Traditional Chinese prescriptions are characterized by complex chemical constituents and wide variations in constituent content, which pose a substantial challenge to their comprehensive characterization. As a classic traditional Chinese prescription known for its heat-clearing and detoxifying properties, Huangqintang Decoction (HQD) is composed of Scutellariae Radix, Paeoniae Radix Rubra, Glycyrrhizae Radix et Rhizoma, and Jujubae Fructus. In this study, we developed an off-line two-dimensional liquid chromatography that addressed the limitations of traditional analysis of unfractionated extracts, such as restricted peak capacity, which often obscured trace components. By coupling with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS), this study successfully performed rapid identification or characterization of the complete chemical profile of HQD. Notably, beyond high-throughput identification, this approach leveraged characteristic fragment ions and reversed-phase chromatographic behaviors to differentiate some isomers of flavonoid glycosides and triterpenoid saponins, demonstrating its depth in structural identification. Flavonoid glycoside isomers were distinguished by diagnostic neutral losses, while flavanones and chalcones were characterized by retro-Diels–Alder (RDA) and β-rearrangement, respectively. Isomers of triterpenoid saponins were inferred from aglycone-specific pathways alongside RDA cleavages. Ultimately, a total of 192 compounds were identified, including 88 flavonoids, 80 triterpenoids, 7 monoterpene glycosides, 3 fatty acid amides, 3 phenylethanoid glycosides, 4 coumarins, 3 saccharides, 1 organic acid, and 3 others. This study demonstrated that the off-line two-dimensional liquid chromatography analysis strategy significantly enhanced chromatographic resolution and expanded the coverage of trace components. It presented an effective strategy for comprehensive compound identification in complex traditional Chinese medicine prescriptions.

Six oligosaccharides with the degree of polymerization (DP) 2 to 7 were isolated from the active fraction of Trillium tschonoskii using a high-temperature semi-preparative porous graphitic carbon (PGC) column based on high-performance liquid chromatography coupled with a charged aerosol detector and vanquish fraction collector (HPLC-CAD-VFC) system. The structures of DP2 − 7 were determined as maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose with (1→4)-α-d-glucopyranose residues by relative quantitative 1H nuclear magnetic resonance (qHNMR) spectroscopy with anomeric protons integration and NMR structural-reporter-group resonances. The immunomodulatory activities of this series of maltooligosaccharides (MOS) were tested on RAW264.7 cells. All samples on cells had no cytotoxicity, and the oligosaccharide fraction (TOS1) could increase the phagocytic capacity and production of nitric oxide (NO) and cytokines to a much higher level than monomers. In particular, maltopentaose (DP5) showed a relatively higher capacity for stimulating proliferation, NO, and cytokine production than other purified oligosaccharides. The immunomodulatory activity of MOS investigated in this study is beneficial for utilizing MOS as a functional ingredient in novel product development.

ObjectiveThe dried succulent stem of C. tubulosa (Schenk) Wight has long been used as herbal medicine in China and other regions of Asia for its tonifying properties. This study aimed to elucidate the pharmacological mechanisms of the total glycosides from Cistanche tubulosa (GCT) in ameliorating cognitive decline, with a focus on gut microbiota remodeling and metabolic regulation.MethodsSix-month-old APP/PS1 double-transgenic mice received oral GCT at three doses or donepezil for 60 days. Cognitive function was assessed by the Morris water maze. Aβ burden and inflammatory factors were evaluated by immunohistochemistry and ELISA. Gut microbiota was analyzed using 16S rRNA sequencing. Metabolomic profiles of mice serum and brain were profiled by a targeted metabolomics approach that enabled simultaneous quantitation of 306 metabolites. The effect of GCT on pure-cultured bacterial strain was assessed via growth curve analysis in vitro.ResultsGCT treatment significantly improved spatial memory and reduced the protein levels of Aβ and proinflammatory factors in APP/PS1 mice. Multi-omics analyses revealed that GCT rapidly enriched beneficial taxa like Akkermansia and suppresses Firmicutes since the seventh day of intervention, leading to increased neuroprotective short-chain fatty acids (e.g., β-hydroxybutyrate) and decreased pro-inflammatory long-chain fatty acids in both serum and brain. Crucially, in-vitro experiments demonstrated that GCT directly promoted the proliferation of Akkermansia muciniphila, a key probiotic implicated in AD amelioration.ConclusionThis work uncovers a novel “gut microbiota-fatty acid metabolism-neuroinflammation” axis as the primary mechanism underlying GCT’s anti-AD effects. These findings highlight GCT’s therapeutic potential and offer new mechanistic insights into how low-bioavailability phytochemicals exert systemic benefits via the gut-brain axis.

