Objective To explore the key enzyme genes of the pentacyclic triterpene esters biosynthesis pathway in Balanophora subcupularis, which lays a certain research idea and theoretical foundation for its biosynthesis mechanism and the development and utilization of medicinal plant resources in the genus Balanophora. Methods Transcriptome sequencing and analysis was performed on the three tissues of the flowerstalk, rhizome and umbel of B. subcupularis, then searching the candidate enzyme genes involved in triterpene ester biosynthesis by PfamScan, constructing the yeast expression vector of BsubOSCs gene which was introduced into the Saccharomyces cerevisiae lanosterol synthase gene（erg7）-deficient mutant strain GIL77 for functional characterization. Results A total of 87 candidate genes involved in pentacyclic triterpene esters biosynthesis were identified from the transcriptome of B. subcupularis, most of them were highly expressed in umbel, suggesting that umbel may be the main site of synthesis of pentacyclic triterpene esters. The enzyme function of two BsubOSC genes were characterized, which was found that BsubOSC2 could catalyze 2, 3-oxidosqualene to form β-amyrin, but no catalytic product of BsubOSC1 was detected. Conclusion Based on transcriptome analysis, the key enzyme genes involved in pentacyclic triterpene esters biosynthesis were found in B. subcupularis, and BsubOSC2 is a β-amyrin synthetase gene, and is one of the key enzyme genes for the biosynthesis of balanophorin A. This study provides gene resources for further analysis of the biosynthesis pathway of triterpene ester compounds such as balanophorins, and facilitate the research on molecular breeding of B. subcupularis.

Objective To investigate the secondary metabolites produced by marine-derived fungi Talaromyces sp. 13-z-02 and their anti-inflammatory activities were studied. Methods The isolation and purification of compounds were performed using silica gel and ODS column chromatography, semi-preparative HPLC. And their structures were elucidated by MS and NMR analyses. The isolated compounds were evaluated for their anti-inflammatory activity in zebrafish. Results One new isochromane and eleven known compounds were isolated and identified as 8-hydroxy-6-methoxy-7-methylisochromane (1) from the fermentation broth of this strain, bacillisporin B (2), macrosporusone D (3), bacillisporin F (4), 1-epi-bacillisporin F (5), macrosporusone E and 1-epi-macrosporusone E (6), bacillisporin A (7), xenoclauxin (8), Sch 725680 (9), aculene C (10), 6-ethyl-2-hydroxy-4-methoxy-3-methylbenzaldehyde (11), 6-ethyl-2,4-dihydroxy-3-methylbenzaldehyde (12). Compounds 1 and 12 significantly reduced the migration of immune cells with the same concentration of 20 μmol/L and exhibited anti-inflammatory activity. Conclusion Compound 1 is a new isochromane, designated as talaroslisochromane. Compounds 1 and 12 showed anti-inflammatory activity.

Objective Taking the glycosyltransferase in Platycodon grandiflorus as the research object, the bioinformatics analysis, gene cloning and recombinant plasmid construction, protein expression and purification, catalytic function validation, enzymatic properties, and substrate promiscuity of the glycosyltransferase gene PgUGT72B21 were investigated. Methods The key glycosyltransferase PgUGT72B21 was screened from the transcriptome data of P. grandiflorus and subsequently cloned. A prokaryotic expression vector, pET-28a-PgUGT72B21, was constructed using gene recombination technology and transformed into Escherichia coli BL21（DE3） competent cells. Protein purification was performed according to the instructions of the His-tagged protein purification kit, and protein expression was detected by SDS-PAGE electrophoresis. The enzymatic reaction products were analyzed using HPLC and LCMS systems. Results P. grandiflorus glycosyltransferase gene Pg UGT72B21 obtained through cloning had an open reading frame（ORF） of 1 407 bp, encoding 468 amino acid residues with a relative molecular mass of approximately 51 000. A highly conserved plant secondary product glycosyltransferase（PSPG） motif was identified at the C-terminus. Phylogenetic analysis indicated that this glycosyltransferase belongs to the UGT72 family. In vitro enzymatic assays demonstrated that PgUGT72B21 could catalyze the glycosylation of the C3 hydroxyl group of quercetin to produce isoquercitrin. After heterologous expression and purification, the recombinant protein was obtained, and its enzymatic properties were analyzed. The optimal pH and temperature for the catalytic reaction were determined to be 6.0 and 60 ℃, respectively, with the highest substrate conversion rate achieved after 2 h of reaction. The kinetic parameters for quercetin catalysis were K_m = 390.10 μmol/L and kcat = 11.10/min. Further substrate promiscuity studies revealed that PgUGT72B21 could not only catalyze flavonols such as quercetin but also flavonoids such as apigenin. Conclusion The newly discovered glycosyltransferase PgUGT72B21 holds significant importance for enriching the glycosylation tool enzyme library and provides a foundation for further elucidating the glycosylation process of flavonoid glycosides in P. grandiflorus.

