outlines current challenges and prospective directions for the field. Key challenges include the lack of clear selection criteria for carrier materials， potential detrimental effects of immobilization on target structure and function， and the need to balance generality with cost-effectiveness. Future efforts should focus on developing more efficient and targeted immobilization protocols， creating multifunctional composite materials that combine separation， detection， and screening capabilities， fostering interdisciplinary collaborations integrating nanotechnology and bioinformatics， and adhering to green chemistry principles to ensure sustainability. As technological progress continues， bioaffinity fishing is poised to play an increasingly vital role in elucidating the material basis of natural medicine efficacy and accelerating the discovery of new therapeutic agents.

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Analysis of chemical constituents and bioavailable components in Yupingfeng Granules using ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry LU Wensen, XIAO Guanlin, WU Minshan, WU Jiaying, LIU Quan, XU Aili 2026, 44 (8):  911-923.  DOI: 10.3724/SP.J.1123.2025.11008 Abstract ( 118 )   HTML ( 23 )   PDF (1332KB) ( 26 )   Yupingfeng Granules， a classical and widely prescribed formula in traditional Chinese medicine， is extensively employed in clinical practice for its well-documented immunomodulatory and preventive activities. However， the highly complex chemical composition of this multi-herb formula and the specific material basis responsible for its therapeutic effects in vivo remain inadequately characterized and systematically elucidated. Research on its pharmacokinetically critical blood-entering components， which are indicative of systemic exposure and potential direct activity， particularly under representative pathological conditions， remains notably scarce and insufficient. This study therefore aims to systematically characterize the comprehensive chemical profile of Yupingfeng Granules by employing advanced analytical techniques such as HPLC-MS. Furthermore， it seeks to comprehensively investigate and characterize its blood-entering components specifically in cyclophosphamide-induced immunodeficient rat models， comparing them with normal physiological conditions where applicable. The overarching objective is to identify， quantify， and shed light on the formula′s potential active constituents that reach the systemic circulation. This work is expected to provide a more solid and detailed scientific basis for a deeper， molecular-level understanding of the pharmacodynamic mechanism of this important traditional Chinese medicine preparation and to inform its future rational clinical application and quality control. An ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry （UPLC-Q-TOF MS） technology was used to identify the chemical composition of Yupingfeng Granules and the plasma-entering components after administration to immunodeficient model rats. The prepared granule and serum samples were analyzed using a Waters CORTECS UPLC C18 column （150 mm×2.1 mm， 1.6 μm） with gradient elution of 0.1% formic acid aqueous solution and acetonitrile as the mobile phases. The flow rate was set at 0.3 mL/min， the column temperature was maintained at 35 ℃， and the injection volume was 3 µL. Data were acquired in both positive and negative ion modes using an electrospray ionization source. The identification of chemical constituents was performed by comparing retention times， accurate relative molecular masses， and MS/MS fragment ions with those of reference standards， along with reference to relevant literature. Through a systematic analysis involving comparison of retention times， accurate molecular weights， and MS/MS fragment ions， combined with cross-referencing against relevant literature and authenticated reference standards， the chemical profile of Yupingfeng Granules and the bioactive components absorbed in vivo were comprehensively characterized. The investigation successfully identified a total of 52 chemical constituents in Yupingfeng Granules. Furthermore， 11 bioavailable components were detected in rat plasma after administration. These compounds encompass a diverse range of chemical classes， specifically including 11 flavonoids， 13 organic acids， 13 phenylpropanoids， 12 terpenoids， and 3 additional components categorized as aldehydes， nucleosides， and amino acids. This detailed identification provides a thorough overview of the chemical composition of the granules and elucidates the key constituents that are systemically absorbed. This study systematically analyzed the blood-entering components of Yupingfeng Granules under immunodeficient pathological conditions. This distinction is critically important， as the altered internal environment in a disease model may significantly influence the absorption， metabolism， and distribution of herbal compounds. The research not only clarifies the comprehensive chemical material basis of the formula in vitro by establishing a detailed chromatographic fingerprint but also， more significantly， bridges the gap to in vivo pharmacology by identifying which specific compounds from the complex mixture actually enter the systemic circulation. By correlating the in vitro chemical profile with the in vivo exposure data， the study effectively pinpoints a refined set of potential direct-acting substances that are most likely to contribute to the observed therapeutic effects. Consequently， these findings provide crucial experimental clues and a reliable analytical foundation for subsequent， more targeted research. This includes in-depth investigations into efficacy correlation—linking specific blood components to immunomodulatory activities—and the detailed exploration of its molecular mechanisms of action， thereby significantly advancing the scientific understanding of this classic formula.

