Objective To systematically investigate the intestinal absorption characteristics of baicalin, glycyrrhizic acid, aloe-emodin, wogonin, emodin and chrysophanol in Jinzhen Oral Liquid (金振口服液) using the rat in situ single-pass intestinal perfusion model and Caco-2 cell model. Methods In vitro experiments were conducted using Caco-2 monolayer cell model to preliminarily evaluate the absorption capacity of the six components through apparent permeability coefficient (P_app). In situ experiments were conducted using rats in situ intestinal perfusion to further verify the absorption of the six components in rat intestinal segments (duodenum, jejunum, ileum, and colon) through the absorption rate constant (K_a) and effective permeability coefficient (P_eff). The in vitro and in situ absorption rules were compared and analyzed to clarify their absorption mechanisms. Results The in vitro and in situ experimental results showed good consistency. Under low-, medium-, and high-concentration perfusion conditions, all six components were absorbable in various intestinal segments of rats. Among them, the P_eff values in the duodenum, jejunum, and colon were all greater than 1.2 × 10⁻³ cm/min, indicating that the six components were well absorbed in these three intestinal segments. In the ileum segment, the P_eff values of baicalin, glycyrrhizic acid and wogonin were 1.8 × 10⁻⁴—1.2 × 10⁻³ cm/min, indicating moderate absorption of these three components in the ileum. The P_eff values of aloe-emodin, emodin and chrysophanol were greater than 1.2 × 10⁻³ cm/min, indicating good absorption of these three components in the ileum. In addition, there were differences in the absorption mechanisms of these six components, and most of them exhibited absorption characteristics that required carrier involvement, such as active transport and diffusion promotion (some of which were concentration dependent or saturated). At the same time, there were also components that mainly participate in passive diffusion or both active and passive transport. Conclusion In situ and in vitro intestinal absorption experiments reveal the segment-specific absorption patterns of six major components in Jinzhen Oral Liquid, with good overall absorption that meets the requirements for oral preparations.

Objective Avoltammetric electronic tongue was applied to establish Bizhunziwei for evaluating the quality consistency between classical formulas Qingxin Lianzi Yin (QLY, 清心莲子饮) intermediate and explore its application in the pretreatment process optimization of QLY. Methods By comprehensively analyzing the precision of Euclidean distance (Ed), mean Euclidean distance (M-Ed), and standardized Euclidean distance (S-Ed) between the taste PCA score plots of intermediates and substance benchmark in combination with the discrimination index (DI), to construct the standard taste profile. The effects of the sample’s pH value, the salt ions and redox-active substances on Bizhunziwei were studied and the precision, repeatability and stability of Bizhunziwei were investigated. The application of Bizhunziwei in the pretreatment processes of QLY such as extraction and drying of classic famous prescriptions was explored and the correlation between Bizhunziwei and the similarity of fingerprint profiles was analyzed. Results The Bizhunziwei could be defined as “S-Ed*DI” which was the product of standardized Euclidean distance and the discrimination index of the taste PCA score plots. The RSD values for the precision, repeatability and stability of Bizhunziwei were 2.355%, 5.630%, 3.573%, respectively. The sample’s pH value, the salt ions and redox-active substances had a significant impact on Bizhunziwei (P < 0.05). The Bizhunziwei could distinguish the extraction and drying process parameters of QLY. The Bizhunziwei had a good correlation with the similarity of the fingerprint profile of QLY. Conclusion The Bizhunziwei provides a new approach for evaluating the quality consistency between intermediates and substance benchmark of QLY, and offers a new perspective for the development of classic famous prescriptions.

