Objective To prepare different polysaccharide fractions of Xiaoyao San (逍遥散, XYS) by graded ethanol precipitation and to clarify the anti-inflammatory activity, structural characteristics, and in vivo absorption and distribution behavior of the major enriched fraction. Methods Different polysaccharide fractions of XYS (40%, 60%, and 80% ethanol-precipitated fractions) were obtained using a water extraction-ethanol precipitation procedure, and the primary enriched fraction was screened based on yield comparison. Based on the inflammation-depression correlation, an LPS-induced microglial inflammation model was established, and anti-inflammatory activity was evaluated by measuring inflammatory markers including TNF-α, IL-1β, IL-6, and iNOS. The composition and structural features of the target polysaccharide were characterized using UV-Vis spectroscopy, FT-IR, and NMR analyses. Fluorescently labeled polysaccharides were further prepared for in vivo pharmacokinetic studies in mice to systematically assess exposure levels and tissue distribution characteristics. Results Significant differences in yield were observed among polysaccharide fractions obtained at different ethanol precipitation ratios. The 60% ethanol fraction (XYSP-60) was identified as the major enriched fraction, with an extraction recovery of 37.5%. In the LPS-induced microglial inflammation model, XYSP-60 significantly downregulated the expression levels of TNF-α, IL-1β, IL-6, and iNOS, demonstrating clear anti-inflammatory activity. Structural characterization indicated that the polysaccharide mainly consisted of galacturonic acid, glucose, and fructose, with relatively stable structural features. Pharmacokinetic results showed that XYSP-60 exhibited high exposure in the intestine but limited entry into systemic circulation. Conclusion The 60% ethanol-precipitated fraction represents the principal enriched polysaccharide fraction of XYS, with stable structural characteristics and definite anti-inflammatory activity. Its in vivo behavior is characterized by high intestinal exposure and limited systemic absorption, suggesting that this fraction may serve as one of the key material bases underlying the anti-inflammatory effects of XYS.

Objective To clone the CtFD1 and CtFD2 genes from the genome of Carthamus tinctorius and perform bioinformatics, expression specificity, and subcellular localization analyses. Methods The CtFD1 and CtFD2 genes were screened and cloned based on the C. tinctorius genome. Bioinformatics online tools were used to analyze physicochemical properties, conserved domains, and other features. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to examine tissue-specific expression patterns. Subcellular localization was observed using a tobacco transient expression system. Soluble expression of the proteins was achieved by optimizing inducer concentration and temperature. Results The coding sequences (CDS) of CtFD1 and CtFD2 were 726 bp and 540 bp in length, encoding 241 and 179 amino acids, respectively. Both proteins contained a typical bZIP transcription factor binding domain, which were hydrophilic, and localized to the nucleus. Phylogenetic analysis indicated a close evolutionary relationship with Artemisia annua. Tissue expression analysis showed that CtFD1 and CtFD2 were most highly expressed in shoot apical meristem (SAM). Prokaryotic expression analysis revealed that both proteins were abundantly expressed in the supernatant at 16  ℃ with an IPTG concentration of 0.2 mmol/L. Conclusion The CtFD1 and CtFD2 genes belong to the bZIP family and exhibit the highest expression levels in the SAM of C. tinctorius, suggesting that they may play important roles in regulating flowering. This study provides a scientific basis for further functional characterization of these genes and elucidation of the flowering mechanism in C. tinctorius.

