Objective To investigate the chemical constituents of the leaves of Tectona grandis, and search for antifungal active ingredients. Methods The compounds were obtained by silica gel, MCI, and prep-HPLC column chromatography. Their structures were identified by HRESIMS, IR, 1D- and 2D NMR analysis. The isolates were tested for their antifungal activity by 96 well plate microbroth dilution method. Results Two new naphthoquinones, (E)-2,5,8-trihydroxy-3-(4-acetoxy-3-methylbut-2-en-1-yl)-1,4-naphthoquinone (1) and (E)-2,5,8-trihydroxy-3-(4-hydroxy-3-methylbut-2-en-1-yl)-1,4-naphthoquinone (2), were isolated from the 90% MeOH extract of the leaves of T. grandis. Compounds 1 and 2 exhibited significant antifungal activity when combined with fluconazole, against fluconazole-resistant Candida albicans with MIC₅₀ values of 3.28 and 5.32 μg/mL, respectively. Conclusion Compounds 1 and 2, named tectonagrandiones A and B, were two new naphthoquinones, and the naphthoquinones might be the active antifungal constituents from T. grandis.

Objective To isolate structurally defined bioactive oligosaccharide fractions from Prunella vulgaris (PV) polysaccharides and evaluate their in vitro antioxidant activity and intestinal microbiota-modulating effects. Methods Total polysaccharides were extracted via water extraction and alcohol precipitation, followed by deproteinization using the Sevag method. Partial acid hydrolysis was performed under two conditions (80 ℃ and 100 ℃, 0.5 mol/L trifluoroacetic acid). The hydrolysates were fractionated and purified by hydrophilic interaction chromatography (HILIC). Structural characterization of the dominant components in each fraction was performed using HILIC/IM-QTOF-HDMSE, UHPLC-UV and 1D/2D-NMR (1H/13C-NMR, COSY, HSQC, HMBC). Antioxidant activity of three oligosaccharide components was assessed via a DPPH free radical scavenging assay, while their modulatory effects on gut microbial abundance were evaluated using an in vitro anaerobic co-culture model of mouse fecal microbiota coupled with real-time quantitative PCR. Results Three oligosaccharide fractions with degrees of polymerization (DP) of 2, 3, and 5 were obtained, namely DP2 (yield: 5.60%), DP3 (1.19%), and DP5 (1.70%). The dominant structural features of three fractions were elucidated as follows: DP2 is mainly composed of a disaccharide composed of 3-O-methyl-α-glucuronic acid linked to xylose, with α/β anomerism at the xylose terminus; DP3 mainly contains a trisaccharide consisting of xylose-arabinose-uronic acid, terminating in either 4-O-methyl-β-glucuronic acid or α-galacturonic acid; DP5 is primarily a linear pentasaccharide starting with 2-O-methyl-α-glucuronic acid, linked through three xylose units, and ending with 3-O-methyl-glucuronic acid existing in both α- and β-forms. Antioxidant assessment revealed that all three fractions exhibited significant DPPH radical-scavenging activity, with DP5 being the most potent (IC₅₀ = 0.36 μg/mL). At 0.1 mg/mL, both DP3 and DP5 showed scavenging rates exceeding 84%, comparable to the positive control VC (IC₅₀ = 0.11 μg/mL, P > 0.05). Microbiota analysis indicated that all three fractions modulated the intestinal flora: DP2 significantly promoted the growth of the probiotic genus Lactobacillus; DP3 increased the abundance of the beneficial phylum Bacteroidetes while suppressing the potentially pathogenic phylum Actinobacteria; DP5 primarily exerted inhibitory effects on Actinobacteria. Conclusion This study successfully isolated three oligosaccharide fractions (DP2, DP3, and DP5) from the PV polysaccharides and characterized their predominant structural features, revealing the antioxidant and gut microbiota-regulating activities.