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Isolation of angular-type pyranocoumarins from Peucedani Radix guided by liver microsomal metabolites and structural identification of the metabolites YANG Yang, LIU Minghao, JIN Hui, SUN Runze, GONG Xingcheng, LIU Wenjing, SONG Yuelin 2026, 44 (8):  924-934.  DOI: 10.3724/SP.J.1123.2025.12018 Abstract ( 115 )   HTML ( 21 )   PDF (1282KB) ( 14 )   Identifying the structures of metabolites is of great importance towards elucidating the therapeutic material basis of traditional Chinese medicines （TCMs）. However， current studies lack sufficient reference standards， hindering the structural identification of metabolites. This study establishes a strategy guided by the retention times and mass spectrometric information of microsomal metabolites. By coupling liquid chromatography-mass spectrometry （LC-MS） to trace and isolate target compounds from the raw materials， these compounds served as reference standards to confirm the structures of microsomal metabolites， thereby elucidating the metabolic pathways. Peucedani Radix （PR， Chinese name： Qianhu） consists of the dried roots of Peucedanum praeruptorum Dunn and contains abundant angular-type pyranocoumarins （APs）， which serve as primary chemical constituents and effective compound clusters as well. From the structural standpoint， C-3′ and C-4′ sites of APs are frequently substituted by acetoxyl， angeloxyl， isovaleroxyl and some other moieties， leading to the extensive occurrences of regio-isomers， which not only possess comparable structures but also similar chemical properties. Previous studies have preliminarily unveiled that APs primarily undergo hydrolysis and acyl migration in vivo， and these hydrolytic metabolites can be observed in both in vitro metabolism and the raw materials. In this study， a pair of regio-isomers from PR， i.e.， （+）-（3′S，4′S）-praeruptorin A （（+）-PA） and （+）-（3′R，4′R）-pteryxin （（+）-Pte）， was incubated separately with human liver microsomes （HLMs） in the presence of necessary cofactors to simulate the metabolic pathways occurring in vivo. After carefully processing LC-MS datasets and applying the well-defined mass fragmentation rules of APs， eight metabolites were detected and putatively identified for （+）-PA， including three hydrolysis products together with five oxidation products， whereas nine metabolites were observed and putatively identified for （+）-Pte， encompassing five hydrolysis products and four oxidation products. Noteworthily， phase I metabolism of （+）-Pte in HLMs was profiled for the first time. Various column chromatographic tools， such as silica gel column chromatography， C18 reversed-phase chromatography （ODS）， Sephadex LH-20 and semi-preparative high performance liquid chromatography equipped with achiral or chiral columns， were employed to isolate compounds-of-interest from PR extracts under the guidance of LC-MS through taking both retention time and MS/MS information into account. A total of nine AP compounds were successfully purified from PR extracts， and notably， four pairs of AP enantiomers were involved. By matching 1H- and 13C-NMR spectral information with the data archived in the literature， these compounds were identified as （3′S，4′S）-qianhucoumarin C （1a）， （3′R，4′R）-qianhucoumarin C （1b）， （3′S，4′S）-qianhucoumarin B （2a）， （3′R，4′R）-qianhucoumarin B （2b）， （3′S，4′S）-3′-angeloylkhellactone （3a）， （3′R，4′R）-3′-angeloylkhellactone （3b）， （3′S，4′S）-4′-angeloylkhellactone （4a）， （3′R，4′R）-4′-angeloylkhellactone （4b） and （3′S，4′S）-isoepoxypteryxin （5）. Thereof， compounds 1b and 2b represent new configurational structures of known planar structures. The study summarized mass fragmentation rules for APs and achieved accurate identification of two pairs of regio-isomers： qianhucoumarin B and C， and 3′- and 4′-angeloylkhellactone. Thereafter， in vitro metabolites of （+）-PA and （+）-Pte were successfully identified by comparing the retention times and MS/MS information with authentic compounds， including cis-khellactone， qianhucoumarin B， qianhucoumarin C， 3′-angeloylkhellactone， 4′-angeloylkhellactone and （3′S，4′S）-isoepoxypteryxin. Regarding the three hydrolytic metabolites， i.e.