Objective To split the classic prescription Baoyuan Decoction (保元汤) into different phases, study the relieving effects of different phases on exercise fatigue in rats, and screen for their effective phases. Methods The gradient centrifugation dialysis method was used to separate Baoyuan Decoction into the nano phase (BD nano phase, BD-N), precipitation phase (BD-P) and solution phase (BD-S). The appearance was physically characterized and contents of main ingredients was determined. A rat swimming fatigue model was established, control group, model group, positive control (Dazhu Hongjingtian Capsule) group, Baoyuan Decoction group, BD-N group and BD-P group were set up, active phases was screened by measuring behavioral indicators, organ indices, serum biochemical indicators, and pathological changes in liver and muscle tissues. Based on combined analysis of gut microbiome, metabolomics and transcriptomics, the potential mechanism of Baoyuan Decoction and its effective phase in alleviating exercise fatigue in rats was explored. Four differentially expressed genes (Gck, Ackr3, Pde4b, Ppp1r3b) were randomly selected for qRT-PCR validation. Results The particle morphology of BD-N was spherical and Tyndall effect was significant. The particle size was in the order of BD-P > Baoyuan Decoction > BD-N. The polydispersity index (PDI) of each phase was in the order of Baoyuan Decoction > BD-N > BD-P. The absolute value of zeta potential for each phase was BD-P > BD-N > Baoyuan Decoction. Ginsenoside Rb₁, calycosin-7- O-β-D-glucoside, liquiritin, ammonium glycyrrhizinate, cinnamic acid, cinnamaldehyde and 6-gingerol were mainly distributed in BD-N. The comprehensive efficacy ranking was Baoyuan Decoction ≈ positive control drug > BD-N > BD-P. The ability of BD-N in regulating the abundance of gut microbiota, increasing the level of Lactobacillus genus and regulating 45 differential metabolites related to fatigue was similar to Baoyuan Decoction. The qRT-PCR results showed that the regulatory trends of BD-N and Baoyuan Decoction on four genes were consistent with the transcriptome sequencing results. Conclusion From the perspective of physics, chemistry and pharmacological effects, the effective phase of Baoyuan Decoction in relieving fatigue is screened, and BD-N is the effective phase of Baoyuan Decoction in relieving fatigue.

Objective To investigate the mechanism of Wumei Pill (乌梅丸) in delaying the transformation of colitis cancer using microbiome and metabolomics. Methods A colitis cancer transformation animal model was constructed using azoxymethane (AOM)/dextran sulfate sodium (DSS). A total of 32 C57BL/6J mice were randomly divided into control group, model group, and Wumei Pill low-, high-dose (11.18, 22.36 g/kg) groups. Colonic tumor progression and histopathological changes were evaluated by intestinal endoscopic observation, macroscopic specimen assessment and hematoxylin-eosin (HE) staining. Levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in serum, and levels of total bile acids (TBA), cholic acid (CA), deoxycholic acid (DCA), lithocholic acid (LCA) in serum and in feces were measured by ELISA. 16S rRNA sequencing technology was used to analyze gut microbial diversity and species abundance. The abundance of metabolites was assessed by non-targeted metabolomics analysis. qRT-PCR was used to verify the expression changes of Clostridium scindens (C. scindens), Clostridium hylemonae (C. hylemonae), Clostridium hiranonis (C. hiranonis) and the coenzyme A transferase encoding gene BaiF. Results Compared with model group, the intervention of Wumei Pill could delay the transformation of colitis cancer, and the therapeutic effect was better at low dose of Wumei Pill. Low dose Wumei Pill could significantly reduce the number of colon tumors (P < 0.001), lower colitis and tumor pathology scores (P < 0.05, 0.01), and lower levels of IL-6 and TNF-α in serum (P < 0.01, 0.001). The microbiological results showed that Wumei Pill could improve the structure of intestinal microbiota, reduce the abundance of opportunistic pathogenic bacteria such as Clostridium, and increase the abundance of probiotics such as Lactobacillus and Roseburia. The non-targeted metabolomics results showed that there were 82 differential metabolites between Wumei Pill low-dose group and model group, significantly enriched in secondary bile acid metabolism pathway. Wumei Pill could significantly reduce the levels of TBA, CA,and DCA in serum and feces (P < 0.05, 0.01, 0.001). qRT PCR results further confirmed that Wumei Pill could inhibit bacteria involved in DCA synthesis such as C. scindens and C. hylmonae (P < 0.05), and lead to a decrease in BaiF gene expression level (P < 0.001). Conclusion Wumei Pill could alleviate colitis and delay the progression of inflammatory cancer by modulating the bile acid homeostasis mediated by gut microbiota, reducing the abundance and dehydroxylation function of bacteria such as C. scindens, and decreasing the production of DCA.