Objective To systematically characterize the metabolites of polygala oligosaccharide esters (PROEs) in rats, thereby providing evidence for elucidating the pharmacodynamic material basis of Polygala tenuifolia. Method Plasma samples from rats after oral administration of PROEs were analyzed using UPLC-QTOF-MS/MS. Feature-based molecular networking (FBMN) was employed to visually cluster and screen for potential metabolites. The identities of predicted metabolites were further confirmed by comparison with reference standards prepared via in vitro enzymatic incubation and chemical isomerization synthesis. Results FBMN effectively clustered structurally similar metabolites, such as glucuronidated and sulfated products, from the mass spectrometry data. A total of 59 metabolites were identified in rat plasma, including 14 free phenolic acids, 21 sulfated organic acids, 17 glucuronidated organic acids, and seven other compounds. No intact PROEs were detected. The metabolic pathway analysis revealed that PROEs were rapidly hydrolyzed in vivo to generate various phenolic acids (e.g., sinapic acid, ferulic acid, syringic acid), which subsequently underwent extensive phase I metabolism (reduction, demethylation, dehydroxylation) and predominantly phase II metabolism (sulfation and glucuronidation). Conclusion Following oral administration, PROEs enter the systemic circulation primarily as hydrolyzed organic acids and their extensive phase II conjugates. This study demonstrates that the combination of UPLC-Qtof-MS/MS and FBMN provides an efficient and comprehensive strategy for identifying the in vivo metabolites of PROEs. This approach offers a basis for revealing the active constituents of P. tenuifolia and serves as an effective method for elucidating the metabolic profiles of traditional Chinese medicines.

Objective To optimize the preparation process of nitidine chloride liposomes (NC-Lips), characterize their physicochemical properties, and evaluate their in vitro anti-cholangiocarcinoma activity. Methods NC-Lips were prepared by the thin-film dispersion method. Three key formulation variables, namely DSPE-mPEG₂₀₀₀ dosage, drug-to-lipid ratio, and phospholipid-to-cholesterol ratio, were selected. The formulation was optimized via single-factor experiments combined with Box-Behnken design-response surface methodology (BBD-RSM), with encapsulation efficiency, drug loading, PDI, and ζ potential as the evaluation indices. The drug content was determined by HPLC. The physicochemical properties of NC-Lips were characterized using a particle size analyzer and transmission electron microscopy. The in vitro drug release profile was investigated by the dialysis bag method. CCK-8 assay, flow cytometry, and cell cycle analysis were employed to evaluate the proliferation inhibition, apoptosis induction, and cell cycle arrest effects of NC-Lips on cholangiocarcinoma TFK-1 cells. Results The optimal formulation was as follows: DSPE-mPEG₂₀₀₀ dosage of 34.79 mg, drug-to-lipid ratio of 1:15.15, and phospholipid-to-cholesterol ratio of 17.88. The prepared NC-Lips exhibited typical liposomal vesicular morphology, with an average particle size of (77.67 ± 0.75) nm, PDI of 0.212 ± 0.01, ζ potential of (&8722;11.43 ± 0.12) mV, encapsulation efficiency of (85.96 ± 0.61)%, and drug loading of (4.37 ± 0.03)%. Among the in vitro drug release models, the Ritger-Peppas model showed the highest goodness of fit, indicating that NC-Lips had obvious sustained-release characteristics. The liposomes remained stable during 30 d storage at 4 ℃, with a drug leakage percentage of < 30%. In vitro cell experiments demonstrated that NC-Lips exerted significantly better effects on proliferation inhibition, apoptosis induction, and G₂/M phase arrest in TFK-1 cells than free nitidine chloride (P < 0.01). At a concentration of 12 μmol/L, the cell viability was only (8.17 ± 1.90)% after 48 h of treatment; at the same concentration, the apoptosis rate reached (28.90 ± 2.38)% at 24 h, and the proportion of cells in the G₂/M phase increased to (66.23 ± 3.31)%. Conclusion A stable and optimized NC-Lips formulation with excellent physicochemical properties was successfully prepared. NC-Lips exerted excellent in vitro anti-cholangiocarcinoma activity by inducing apoptosis and G₂/M phase arrest. These findings provide an experimental basis for formulation optimization and subsequent mechanistic and in vivo studies of nitidine chloride.

