Objective Compare the differences in material basis and anti-insomnia and anti-anxiety efficacy between Suanzaoren Decoction (SZRD, 酸枣仁汤) and Suanzao Decoction (SZD, 酸枣汤) in the Jin Gui Yao Lue. Methods This study quantifies 20 chemical ingredients as evaluation indicators to establish the boiling methods of SZRD and SZD, based on ultra-high performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (UPLC-Qtrap-MS) technology. Moreover, the ultra-high performance liquid chromatography-orbitrap tandem mass spectrometry (UPLC-Orbitrap-MS/MS) metabolite identification method specific to the Chinese herbal medicine was used to identify and compare the chemical profile of the two decoctions. Lastly, the p-chlorophenylalanine (PCPA) induced insomnia model mice were used to evaluate and compare the efficacy of the two decoctions in anti-insomnia and anti-anxiety. Results The method of boiling Suanzaoren in ceramic-coated iron pot for 25 min and adding other herbs then continuing to boil for 40 min can match well with the original records in Jin Gui Yao Lue. When Suanzaoren and Suanzao fruit were both used with the amount of 2 litters in Han Dynasty, the contents of jujuboside A, jujuboside B, spinosin, 6‴-feruloylspinosin, magnoflorine, pachymic acid and ligustilide in SZRD were over 6-fold higher than those in SZD, whereas the contents of liquiritigenin, isoliquiritin, isoliquiritigenin and timosaponin AIII in SZD were over 2 times than those in SZRD. Among the top 100 compounds with the strongest mass spectrometric signals detected in the two decoctions, 42 compounds exhibited higher contents in SZD and 21 compounds were enriched in SZRD. Among the differential compounds, three isoquinoline alkaloids [(-)-isocorypalmine, cheilanthifoline and asimilobine], one pyrrolidine alkaloid (stachydrine) and one steroidal alkaloid (zhebeinine) were further identified. The relative abundances of cheilanthifoline and asimilobine in SZD were 5.2 times and 4.8 times higher than those in SZRD, respectively; meanwhile, the relative abundances of (-)-isocorypalmine, stachydrine and zhebeinine in SZD only reached 0.3, 0.4 and 0.4 times the corresponding values in SZRD. The anti-insomnia and anti-anxiety efficacies of the two decoctions in the PCPA induced insomnia mice are similar to each other, and no statistically significant intergroup difference in their pharmacological efficacy was observed.. Conclusion Under the original decoction method recorded in Jin Gui Yao Lue, SZRD and SZD show significant differences in material basis, yet their similar anti-insomnia and anti-anxiety effects under specific condition may result from the combined actions of multiple active components.

Objective To explore the self-assembly behavior and molecular association patterns of glycyrrhizic acid (GA) and genkwanin (Gen) after combination, and to analyze the correlation between the incompatibility and the molecular state of Gancao (Glycyrrhizae Radix et Rhizoma)-Yuanhua (Genkwa Flos) from the microscopic perspective. Methods Employ dynamic light scattering (DLS), scanning electron microscopy (SEM), and ultrafiltration to characterize the particle size, morphology and ultrafiltration behavior of GA-Gen mixed solutions. Results When GA and Gen were combined at mass ratios of 55:1 and 45:1, the particle size decreased significantly from 100—150 nm (GA monomer solutions) to approximately 50 nm. After combination, DLS showed a light intensity signal that was not present in the monomer solution at 10—100 nm. SEM images showed that the GA-Gen compatible solutions formed well-defined cubic particles, distinctly different from the globular and long needle-like morphologies of the individual components. When ultrafiltration membranes with molecular weight cut-off (M_w) of 3 000, 10 000, 30 000, 50 000 and 100 000 were used for separation, it was found that with the increase of GA concentration, the transmittance of GA gradually decreased, and the molecular weight at 10% transmittance (T₁₀) and the association coefficient (N) also gradually increased. At the same concentration, the transmittance of GA in the compatibility system was higher than that in the GA monomer system, and T₁₀ and N were lower than those in the monomer system. In addition, the transmittance of Gen in GA-containing system was significantly improved. The decoction system of medicinal materials is consistent with the trends of monomer system. Comparing the SEM of single decoction and co-decoction, the particle size of Glycyrrhizae Radix et Rhizoma single decoction was regular and the size was relatively uniform, and the particle size of Genkwa Flos single decoction was greatly different. The co-decoction failed to reproduce the regular cube of monomer compatibility, but the particle uniformity was between the two single decoctions. Conclusion GA and Gen do not simply coexist in aqueous solution. Instead, Gen is incorporated into GA micelles via molecular substitution, leading to the formation of new self-assembled associates. These findings provide a novel perspective for understanding the mechanism of “reverse” of Glycyrrhizae Radix et Rhizoma-Genkwa Flos herb pair.