Enzymatically modified isoquercitrin (EMIQ) is a highly water-soluble flavonoid derivative produced through enzymatic glycosylation of isoquercitrin. Compared with quercetin and rutin, EMIQ exhibits markedly improved bioavailability. A growing body of research has demonstrated that EMIQ possesses a broad spectrum of pharmacological activities, including antioxidant, anti-inflammatory, anti-allergic, cardiovascular-protective, and muscle-enhancing effects. In addition, EMIQ can precisely regulate glucose and lipid metabolism, primarily through activation of the AMPK signaling pathway. Both animal experiments and clinical studies have confirmed its safety and reliability, and EMIQ has been approved for use as a food additive in Japan and the United States. This article provides a comprehensive review of the structural features, physicochemical properties, preparation methods, pharmacological activities, and safety evaluations of EMIQ, with the aim of offering a solid theoretical foundation and practical guidance for future research and industrial development of this compound.

Objective To investigate the chemical constituents from the ethanol extract of the bark of Spondias pinnata and preliminarily evaluate their anti-inflammatory activity. Methods Various chromatographic techniques were employed to isolate and purify the constituents from the extract. Their structures were elucidated by spectroscopic methods including NMR spectroscopy, as well as GFN2NMR and computational chemistry approaches. The anti-inflammatory activity of the new compound was evaluated using an LPS/IFN-γ-induced MH-S cell inflammation model in vitro. Results Ten compounds were isolated from the ethanol extract of the bark of S. pinnata and identified as (5R,6S)-6-[(7E,9R)-9-hydroxy-10-({(15R,16R)-17-butoxycarbonyl-15,16-dihydroxypropoxy})but-7-en-7-yl]-6-hydroxy-1,1-dimethylcyclohexan-3-one (1), ampelopsisionoside (2), (2E,4E,1'R,3'S,5'R,8'S)-dihydrophaseic acid 3'-O-β-D-glucopyranoside (3), N-benzoyl-D-phenylalanine (2R)-2-(benzoylamino)-3-phenylpropyl ester (4), isolariciresinol-4-O-β-D-glucopyranoside (5), (+)-lyoniresin-4-yl-β-D-glucopyranoside (6), demethoxymatteucinol (7), 4'-hydroxy-3-O-β-D-glucopyranoside flavone (8), (2S)-2-hydroxynaringenin-4'-O-β-D-glucoside (9), and (S)-(-)-N-benzoylphenylalaninol (10). Compound 1 significantly inhibited the expression of TNF-α (P < 0.05) and IL-6 (P < 0.01) at a concentration of 10 μmol/L. Moreover, at 20 μmol/L, it markedly ameliorated the LPS/IFN-γ-induced morphological changes in MH-S cells. Conclusion Compound 1 is a new compound, named as spondias A. Compounds 2—4 and 7—10 were isolated from the genus Spondias for the first time, while compounds 5—6 were obtained from this species for the first time. The bioassay results indicate that compound 1 inhibits the transformation of MH-S cells to the M1 phenotype, suggesting its potential anti-inflammatory activity.