， cis-khellactone， qianhucoumarin B and qianhucoumarin C， they were shared by （+）-PA and （+）-Pte. Hydrolysis and oxidation reactions， as well as acyl migration， served as the primary metabolic pathways for APs in HLMs. In this study， （+）-PA and （+）-Pte were selected as representatives to demonstrate that it is feasible to obtain their metabolites from PR extracts through LC-MS-guided isolation and purification. After structural identification through analyzing 1H- and 13C-NMR spectral information， these pure compounds were eligible authentic compounds enabling confirmative structural identification of metabolites， providing pronounced insights into the primary metabolic pathways of APs and offering an effective approach for the accurate identification of metabolite structures through isolating authentic compounds that are available in the original TCM. However， due to differences in metabolism between in vitro and in vivo， the study failed to construct a definitive structure-activity relationship for the metabolites. Future studies should be devoted to validating these findings through performing in vivo investigations.

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Non-targeted metabolomics analysis of exosomes derived from Glycyrrhiza uralensis YAO Yunjun, QIAN Xu, SHU Fuxing, SONG Xichao, DONG Yuanyuan, JIN Leilei, LIU Fengjian, JI Zuen, LIU Jia, CHEN Jishuang 2026, 44 (8):  935-945.  DOI: 10.3724/SP.J.1123.2025.12030 Abstract ( 127 )   HTML ( 15 )   PDF (1637KB) ( 13 )   Exosomes are nanometer-sized secretory vesicles that play a pivotal role in mediating intercellular communication. Recently， exosomes derived from herbal medicine have garnered significant attention owing to their unique composition features and therapeutic potential. Glycyrrhiza uralensis， a traditional Chinese medicinal and edible plant， is rich in flavonoids， terpenoids， polyketides， and phenolic acids. However， the long-term consequences of overharvesting and artificial cultivation have led to inconsistent quality of licorice materials. Plant tissue culture technology offers a sustainable approach to preserve medicinal plant resources， enabling year-round production independent of seasonal and environmental constraints. To address these challenges and explore the therapeutic potential of herb-derived exosomes， this study employed a laboratory-constructed temporary immersion bioreactor system （TIBS） for G. uralensis cultivation and exosomes isolation. The key experimental procedures are summarized as follows： sterile buds derived from G. uralensis seeds were used as explants. The callus proliferation medium consisted of Murashige and Skoog medium （MSM） supplemented with 1.18 mg/L 6-benzylaminopurine （6-BA）， 1.44 mg/L naphthaleneacetic acid （NAA） and 1.44 mg/L 2，4-dichlorophenoxyacetic acid （2，4-D）， yielding a maximum proliferation coefficient of 10.81 after 21 days. For shoot proliferation， sterile plantlets were generated on MSM medium supplemented with 1 mg/L 6-BA， 0.05 mg/L NAA， and 150 mL/L coconut milk （CM）， which produced sterile plantlets within 28 days of culture. When cultured in the TIBS system with an immersion frequency of 5 min every 6 h， the callus proliferation coefficient reached 13.12 after 28 days of culture. Exosomes were successfully isolated from the TIBS-cultured G. uralensis calli using differential centrifugation and ultracentrifugation protocols. Comprehensive characterization was performed to validate the identity， purity， and stability of isolated exosomes. Morphological observation via transmission electron microscopy （TEM） revealed typical cup-shaped vesicles with a distinct lipid bilayer structure， which is consistent with the canonical exosomes morphology. Dynamic light scattering （DLS） analysis determined an average hydrodynamic diameter of 56 nm. The exosomes preparation exhibited a particle concentration of 1.07×1012 particles/mL， and a zeta potential of （-15.030±3.815） mV， indicating excellent colloidal stability suitable for biological applications. To decipher the molecular cargo of these exosomes， untargeted metabolomic profiling was conducted. A total of 1 760 metabolites were identified across callus and exosomes samples， of which 1 068 differential metabolites （DMs） distinguished exosomes from parental calli. Notably， 195 DMs were significantly enriched in exosomes. KEGG （Kyoto Encyclopedia of Genes and Genomes） pathway enrichment analysis revealed enrichment in 11 metabolic pathways， primarily involving terpenoids， flavonoids， alkaloids， fatty acids， and phenylpropanoids. Bioactive compounds of interest included azelaic acid， phloretin， and senkyunolide C， highlighting their potential pharmacological