Objective To investigate the antifungal secondary metabolites from the rice fermentation products of the Valeriana officinalis endophytic fungus Chaetomium elatum FH-3. Methods The one strain many compounds OSMAC (OSMAC) strategy and global natural products social molecular networking (GNPS) molecular networking technology were employed for targeted separation. Chromatographic and spectroscopic methods were used for structural identification. The antifungal activity of the compounds against Colletotrichum gloeosporioides was evaluated using the mycelial growth rate method. Results A total of 12 compounds were targeted and isolated from the ethyl acetate extract of Chaetomium elatum FH-3. These compounds were identified as 4′′-methoxy-asperianas A (1), (3S,4S)-4-hydroxy-6-methoxymellein (2a), (3R,4R)-4-hydroxy-6-methoxymellein (2b), cis-4,6-dihydroxymellein (3), cis-4-hydroxymellein (4), 6-methoxymellein (5), 6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin (6), 3-methyl-6-hydroxy-8-methoxy-3,4-dihydroisocoumarin (7), xenofuranone B (8), flavipesin B (9), p-hydroxybenzaldehyde (10), and p-hydroxybenzoic acid methyl ester (11). Antifungal testing showed that compounds 1—4 exhibited significantly stronger inhibitory activity against C. gloeosporioides than the positive control carbendazim, with half-maximal effective concentration (EC₅₀) values of 15.02, 25.31, 37.89, and 47.86 μg/mL, respectively. Conclusion Compounds 1 and 2a are new butenolide and dihydroisocoumarin derivatives, named butenolide A and dihydroisocoumarin C respectively, and compounds 8 and 9 are reported for the first time from this strain. Compounds 1—9 demonstrate significant inhibitory activity against C. gloeosporioides.

Objective To investigate the chemical compositions of the ethyl acetate (EtOAc) fraction of Sophora flavescens and evaluate their anti-liver fibrosis activity. Methods The EtOAc extraction of S. flavescens was separated and purified by various column chromatography methods, including silica gel, polyamide, Sephadex LH-20 and C₁₈reversed-phase column chromatography. The structures of the isolated compounds were elucidated based on modern spectroscopic techniques and physical and chemical identification method. Transforming growth factor beta-1 (TGF-β1)-induced LX-2 cells were used as an in vitro model of hepatic fibrosis, and the anti-fibrotic activities of the compounds were evaluated using the CCK-8 assay. Additionally, cell wound scratch assay, real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and flow cytometry assays were employed to investigate the anti-liver fibrosis activity and mechanism of compound 14 (kushenol T). Results A total of 28 compounds were isolated from the EtOAc of S. flavescens, and they were identified as follows: ethyl 2,4-dihydroxybenzoate (1), trifohrhizin (2), 2,4- dihydroxybenzoic acid (3), kuraridin (4), kushenol N (5), kurarinol A (6), kushenol C (7), 8-isopentenyl-5-methoxy-7,2′,4′-trihydroxy flavanonol (8), 2′-hydroxy-isoxanthohumol (9), kurarinone (10), methyl 2,4-dihydroxybenzoate (11), sophoflavescenol (12), umbeliferone (13), kushenol T (14), sophophenoside B (15), 6′′-β-D-xylosegenistin (16), 3′,4′-methylenedioxyisoflavone-7-O-β-D-apiofuranosyl-(l→6)-β-D-glucopyranoside (17), ambocin (18), kushenol O (19), 4′-hydroxy-3′-methoxyisoflavone-7-O-β-D-xylopyranosyl-(l→6)-β-D-glucopyranoside (20), 3′-hydroxy-4′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (21), genistein (22), isoxanthohumol (23), 8-isopentenyl-5-methoxy-7,4′-dihydroxyflavanonol (24), 2-(2¢,4¢-dihydroxyphenyl)-5,6-methylenedioxy- benzofuran (25), calycosin (26), daidzein (27), narirutin (28). Bioactivity screening revealed that compounds 9, 11—14, 17, 20, 23—25 and 28 exhibited significant inhibitory effects on LX-2 cells. Among these, 14 demonstrated the most potent activity, markedly downregulating the expression of hepatic fibrosis markers, such as α-smooth muscle actin (α-SMA), fibronectin (FN) and Collagen I, at both the gene and protein levels. Furthermore, 14 promoted cell apoptosis and suppressed TLR4 in the Toll-like receptors 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway, myeloid differentiation primary response 88 (MyD88), and TGF-β-activated kinase 1 (TGF-β-activated kinase 1), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β). Conclusion Compounds 18, 25 and 28 were isolated from this species for the first time. Compounds 9, 11—14, 17, 20, 23—25 and 28 significantly inhibited LX-2 cell proliferation. Among them, compound 14 had the most significant activity and exerts an anti-liver fibrosis effect through the TLR4/NF-κB signaling pathway.