Objective To investigate the protective effect and mechanism of small RNAs (sRNA) derived from Xuanfei Baidu Formula (宣肺败毒方, XFBD) targeting angiotensin-converting enzyme (ACE) in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Methods The modified CTAB method was employed to extract sRNA from XFBD and a library was constructed, followed by prediction and screening of sRNAs targeting ACE. The targeting specificity was verified using a dual-luciferase reporter system, and sRNAs capable of suppressing ACE expression were screened in human pulmonary microvascular endothelial cells (HPMEC). The effects of sRNA on angiotensin Ⅱ (Ang II) generation, inhibitor of inhibitor of nuclear factor-κB α (IκBα) and inflammatory cytokine expressions were examined by ELISA, Western blotting and qRT-PCR. An LPS-induced ALI mice model was established, control group, model group, captopril (10 mg/kg) group, XFBD (9.2 g/kg) group, NC-sRNA (10 nmol/animal) group, ACE-sRNA-1 (10 nmol/animal) group and ACE-sRNA-26 (10 nmol/animal) group were set up, with six mice in each group. Hematoxylin-eosin (HE) staining and Micro CT were used to evaluate the pathological and imaging changes of lung tissue; The number of white blood cells and neutrophils in peripheral blood, as well as the total protein concentration, white blood cells, neutrophils and lymphocytes numbers in bronchoalveolar lavage fluid (BALF) were detected; Immunohistochemistry was used to detect the expressions of vascular endothelial-cadherin (VE-cadherin) and intercellular adhesion molecule-1 (ICAM-1) in lung tissue; Western blotting and qRT-PCR were used to detect the expressions of ACE-Ang Ⅱ-Ang Ⅱ type 1 receptor (AT1R) pathway, IκBα and inflammatory factors in lung tissue; ELISA was used to detect the levels of Ang II and inflammatory factors in serum. Results A total of 50 potential ACE-targeting sRNAs were screened from XFBD, with 26 sRNAs validated to exhibit targeting effects, among which 12 sRNAs significantly inhibited ACE expression in HPMEC cells (P < 0.05, 0.01, 0.001). Seven sRNAs significantly suppressed Ang II generation, with ACE-sRNA-1/26 demonstrating the most potent effect (P < 0.001), inhibited IκBα protein and inflammatory factor expressions (P < 0.01, 0.001). In animal experiments, mice in model group had severe lung injury (P < 0.001); Compared with model group, ACE-sRNA-1 and ACE-sRNA-26 significantly improved lung injury in mice (P < 0.05, 0.01, 0.001), up-regulated VE-cadherin expression in lung tissue (P < 0.001), down-regulated ICAM-1, ACE, AT1R, IκBα and inflammatory factor expressions in lung tissue (P < 0.05, 0.01, 0.001), and reduced Ang II and inflammatory factor levels in serum (P < 0.05, 0.01, 0.001). Conclusion ACE-sRNA-1 and ACE-sRNA-26 derived from XFBD could target the inhibition of ACE expression and activity, alleviate pulmonary endothelial inflammation and barrier damage, and improve LPS-induced ALI in mice. The mechanism may be related to the regulation of ACE-Ang Ⅱ-AT1R pathway.

Objective To investigate the components of Isodon lophanthoides var. gerardianus aqueous extract (ILAE) and its protective effects against ethanol-induced alcoholic liver disease (ALD) in zebrafish, and to analyze its potential active components and targets using network pharmacology. Methods Ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was employed to identify the components of ILAE. Wild-type AB strain and transgenic neutrophil zebrafish Tg (lyz: DsRed) at 4 d post-fertilization (4 dpf) were treated with 36, 73, 146 μg/mL ILAE or 70 μg/mL silymarin for 16 h, followed by induction of ALD with 2% ethanol for 32 h. The protective effects of ILAE on liver were assessed by observing developmental status, measuring biochemical indicators and conducting behavioral tests. Network pharmacology was used to analyze the potential components and targets of ILAE in treating ALD. Validation was performed through molecular docking and Western blotting. Results A total of 56 components were identified in ILAE, primarily flavonoids and phenolic acids. ILAE significantly alleviated ethanol-induced developmental impairment in zebrafish, the delayed absorption area of yolk sac and the area of liver enlargement were significantly reduced (P < 0.05, 0.001), lipid accumulation was significantly reduced (P < 0.001), liver pathological damage was relieved, liver cells were arranged in an orderly manner, and fat vacuoles were reduced. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly decreased (P < 0.05, 0.01, 0.001), while the activity of alcohol dehydrogenase (ADH) and reduced glutathione (GSH) level were significantly increased (P < 0.05, 0.01, 0.001). The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and triglycerides (TG) were significantly decreased (P < 0.05, 0.01, 0.001), and the number of neutrophils was significantly reduced (P < 0.01, 0.001). The excitement state of zebrafish was decreased, and the rapid movement trajectory and total movement distance were decreased (P < 0.05, 0.001). The core targets associated with ILAE and ALD were protein kinase Bα (AKT1), nuclear factor-κB subunit 1 (NF-κB1), signal transducer and activator of transcription 3 (STAT3), key components were chrysin, eupatilin and rosmarinic acid, which exhibited strong binding affinity to the targets. Kyoto encyclopedia of genes and genomes (KEGG) analysis and gene ontology (GO) analysis showed that the process involved lipid metabolism, insulin resistance, bile acid secretion, and exogenous substance metabolism pathways. Western blotting results showed that ILAE significantly reduced the expression levels of NF-κB1, STAT3 proteins and their phosphorylated forms in ethanol-induced zebrafish (P < 0.05, 0.01, 0.001), and increased the expression levels of AKT1 protein and its phosphorylated forms (P < 0.05, 0.01). Conclusion ILAE may improve ALD by targeting AKT1, NF-κB1, STAT3 through rosmarinic acid, chrysin, caffeic acid, 7-hydroxycoumarin and other components. Its protective effect may be related to reducing inflammatory response, decreasing lipid accumulation and enhancing antioxidant activity.