Aim of the studyDiabetic ulcer (DU), a major complication of diabetes, is characterized by complex pathophysiology and limited therapeutic options. Chuang Ling Ye (CLY) has demonstrated significant therapeutic efficacy in the treatment of DU. This study investigates the therapeutic potential of CLY for DU in vivo and in vitro, examining its mechanisms at tissue, cellular, and molecular levels.MethodsA UHPLC-MS/MS methodology was employed to identify metabolites in CLY. Network pharmacology was utilized to construct a metabolite-target network, and GO and KEGG enrichment analyses were performed to predict potential biological mechanisms of CLY in DU treatment. Furthermore, the therapeutic efficacy of CLY against DU was validated through in vitro and in vivo experiments. The mechanism of action of CLY in DU treatment was further verified using Western blot and flow cytometry.ResultsA total of 357 metabolites were identified in CLY using UHPLC-MS/MS. Our data indicate that CLY significantly promoted DU healing in vivo. In vitro experiments revealed that CLY ameliorated high glucose-induced dysfunction in HUVEC cells and upregulated the expression of angiogenesis-related proteins VEGF and CD31. Additionally, CLY significantly reduced the release of inflammatory factors in lipopolysaccharide-induced THP-1 cells, confirming its anti-inflammatory properties. These effects were attenuated following inhibition of the PI3K-Akt signaling pathway with LY294002. Consistent results were observed in vivo, elucidating the therapeutic efficacy and mechanistic basis of CLY in DU treatment.ConclusionCLY promotes DU healing by attenuating inflammatory responses and enhancing angiogenesis through activation of the PI3K-Akt signaling pathway.

Dai-Bai-Jie, the root of the plant Marsdenia tenacissima from the Asclepiadaceae family, is well-known for its therapeutic effects in clearing heat, detoxifying, reducing swelling, and relieving pain as one of the most commonly used Dai medicine. Due to numerous structurally similar C21 steroidal compounds in Dai-Bai-Jie, chemical composition profiling has been substantially challenged. In this study, an offline two-dimensional chromatographic method (LC x SFC separation system) was developed to address these issues. Using the Hypersil Gold (1stD LC column) and 2-PIC (2ndD SFC column) based on 40 reference standards, the orthogonality was as high as 83.83 %. Most profiled ion peaks were tentatively identified through quadrupole time-of-flight mass spectrometry and a self-built compound virtual library. Consequently, the integrated method effectively addressed and resolved the issues associated with co-elution, thus significantly expanding the peak capacity. This advancement identified 362 C21 steroidal components, 319 of which were speculated to be potentially novel compounds. Furthermore, 86 groups of isomeric compounds were distinguished. This method provides a comprehensive understanding of chemical composition of Dai-Bai-Jie and an integrated qualitative analysis method for the C21 steroids.

Species of the genus Marsdenia have a long history of medicinal use in China, with C21 steroids serving as the principal bioactive constituents. Marsdenia cavaleriei remains an unexplored phytochemical resource, particularly regarding the material basis in different plant parts. In this study, an ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) analytical method was developed to thoroughly characterize the chemical constituents of this plant. A total of 68 compounds were identified, 48 of which were tentatively identified as novel. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed to screen 18 potential chemical markers, elucidating compositional differences across various plant parts. Quantification of Tenacissoside H was conducted using ultra-high-performance liquid chromatography with charged aerosol detection (UHPLC-CAD), revealing its absence in the leaves. The average content of Tenacissoside H in the roots (0.281 %) exceeded that in the stems. A semi-quantitative analytical method was developed under identical gradient conditions with inverse gradient compensation. The relative standard deviation (RSD) of the average response factors for five references was 2.46 %. The semi-quantitative analysis of nine primary C21 steroids showed that Tenacigenoside K and Tenacissoside A were most abundant in the leaves, Tenacissoside A and Marsdenoside H dominated in the stems, and Marsdenoside H and Tenacissoside D were the most prevalent in the roots. This study presents a comprehensive approach for qualitative and quantitative analysis, thereby enhancing our understanding of the chemical composition of M. cavaleriei across its various parts and providing a foundation for its broader application.