Objective To systematically analyze the blood-absorbed and brain-absorbed components of the classical formula Kaixin San (KXS) in normal and depression model rats, and preliminarily elucidate the pharmacological material basis of its antidepressant effect. Methods A depression model rat was established using the chronic unpredictable mild stress (CUMS) method, and behavioral verification was performed through open field test, novel object recognition test, sucrose preference test, and forced swimming test. The components in the serum and brain tissue from normal and model rats after intragastric administration of KXS were comprehensively analyzed by UPLC-Q-Exactive Orbitrap MS/MS. Results In the control rats, 143 (including 47 prototypes and 96 metabolites) and 11 components were identified in serum and brain tissue, respectively. In contrast, the overall number in the depression model rats significantly decreased, with only 83 (including 28 prototypes and 55 metabolites) and 4 components detected in serum and brain tissue, respectively. Among them, four prototype components were confirmed to cross the brain in serum and brain tissue samples of the CUMS model rats, including β-asarone, ginsenoside Rg₁, ginsenoside Rb₁, and polygalaxanthone III. Conclusion This study identified four key active components from the blood-absorbed and brain-absorbed components of KXS in normal and depression model rats, providing experimental evidence for screening quality markers of KXS and in-depth analysis its antidepressant mechanism.

Objective To screen the optimal purification and drug-loading processes of exosomes (Exo), construct an in vitro blood-brain barrier (BBB) cell model and an Alzheimer’s disease (AD) mouse model, and investigate the efficiency of human embryonic kidney 293T (HEK 293T) Exo-mediated ginsenoside Rg₁ (Rg₁) across the BBB, so as to provide a basis for the treatment of brain diseases. Methods Exosomes were purified using ultracentrifugation, tangential flow filtration, and dual-coupled harmonic oscillation technology. The optimal purification method was selected based on concentration, particle size, and polydispersity index (PDI). On the basis of single-factor screening of voltage, pulse duration, pulse number, and Rg₁ concentration, the electroporation process for constructing Exo-Rg₁ complexes was optimized using orthogonal design with drug encapsulation efficiency as the evaluation index. An in vitro BBB model was established using brain endothelial cell line 3 (bEnd.3). Model integrity was verified by measuring transendothelial electrical resistance (TEER), liquid level permeability, and sodium fluorescein permeability. The Exo-Rg₁ complex group (experimental group), free Rg₁ group (control group), exosome alone group, and empty exosome + free Rg₁ group were set up. The concentration of Rg₁ penetrating the in vitro BBB model at different time points was detected by HPLC, and the permeability efficiency was calculated. In vivo therapeutic effects were evaluated through behavioral tests, histological observation, and detection of protein and inflammatory factor levels in AD model mice. Results The optimal exosome purification process was dual-coupled harmonic oscillation technology, yielding an exosome concentration of 2.9 × 10¹¹ particles/mL, a particle size of (91.8 ± 1.5) nm, and a PDI of 0.21 ± 0.01. The optimized electroporation conditions were voltage of 100 V, pulse duration of 15 ms, pulse number of four times, and Rg₁ concentration of 0.4 mg/mL. Under these conditions, the encapsulation efficiency of the complexes was 61.58%, which was superior to that of