Objective To investigate the effects and underlying molecular mechanisms of glycyrrhetinic acid (GLG) and glycyrrhizic acid (GLA) on alleviation of cisplatin (CP)-induced acute liver injury (ALI) based on proteomics. Methods A total of 50 SPF male Wistar rats were randomly divided into control group, model group, amifostine (200 mg/kg) group, GLG (100 mg/kg) group and GLA (200 mg/kg) group. Rats in GLG group and GLA group were given intragastric administration for eight consecutive days. Rats in amifostine group was given intraperitoneal administration from the 5th to the 8th day of the experiment, control group and model group were given intragastric administration of physiological saline. On the 5th day of the experiment, except for the control group, all other rats were induced to ALI with a single dose of cisplatin solution (8 mg/kg) via ip. Liver index, liver function and pathological changes in liver tissue were detected. Label free non-standard quantitative proteomics technology was used to analyze protein expressions in liver tissue, differentially expressed proteins were screen, and bioinformatics analysis was performed. The protein-protein interaction (PPI) integration network of drug and disease prediction intersection targets and differentially expressed proteins was constructed by integrating network pharmacology methods. The core targets were screened by cytoNCA, MCODE and cytoHubba, gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed. Key target expressions were validated using Western blotting, immunohistochemistry and other techniques. Results Compared with model group, the liver index and activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of rats in GLG group and GLA group were significantly reduced (P < 0.05, 0.01, 0.001), and the pathological damage of liver tissue was significantly improved. Proteomic identification identified 23 potential key differentially expressed proteins each in cisplatin-induced ALI treated with GLG and GLA, enriched in multiple biological processes related to redox reactions, inflammation and apoptosis. PPI integrated network showed that signal transducer and activator of transcription 3 (STAT3), heme oxygenase 1 (HMOX1), protein kinase B1 (AKT1), B-cell lymphoma-2 (Bcl-2), cysteine aspartate protease-3 (Caspase-3) and tumor protein p53 (TP53) were key targets of GLG action network, HMOX1, Bcl-2, epidermal growth factor receptor (EGFR) and TP53 were key targets of GLA action network, and they can be enriched in phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, tumor necrosis factor-α (TNF-α) signaling pathway, interleukin-17 (IL-17) signaling pathway, p53 signaling pathway, etc. The validation results showed that both GLG and GLA could significantly upregulate the p-Akt/Akt value and Bcl-2, superoxide dismutase (SOD) levels in liver tissue of ALI rats (P < 0.01, 0.001), and downregulate the expressions of Akt, HMOX1, STAT3, high mobility group box 1 protein (HMGB1) and levels of TNF-α, IL-1β, malondialdehyde (MDA) in liver tissue (P < 0.05, 0.01, 0.001). Conclusion GLG and GLA could effectively alleviate cisplatin-induced ALI, and their protective effects may be closely related to regulating Akt/STAT3/HMOX1 signaling axis, thereby inhibiting liver oxidative damage, cell apoptosis and inflammatory response.

Objective To optimize the extraction and β-cyclodextrin (β-CD) inclusion process for the volatile oil from the classical formula Dihuang Yinzi (地黄饮子) based on the quality by design (QbD) concept and the analytic hierarchy process-entropy weight (AHP-entropy) method. Methods Failure mode and effects analysis (FMEA) was employed for risk identification in the volatile oil extraction process. Heating time, material-to-liquid ratio, and soaking time were determined as the critical process parameters (CPPs), while cinnamaldehyde content, β-asarone content, and volatile oil extraction yield were defined as the critical quality attributes (CQAs). On the basis of single-factor tests, the Box-Behnken design-response surface methodology (BBD-RSM) was used for process optimization. The AHP-entropy weight method was applied to calculate a comprehensive score for the CQAs, which was then used to establish and validate an optimized design space. The inclusion process of volatile oil was investigated using the saturated aqueous solution method. The inclusion conditions were comprehensively optimized through orthogonal experimental design combined with the AHP-entropy weight method for multi-index evaluation. Finally, the optimal inclusion process parameters were determined and validated. Results The optimized volatile oil extraction process was as follows: soaking for 30 to 45 min, adding 9- to 10-fold volume of water, and extracting for 3.0 to 3.5 h. The optimal inclusion process parameters were: inclusion time of 2.0 h, β-cyclodextrin to volatile oil ratio of 10:1, and inclusion temperature of 30 ℃. Conclusion The volatile oil extraction and inclusion processes optimized based on the QbD concept and the AHP-entropy weight method can reliably ensure a stable extraction yield and consistent content of key active components. The resulting inclusion complex exhibits excellent quality. This study provides reliable data support and a solid process foundation for the industrial production and development of related Chinese medicinal preparations, while also offering a feasible pathway for the modern dosage form development of classical formulas.