Ziziphi Spinosae Semen refers to the dried seed of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou. The seed is composed of a reddish brown coat and a yellow kernel. A comparative study was conducted to investigate differences in the chemical composition and their relative contents between the seed coat and kernel of Ziziphi Spinosae Semen. First, the chemical compounds found in the seed coat and kernel were characterized and identified using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). The analytical results tentatively identified 57 chemical compounds based on reference-compound comparison, literature retrieval, and chemical-database (e. g., MassBank) searches; these compounds included 14 triterpenes, 23 flavonoids, 7 alkaloids, 6 carboxylic acids, and 7 other types of compounds. The mass error of the identified compounds was within the mass deviation range of 5×10-6 (5 ppm). Next, two methods of multivariate statistical analysis, namely, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), were used to compare the differential compounds between the two seed parts. A total of 17 differential compounds were screened out via OPLS-DA based on a variable importance in projection (VIP) value of >5. The results revealed that betulinic acid, betulonic acid, alphitolic acid, and jujuboside Ⅰ mainly existed in the seed coat whereas the 13 other compounds, such as spinosin, jujuboside A, and 6‴-feruloylspinosin, mainly existed in the seed kernel. Therefore, these 17 differential compounds can be used to distinguish between the two seed parts. Finally, a semiquantitative method was established using UPLC and a charged aerosol detector (CAD) with inverse gradient compensation in the mobile phase. Six representative compounds with different types were selected to examine the CAD response consistency: magnoflorine (alkaloid), spinosin (flavone), 6‴-feruloylspinosin (flavone), jujuboside A (triterpenoid saponin), jujuboside B (triterpenoid saponin), and betulinic acid (triterpenoid acid). The results showed that the relative standard deviation (RSD) of the average response factors at different levels of these six compounds was 7.04% and that their response intensities were similar. Moreover, each compound in the fingerprint demonstrated good response consistency, and the peak areas obtained directly reflected the contents of each compound. Based on the semiquantitative fingerprints obtained, betulinic acid and oleic acid were considered the main components of the seed coat. The betulinic acid content in the seed coat was approximately 7 times higher than that in the seed kernel. Spinosin, jujuboside A, linoleic acid, betulinic acid, and oleic acid were the main components of the seed kernel. The spinosin content in the seed kernel was 18 times higher than that in the seed coat. In addition, the jujuboside A content in the seed kernel was 24 times higher than that in the seed coat. The proposed method can accurately determine the main components and compare the relative contents of these components in different seed parts. In summary, this study identified the differences in chemical components between the seed coat and kernel of Ziziphi Spinosae Semen and clarified the main components and their relative contents in these parts. The findings can not only provide a basis for the identification of chemical compounds and quality research on different parts of Ziziphi Spinosae Semen but also promote the development and utilization of this traditional Chinese medicine.

Fufang Yinhua Jiedu granules (FYJG) is a Traditional Chinese Medicine (TCM) compound formulae preparation comprising ten herbal drugs, which has been widely used for the treatment of influenza with wind-heat type and upper respiratory tract infections. However, the phytochemical constituents of FYJG have rarely been reported, and its constituent composition still needs to be elucidated. The complexity of the natural ingredients of TCMs and the diversity of preparations are the major obstacles to fully characterizing their constituents. In this study, an innovative and intelligent analysis strategy was built to comprehensively characterize the constituents of FYJG and assign source attribution to all components. Firstly, a simple and highly efficient ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MSE) method was established to analyze the FYJG and ten single herbs. High-accuracy MS/MS data were acquired under two collision energies using high-definition MSE in the negative and positive modes. Secondly, a multistage intelligent data annotation strategy was developed and used to rapidly screen out and identify the compounds of FYJG, which was integrated with various online software and data processing platforms. The in-house chemical library of 2949 compounds was created and operated in the UNIFI software to enable automatic peak annotation of the MSE data. Then, the acquired MS data were processed by MS-DIAL, and a feature-based molecular networking (FBMN) was constructed on the Global Natural Product Social Molecular Networking (GNPS) to infer potential compositions of FYJG by rapidly classifying and visualizing. It was simultaneously using the MZmine software to recognize the source attribution of ingredients. On this basis, the unique chemical categories and characteristics of herbaceous plant species are utilized further to verify the accuracy of the source attribution of multi-components. This comprehensive analysis successfully identified or tentatively characterized 279 compounds in FYJG, including flavonoids, phenolic acids, coumarins, saponins, alkaloids, lignans, and phenylethanoids. Notably, twelve indole alkaloids and four organic acids from Isatidis Folium were characterized in this formula for the first time. This study demonstrates a potential superiority to identify compounds in complex TCM formulas using high-definition MSE and computer software-assisted structural analysis tools, which can obtain high-quality MS/MS spectra, effectively distinguish isomers, and improve the coverage of trace components. This study elucidates the various components and sources of FYJG and provides a theoretical basis for its further clinical development and application.