Objective To establish a method for differentiating gelatinous Chinese medicines (GCMs) based on supramolecular sensing technology. Methods An indicator displacement assay (IDA) strategy was employed where test sample solutions of GCMs served as analytes and macrocyclic molecule-dye host-guest pairs with specific fluorescence signals were used as sensing pairs to construct a sensor array. The fluorescence intensity changes resulting from the interaction between different GCMs and the sensing units were detected and combined with pattern recognition methods for differentiation. Results Through screening host-guest sensing units with selective fluorescence responses to GCMs from a supramolecular library consisting of cyclodextrins, cucurbiturils, calixarenes, and biphen[n]arenes hosts combined with dyes, a quaternary supramolecular sensor array was constructed including quaternary ammonium salt calix[5]arene (QAC5A)·eosin Y (EY) (0.4 μmol/L vs 0.5 μmol/L), quaternary ammonium modified azocalix[4]arene (QAAC4A)·EY (1 μmol/L vs 1 μmol/L), dodecyl quaternary ammonium salt azo calix[4]arene (QAAC4A12C)·EY (0.6 μmol/L vs 1 μmol/L), and terphen[3]arene (WTP3)·fluorescein (Fl) (0.3 μmol/L vs 0.6 μmol/L). Principal component analysis (PCA) demonstrated that Ejiao (Colla Corii Asini), Xin’ejiao (Colla Corii Suilli), Biejiajiao (Colla Carapacis Trionychis), Lugujiao (Colla Ossis Cervi), Guijiajiao (Colla Carapacis et Plastri Testudinis) or Lujiajiao (Colla Cornus Cervi) could be significantly distinguished within a 95% confidence interval. Conclusion This study developed a quaternary supramolecular sensor array integrated with pattern recognition analysis which enables rapid discrimination of various GCMs.

Objective To screen for the most effective antimicrobial carbon dots from the traditional Chinese medicines Baiji (Bletilla striata), Cili (Rosa roxburghii), Lingzhi (Ganoderma lucidum) and Tianma (Gastrodia elata), and to systematically investigate the physicochemical properties, photothermal performance, antibacterial activity and mechanism of manganese-doped G. lucidum carbon dots (Mn-GLCDs). Methods Four kinds of herbal carbon dots derived from B. striata, R. roxburghii, G. lucidum and G. elata were prepared by a conventional hydrothermal method. The carbon dots with optimal antibacterial activity were screened out, and the morphology, structure and in vitro photothermal performance of the manganese-doped carbon dots were characterized. The antibacterial performance against Staphylococcus aureus and Escherichia coli under near-infrared (NIR) irradiation was evaluated through zone of inhibition assays, bacterial co-culture experiments, and biofilm inhibition and disruption tests. The antibacterial mechanism was preliminarily investigated by intracellular active oxygen fluorescence detection using a DCFH-DA probe. Biosafety was assessed by hemolysis assay and cytotoxicity test. Intracellular reactive oxygen species (ROS) fluorescence measurement using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was adopted to preliminarily elucidate its antibacterial mechanism. Hemolysis assays and cytotoxicity evaluations were conducted to verify their biological safety. Results GLCDs possessed remarkably stronger antibacterial activity than carbon dots prepared from B. striata, R. roxburghii and G. elata carbon dots. Mn-GLCDs had a particle size of approximately 5 nm, and their surfaces were rich in hydrophilic functional groups including -OH, -NH₂, C=O, C-N-C and C-O-C. X-ray photoelectron spectroscopy (XPS) and inductively coupled plasma mass spectrometry (ICP-MS) confirmed that the mass fraction of Mn²⁺ reached 0.83%, and Mn²⁺ was stably present in the carbon dot framework in a coordinated form. The red shift of the UV-Vis absorption peak and the Raman I_D/I_G > 1 indicated that Mn²⁺ doping narrowed the bandgap and increased the density of structural