the traditional ultrasonic method. The in vitro BBB model using bEnd.3 cells was successfully established, with TEER values stably above 300 Ω·cm². A significant liquid level difference was maintained in the in vitro BBB model group after 4 h. The sodium fluorescein permeability in the model group was significantly lower than that in the blank control group [(41.34 ± 1.83)% vs (98.87 ± 2.09)%, P < 0.01], meeting the requirements for barrier function. In vitro penetration experiments showed that the penetration efficiency of Rg₁ in the experimental group (15.3%) was significantly higher than that in the control group (3.3%) at 8 h (P < 0.05). Animal experiments demonstrated that the complex significantly improved learning and memory abilities in model mice, protected hippocampal neurons, upregulated the expression of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor (α7 nAChR) proteins, and reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), with effects superior to those of the free Rg₁ group and the exosome-only group. Conclusion Exosomes can effectively enhance the efficiency of Rg₁ in crossing the BBB, providing an experimental basis for improving the therapeutic effect of brain diseases.

Objective To prepare paeoniflorin-glycyrrhiza protein self-assembled nanoparticles (Pae-GP/SAN) with good bioavailability and investigate its intestinal absorption mechanism. Methods In this study, glycyrrhiza protein (GP) was used as the carrier to prepare Pae-GP/SAN by ultrasonic dispersion method. The formulation and preparation process were optimized using average particle size, polydispersity index (PDI), encapsulation efficiency, and drug loading as evaluation indicators, and the prepared nanoparticles were characterized. A rat in situ single-pass intestinal perfusion model was established to investigate and compare the intestinal absorption behaviors of paeoniflorin solution (Pae/Sol), paeoniflorin-glycyrrhiza protein physical mixture (Pae-GP/PM), and Pae-GP/SAN. Additionally, an aggregation-caused quenching (ACQ) fluorescent probe was used to observe the intestinal absorption of GP/SAN by confocal laser scanning microscopy (CLSM), aiming to initially clarify its penetration-enhancing mechanism. Results The optimized Pae-GP/SAN had an average particle size of (178.2 ± 6.3) nm, a PDI of 0.152 1 ± 0.011 2, a ζ potential of (−14.91 ± 1.13) mV, an encapsulation efficiency of (36.45 ± 2.32)%, and a drug loading of (21.70 ± 1.30)%, with a uniform spherical micromorphology. Intestinal perfusion experiments indicated that the absorption efficiency of Pae-GP/SAN in the ileum was superior to that in the jejunum (P < 0.05, 0.01). The formation of nanoparticles significantly promoted the absorption of paeoniflorin. Moreover, the absorption parameters of high mass concentration Pae-GP/SAN were significantly higher than those of low mass concentration Pae-GP/SAN (P < 0.001), while mass concentration had no significant effect on the absorption of Pae/Sol and Pae-GP/PM. Transporter inhibitor intervention experiments showed that indomethacin and reserpine had no significant effect on the absorption of the three formulations. Verapamil significantly increased the absorption of Pae/Sol and Pae-GP/PM (P < 0.01, 0.001) but had no significant effect on Pae-GP/SAN. CLSM observations confirmed that GP/SAN can be absorbed by the intestine in the form of intact nanoparticles. Conclusion Pae-GP/SAN can be endocytosed and absorbed by the intestine as intact nanoparticles, effectively evading the barrier effect of P-gp efflux protein, thereby significantly improving the oral bioavailability of Pae.