Oligosaccharides constitute fundamental components in numerous traditional Chinese medicines (TCMs). Conventional chromatographic methods for natural product analysis are not suitable for oligosaccharides due to their large polarity and structural similarity. Herein, an ultra-high performance liquid chromatography with charged aerosol detector (UHPLC-CAD) method was developed for the profiling of oligosaccharides using 9 neutral (DP3-DP11) reference oligosaccharides. Various factors, including columns, mobile phase, elution conditions, flow rate, and column temperature were systematically examined. Optimal separation was achieved using an Amide column with gradient elution within 18min, at 0.5mL/min flow rate and 30℃ column temperature. Moreover, an ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) method was also optimized to provide structural information. The developed method was applied to detect oligosaccharides in several TCMs, including Morindae Officinalis Radix (MOR), Ziziphi Spinosae Semen (ZSS), Menthae Haplocalycis Herba (MHH) and Chrysanthemi Indici Flos (CIF), revealing 9 and 16 oligosaccharides being uncovered from MHH and CIF respectively for the first time. This study presents a versatile UHPLC-CAD and UHPLC-Q-TOF/MS method with the potential for advancing oligosaccharides discovery and contributing to the quality analysis of TCMs.

Leptadenia hastata (Pers.) Decne is a commonly used food source and prescribed as a traditional African medicine for treatment of various diseases, such as diabetes, skin disorders, wounds, and ulcers. However, quality control has become a bottleneck restricting the therapeutic development and utilization of this plant. In this study, a reliable method for qualitative and quantitative determination of components in Leptadenia hastata was established. The components of L. hastata were profiled using ultra-high performance liquid chromatography coupled with quadruple time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS). Subsequently, an ultra-high performance tandem diode array detector (UHPLC-DAD)-based method was used for simultaneous quantitative analysis of five major constituents in six batches of L. hastata samples. As a result, 35 compounds were tentatively identified. The quantities of the five constituents (vicenin-Ⅱ, orientin, schaftoside, chrysin 6-C-arabinoside 8-C-glucoside, chrysin 6-C-glucoside 8-C-arabinoside) were determined as 124.8–156.9 μg/g, 170.5–216.0 μg/g, 61.31–93.73 μg/g, 85.13–119.3 μg/g and 99.82–129.4 μg/g, respectively. This method offers a successful strategy for precise and effective evaluation of the constituents of L. hastata, providing a robust foundation for holistic quality assessment of medicinal plants.

Jiaogulan herbal tea, known for its pharmacological benefits, is derived from various species within the genus Gynostemma, particular for G. pentaphyllum (GP) and G. longipes (GL). Herein, a global approach integrating chemometrics with qualitative and quantitative methods was utilized to investigate the chemical profiles and variations in GP and GL from different geographic origins. A total of 227 triterpenoid saponins were identified in two Gynostemma species, among which 23 differential markers ware screened out by multivariate statistical analysis. Furthermore, the contents of significant markers were determined after the demalonylation process. The chemical composition of GP samples sourced from diverse regions were firstly summarized into three chemotypes (I-III), along with an additional chemotype (IV) for GL. This study contributes to a deeper understanding of Gynostemma plants and provides valuable theoretical basis for the quality control in the production of Jiaogulan herbal tea.

The rhizoma of Polygonatum odoratum (PO) is used to treat yin injuries of the lung and stomach in traditional Chinese medicine. The chemical constituents of this herb are steroidal saponins, homoisoflavanones, and alkaloids. Xiangyuzhu (XPO) and Guanyuzhu (GPO) are available in the market as two specifications of the commodity. Nonetheless, systematic research on the identification and comparison of chemical constituents of these two commercial specifications is yet lacking. Herein, an integrated method combing ultra-high-performance liquid chromatography-quadruple time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) with ultra-high-performance liquid chromatography-charged aerosol detection (UHPLC-CAD) was employed for the comprehensively qualitative and quantitative analyses of PO. A total of 62 compounds were identified by UHPLC-Q-TOF/MS, among which 13 potential chemical markers were screened out to distinguish two commercial specifications. Subsequently, the absolute determination method for polygodoraside G, polygonatumoside F, and timosaponin H1 was established and validated by UHPLC-CAD. The contents of the three compounds were 13.33–236.24 μg/g, 50.55–545.04 μg/g, and 13.34–407.83 μg/g, respectively. Furthermore, the ratio of timosaponin H1/polygodoraside G could be applied to differentiate the two specifications. Samples with a ratio <2 are considered XPO and >5 are considered GPO. Therefore, the above results provide a valuable means for the quality control of PO.