defects. Photothermal performance evaluation showed that Mn-GLCDs could rapidly heat up under 808 nm laser irradiation (1.5 W/cm²), with a photothermal conversion efficiency of 35.03%. The maximum temperature fluctuation in six cycles of heating-cooling stability test was less than 6%, indicating good photothermal stability. Fluorescence performance test showed a fluorescence quantum yield of 20.51% and excitation-dependent fluorescence emission. In vitro antibacterial experiments demonstrated that, compared with undoped G. lucidum carbon dots, Mn-GLCDs exhibited the strongest antibacterial effect against S. aureus and E. coli under near-infrared irradiation, and could significantly inhibit and disrupt bacterial biofilms. Intracellular active oxygen detection showed that upon near-infrared irradiation, Mn-GLCDs elevated the active oxygen levels in S. aureus and E. coli to 6.75-fold and 5.44-fold of the negative control group, respectively. Biosafety evaluation showed that the hemolysis rate of Mn-GLCDs was below 5% within the antibacterial concentration range, and Mn-GLCDs exhibited no obvious toxicity to L929 and RAW264.7 cells. Conclusion This study constructs for the first time Mn-GLCDs, which significantly enhance the photothermal conversion capability and antibacterial activity. The mechanism is related to the induction of intracellular active oxygen. This work provides an experimental basis for the development of novel traditional Chinese medicine-based nanoscale antibacterial agents.

Objective This study focused on the concentration process in the manufacturing of Guizhi Fuling Capsules (GFC, 桂枝茯苓胶囊). By analyzing the correlations among effective components, physical properties, and process parameters, it aimed to identify critical quality attributes (CQAs) and critical process parameters (CPPs), explore quality control indicators and their acceptable ranges, and assess the potential for using physical properties of intermediate to guide its quality control. Methods The variations in process parameters (temperature, relative density at endpoint), physical properties (particle size, polydispersity index, ζ potential, conductivity, pH value, viscosity and refractive index), and effective components and total solid content during the concentration process were systematically investigated. Chemometric methods, including Pearson correlation coefficient, grey relational analysis, and partial least squares regression, were applied. Linear models between effective components and physical properties under different process parameters were established to elucidate the correlations among them. Results Analysis of effective component content and physical properties revealed strong correlations between effective components and ζ potential, conductivity, viscosity, and total solids content. For instance, viscosity was positively correlated with total solids, and the contents of amygdalin and paeoniflorin were both positively correlated with total solid content. Analysis of the active ingredients and physical property parameters of samples under various process parameters revealed that the concentration temperature is a key factor influencing the correlation between active ingredients and physical property parameters. Conclusion Correlation analysis of process parameters, physical properties, and effective components revealed interactive and dynamically linked relationships among these variables. Precise control of the concentration temperature during the manufacturing is essential to improve the quality and batch-to-batch consistency of the extract. To a certain extent, parameters such as viscosity, relative density, refractive index and pH value can reflect the quality of intermediates during concentration, providing a reference for the development of online intelligent monitoring in the concentration process of traditional Chinese medicine extracts.

