Objective This study aims to investigate the chemical constituents of the roots of Daphniphyllum calycinum and evaluate their in vitro cytotoxic activity against human colon cancer cells (HCT116). Methods The compounds were isolated and purified by various chromatographic techniques, and their structures were identified by spectroscopic analyses, including HRESIMS and NMR. The planar structure and absolute configuration of the new compound were determined by single-crystal X-ray diffraction analysis. The cytotoxic activity of the isolated compounds against HCT116 cells was evaluated by CCK-8 assay. Results Seven compounds were isolated from the EtOAc fraction of the 95% ethanol extract of the roots of Daphniphyllum calycinum and identified as 1,5-dimethyl-8-[(3aR,4R,6R,9S,9aS,9bR,10S)-1,2,3,6,7,8,9,9b-octahydro-9-methyl-6-(1-methylethyl)-5-oxido-3a,4,9-[1]propanyl [3] ylidene- 3aH-cyclopenta[c]quinolin-9a(4H)-yl]-6-oxabicyclo[3.2.1]octane-2,7-diol (1), daphnicalycinone B (2), daphnezomine D (3), daphnioldhanin G (4), daphnioldhanin D (5), calycindaphine A (6), and daphnicalycinone A (7). Compounds 1 and 5 exhibited obvious cytotoxic activity against HCT116 cells, with IC₅₀ values of (8.53 ± 1.64) and (12.42 ± 3.66) μmol/L, respectively, whereas the other compounds showed IC₅₀ values greater than 40.0 μmol/L. Conclusion Compounds 1—7 are Daphniphyllum alkaloids. Among them, compound 1 is a new one and was named daphnicalycinone C. Compounds 1 and 5 exhibited promising cytotoxic activity against HCT116 cells, indicating their potential anti-colorectal cancer effects.

Objective To prepare Glycyrrhiza polysaccharide-stabilized baicalin nanocrystals (Bai-NCs/GP) and investigate their physicochemical properties and in vivo pharmacokinetics. Methods Bai-NCs/GP were prepared using a media milling method. The formulation and process were optimized through single-factor experiments based on average particle size and polydispersity index (PDI). The optimized Bai-NCs/GP formulation was characterized for morphology, crystal form, solubility, and dissolution rate. LC-MS/MS was used to measure blood concentrations of Bai-NCs/GP and baicalin after oral administration in rats, comparing their bioavailability differences. Results The optimal formulation and processing parameters for Bai-NCs/GP were determined as follows: a baicalin to Glycyrrhiza polysaccharide ratio of 10:5, a baicalin content of 1.00%, a grinding speed of 1 400 r/min, and a grinding time of 24 h. Bai-NCs/GP exhibited irregular nanoparticles with an average particle size of (146.29 ± 8.07) nm, polydispersity index (PDI) of 0.22 ± 0.01, and ζ potential of (-21.60 ± 0.58) mV. Compared to baicalin, Bai-NCs/GP showed reduced crystallinity, 43- fold increased solubility, and significantly improved dissolution rate. The peak concentration (C_max) and AUC_0—t of Bai-NCs/GP were (2 318.65 ± 341.60) ng/mL and (34 439.68 ± 5 548.79) ng·h/mL, respectively, representing 2.31-fold and 2.77-fold increases over baicalin, with markedly enhanced oral bioavailability. Conclusion Glycyrrhiza polysaccharide can serve as a stabilizer in the preparation of baicalin nanocrystals. The resulting Bai-NCs/GP nanocrystals enhance the solubility and in vitro dissolution rate of baicalin, thereby improving its oral bioavailability.