Six oligosaccharides were discovered and isolated for the first time from Ziziphi Spinosae Semen. On the basis of spectroscopic analysis, their structures were determined to be verbascose (1), verbascotetraose (2), stachyose (3), manninotriose (4), raffinose (5), and melibiose (6). The prebiotic effect of the oligosaccharide fraction was assayed by eight gut bacterial growth in vitro, revealing a significant increase in cell density, up to 4-fold, for Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus johnsonii. The impact of six oligosaccharides with different degrees of polymerization (DPs) and structures on the growth of Lactobacillus acidophilus was evaluated. As a result, stachyose and raffinose demonstrated superior support for bacterial growth compared to the other oligosaccharides. This study explored the structure-activity relationship of raffinose family oligosaccharides (RFOs) and showed that the more the monosaccharide type, the more supportive the gut bacteria growth when oligosaccharides have the same molecular weight.

利用超高效液相色谱-四极杆-飞行时间质谱法(UHPLC-Q-TOF/MSE)结合UNIFI数据分析平台在线鉴定了武夷岩茶中104个化合物,包括茶多酚、茶色素、生物碱、氨基酸和脂质.通过主成分分析(PCA)及正交偏最小二乘判别分析(OPLS-DA)发现了水仙和肉桂的化学成分具有明显差异,并筛选出19个显著差异性成分,其中水仙样品中表儿茶素、槲皮素-3-O-葡萄糖基-鼠李糖基-(对香豆酰基-阿拉伯糖基)葡萄糖苷、茶黄素-3-没食子酸酯等成分的相对含量较高;肉桂样品中5-O-对香豆酰基奎宁酸、表没食子儿茶素-3-O-(3-O-甲基)-没食子酸酯、杨梅素-3-O-半乳糖苷、山柰酚-3-O-葡萄糖苷等成分的相对含量较高.此方法可用于区分水仙和肉桂成品茶.该研究了解了武夷岩茶的化学组成并阐明了其两个主栽品种水仙和肉桂的化学成分差异性,对合理开发武夷岩茶的食用及药用价值具有指导意义,为武夷岩茶品种的鉴别提供科学依据.

Xanthoceras sorbifolia, an excellent oil-rich woody species, has high comprehensive economic value in edible, medicinal, and ornamental fields. The chemical composition, pharmacological effect, and quality control of X. sorbifolia were introduced, and its development and application were reviewed in this study. As revealed by the previous research, the main chemical constituents of X. sorbifolia were triterpenoids, flavonoids, fatty acids, phenylpropanoids, steroids, phenolic acids, organic acids, etc. It possesses pharmacological effects, such as neuroprotection, bacteriostasis, anti-oxidation, anti-tumor, anti-inflammation, analgesia, anti-HIV, and anti-coagulation. X. sorbifolia is widely applied in medical, food, chemical industry, and other fields, and deserves in-depth research and development.

目的:建立HPLC-CAD方法同时测定知母中芒果苷和知母皂苷BⅡ的含量.方法:采用Acclaim-C18色谱柱(150 mm × 4.6 mm,3 μm),以乙腈(A )-0.2％醋酸水溶液(B)为流动相,梯度洗脱(0～10 min,15％A → 21％A;10～12 min,21％A → 23％A,12～30 min,23％A → 25％A ),流速 1.0 mL · min-1,柱温30 ℃;电雾式检测器(CAD)参数:采集频率10 Hz,雾化温度55℃,滤波为5 s.结果:知母中芒果苷和知母皂苷BⅡ均具有良好的线性关系(r＞0.999 ),LOD分别为0.43 ng和1.20 ng,LOQ分别为1.28 ng和4.80 ng,精密度、重复性、24 h稳定性试验的RSD均小于3.0％,平均加样回收率分别为102.3％和95.2％.11批样品中芒果苷和知母皂苷B Ⅱ的含量范围分别为0.54％～1.69％和3.27％～5.57％.结论:建立的HPLC-CAD法可同时对知母中芒果苷和知母皂苷B Ⅱ进行含量测定,为知母药材和饮片的检测分析和质量控制提供了新的方法.