significance. In summary， this study demonstrates that the TIBS-based in vitro platform enables efficient， scalable， and consistent production of G. uralensis-derived exosomes with homogeneous morphology and specialized metabolite profiles. The successful isolation， comprehensive characterization， and identification of bioactive metabolite enrichment establish a robust foundation for mechanistic investigations and translational applications. These exosomes exhibited promising therapeutic potential in anti-inflammatory， antioxidant， antitumor， and antibacterial biomedicine. Furthermore， this bioreactor system addresses the quality inconsistency issue in natural G. uralensis resources， paving new ways for developing innovative herbal exosome-based nanomedicines.

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Deep eutectic solvents modified reversed-phase liquid chromatography for the separation of natural products ZHANG Hui, WU Huimin, HE Qun, QIN Tianxue, GUO Suying, LI Wenjing, JIANG Rong, SUN Guangying 2026, 44 (8):  946-959.  DOI: 10.3724/SP.J.1123.2026.04013 Abstract ( 125 )   HTML ( 24 )   PDF (1720KB) ( 31 )   The composition of natural products is usually very complex. During the separation of natural products， high loading capacity and satisfactory chromatographic resolution are often hardly achieved. To address this limitation， various modifiers were added in the mobile phase to alter the chromatographic behavior. Here， deep eutectic solvents （DESs） were chosen to accomplish this objective on C18 column. Changes in retention factor， overloaded peak shape and adsorption capacity were systematically investigated and interpreted. The application potential of DESs in the separation of natural products was then evaluated. For ionizable compounds， the addition of DESs could compress the peak width and enhance the resolution. This is closely related to the electrostatic interactions provided by hydrogen bond acceptor （HBA） of DESs. For ionizable basic compounds like nortriptyline hydrochloride， they could remain in a cationic state constantly. Consequently， ion-exchange interactions occurred between nortriptyline cation and residual silanol anion， which resulted in severe peak tailing. However， this interaction could be shielded by the addition of HBA of DESs （such as choline chloride （ChCl））. As the pH of the mobile phase increased， the dissociation of silanol groups was enhanced. This rendered the shielding effect more prominent and led to a higher peak compression of nortriptyline hydrochloride. Notably， although the mobile phase was acidic， the dissociation of silanol groups cannot be completely suppressed. It means that this modulating effect of HBA never disappears. Based on these principles， the semi-preparative separation of berberine from Coptis chinensis extracts was successfully improved. The improvement in the separation of acidic compounds by DESs depends on the ion-pair formation. This effect is highly correlated with the mobile phase pH and salt concentration. Here， benzoic acid was chosen as a model to investigate the overload behavior and adsorption. The results indicated that under neutral conditions， ChCl addition converted the sigmoidal adsorption to a linear type. It can be attributed to the ion-pair formation between benzoate anions and HBA cations. Mutual repulsion between benzoate anions was therefore reduced and adsorption capacity correspondingly enhanced. Furthermore， the results of the overload experiment indicated that ChCl can effectively weaken the solute-solute interactions. It is worth noting that the mobile phase pH is barely influenced after the addition of ChCl. Remarkably， these features are of critical importance for the optimization of preparative chromatographic methods. The beneficial effect of HBAs was validated in the semi-preparative separation of chlorogenic acid from Lonicera japonica extracts. For neutral compounds like morroniside， the HBA in DESs cannot affect their chromatographic behavior through electrostatic interactions. However， there are usually many ionizable impurities surrounding the neutral compounds in natural products. Their chromatographic behavior can be modulated by the HBAs in DESs. The separation of neutral compounds in natural products could be indirectly optimized by modulating their chromatographic behavior. This view was also confirmed by the separation of morroniside from Cornus officinalis extracts.