Objective To explore the mechanism by which Taohong Siwu Decoction (桃红四物汤, THSWD) alleviates cerebral ischemia-reperfusion injury (CIRI) through protecting the integrity and functionality of glycocalyx of brain microvascular endothelial cells. Methods A model of middle cerebral artery occlusion-reperfusion (MCAO/R) was established to simulate CIRI injury. SD rats were randomly divided into sham group, model group, THSWD low-, medium-, high-dose (4.5, 9.0, 18.0 g/kg) groups and nimodipine (20 mg/kg) group, and drugs were given for intervention. After 7 d of modeling, Longa grading system was used for neurological function evaluation, 2,3,5-triphenyltetrazolium chloride (TTC) staining was used to assess cerebral infarction volume. The motion function was evaluated through corner experiments and balance beam experiments. Laser speckle and small animal super-resolution imaging were used to detect cerebral blood flow and cerebral vascular density, Evans blue leak assay combined with Western blotting were to evaluate blood-brain barrier integrity. Glycocalyx morphology was observed using transmission electron microscopy. The expression of glycocalyx-related indicator such as syndecan-1 (SDC-1), hyaluronic acid (HA), heparan sulfate (HS), chondroitin sulfate (CS) and core metabolic enzymes of glycocalyx such as heparanase (HPSE), hyaluronidase 2 (Hyal2), neuraminidase 1 (Neu1), matrix metalloproteinase-9 (MMP-9) were detected by immunofluorescence, immunohistochemistry, Western blotting and ELISA, and the effects of HPSE and MMP-9 were verified by enzyme inhibitors. Results Compared with model group, THSWD significantly reduced the cerebral infarction volume in MCAO/R rats (P < 0.05, 0.001), improved neurological function and limb coordination ability (P < 0.05, 0.01, 0.001), alleviated weight loss, improved cerebral blood flow and microvascular generation in the infarct area, reduced blood-brain barrier leakage (P < 0.01, 0.001), regulated vascular dilation and contraction function (P < 0.01, 0.001). Meanwhile, THSWD could significantly reduce the shedding of core components of glycocalyx (CS, HA, HS, SDC-1) into the serum (P < 0.01, 0.001), and maintain the integrity of glycocalyx by inhibiting the activities of HPSE and MMP-9. Conclusion THSWD reduces the shedding of glycocalyx by inhibiting the activity of HPSE and MMP-9, thereby maintaining the integrity of blood-brain barrier and minimizing stroke damage.

Objective To investigate the effect of catalpol on lipopolysaccharide (LPS)-induced inflammatory injury, extracellular matrix (ECM) metabolic dysregulation and pyroptosis in chondrocytes, and to explore whether these effects involve the AMP-activated protein kinase (AMPK)/nuclear factor-κB (NF-κB)/NOD like receptor family pyrin domain containing 3 (NLRP3) signaling axis. Methods Network pharmacology was used to screening the intersection targets of catalpol, knee osteoarthritis and pyroptosis and enrichment analysis were performed. LPS was used to establish an inflammatory injury and induce pyroptosis model, followed by pretreatment with catalpol (20, 50 μmol/L) for 1 h. Protein expressions of collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), matrix metalloproteinase 3 (MMP3), MMP13 and key molecules in AMPK/NF-κB/NLRP3 signaling axis were detected by Western blotting. Lactate dehydrogenase (LDH) release assay was performed to assess cell membrane integrity-related injury. The levels of interleukin-1β (IL-1β) and IL-18 in supernatants were detected by ELISA. The mRNA levels of NLRP3, IL-1β, IL-6 and tumor necrosis factor-α (TNF-α) were detected by qRT-PCR. The cystein-asparate protease-1 (Caspase-1) inhibitor VX-765 was used as a positive control, the mechanism was further validated using AMPK inhibitor Compound C. Results Compared with control group, LPS stimulation could induce a decrease in ECM synthesis and an increase in ECM decomposition, significantly activate the NLRP3 related pyroptosis pathway (P < 0.001). Compared with model group, catalpol could significantly upregulate the expressions of COL2A1, ACAN and downregulate the expressions of MMP3, MMP13 (P < 0.05, 0.01, 0.001), while significantly reduce the release of LDH, IL-1β and IL-18 (P < 0.01, 0.001), and inhibit NLRP3, cleaved Caspase-1, N-terminal fragment of gasdermin D (GSDMD-N) and other pyroptosis related indicators (P < 0.05, 0.001). In addition, catalpol could significantly upregulate the expressions of p-AMPK/AMPK and phosphorylated acetyl CoA carboxylase (p-ACC)/ACC (P < 0.05, 0.001), and downregulate the expression of p-p65/p65 (P < 0.001), while downregulate the expressions of NLRP3, IL-1β, IL-6, TNF-α inflammation related genes (P < 0.05, 0.001). AMPK inhibitor Compound C could partially reverse the above-mentioned effects of catalpol (P < 0.05, 0.01, 0.001). Conclusion Catalpol may protect the matrix homeostasis of chondrocytes by activating AMPK and promoting downstream ACC phosphorylation, inhibiting NF-κB/NLRP3 related pathways.