Objective To optimize the processing technology for ginger-steamed Sharen (Amomi Fructus, AF) kernels and establish UPLC fingerprints before and after ginger-steaming. Methods Using the contents of volatile oil, bornyl acetate, extracts, 6-gingerol, vanillic acid, epicatechin, and quercitrin as evaluation indicators, the combined AHP-entropy weight method composite weighting method was employed to determine the weight coefficients of each indicator. Combined with the Box-Behnken design-response surface methodology (BBD-RSM), the effects of ginger juice dosage, moistening duration, steaming duration, and drying temperature on the ginger-steaming process of AF kernels were investigated to determine the optimal process parameters. UPLC fingerprints of AF kernels and ginger-steamed AF kernels were established and imported into the “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System” (2012 edition) for pattern analysis. Chemometrics was used to compare the differences before and after processing. Results The optimal processing conditions for ginger-steamed AF kernels were determined as follows: ginger juice dosage 15%, moistening duration 3 h, steaming duration 38 min, drying temperature 60 ℃. Comparative analysis of the fingerprints before and after processing identified two differential components, which were ranked by variable importance projection (VIP) value as peak 6 (vanillic acid) > peak 8. Conclusion The optimized processing technology of ginger-steamed AF kernels is stable, reliable and reproducible. The method of UPLC fingerprint combined with chemometrics to screen quality difference markers before and after processing is simple and convenient, which can provide a scientific reference for the quality control and evaluation of ginger-steamed AF kernels.

Objective To investigate the chemical constituents of Buxus microphylla and their anti-hepatocarcinoma activity in vitro. Methods The compounds were isolated and purified by column chromatography of silica gel, reversed-phase C₁₈, Sephadex LH-20, semi-preparative HPLC and preparative TLC. Their structures were elucidated by physicochemical properties and spectral analyses. The cytotoxicity of the isolates against human hepatocellular carcinoma HepG2 and Hep3B cells was assessed using the MTT assay. Flow cytometry was employed to analyze cell apoptosis. Western blotting was used to detect the expression of proteins related to cell apoptosis. Results Eighteen compounds were isolated and identified from the 70% ethanol extract of B. microphylla, including hederagenin (1), 3-O-β-D-glucopyranosylspinasterol (2), fagraeanolide (3), rel-(2α,3β)-7-O-methylcedrusin (4), caruilignan D (5), (7′S,8R,8′S)-isolariciresinol (6), 3-hydroxy-1,2-dimethoxyanthrone (7), 1-methoxy-2,3-methylenedioxyanthrone (8), 1,2,3-trimethoxyanthrone (9), syringaldehyde (10), syringic acid (11), vanillic acid (12), p-hydroxybenzoic acid (13), polygital (14), 9-pentacosenoic acid (15), rhamnakoside B (16), lupeol (17), and betulin (18). The in vitro activity screening revealed that compounds 2, 3, 16—18 exhibited cytotoxicity in HepG2 and Hep3B cells, among which compound 2 was the most potent. compound 2 significantly increased the proportion of apoptotic cells and elevated the expression of apoptosis-related proteins such as Bax, decreased the expression of Bcl-2, all of which were statistically significant compared with control group (P < 0.001) at 10 μmol/L. Conclusion Compound 1 and 17—18 are triterpenoids; Compound 2 is a steroidal glycoside; compounds 3—6 are lignans, compounds 7—9 are anthrones, compounds 10—13 are phenolic acids, and compounds 14—16 are other types. Among them, compounds 2—5, 7—9, and 14—16 are isolated from the genus Buxus for the first time. In the anti-hepatocarcinoma activity assay, compound 2 demonstrated significant effects, with IC₅₀ values of 5.87 μnol/L and 6.42 μnol/L against HepG2 and Hep3B cells, respectively. Moreover, it effectively induced apoptosis in HepG2 cells.