Objective Vitexin liposomes (Vit-Lips), chitosan modified Vit-Lips (CS-Vit-Lips) and deoxycholic acid and chitosan co-modified Vit-Lips (DA/CS-Vit-Lips) were prepared, and their oral pharmacokinetic behavior were compared. Methods Deoxycholic acid-chitosan complex (DA/CS) was synthesized. Film-ultrasonic method was used to prepare Vit-Lips. Encapsulation rate, drug loading and particle size were used as evaluation indexes, single factor investigation combined with Box-Behnken design-response surface methodology (BBD-RSM) were used to investigate the optimal prescriptions of Vit-Lips. CS-Vit-Lips and DA/CS-Vit-Lips were prepared by introducing chitosan and DA/CS, respectively. Transmission electron microscopy (TEM) was used to observe the microstructure of the three kinds of liposomes, and their particle stability in gastrointestinal fluids were compared. Using vitexin as reference, the drug release of Vit-Lips, CS-Vit-Lips and DA/CS-Vit-Lips in simulate gastrointestinal fluid were compared, and their drug release mechanism were also studied. The absorption of the vitexin and its three kinds of liposomes in different intestinal segments was investigated, and the intestinal absorption parameters were calculated. Blood samples were collected after gastric administration of vitexin and its three kinds of liposomes at a dose of 20 mg/kg (vitexin), respectively. Main pharmacokinetic parameters and relative oral bioavailability were also calculated. Results The optimal formulation of Vit-Lips: phospholipids to cholesterol dose ratio was 7.95:1, lipids to drug dose ratio was 9.63:1 and hydration time was 30.00 min. CS-Vit-Lips and DA/CS-Vit-Lips were prepared using chitosan and DA/CS solution with mass fraction of 0.8%, respectively. Entrapment efficiency of Vit-Lips, CS-Vit-Lips and DA/CS-Vit-Lips were (82.31 ± 0.96)%, (84.93 ± 1.17)%, (85.15 ± 1.38)%, drug loading were (7.33 ± 0.09)%, (6.24 ± 0.08)%, (6.30 ± 0.11)%, particles size were (197.70 ± 5.07), (233.06 ± 7.19), (237.93±6.96) nm, and ζ potential were (−28.81 ± 0.86), (27.75 ± 1.10), (26.14 ± 1.13) mV, respectively. The appearance of three kinds of liposomes was spherical vesicular, particle stability of CS-Vit-Lips and DA/CS-Vit-Lips was higher than that of Vit-Lips in simulated gastrointestinal fluid. Vit-Lips, CS-Vit-Lips and DA/CS-Vit-Lips enhanced cumulative release rate to 91.02%, 86.74% and 83.15%, respectively. The drug release process of the three kinds of liposomes conformed to Weibull model. The three kinds of liposomes effectively improved the absorption rate constant (K_a) and apparent absorption coefficient (P_app) of vitexin. Compared to vitexin, Vit-Lips, CS-Vit-Lips and DA/CS-Vit-Lips increased relative oral bioavailability to 1.10-fold, 3.08-fold and 4.70-fold, respectively. The half-life time (t_1/2) of CS-Vit-Lips and DA/CS-Vit-Lips had significantly prolonged (P < 0.01) and the peak concentration (C_max) was significantly increased (P < 0.01). Conclusion CS-Vit-Lips and DA/CS-Vit-Lips effectively promoted the oral absorption of vitexin, and the advantage of DA/CS-Vit-Lips were more obvious, laying an experimental foundation for further research.