Objective To screen the core active components of Shenfu Injection (参附注射液, SFI) using UHPLC-MS and molecular docking techniques, and to verify the effects of SFI on expressions of post-translational modifications of tubulin in chronic heart failure of heart-yang deficiency syndrome through in vivo and in vitro experiments. Methods UHPLC-MS was applied to identify the main components of SFI, and molecular docking was performed to analyze their interactions with tubulin-related targets. SD rats were randomly divided into control group, model group, colchicine (0.1 mg/kg) group, SFI low- and high-dose (3, 6 mL/kg) groups, with eight rats in each group. The CHF heart-yang deficiency model was replicated using sc isoproterenol, and drug intervention was given for two weeks. Echocardiography was used to detect cardiac function. ELISA was used to detect N-terminal pro-B-type natriuretic peptide (NT-proBNP) level in serum. Hematoxylin-eosin (HE) staining was used to detect pathological changes in myocardial tissue. Immunofluorescence was used to detect the expressions of acetyl α-tubulin, polyglut α-tubulin and detyr α-tubulin in myocardial tissue. Immunohistochemistry was used to detect the expressions of tubulin tyrosine ligase (TTL) and histone deacetylase 6 (HDAC6) in myocardial tissue. H9c2 cardiomyocytes were treated with isoproterenol to establish a cell injury model, and after intervention with SFI, the level of microtubule protein modification was detected. Results A total of 12 main active ingredients in SFI were obtained through UHPLC-MS screening, and molecular docking results showed strong binding affinity with tubulin and modification related proteins. The animal experiment results showed that compared with model group, SFI could significantly improve cardiac function of rats (P < 0.01), reduce the level of NT-proBNP in serum (P < 0.01), alleviate the degree of myocardial interstitial fibrosis, reduce the expressions of microtubule protein α-tubulin and microtubule density in myocardial tissue (P < 0.05, 0.01), reduce the expressions of microtubule protein tyrosine and glutamate, increase acetylation expression, down-regulate HDAC6 positive expression (P < 0.01), and up-regulate TTL positive expression (P < 0.01). The results of cell experiments were consistent with animal experiments, further verifying that SFI could improve post-translational modifications of microtubule proteins. Conclusion SFI could effectively reduce the levels of tyrosinization and glutamatzation of microtubules, increase acetylation levels, correct the imbalance of post-translational modifications of microtubules, thereby improving the abnormal microtubule network in cardiac myocytes of rats with CHF heart-yang deficiency, enhancing microtubule stability and improving heart function. This provides experimental evidence for the molecular mechanism of SFI treatment for CHF heart-yang deficiency.

Objective To investigate the effect and potential mechanism of bufotalin on growth of human head and neck squamous cell carcinoma (HNSCC). Methods The effect of bufotalin on viability and proliferation of Cal-27 and FaDu cells were observed by CCK-8 method and plate clone formation experiment. The effect of bufotalin on cell cycle distribution of Cal-27 and FaDu cells were detected by flow cytometry. Western blotting was used to detect the effect of bufotalin on expressions of cyclin-dependent kinase 4 (CDK4) and cyclin D1 in Cal-27 and FaDu cells. To further explore the anti-tumor effects of bufotalin in vivo, a xenograft tumor model of HNSCC was established in nude mice. Proteomics technology was applied to identify differentially expressed proteins in bufotalin-treated Cal-27 cells, and the associated signaling pathways underlying its anti-tumor effect were analyzed. Western blotting was performed to assess the expressions of protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) signaling pathway related proteins. Results In vitro experimental results showed that bufotalin significantly reduced the viability of Cal-27 and FaDu cells (P < 0.01, 0.001), inhibited their proliferation (P < 0.01, 0.001), induced cell cycle arrest in G₀/G₁ phase (P < 0.05, 0.01, 0.001), down-regulated the protein expressions of CDK4 and cyclin D1 in FaDu cells (P < 0.05, 0.001), and down-regulated the protein expression of CDK4 in Cal-27 cells (P < 0.05). The in vivo experimental results showed that bufotalin could significantly inhibit the growth of tumors in tumor bearing mice (P < 0.01, 0.001). Proteomics and IPA analysis showed that bufotalin could induce endoplasmic reticulum stress and up-regulate the expression levels of p-PERK and p-eIF2α in Cal-27 and FaDu cells (P < 0.05, 0.001). Conclusion Bufotalin could induce endoplasmic reticulum stress, activate EIF2 signaling pathway, and induce the arrest of HNSCC cells in G₀/G₁ phase, thereby inhibiting tumor cell proliferation and tumor growth in tumor-bearing mice.