Objective To investigate the mechanism of tannic acid in promoting mitochondrial autophagy in chondrocytes and improving knee osteoarthritis (KOA) in mice based on PINK1/Parkin signaling pathway. Methods A mouse KOA model was established via destabilization of medial meniscus surgery. Sham group, model group and tannic acid low-, high-dose (25, 50 mg/kg) groups were set up, drugs were given for intervention for eight weeks. Joint cartilage pathology was assessed by imaging, safranin O-fast green staining and toluidine blue staining. Immunohistochemistry was used to detect the expressions of collagen II (Col-II), matrix metalloproteinase 13 (MMP13), interleukin-1β (IL-1β), PINK1, Parkin, p62 and microtubule-associated protein light chain 3B (LC3B) in cartilage tissue. ATDC5 mouse chondrocytes were stimulated with IL-1β and then treated with tannic acid. Cell viability was measured by CCK-8 assay. Western blotting and qRT-PCR were used to detect the protein and mRNA expressions of Col-II, MMP13 and IL-1β. Western blotting was used to detect the expressions of mitophagy-related proteins PINK1, Parkin, p62 and LC3-II/I. Changes in reactive oxygen species (ROS) and mitochondrial membrane potential were detected by immunofluorescence. Mitochondrial autophagy level was observed by transmission electron microscopy. And reverse mechanism validation was performed using the mitochondrial autophagy inhibitor Mdivi-1. Results Compared with sham group, the cartilage of mice in model group was severely worn, OARSI score was significantly increased (P < 0.01), the protein expression levels of Col-II, PINK1, Parkin and LC3B in cartilage tissue were significantly reduced (P < 0.01), while the protein expression levels of IL-1β, MMP13 and p62 were significantly increased (P < 0.01). Compared with model group, the cartilage morphology of mice in tannic acid group was improved, OARSI score was significantly reduced (P < 0.05), the protein expression levels of Col-II, PINK1, Parkin and LC3B in cartilage tissue were significantly increased (P < 0.01), while the protein expression levels of IL-1β, MMP13 and p62 were significantly decreased (P < 0.01). The cell experiment results showed that IL-1β-induced chondrocyte viability was significantly reduced (P < 0.01), IL-1β, MMP13 protein and mRNA expression levels were significantly increased (P < 0.01), Col-II protein and mRNA expression levels were significantly reduced (P < 0.01), LC3-II/I, PINK1, Parkin protein expression levels were significantly reduced (P < 0.05, 0.01), p62 protein expression level was significantly increased (P < 0.05), intracellular ROS accumulation was increased (P < 0.01), mitochondrial membrane potential was decreased (P < 0.01), and autophagosome was decreased. Compared with model group, cell viability in tannic acid group was significantly increased (P < 0.05, 0.01), ROS accumulation significantly was decreased (P < 0.01), mitochondrial membrane potential was recovered (P < 0.05), IL-1β, MMP13 protein and mRNA expression levels were significantly decreased (P < 0.05, 0.01), Col-II protein and mRNA expression levels were significantly increased (P < 0.05, 0.01), p62 protein expression level was significantly decreased (P < 0.01), LC3-II/I, PINK1 and Parkin protein expression levels were significantly increased (P < 0.01). After the combined use of mitochondrial autophagy inhibitor Mdivi-1, the therapeutic effect of tannic acid on chondrocytes was reversed (P < 0.05, 0.01). Conclusion Tannic acid alleviates articular cartilage damage and delays the progression of KOA by promoting chondrocyte mitophagy through PINK1/Parkin signaling pathway.