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Iron porphyrin metal-organic framework material for efficient enrichment of earthworm polypeptides ZHAN Kunsong, TANG Mingzhu, LIN Ying, NI Linjun, YU Lishuang 2026, 44 (8):  960-967.  DOI: 10.3724/SP.J.1123.2025.11018 Abstract ( 81 )   HTML ( 20 )   PDF (1192KB) ( 10 )   Earthworms are a commonly used animal-derived traditional Chinese medicine in China， with proteins and polypeptides as their main active ingredients. However， the existing methods for separating and detecting earthworm polypeptides have problems such as insufficient preparation capacity and poor separation reproducibility. To address these challenges， this study used the iron porphyrin metal-organic framework material PCN-222（Fe） to specifically adsorb the earthworm polypeptide component L-PeakⅡ， with an adsorption capacity of 139 mg/g. First of all， PCN-222（Fe） material was successfully prepared via the one-pot method. The earthworm protease hydrolysate was obtained through alkali extraction and acid precipitation， and trypsin enzymatic hydrolysis. Then， PCN-222（Fe） was further used as a solid-phase adsorbent and combined with liquid chromatography to rapidly separate and prepare the polypeptide component L-PeakⅡ from complex earthworm enzymatic hydrolysate of the Chinese medicine. Qualitative analysis was performed by HPLC， and quantitative analysis was conducted with a BCA assay kit. HPLC analysis of the earthworm protease hydrolysate revealed multiple chromatographic peaks. After enrichment with PCN‑222（Fe） and subsequent elution， the eluted sample exhibited fewer peaks， with a well‑defined peak appearing at a retention time of approximately 15 min. In order to obtain the optimal experimental conditions for the enrichment of earthworm polypeptides， influencing factors including enzyme hydrolysate mass concentration， pH value， elution solvent type， and elution time were investigated and optimized. The results showed that optimal enrichment was achieved by incubating at an enzymatic hydrolysate mass concentration of 3.0 mg/mL and pH of 9， followed by elution with 50% acetonitrile containing 0.2% trifluoroacetic acid for 2 h. Finally， the earthworm-derived polypeptide component was obtained using a high-pressure preparative chromatographic system， achieving a purity exceeding 95%. The integrated approach employed in this study， which combines PCN-222（Fe） material with preparative chromatography， enables the efficient preparation of the target polypeptide L-PeakⅡ from medicinal earthworm samples. This strategy provides a valuable reference for the separation and purification of polypeptide components from complex animal-derived traditional Chinese medicines. Moreover， it offers a more robust， scalable， and reproducible pathway for harnessing the therapeutic potential of natural product peptides， thereby bridging the gap between traditional medicine and modern pharmaceutical analysis.