Objective To develop an icariside II nanoemulsion (NE-ICS II) to enhance oral bioavailability and improve efficacy against cerebral ischemia-reperfusion injury (CIRI). Methods NE-ICS II was prepared by ultrasonic emulsification and its physicochemical properties were characterized. Pharmacokinetic parameters and brain distribution were systematically evaluated. Therapeutic efficacy was assessed in rats subjected to middle cerebral artery occlusion (MCAO) by measuring infarct volume, neurological deficit scores, and oxidative stress and inflammatory markers. RNA sequencing was performed to elucidate the underlying mechanisms. Results NE-ICS II showed a mean particle size of (147.59 ± 0.71) nm and an encapsulation efficiency of (84.85 ± 5.47) %. NE-ICS II increased the oral bioavailability of ICS II by 2.4-fold and significantly enhanced its accumulation in the brain. Therapeutic oral administration of NE-ICS II markedly improved neurological function, reduced infarct volume, and attenuated oxidative stress and inflammatory responses in MCAO rats. RNA sequencing revealed that NE-ICS II may downregulate signaling pathways associated with oxidative stress and inflammation. Conclusion NE-ICS II substantially improves the oral bioavailability of ICS II and enables effective treatment of CIRI at a low dose, providing a promising strategy for efficient brain delivery of ICS II and for the treatment of central nervous system diseases.

Objective To establish a multi-index weighted optimization strategy for wine-processed Xiangfu (Cyperi Rhizoma) by integrating CRITIC weighting, Box-Behnken design-response surface methodology (BBD-RSM), and an artificial neural network (ANN), and define a robust operating space based on model prediction, thereby providing a reference for process parameter fixation and quality consistency control. Methods The contents of cyperotundone, α-cyperone, total flavonoids, and total volatile oil were used as comprehensive evaluation indices. Objective weights were assigned using the CRITIC method, and an overall desirability (OD) value was calculated. Based on single-factor experiments, a four-factor, three-level Box-Behnken design (BBD) was conducted with processing temperature, processing time, moistening time, and dosage as independent variables, and a regression model was established. An ANN prediction model was then developed, and the Garson algorithm was applied to interpret the relative contributions of factors. In addition, the Pareto non-dominated solution set was used to screen the process-parameter space to obtain a robust operating interval (model-predicted). Results The CRITIC-derived weights for cyperotundone, α-cyperone, total volatile oil, and total flavonoids were 0.274 9, 0.255 4, 0.253 4, and 0.216 3, respectively. The optimal processing conditions were determined as follows: processing temperature 140 ℃, processing time 19 min, moistening time 6.9 h, and dosage 32 g/L. The OD obtained from validation experiments was 0.655 1, RSD was 2.97%, which was close to the predicted value (0.653 3). The robust parameter interval defined based on ANN prediction and the Pareto non-dominated solution set was as follows: processing temperature 140—150 ℃, processing time 18—22 min, moistening time 6.5—7.5 h, and dosage 30—35 g/L. The model suggested that OD could remain relatively stable under ± 5% parameter fluctuations within this interval. Conclusion The constructed BBD-ANN-Pareto coupling model demonstrated good predictive ability and stability, which may provide a reference for optimizing the processing technology and ensuring the quality consistency of wine-processed Cyperi Rhizoma.

Objective To address the challenges of insufficient standardization in the processing of Orpiment (arsenic disulfide, As₂S₂) and the difficulty in controlling its toxicity, this study elucidated the interactions among “particle size-morphology-porosity-crystal defects-arsenic (As) release”. It analyzed the effects of different processing methods on elemental composition and microstructure, and established a rapid identification model based on multimodal spectral fusion. Methods A multi-dimensional “element-morphology-spectrum” data matrix was constructed. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine elemental content. Scanning electron microscopy (SEM) was employed to observe morphology and analyze surface porosity, combined with dissolution experiments to quantify As release. Furthermore, Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy (RS) data were integrated. Partial least squares-discriminant analysis (PLS-DA) and support vector machine (SVM) discrimination models were built using both low-level and mid-level data fusion strategies. Results The zirconia-ball water-grinding method reduced As content and increased sulfur (S) content in Orpiment. SEM analysis revealed that particles processed by this method exhibited a more concentrated size distribution, their morphology became more rounded and blunt, and surface porosity significantly increased, forming a hierarchical pore structure. In simulated gastric and intestinal fluids, As dissolution decreased by 72.8% and 81.4%, respectively, compared to dry crushing (P < 0.001). The SVM model based on mid-level spectral fusion achieved 100% classification accuracy. Conclusion The water-grinding method achieves detoxification by removing As³⁺ from Orpiment, regulating the particle morphology and pore structure of Orpiment particles, and enhancing the stability of Orpiment crystals. It establishes a multidimensional data matrix integrating elemental composition, microscopic morphology, pore structure, and spectral characteristics, providing efficient and reliable technical support for quality control of the mineral drug Orpiment.