Objective To prepare trifolin-phospholipids complex/chitosan nanoparticles (Tri-PC/CS-NPs), and investigate its in vivo pharmacokinetic behavior and protective effect on acute liver injury. Methods Trifolin-phospholipids complex (Tri-PC) was prepared by solvent evaporation method. Single factor experiments combined Box-Behnken design-response surface methodology (BBD-RSM) were used to optimize the prescriptions of Tri-PC/CS-NPs. Transmission electron microscope (TEM) was employed to observe microscopic appearance of Tri-PC/CS-NPs, X-ray powder diffraction (XRPD) was used to analyze crystal form of Tri-PC/CS-NPs. Saturated solubility, oil/water partition coefficient and drug release behavior in simulated gastrointestinal fluids of trifolin, Tri-PC and Tri-PC/CS-NPs were compared. SD rats were administered intragastrically with trifolin, Tri-PC and Tri-PC/CS-NPs, respectively. Blood drug concentration was determined, and the main pharmacokinetic parameters were calculated. The model of acute liver injury was established, and the protective effect of trifolin, Tri-PC, Tri-PC/CS-NPs on acute liver injury were compared. Results Optimal formulations of Tri-PC/CS-NPs: the volume of water phase was 20.50 mL, the mass ratio of Tri-PC to chitosan was 4.15:1, and the mass fraction of Poloxamer 188 was 0.10%. Envelopment efficiency, drug loading, particle size and ζ potential were (84.80 ± 1.21)%, (15.56 ± 0.10)%, (197.74 ± 5.58) nm and (32.69 ± 1.14) mV, respectively. The appearance of Tri-PC/CS-NPs were spherical. The state of trifolin changed into amorphous form in Tri-PC and Tri-PC/CS-NPs. Tri-PC and Tri-PC/CS-NPs significantly enhanced the saturated solubility and oil/water partition coefficient of trifolin (P < 0.01). Cumulative release rate of Tri-PC and Tri-PC/CS-NPs were 56.68% and 88.45% in 18 h, respectively. The drug release process of Tri-PC/CS-NPs conformed to the Weibull model. Compared with trifolin, the relative bioavailability of Tri-PC and Tri-PC/CS-NPs was enhanced to 2.41-fold and 5.36-fold, respectively. Tri-PC and Tri-PC/CS-NPs effectively enhanced the protective effect of trifolin against acute liver injury, and the protective effect of Tri-PC/CS-NPs was superior to that of Tri-PC. Conclusion Tri-PC/CS-NPs changed the pharmacokinetic behavior of trifolin in vivo, effectively promoted the absorption of trifolin, and enhanced the protective effect of trifolin on acute liver injury.

Objective To optimize the processing technology of Shanzhatan (Crataegi Fructus Charcoal, CFC), evaluate it objectively by electronic sensory technology, and verify its antidiarrheal efficacy. Methods TG-DTG thermal analysis was used to investigate the pyrolysis behavior of total flavonoids, total organic acids, polysaccharides, and tannins in hawthorn, and to determine the processing temperature range. With tannin content as the key index, frying temperature and time were selected as process variables, and the technology was optimized through single-factor experiments combined with central combination design-response surface methodology (CCD-RSM). An electronic eye, electronic nose, and electronic tongue were applied to quantitatively evaluate the color, odor, and taste profiles of hawthorn before and after processing. A Fanxieye (Sennae Folium)-induced mouse diarrhea model was established to validate the antidiarrheal efficacy of the CFC prepared under the optimized process, with body weight change, diarrhea index, gastrointestinal hormone (gastrin, VIP) levels, small intestinal propulsion rate, and gastric residual rate as indicators. Results Thermal analysis indicated that the optimal processing temperature range was 307.4—332.0 ℃. The optimal parameters were frying temperature 311 ℃ and frying time 3.64 min (218 s). Electronic sensory analysis showed obvious differences in lightness (L), red-green degree (a), yellow-blue degree (b), total color difference (E_ab), odor, and taste characteristics between raw and processed hawthorn. The e-nose response signals were positively correlated with e-eye chromaticity values. Compared with the model group, the CFC group attenuated the trend of body weight loss (P < 0.05), reduced diarrhea index (P < 0.05), reversed serum gastrin and vasoactive intestinal peptide (VIP) levels (P < 0.05), increased gastric residual rate (P < 0.01), and a numerically lower but statistically non-significant small intestinal propulsion rate. Conclusion The processing technology was optimized by thermal analysis and response surface methodology. Electronic sensory technology enabled objective quantification of sensory attributes, and pharmacodynamic validation further confirmed the rationality of the process. This study provides a methodological reference for parameterizing fire intensity and duration in the preparation process of hawthorn charcoal and the construction of its quality standards.