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Determination of the key characteristic components in ginger from different origins by internal extractive electrospray ionization mass spectrometry WANG Huiting, XIE Siyu, XIE Jingjing, WU Ruotian, QIN Manman, CHEN Huanwen 2026, 44 (8):  968-978.  DOI: 10.3724/SP.J.1123.2025.09001 Abstract ( 59 )   HTML ( 25 )   PDF (2498KB) ( 13 )   Ginger， the fresh rhizome of Zingiber officinale Rosc. from the Zingiberaceae family， is highly valued worldwide for its unique aromatic profile and distinct pungent flavor. It is widely utilized both as a culinary spice and a natural food additive. Globally， ginger comprises 53 known genes and approximately 1 300 varieties， predominantly cultivated in tropical and subtropical regions. Major producing countries include India， China， Indonesia， and Nigeria， with China ranking as the second-largest producer. In many Asian countries， such as China， Korea， and Japan， ginger has long been recognized not only for its culinary applications but also for its medicinal properties. It is traditionally employed to alleviate cold symptoms， promote body warmth， reduce nausea， suppress coughs by clearing phlegm， and even neutralize toxins from seafood consumption. The efficacy and safety of ginger as a medicinal herb are largely determined by the content and purity of its active constituents， such as gingerols and shogaols. Thus， accurate quality assessment is essential to ensure its therapeutic value. Moreover， the contents of these bioactive compounds can vary considerably depending on the geographical origin. In China， high-quality medicinal ginger is primarily cultivated in provinces such as Yunnan， Sichuan， Guizhou and Henan. Given the complexity and diversity of ginger’s chemical composition， it is crucial to understand its characteristic components and their variation with origin. This knowledge supports not only accurate quality evaluation but also reliable traceability of ginger sources. Ultimately， it facilitates the selection of ginger with superior medicinal properties for direct use in traditional medicine or as high-quality raw material in pharmaceutical development. However， conventional analytical techniques for determining origin and quality， such as high performance liquid chromatography （HPLC） and gas chromatography-mass spectrometry （GC-MS）， are often time-consuming， require extensive sample preparation， and consume large volumes of solvents. To overcome these limitations， this study developed a high-throughput method based on internal extractive electrospray ionization mass spectrometry （iEESI-MS） for rapid compositional profiling and origin authentication of ginger. Samples were collected from four major ginger-producing regions in China： Yunnan， Sichuan， Guizhou， and Henan. Each sample was minimally processed into fragments， with only 5.0 mg used for analysis. The fragments were placed on a filter membrane inside a custom-built iEESI-MS device. Key instrumental parameters such as the extraction solvent composition， ion transfer tube temperature， spray voltage， and solvent flow rate were systematically optimized to enhance detection sensitivity and reproducibility. Under optimal conditions， real-time extraction and ionization of chemical constituents from ginger tissue were achieved， yielding representative mass spectral fingerprints for each geographical origin. Multivariate statistical tools were applied to interpret the complex mass spectrometry data. Principal component analysis （PCA） provided an overview of sample distribution and revealed inherent clustering trends according to origin. Partial least squares discriminant analysis （PLS-DA） further improved the classification accuracy by filtering out unrelated variations and emphasizing the ions that contributed most to inter-regional differences. Through this approach， 27 compounds were consistently detected and identified， among which three key markers， such as 6-gingerol， 8-gingerol， and L-serine， were selected as characteristic of origin-related variation. A quantitative method was developed for these three markers， demonstrating excellent linearity across a broad content range （2.0-20 000.0 μg/g） with coefficients of determination （R²） no less than 0.996. Sensitivity was assessed in terms of limits of detection and limits of quantification， which ranged from 3.0 μg/g to 20.0 μg/g and 10.0 μg/g to 50.0 μg/g， respectively. Recoveries varied between 98.8% and 100.9%， indicating high accuracy， while repeatability was excellent， with relative standard deviations （RSDs） no more than 1.6%. When the established method was applied to ginger samples from different regions， heatmap visualization clearly illustrated the correlation between geographic origin and the abundance of the three characteristic compounds. For instance， Yunnan samples exhibited notably higher contents of certain gingerols， whereas those from Henan were richer in L-serine. This chemical fingerprinting strategy offers a reliable and efficient means for rapid origin verification and quality assessment. In conclusion， the iEESI-MS platform developed in this study combines minimal sample preparation， rapid analysis， and high sensitivity， making it well-suited for high-throughput applications. It holds significant potential not only for ginger authentication but also for quality control of other medicinal plants， food traceability systems， and the protection of geographically indicated products. Future studies may expand the database to include more regions and varieties， further improving the robustness and general applicability of the model.