Objective The quality research system of the classic formula Tiaowei Chengqi Decoction (TCD, 调胃承气汤) was constructed by establishing HPLC fingerprinting and index component content determination methods, and the quantity value transfer law of its reference samples were explored. Methods Prepare TCD reference samples from 15 batches of herbal materials sourced from different regions using traditional methods. Establish fingerprint profiles via HPLC and evaluate batch-to-batch consistency using chemometric methods such as hierarchical cluster analysis (HCA) orthogonal partial least squares-discriminant analysis (OPLS-DA) and principal component analysis (PCA) to identify the batch-to-batch consistency and differential markers. Simultaneously, Hotelling’s T² and DModX were calculated based on the PCA model, with 95% and 99% confidence limits serving as the warning and control limits [T²_Crit (95%) = 31.49, T²_Crit (99%) = 54.15, D_Crit = 1.97] for batch consistency evaluation. A method was established to simultaneously determine the content of six target components gallic acid, glycyrrhizin, aloe-emodin, emodin, glycyrrhetic acid, and rhein in 15 batches of samples and their corresponding crude drug slices. The transfer rate of these components from crude drug slices to samples was calculated. Results A robust HPLC fingerprint profile of TCD was successfully established, allowing for the identification of 16 common peaks. Chemometric analysis confirmed effective batch-to-batch differentiation, with gallic acid and glycyrrhizic acid identified as key chemical markers contributing to the discrimination of batch variations. Quantitative analysis of six marker components across 15 batches revealed mass fractions ranging from 0.521 to 1.581 mg/g for gallic acid, 0.143 to 0.711 mg/g for glycyrrhizin, 0.025 to 0.071 mg/g for aloe-emodin, 0.016 to 0.182 mg/g for emodin, 0.487 to 2.740 mg/g for glycyrrhizic acid, and 0.007 to 0.039 mg/g for rhein. Furthermore, component tracking from crude drug to final sample demonstrated average transfer rates of 17.625%,2.878%,5.929%,5.159%,1.689% and 1.088% for these compounds, respectively. Conclusion Combined with multi-component quantitative analysis with chemometric methods to develop an HPLC fingerprint profile for TCD, systematically investigating the compositional variation patterns from herbal materials to reference samples. The findings provide a scientific foundation for quality evaluation research on TCD.

Objective To conduct qualitative and quantitative analysis of the chemical constituents in Tiaojing Cuyun Pills (TCP, 调经促孕丸) using high-resolution mass spectrometry and to evaluate its quality comprehensively. Methods Ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap HRMS) was employed in this study. Combined with reference standard comparison, mass spectral fragmentation rule analysis and database retrieval, a method for identifying and analyzing the in vitro chemical constituents of TCP was established, and the key constituents were quantified. Multivariate statistical methods were applied to analyze the quantitative data for comprehensive quality evaluation of TCP. Results A total of 105 compounds were identified by collecting mass spectral data in Full-scan data-dependent MS/MS (Fullmass ddMS²) mode, covering various structural types such as flavonoids, organic acids, quinones, glycosides, terpenoids, alkaloids and others. Meanwhile, a quantitative analysis method was established for 24 key constituents in the drug using the full-scan accurate quasi-molecular ion mode to achieve precise content determination. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) analysis showed that the overall quality of 10 batches of drugs was stable, and catechin, tanshinone I, baohuoside I, daidzein, paeoniflorin and caffeic acid might be the key constituents affecting drug quality. Conclusion Based on high-resolution mass spectrometry technology, a variety of chemical components in TCP can be qualitatively and quantitatively analyzed in a systematic, accurate and rapid manner, which lays a foundation for the research on its pharmacodynamic material basis and quality control.