Objective To investigate the change patterns of flavonoid and saponin contents in Hongqi (Hedysari Radix, HR) before and after honey-processing, and to provide a scientific basis for quality control of the honey-processing technology and its processing mechanism. Methods UV-Vis spectrophotometry was employed to determine the contents of total flavonoids and total saponins in 10 batches of HR and Zhihongqi (Hedysari Radix Praeparata Cum Melle, HRPCM). HPLC was used to establish flavonoid-based and saponin-based fingerprints, and similarity evaluations were conducted. The contents of six marker components calycosin-7-O-β-D-glucoside, ononin, calycosin, formononetin, astragaloside IV, and isoastragaloside II were determined by HPLC. Results After honey-processing of 10 batches of HR, the total flavonoid contents were significantly decreased (P < 0.01), with an average decrease of 25.8%; the total saponin contents were significantly increased (P < 0.01), with an average increase of 37.93%. Similarity evaluation of fingerprints indicated that honey-processing did not alter the basic chemical profile of HR. HPLC content determination showed that after honey-processing, formononetin content was significantly increased in 8 out of 10 batches (P < 0.05, 0.01), with an average increase of 30.49%; astragaloside IV and isoastragaloside II contents were significantly increased in 8 and 7 batches, respectively (P < 0.05, 0.01), with average increases of 34.79% and 26.22%; calycosin-7-O-β-D-glucoside and ononin contents were mainly decreased; calycosin content was mainly increased. Conclusion Honey-processing of Hedysari Radix can alter the active flavonoid and saponin components, thereby adjusting the structure of its pharmacodynamic material basis.

Objective Lappaconitine-self-micellizing solid dispersion sustained-release tablets (Lap-SMSD-SRT) was prepared, and investigated its pharmacokinetics and toxicokinetics in vivo. Methods Lap-SMSD powder was prepared by solvent evaporation method, and its basic physicochemical properties were also studied. The influence of hydroxypropyl methyl cellulose (HPMC) K4M dosage, carboxymethyl starch sodium (CMS-Na) dosage and lactose dosage on the drug release behavior of Lap-SMSD-SRT were investigated by single factor experiments. Composite score of cumulative release rate at 2, 6, 12 h was used as response value, Box-Behnken design-response surface method (BBD-RSM) was employed to optimize formulation of Lap-SMSD-SRT. The release behavior of lappaconitine hydrobromide tablets, lappaconitine tablets and Lap-SMSD-SRT were compared. Six beagle dogs were used to study the pharmacokinetic behavior by a three-period crossover trial, and the relative bioavailability of Lap-SMSD-SRT was calculated. Three level of low, medium, high (0.75, 1.50, 2.25 mg/kg) dose groups of Lap-SMSD-SRT were set up, and compared the plasma concentration of lappaconitine after the first and last administration with a 4-week dosing period and a 4-week recovery period. Then the toxicokinetic parameters were calculated. The living condition of Beagle dogs was observed, and the hematology and biochemical indexes were also examined. Results The dosage ratio of polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer (Soluplus) to lappaconitine in Lap-SMSD powder was 5:1, and quickly became into micelles with particle size of (29.44 ± 2.01) nm when dispersed in water. Optimal formulation of Lap-SMSD-SRT: HPMC K4M dosage was 19.09%, CMS-Na dosage was 15.59%, and lactose dosage was 22.63%. The cumulative release rate of Lap-SMSD-SRT at 2, 6, 12 h were 26.50%, 61.67% and 93.67%, respectively. Pharmacokinetic results showed that the