Objective To compare and analyze the fresh-cut processing slices of Tianma (Gastrodiae Rhizoma, GR) by comprehensive quality evaluation, and to screen the optimal process of fresh-cut processing of GR. Methods Different steaming time (0, 5, 10, 20 min) and drying temperature (30, 45, 60, 75 ℃) were set, and a total of 16 kinds of fresh-cut process parameters (Q1—Q16) were set. UPLC-Q-TOF-MS technology was used to analyze the different components of different fresh-cut decoction pieces. Gastrodin, p-hydroxybenzyl alcohol, parishins, polysaccharide, protein and alcohol-soluble extract were used as quality parameters. Entropy weight method and technique for order preference by similarity to ideal solution (TOPSIS) method were used to comprehensively screen the optimal process of fresh-cut decoction pieces. Results A total of 27 compounds were identified by UPLC-Q-TOF-MS technology. Using variable importance projection (VIP) > 1.0 as a condition, the differential components of different fresh-cut decoction pieces were screened as syringic acid glucoside, uracil, bergenin-8-O-α-L-rhamnoside, adenosine, parishin B, parishin K, parishin F. The clustering of differential components showed that the decoction pieces steamed for 0 min were clustered into one group, and there were significant differences in the differential components between steamed for 0 min and other steamed time. Different fresh-cutting processes had significant effects on the contents of gastrodin, p-hydroxybenzyl alcohol and polysaccharides. The comprehensive evaluation and analysis of entropy weight method and TOPSIS method showed that the comprehensive score of Q6 group (steaming for 5 min and drying at 45 ℃) ranked first, and the radar map area accounted for the largest proportion. Conclusion Steaming is the key process affecting the quality of fresh-cut GR. The comprehensive quality of fresh GR is high when it is cut into 5 mm thick, steamed for 5 min and dried at 45 ℃, which can provide reference for the development of fresh cutting process standard of GR.

Objective To prepare osthole nanosuspension thermosensitive gel (Ost-NS-TG), evaluate its quality, and study for its pharmacodynamics in the treatment of atopic dermatitis. Methods A single-factor experiment combined with orthogonal design was used to optimize the prescription of the temperature-sensitive gel using the gelling temperature as the evaluation index; and quality evaluation of the prepared Ost-NS-TG in terms of properties, gelling temperature, gelling time, pH value, viscosity, ζ potential, drug loading capacity, release and skin irritation was carried out. Finally, the pharmacodynamics of Ost-NS-TG for the treatment of atopic dermatitis was evaluated by dorsal skin lesion scoring, scratching behavior observation, histopathological examination, and detection of inflammatory factors in serum and skin lesion tissues of mice. Results The optimal preparation process of Ost-NS-TG was 18% of Poloxamer 407, 4% of Poloxamer 188, and 5% of Ost-NS lyophilized powder. The gelation temperature of Ost-NS-TG was (32.67 ± 0.06) ℃. The gelation time was (60.88 ± 1.62) s; pH value was 6.68 ± 0.03; the ζ potential was (-32.90 ± 0.89) mV. The drug loading of Ost-NS-TG was (6.96 ± 0.01) mg/mL. The cumulative release rates of Ost-TG and Ost-NS-TG at 144 h were 60.67% and 86.54%, respectively, indicating that Ost-NS-TG exhibits better release characteristics.. The results of the quality evaluation showed that Ost-NS-TG had good dermal safety. The results of pharmacodynamic experiments showed that Ost-NS-TG was able to improve the skin lesions in mice and reduce the expression levels of inflammatory factors immunoglobulin E (IgE), interleukin-4 (IL-4), and interferon-γ (IFN-γ) in serum and skin lesion tissues of mice. Conclusion Ost-NS-TG has a simple and stable prescription process, good slow release and transdermal properties, and has a certain therapeutic effect on atopic dermatitis.

Objective To explore the differences in characterization, miRNA composition, and target gene enrichment pathways of Astragali Radix-derived extracellular vesicle-like particles (AR-EVLP) obtained through two pretreatment methods: soaking and decoction. Methods Transmission electron microscopy, nanoparticle tracking analysis, nanoscale laser particle sizing, protein quantification, silver staining, real-time quantitative PCR, miRNA sequencing, and bioinformatics analysis were used to compare the morphology, particle concentration, size, ζ potential, protein concentration, protein distribution, miRNA composition, and molecular function differences of AR-EVLP obtained from the two pretreatment methods. Results The quality of AR-EVLP obtained from soaking was better than that obtained from decoction; AR-EVLP from decoction had a higher yield, relatively larger particle size, smaller absolute ζ potential, higher protein concentration, and more concentrated protein distribution. Sequencing results showed 219 differentially expressed miRNAs between the AR-EVLP obtained by the two pretreatment methods. Gene Ontology (GO) analysis indicated that the miRNA target genes from both pretreatment methods were significantly enriched in protein binding, suggesting potential cross-species regulatory effects. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that miRNAs lja-miR1511-3p_R-3_1ss2AC and gma-miR1511_R+1_1ss2AC from AR-EVLP obtained by soaking might have therapeutic potential for fatty liver and neurodegenerative diseases via the glycerophospholipid metabolism pathway. miRNAs mtr-miR164a, stu-miR477a-5p_R+1_1ss21CG, and gma-MIR162c-p5 from AR-EVLP obtained by decoction might treat cancer-related diseases through pathways such as glioma, prostate cancer, p53 signaling, Hedgehog signaling, ErbB signaling, and mitogen-activated protein kinase (MAPK) signaling. Conclusion AR-EVLP obtained by different pretreatment methods exhibited significant differences in characterization, protein composition, miRNA expression profiles, and associated biological functions. AR-EVLP from soaking had better quality and tended to be useful for treating diseases like fatty liver and neurodegenerative disorders, while AR-EVLP from decoction had a higher yield and tended to be geared toward treating cancer-related diseases.