Objective To analyze the structural characteristics of water-eluted fraction of Sanghuangporus vaninii polysaccharides (SHP-W) and explore the mechanism of SHP-W on improving carbon tetrachloride (CCl₄)-induced liver fibrosis in mice by regulating gut microbiota. Methods SHP-W was extracted from S. vaninii using water extraction and ethanol precipitation, followed by purification through weak anion-exchange chromatography. The total sugar, uronic acid and protein contents of SHP-W were determined by phenol sulfuric acid method, m-hydroxybiphenyl method and BCA method. The structural characterization of SHP-W was carried out by high performance size exclusion chromatography (HPSEC), Fourier transform infrared spectrophotometer (FT-IR), ion chromatography (IC) and methylation combined gas chromatography-mass spectrometry (GC-MS). C57BL/6J mice were prepared with a 15% CCl₄ olive oil solution via ip for six weeks to establish a liver fibrosis model. SHP-W (25, 50, 100 mg/kg) or sorafenib (10 mg/kg) were administered for three weeks, and the pathological morphological changes in liver tissues were observed using hematoxylin-eosin (HE), sirius red and Masson staining. A fully automated biochemical analyzer was used to analyze the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acids (TBA), total bilirubin (TBIL) and direct bilirubin (DBIL) in serum. Immunohistochemistry was used to detect the expressions of collagen-I (Col-I) and α-smooth muscle actin (α-SMA) in liver tissue. Western blotting was used to detect the expressions of Col-I, α-SMA and transforming growth factor-β1 (TGF-β1) proteins in liver tissue. 16S rDNA sequencing was used to analyze the gut microbiota, and antibiotic interference was used to verify the role of the microbiota. Results SHP-W was a refined polysaccharide composed of two polysaccharides with different relative molecular weights (1.818 × 10⁴, 2.040 × 10³), with a total sugar content of (88.27 ± 3.76)%, and without uronic acid and protein. The monosaccharide composition results showed that SHP-W was composed of galactose, mannose, fucose and glucose, with a molar ratio of 34.6∶23.0∶22.7∶19.7, and also contained 3-O-methylgalactose. Methylation analysis showed that SHP-W was composed of 14 glycosidic bonds, including 1,3,6-Glcp.The animal experiment results showed that compared with control group, the levels of AST, ALT, TBIL, DBIL and TBA in serum of mice in model group were significantly increased (P < 0.001), the arrangement of liver cell cords was disordered, inflammatory cell infiltration was obvious, and a large amount of fibrous tissue proliferation was appeared, the expressions of Col-I, α-SMA and TGF-β1 protein in liver tissue was significantly up-regulated (P < 0.05, 0.001). Compared with model group, the collagen fiber deposition in pseudo lobules and portal area of liver tissue of mice in each treatment group was improved to varying degrees, and the serum liver function level was significantly reduced (P < 0.05, 0.01, 0.001), the expressions of Col-I, α-SMA and TGF-β1 protein in liver tissue was down-regulated (P < 0.05, 0.01, 0.001). After antibiotic interference, the effect of SHP-W on improving liver fibrosis was significantly inhibited. Conclusion SHP-W were prepared from S. vaninii fruiting bodies, the determination of uronic acid content, infrared spectroscopy and monosaccharide composition showed that SHP-W were neutral polysaccharide. SHP-W could significantly improve serum liver function, inhibit HSCs activation, reduce collagen fiber deposition, and thus improve CCl₄-induced liver fibrosis in mice. Its anti-fibrotic effect depends on the regulation of gut microbiota.