peak time (t_max) of Lap-SMSD-SRT was delayed to (6.20 ± 1.61) h, the half-life (t_1/2) was prolonged to (9.13 ± 2.64) h, the peak concentration (C_max) was enhanced to (123.94 ± 46.77) ng/mL, and the relative bioavailability was enhanced to 5.21-fold. No significant accumulation of lappaconitine was observed in Beagle dogs at each dose of Lap-SMSD-SRT. There might be slight effect on the blood system and liver in the medium-dose and high-dose groups of Lap-SMSD-SRT, but these symptoms disappeared during the 4-week recovery period. Conclusion The preparation process of Lap-SMSD-SRT was reproducible, and the formulation exhibited obvious sustained-release characteristics, significantly enhanced the in vivo absorption of lappaconitine, and showed good safety.

Objective To enhance the hair follicle-targeted delivery efficiency of cedrol, a cedrol nanosuspension (Ce-NS) was prepared. The formulation and process parameters were optimized, the physicochemical properties were characterized, and its therapeutic efficacy in the treatment of androgenetic alopecia (AGA) was evaluated. Methods Ce-NS was prepared using a solvent-antisolvent precipitation method. Based on single-factor experiments, particle size and polydispersity index (PDI) were selected as evaluation parameters to optimize the formulation and process variables using the Box-Behnken design-response surface methodology (BBD-RSM). The physicochemical characteristics and storage stability of the formulation were determined by dynamic light scattering (DLS). In vitro transdermal performance was evaluated using a Franz diffusion cell system. Morphological characteristics were examined by scanning electron microscopy (SEM) to assess the accumulation of nanocrystals within hair follicles. An AGA model was established in C57BL/6 mice. Hematoxylin-eosin (HE) staining and immunohistochemical analysis of dorsal skin tissues were performed to evaluate the in vivo hair growth-promoting efficacy. Results The optimal formulation parameters were as follows: cedrol mass concentration 7.5 mg/mL, sodium dodecyl sulfate (SDS) 170 mg, hydroxypropyl methylcellulose (HPMC) 130 mg, and a polyethylene glycol (PEG) 400-to-water phase ratio of 1∶12. The process parameters included a dropping rate of 20 mL/min, stirring speed of 10 000 r/min, and shearing time of 5 min. The prepared Ce-NS exhibited a mean particle size of (434.47 ± 9.90) nm, a PDI of 0.232 ± 0.005, and a ζ potential of (&8722;15.20 ± 1.15) mV. The experimental values were in good agreement with the predicted values of the model. Ce-NS remained stable for 90 days at room temperature. In vitro transdermal studies showed that the skin retention of Ce-NS within 24 h reached (74.38 ± 3.90) μg/cm2, which was 4.1-fold higher than that of the raw drug group, while no cedrol was detected in the receptor medium. SEM observation of skin sections confirmed the accumulation of cedrol nanocrystals within hair follicles. Pharmacodynamic evaluation demonstrated that, compared with the model group, Ce-NS significantly promoted hair regeneration in AGA model mice, increased hair follicle density and hair length, decreased androgen receptor (AR) expression in dorsal skin tissue, and upregulated the expression of Ki67 and β-catenin. Conclusion Ce-NS was successfully prepared using a green preparation process and exhibited stable physicochemical properties. The formulation significantly enhanced the skin retention and follicular targeting of cedrol, demonstrating promising therapeutic potential for androgenetic alopecia. The underlying mechanism may be associated with the downregulation of androgen signaling and activation of the Wnt/β-catenin pathway.