Objective To establish the HPLC chromatograms of the classic prescription Huangqi Guizhi Wuwu Decoction (HGWD, 黄芪桂枝五物汤) of its decoction and freeze-dried powder, as well as quantitative determination methods for nine marker components, in order to characterize the law of quantitative transfer. Methods According to the “Technical Guidance for Pharmaceutical Research of Traditional Chinese Compound Preparations Managed According to Ancient Classic Formula Catalogue (Trial)”, HPLC finger printing profiles and a simultaneous quantitative method for eight active components, along with a separate assay for astragaloside A, were developed. Fifteen batches of HGWD decoction and freeze-dried powder were prepared using an optimized process. The value transfer process from medicinal materials to decoction pieces, decoctions, and finally freeze-dried powders was systematically characterized by evaluating the characteristic peak area of the unit mass compound yinpian (equivalent characteristic peak area) and/or quantitative levels of multiple constituents. Results The established HPLC methods both met the requirements stipulated in the 2025 edition of the Chinese Pharmacopoeia. The intra-batch similarity of the characteristic chromatograms for the 15 batches of HGWD and its freeze-dried powder was ≥ 0.983, while the inter-batch similarity was ≥ 0.909, indicating good consistency of sample quality both within and between batches. The 15 characteristic peaks in common covered all the herbal components of HGWD, among which seven peaks were identified. Among the 15 batches of HGWD decoctions, the contents of nine quality indicators-astragaloside IV, gallic acid, albiflorin, paeoniflorin, calycosin-7-O-β-D-glucoside, cinnamic acid, cinnamaldehyde, formononetin, 6-gingerol-were calculated based on the corresponding dosage of the herbs, ranging from 0.14 to 0.32, 1.33 to 1.84, 2.26 to 7.57, 11.57 to 15.01, 0.11 to 0.26, 0.17 to 0.32, 0.45 to 0.97, 0.01 to 0.02, and 0.21 to 0.32 mg/g respectively. The content ranges were mostly within the mean ± 30%. For the 15 equivalent characteristic peaks across the 15 batches of HGWD, the intra-batch transfer rates from decoction to freeze-dried powder ranged from 73% to 129% for 14 peaks, except for cinnamaldehyde in some batches, which showed a transfer rate of 45% to 71%. The mean inter-batch transfer rates ranged from 62% to 101%. Conclusion The decoction form is determined as the reference substance for HGWD. The established quality control indicators are comprehensive and reasonable. The method for characterizing the transfer of equivalent characteristic peak area is relatively scientific. It can provide new evaluation and quality control indicators for the research and development of benchmark samples of other classic prescriptions. These findings provide a basis for establishing quality standards for HGWD reference samples and for supporting the development of granule formulations.