Objective To investigate the mechanism by which tetrandrine regulates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) signaling pathway to induce ferroptosis in breast cancer cells based on peroxiredoxin 1 (PRDX1). Methods Human breast cancer MCF-7 cells transfected with PRDX1 siRNA were treated with tetrandrine to examine its effects on cell proliferation, apoptosis, migration and invasion. The levels of malondialdehyde (MDA), Fe²⁺, intracellular and mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential, and mitochondrial membrane permeability were measured. Western blotting and qRT-PCR were used to detect the expressions of voltage-dependent anion channel 1 (VDAC1), p53, PI3K/Akt/GSK-3β signaling pathway and nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway related proteins and genes. Results Tetrandrine dose-dependently inhibited MCF-7 cell proliferation, migration and invasion while inducing apoptosis (P < 0.01), increased intracellular and mitochondrial ROS, MDA and Fe²⁺ levels (P < 0.01), aggravated lipid peroxidation, reduced mitochondrial membrane potential (P < 0.01), enhanced mitochondrial membrane permeability (P < 0.01), upregulated HO-1, p53, GSK-3β and VDAC1 expressions (P < 0.01), downregulated Nrf2, PI3K, p-PI3K, Akt and p-Akt expressions (P < 0.01). Knockdown of PRDX1 gene significantly amplified these effects of tetrandrine (P < 0.01). Conclusion Tetrandrine induces mitochondria-associated ferroptosis in MCF-7 cells via PI3K/Akt/GSK-3β signaling pathway, with PRDX1 playing a critical regulatory role.

Objective To investigate the therapeutic effect of icariin on diabetic kidney disease (DKD) and elucidate its molecular mechanism from the perspective of renal inflammatory response and related inflammatory signaling pathways. Methods A DKD model was established using C57BL/KsJ db/db mice, with age-matched db/m mice on the same genetic background serving as control group. The db/db mice were randomly divided into model group, dapagliflozin (1.6 mg/kg) group, icariin high-, medium-, low-dose (120, 60, 30 mg/kg) groups, with 10 mice in each group. Drugs were given for continuously intervention over four weeks, biochemical parameters including body weight, blood glucose, urinary protein, blood lipids and serum creatinine were measured. Renal pathological changes were observed using hematoxylin-eosin staining and transmission electron microscopy. The expressions of extracellular matrix proteins [fibronectin (FN) and collagen type IV (Col-Ⅳ)] in the glomerular mesangium was assessed by immunofluorescence. Levels of inflammatory cytokines [interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α)] and proteins related to high mobility group box 1 (HMGB1)/Toll-like receptors (TLRs)/nuclear factor-κB (NF-κB) signaling pathway were detected by ELISA assay and Western blotting. In vitro experiments were performed to investigate the effect of icariin on extracellular matrix protein expressions in high glucose-induced SV40 MES-13 cells. Results In vivo experiments results showed that high-dose icariin significantly reduced the body weight, urinary protein, blood lipid and serum creatinine levels in DKD mice (P < 0.01, 0.001), alleviated pathological changes such as mesangial expansion, basement membrane thickening and podocyte fusion (P < 0.001), reduced the levels of IL-1β, IL-6 and TNF-α in plasma and renal tissue (P < 0.01, 0.001), decreased the infiltration of macrophages in the kidney, and significantly inhibited the protein expressions of HMGB1, TLR2, TLR4 and p-p65 in renal tissue (P < 0.001). In vitro experiments results showed that icariin inhibited the expressions of FN and Col-Ⅳ in high glucose-induced glomerular mesangial cells. Conclusion Icariin could effectively inhibit HMGB1/TLRs/NF-κB signaling pathway, thereby attenuating renal inflammatory response and abnormal extracellular matrix accumulation, ultimately improving pathological injury in DKD.