Technical Notes

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Separation and purification of phospholipids from Antarctic krill oil by hydrophilic interaction liquid chromatography PAN Jian, HONG Lin, ZHANG Yan 2026, 44 (8):  979-985.  DOI: 10.3724/SP.J.1123.2026.04010 Abstract ( 85 )   HTML ( 18 )   PDF (743KB) ( 21 )   Antarctic krill has the largest biomass of any wild animal species on Earth. Krill oil extracted from Antarctic krill is rich in phospholipids， and is a major natural source of marine phospholipids. In marine phospholipids， eicosapentaenoic acid （EPA） and docosahexaenoic acid （DHA） are esterified at the sn-2 position of glycerol. This unique structural configuration endows them with the dual physiological functions of both phospholipids and Ω-3 polyunsaturated fatty acids. Compared with terrestrial phospholipids， marine phospholipids exhibit superior bioactivity in several key aspects， including neurotrophic support， anti-atherosclerotic effects， and lipid metabolism regulation， thereby conferring significant application value in pharmaceuticals， health foods， and functional foods. However， pharmaceutical excipient applications require phospholipid purity above 90%， a standard also required for high-end pharmaceutical formulations and active pharmaceutical ingredients. Currently， commercial krill oil products vary significantly in quality. Their total phospholipid content ranges from only 30% to 60%. Therefore， purifying phospholipids from Antarctic krill is essential. This process holds practical value for improving the quality standards of marine phospholipids. Hydrophilic interaction liquid chromatography （HILIC） separates polar compounds through multiple mechanisms， relying on polar interactions between the hydrophilic headgroups of phospholipids and the stationary phase， as well as partitioning within the water-rich layer on the stationary phase surface. In this study， a HILIC-based method was developed for the purification of phospholipids from Antarctic krill oil. Industrial-scale preparative chromatography requires stable stationary phases. Three HILIC-compatible stationary phases were systematically evaluated. These were unmodified bare silica gel， Diol （3% carbon）， and Diol-H （7% carbon）. Diol showed favorable retention and resolution. Therefore， Diol was selected as the optimal stationary phase. The superior performance of the Diol phase compared to Diol-H suggests that a moderate carbon content is crucial. Excessive hydrophobicity in Diol-H likely interfered with the specific hydrogen bonding interactions required for separating polar lipid headgroups. Ethanol and water were chosen as the mobile phases， because these solvents have low toxicity. Ethanol-water proportions were investigated to develop a three-step elution program： washing with 100% ethanol， elution with 95% ethanol， and column re-equilibration with 80% ethanol. Based on chromatographic plate and rate theories， a Diol column （250 mm×4.6 mm） was used. Isocratic elution with 95% ethanol was applied. The relationship between flow rate， theoretical plate number， and peak width was evaluated. An optimal flow rate was selected. Peak broadening under high sample loading was examined. This determined the maximum loading capacity. It was optimized at 8% of the stationary phase mass. Separation remained efficient at this level. Resolution did not decrease significantly. The final purification protocol involved washing with 2 column volumes of 100% ethanol， eluting the target phospholipid fraction with 5.5 column volumes of 95% ethanol， and re-equilibrating the column with 2.5 column volumes of 80% ethanol. Finally， scale-up separation was performed using a DAC50 column. The crude oil contained 57.88% phospholipids. Four fractions were collected based on online chromatograms. Each fraction was concentrated， dried， and then analyzed. The recovery of the purified product was 49.15%. Purity was measured using the molybdenum blue colorimetric method （GB/T 5537-2008）. Purity reached 95.42%. The overall phospholipid recovery was 81%. EPA and DHA were quantified via external standard method （GB 5009.168-2016）. EPA and DHA contents were 18.5% and 7.18%， respectively. This method efficiently prepares pharmaceutical-grade phospholipids. The process is environmentally friendly and easily scalable for industrial manufacturing. This approach supports the purification of marine phospholipids from Antarctic krill oil and the high-purity concentrate serves as a reference material. It supports clinical research on krill-derived lipids and promotes applications in drug delivery and nutraceuticals. This work improves quality standards for marine lipid products and contributes to the sustainable use of Antarctic krill resources. Preparative liquid chromatography is a mature technology widely used for separating complex samples like drugs and natural products. This study investigated HILIC for purifying phospholipids from Antarctic krill oil. The research provides a technical basis for selecting preparation methods.