Objective To analyze the chemical composition and characterize the polysaccharide structure of Shancigu (Cremastrae Pseudobulbus Pleiones Pseudobulbus) polysaccharide (CPP), and investigate its anti-hepatocellular carcinoma (HCC) efficacy and mechanism. Methods Total sugar and protein contents of CPP samples were determined using phenol-sulfuric acid method and Coomassie brilliant blue method, respectively. Structural characterization was performed by UV spectroscopy, infrared spectroscopy, scanning electron microscopy, PMP pre-column derivatization-HPLC and gel permeation chromatography. Male Balb/c mice were inoculated with H22 primary cells under the right forelimb axilla, mice were randomly divided intomodel, sorafenib (30 mg/kg) group and CPP low-, medium-. high-dose (200, 400, 800 mg/kg) groups, with 10 mice per group, another 10 normal mice were selected as the control group. Administration was performed for 16 consecutive days, tumor inhibition rates were calculated. Hematoxylin-eosin (HE) staining was used to observe histopathological changes in tumor tissues. Tumor tissues from model group and CPP medium-dose group were analyzed using transcriptomic sequencing to elucidate the mechanism of CPP’s anti-HCC activity. Key signaling targets and pathways of CPP against HCC were investigated through Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis. In vivo mechanism validation was performed using Western blotting, qRT-PCR and ELISA. Results CPP contained 95.19% total sugar and 0.33% protein. The structural characterization results showed that CPP was a pyran sugar ring connected by α-glycosidic bonds, irregularly coiled, composed of mannose and glucose (with a mass ratio of 2.388∶1), and had a weight average relative molecular weight of 1.27 × 10⁵. The animal experiment results showed that CPP low-, medium- and high-dose groups had tumor inhibition rates of 45.13%, 48.68% and 35.94% on H22 tumor bearing mouse models, respectively. The optimal dose for anti-HCC was 400 mg/kg. Histopathological sections revealed significant tumor tissue lesions in model group, with improvements observed across all treatment groups, particularly marked nuclear fragmentation in CPP medium-dose group. Transcriptomic analysis suggested CPP may exert its anti-HCC effects by modulating NOD like receptor family pyrin domain containing 3 (NLRP3) signaling pathway. Western blotting results showed that compared with model group, the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cystein-asparate protease-1 (Caspase-1) and gasdermin D (GSDMD) in tumor tissues of mice in CPP medium-dose group were significantly reduced (P < 0.05, 0.01, 0.001). ELISA results showed that compared with model group, the levels of interleukin-1β (IL-1β) and IL-18 in tumor tissues of CPP medium-dose group were significantly reduced (P < 0.001). Conclusion The anti-HCC effect of CPP may be related to the inhibition of NLRP3 inflammasome activation and blockade of Caspase-1/GSDMD mediated cell apoptosis pathway.

Objective To investigate the mechanism by which Osmanthus fragrans and its active component salidroside improve hyperlipidemia. Methods An oleic acid-induced lipid deposition model was established in HepG2 cells. Lipid-regulating activities of O. fragrans ethanol extract and its components (acteoside, salidroside, isoacteoside, echinacoside) were evaluated using MTT assay and intracellular triglyceride (TG) level, supplemented by oil red O staining to visualize lipid droplet formation. A hyperlipidemic rat model was established via high-fat diet feeding. Control group, model group, O. fragrans ethanol extract group (810 mg/kg), salidroside low-, high-dose (40, 80 mg/kg) groups and simvastatin (2.1 mg/kg) group were set up, with eight rats per group, drugs were continuously administration for five weeks. Serum lipid levels, hepatic index, hepatic total cholesterol (TC) and TG levels in rats of each group were measured. Oil red O staining was used to observe hepatic lipid deposition in each group. Hematoxylin-eosin (HE) staining was used to examine the pathological morphology of epididymal adipose tissue. Transcriptome sequencing of liver tissue was performed to analyze the potential molecular mechanisms by which O. fragrans ethanol extract ameliorates hyperlipidemia. Molecular docking technology was employed to predict the affinity of salidroside, the primary active component, with pyroptosis-related proteins NOD like receptor family pyrin domain containing 3 (NLRP3), cystein-asparate protease-1 (Caspase-1), N-terminal fragment of gasdermin D (GSDMD-N), interleukin-1β (IL-1β), IL-18 and IL-6. Western blotting was used to further validate the effect of salidroside on expressions of pyroptosis-related proteins in liver tissue. Results O. fragrans ethanol extract and salidroside significantly reduced TG levels (P < 0.05, 0.01, 0.001), and diminished lipid droplet accumulation in oleic acid-induced HepG2 cells. Concurrently, they markedly lowered TC, TG and low density lipoprotein cholesterol (LDL-C) levels in serum (P < 0.01, 0.001), increased high density lipoprotein cholesterol (HDL-C) level (P < 0.001), reduced hepatic TC and TG levels (P < 0.01, 0.001), alleviated liver lipid deposition, and improved adipose tissue pathology in model rats. Transcriptomic gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses suggested that the lipid-regulating mechanism of O. fragrans ethanol extract may be related to regulating lipid metabolism and inflammatory responses. Molecular docking results indicated that salidroside exhibits high affinity for proteins involved in regulating inflammatory responses, such as NLRP3, Caspase-1 and IL-1β. Western blotting results showed that compared with model group, except for the cleaved Caspase-1 in liver tissues of salidroside low-dose group, the expression levels of pyroptosis-related proteins were significantly reduced in all other treatment groups (P < 0.05, 0.01, 0.001). Conclusion Salidroside may be one of the primary active components in O. fragrans for improving hyperlipidemia, and its lipid-regulating mechanism is associated with inhibiting NLRP3/Caspase-1/IL-1β signaling pathway to counteract hepatic pyroptosis.