Objective To investigate the mechanism of Buyang Huanwu Decoction (补阳还五汤) in ameliorating diabetes cognitive impairment (DCI) based on nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway. Methods A rat model of DCI was established by feeding a high-fat and high-sugar diet combined with low-dose streptozotocin (STZ) injection. The successfully modeled rats were randomly divided into model group, Buyang Huanwu Decoction low-, high-dose (7.125, 28.500 g/kg) groups and metformin (0.2 g/kg) group, control group was set up separately. After four weeks of administration, the cognitive and memory abilities of rats were evaluated by Morris water maze. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of hippocampal tissue in rats. The activities of superoxide dismutase (SOD), catalase (CAT) and levels of glutathione (GSH), malondialdehyde (MDA) in serum and ferrous ions (Fe2+) in brain tissue of rats were detected by biochemical methods. Immunohistochemistry was used to observe the expressions of Nrf2 and GPX4 proteins in CA1 region of hippocampus in rats. Western blotting was used to detect the expressions of Kelch-like ECH-associated protein 1 (Keap1), Nrf2, heme oxygenase-1 (HO-1), solute carrier family 7 member 11 (SLC7A11), GPX4, transferrin receptor (TFR), ferroportin 1 (FPN1) and ferritin heavy chain (FTH) proteins in hippocampal tissue of rats. Human neuroblastoma cells SHSY-5Y were cultured in vitro and a damage model was established by inducing with 160 mmol/L glucose. Control group, model group, Buyang Huanwu Decoction low-, high-dose drug containing serum groups and Buyang Huanwu Decoction high-dose drug containing serum + Nrf2 inhibitor ML385 (10 μmol/L) group were set up. Biochemical methods were used to detect the activities of SOD, CAT and levels of GSH, MDA in cells. JC-1 fluorescent probe was used to detect changes in mitochondrial membrane potential in cells. Western blotting was used to detect the expressions of Keap1, Nrf2, HO-1, SLC7A11 and GPX4 proteins in cells. Results The results of in vivo experiments showed that Buyang Huanwu Decoction could delay weight loss of DCI rats (P < 0.05), reduce fasting blood glucose (P < 0.05, 0.01), shorten the escape latency (P < 0.05, 0.01), increase the distance moved, dwell time and number of crossings in target quadrant (P < 0.05, 0.01), alleviate the damage of hippocampal neurons, reduce the levels of MDA in serum and Fe2+ in brain tissue (P < 0.05, 0.01), increase the activities of SOD, CAT and levels of GSH in serum (P < 0.05, 0.01), downregulate the expressions of TFR and Keap1 proteins in hippocampus (P < 0.05, 0.01), upregulate the expressions of Nrf2, HO-1, SLC7A11, GPX4, FTH and FPN1 proteins (P < 0.05, 0.01). The results of in vitro experiments showed that Buyang Huanwu Decoction drug-containing serum could significantly increase the survival rate of SHSY-5Y cells induced by high glucose (P < 0.01), reduce MDA level (P < 0.05), increase the activities of SOD, CAT and level of GSH (P < 0.05, 0.01), increase the mitochondrial membrane potential, downregulate the expression of Keap1 protein (P < 0.01), and upregulate the expressions of Nrf2, HO-1, SLC7A11 and GPX4 proteins (P < 0.05, 0.01). Compared with Buyang Huanwu Decoction high-dose drug containing serum group, the administration of ML385 significantly reversed the changes in the above indicators (P < 0.05, 0.01). Conclusion Buyang Huanwu Decoction could improve cognitive dysfunction, alleviate oxidative stress damage, and improve iron metabolism disorders in DCI rats. Its effects may be related to the activation of Nrf2/GPX4 signaling pathway and the regulation of ferroptosis processes.