Objective To construct and optimize tanshinone II_A (TS II_A)-loaded micelles modified with a cardiomyocyte-targeting peptide CTP (APWHLSSQYSRT) (CTP-Ms/TS II_A), optimize their formulation, and evaluate their physicochemical properties and cardioprotective effects in vitro. Methods CTP-Ms/TS II_A were prepared using the film hydration method. A Box-Behnken response surface design was applied to optimize formulation parameters (Soluplus concentration, TPGS₁₀₀₀ concentration, hydration temperature) with encapsulation efficiency as the evaluation index. Particle size, ζ potential, and morphology of micelles were characterized by dynamic light scattering and transmission electron microscopy (TEM). Critical micelle concentration (CMC) was determined using pyrene as a probe, and storage and serum stability were assessed. Cellular uptake was investigated in H9c2 cardiomyocytes by flow cytometry and fluorescence microscopy. In an isoproterenol-induced H9c2 injury model, intracellular reactive oxygen species (ROS), mitochondrial membrane potential, cell viability, and apoptosis were evaluated by DCFH-DA, JC-1, calcein/PI staining, and CCK-8 assays. Results The optimal preparation parameters were determined as: formulation volume 5 mL, Soluplus concentration 12 g/L, TPGS₁₀₀₀ concentration 5 g/L, and hydration temperature 40 ℃. The CTP-Ms/TS II_A prepared under these conditions (with membrane materials including TS II_A, Soluplus, TPGS₁₀₀₀, DSPE-PEG₂₀₀₀, and DSPE-PEG₂₀₀₀-CTP) achieved a high EE of (90.64 ± 1.01)% (n＝3). The micelles exhibited a particle size of (89.30 ± 0.76) nm, a ζ potential of (−0.033 ± 0.150) mV, and a critical micelle concentration of 42.8 μg/mL, with good stability. Compared with non-targeted micelles, CTP-Ms/TS II_A exhibited significantly enhanced uptake in H9c2 cells, more effective ROS scavenging, restoration of mitochondrial membrane potential, and improved cell survival. Conclusion CTP-Ms/TS II_A with stable physicochemical properties were successfully prepared, and their cardiac-targeting and cardioprotective potential were verified by in vitro experiments. This work provides a new experimental basis and potential delivery strategy for the application of TS II_A in the prevention and treatment of cardiovascular diseases.

Objective To systematically evaluate the effects of soluble excipients on the disintegration behavior of Chinese medicine extract tablets prepared by different processes, thereby providing a scientific basis for the formulation design of Chinese medicine tablets. Methods A total of 11 representative Chinese medicine extract powders were selected, and the impacts of three processes (direct compression, dry granulation compression, and wet granulation compression) on tablet disintegration time and dissolution were compared. On this basis, Rougui (Cinnamomi Cortex) and Quɑnshen (Bistortae Rhizoma) extract tablets with poor disintegration performance were chosen for a factorial experimental design to investigate the modulating effects of soluble excipient type (sugar alcohols, lactose, and starch and its derivatives), proportion (10%, 20%, and 30%), and preparation process (dry granulation compression and wet granulation compression) on tablet disintegration. Results Except for Kushen (Sophorae Flavescentis Radix), the granulation process had minimal impact on the disintegration behavior of single Chinese medicine extract tablets, including Hujisheng (Visci Herba), Dɑnggui (Angelicae Sinensis Radix), Chuɑnxiong (Chuanxiong Rhizoma), Wumei (Mume Fructus), Fuchaocɑngzhu (Atractylodis Rhizoma stir-fried with bran), Zhiheshouwu (Polygoni Multiflori Radix Praeparata), Bohe (Menthae Haplocalycis Herba) with a disintegration time ≤ 30 min. However, for single extract tablets with slower disintegration (> 30 min), such as Xixin (Asari Radix et Rhizoma), Cinnamomi Cortex and Bistortae Rhizoma, granulation prolonged disintegration time and reduced dissolution rate. Under high drug-loading conditions (≥ 70%) sugar alcohols and lactose showed the most significant improvement in disintegration of Cinnamomi Cortex and Bistortae Rhizoma extract tablets, whereas starch and its derivatives exhibited limited effects but enhanced the dissolution of water-soluble components under wet granulation. Wet granulation was more suitable for Cinnamomi Cortex tablets, while dry granulation was preferable for Bistortae Rhizoma tablets. Conclusion In the formulation design of high drug-loading Chinese medicine extract tablets, selecting sugar alcohols or lactose as excipients and optimizing the granulation process based on material properties are effective strategies to enhance disintegration and dissolution. This study provides foundational data for the development of Chinese medicine tablets and related solid dosage forms.