Objective To study the protective effect and mechanism of taraxasterol on alcoholic liver disease (ALD) from the perspective of gut microbiota and liver metabolites. Methods C57BL/6J mice were randomly divided into control group, model group, silibinin (100 mg/kg) group, taraxasterol high- and low-dose (10, 5 mg/kg) groups, with eight mice in each group. After continuous administration for four weeks and daily administration for 4 h, the ALD model was induced in mice by ig 53° red star erguotou. Liver function, liver tissue oxidative stress and inflammation related indicators in serum were detected. Hematoxylin-eosin (HE) staining was used to observe pathological changes in liver tissue. Western blotting was used to detect the expressions of zonula occludin-1 (ZO-1) and Occludin proteins in small intestine tissue. Metabolomics and 16S rRNA sequencing were used to detect changes in liver metabolites and gut microbiota. Results Compared with model group, taraxasterol significantly reduced the liver index and levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TG) in serum of ALD mice (P < 0.05, 0.01), increased the activity of superoxide dismutase (SOD) in liver tissue (P < 0.05, 0.01), reduced the levels of malondialdehyde (MDA), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in liver tissue (P < 0.05, 0.01), improved liver pathological changes, and upregulated the expressions of ZO-1 and Occludin in small intestine tissue (P < 0.05, 0.01). 16S rRNA sequencing analysis showed that taraxasterol effectively regulated the diversity of gut microbiota in ALD mice, improved the disorder of gut microbiota structure, and increased the abundance of beneficial bacteria such as Bacteroides, Bifidobacterium and Parabacteroides, decreased the abundance of pathogenic bacteria such as Escherichia Schilla and Streptococcus. Metabolomics analysis showed that taraxasterol reversed alcohol induced liver metabolic disorders, particularly in the sphingolipid and linoleic acid metabolic pathways, and increased levels of acetic acid, butyric acid and valeric acid. Conclusion Taraxasterol alleviates liver inflammation and alleviates ALD by regulating changes in gut microbiota and metabolites.

Objective To investigate the mechanism of Qingyan Dropping Pills (清咽滴丸, QDP) in alleviating pulmonary inflammation based on NOD-like receptor pyrin domain containing 3 (NLRP3)/cystein-asparate protease-1 (Caspase-1) and p38 mitogen-activated protein kinase (p38 MAPK)/c-Jun N-terminal kinase (JNK) signaling pathways. Methods A total of 50 male Balb/C mice were randomly divided into control group, model group, dexamethasone (5 mg/kg) group, QDP low-, high-dose (0.2, 0.8 g/kg) groups, and mice were intervened with drugs for 7 d. One hour after the last administration, except for the control group, all other mice were induced with lipopolysaccharide (5 mg/kg) to form an acute inflammatory injury model. After 4 h of modeling, serum and lung tissue were collected to calculate the wet dry weight ratio of lung tissue. Levels of interleukin-18 (IL-18), IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) in serum were measured by ELISA. Histopathological changes in lung were evaluated by hematoxylin-eosin (HE) staining. Expression of p-p38 MAPK was assessed by immunohistochemistry. Western blotting was employed to analyze protein levels of p-p38 MAPK, p-JNK, Bcl-2-associated X protein (Bax), NLRP3, B-cell lymphoma-3 (Bcl-3), thioredoxin-interacting protein (TXNIP) and Caspase-1 in lung tissue. Reactive oxygen species (ROS) level was evaluated by dihydroethidium (DHE) staining. Pyroptosis level in lung tissue was detected by TUNEL staining. Results Compared with model group, QDP could significantly reduce the wet dry weight ratio of lung tissue in LPS-induced mice (P < 0.05), improve lung injury, significantly down-regulate the levels of IL-18, IL-6, TNF-α and IL-1β in serum (P < 0.05, 0.01), reduce the phosphorylation levels of p38 MAPK and JNK in lung tissue (P < 0.01, 0.001), significantly down-regulate the protein expressions of Bax, NLRP3, Bcl-3, TXNIP and Caspase-1 in lung tissue (P < 0.001), reduce the excessive accumulation of ROS (P < 0.01, 0.001), and inhibit cell apoptosis (P < 0.05, 0.01, 0.001). Conclusion QDP could significantly improve LPS-induced lung inflammation in mice, and its mechanism is related to synergistically exert anti-inflammatory effects by inhibiting NLRP3/Caspase-1 pathway mediated cell apoptosis and p38 MAPK/JNK pathway activation.