Objective To prepare sinomenine nanoemulsion (Sin-NE) co-stabilized by Bletilla striata polysaccharide and glycyrrhizic acid, systematically characterize its properties, investigate its stability and drug release behavior, and further evaluate its anti-inflammatory effects through in vitro experiments. Methods The Sin-NE was prepared using the ultrasonic emulsification method. Its morphology, particle size, polydispersity index (PDI), and ζ potential were observed and determined. Optimal formulation parameters were screened via single-factor and orthogonal experiments, followed by formulation validation. pH value, turbidity, viscosity, encapsulation efficiency, and drug loading capacity were measured. Stability was investigated under centrifugation, storage, dilution, and heating conditions was evaluated, and in vitro drug release behavior was investigated. A RAW264.7 cell inflammation model was established to validate the effects of the Sin-NE on nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Results The optimized formulation comprised 3% Coicis Semen oil, 0.5% combined emulsifiers, a 1∶9 mass ratio of Bletilla striata polysaccharide to glycyrrhizic acid, 10 min of ultrasonic treatment, and 325 W ultrasonic power. The resulting Sin-NE exhibited an average particle size of (145.30 ±2.35) nm, ζ potential of (-39.51 ±0.43) mV, PDI of 0.095 ±0.022, and pH value of 3.990 ±0.033. Encapsulation efficiency and drug loading capacity reached 88.78% and 26.14 mg/g, respectively. The nanoemulsion demonstrated excellent stability under centrifugation, storage, dilution, and heating conditions and displayed sustained-release characteristics. In vitro, it significantly inhibited NO release and effectively reduced IL-6 and TNF-α levels in inflamed cells. Conclusion The Sin-NE significantly enhances sinomenine’s bioavailability and anti-inflammatory activity, providing experimental evidence for developing novel traditional Chinese medicine nanoemulsion formulations.

Objective To research the chemical constituents from the fruits of Lycium barbarum. Methods Separation and purification were performed using silica gel, D-101 macroporous resin, MCI, Sephadex LH-20, and semi-preparative HPLC. The structures of the compounds were identified based on spectroscopic data and physicochemical properties. The α-glucosidase inhibitory activity of the selected compounds was evaluated using 4-nitrophenol-α-D-glucopyranoside (PNPG) assay. Results A total of 15 compounds were isolated from the fruits of L. barbarum, which were identified as methyl (2R)-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]-4-methylpentanoate (1), 5-epi-acortatarin A (2), 5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde (3), 4-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoic acid (4), (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(4-hydroxyphenyl) propanoate (5), 4-[formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoate (6), 3-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] pentanedioic acid (7), N-trans-feruloyl-3'-O-methyldopamine (8), N-trans-feruloyl-3',4'-dihydroxyphenylethylamine (9), N-cis-p-coumaroyl tyramine (10), N-cis-feruloyl tyramine (11), bungeanoline E (12), N-malonyl-tryptophan (13), 3-hydroxy-4-ethyl ketone pyridine (14), cartorimine (15). Conclusion Compound 1 is a new pyrrole alkaloid, named lycipyrrole A. Compound 2 is a new natural product. Compounds 4, 7 and 12—15 were isolated from L. barbarum for the first time, while compounds 12, 13 and 15 were isolated from this genus for the first time. Notably, compound 9 exhibited significant inhibitory activity against α-glucosidase.

Objective To investigate the subcellular distribution patterns of the combination of cantharidin (CTD) and baicalin (BA) based on cell pharmacokinetics and elucidate the synergistic mechanism against hepatocellular carcinoma. Methods Using human hepatoma HepG2 cells as the model, the dynamic concentrations of the drugs in whole cells and organelles (nucleus, mitochondria, endoplasmic reticulum, and lysosomes) over 12 h after administration of single drugs (CTD 6 μg/mL, BA 30 μg/mL) and their combination (CTD 6 μg/mL + BA 30 μg/mL) were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Pharmacokinetic parameters were calculated using a non-compartmental model with Phoenix WinNonlin software. Results At the whole-cell level, the combination increased the intracellular area under the curve (AUC_0—t) of CTD by 48.9% and decreased its clearance (P < 0.05), but did not significantly affect the pharmacokinetics of BA. At the subcellular level, the combination significantly increased the AUC_0—t of CTD and BA in nucleus, lysosome, mitochondria and endoplasmic reticulum by 93.5%, 46.4%, 38.3%, 52.3%, and 68.4%, 40.0%, 41.0%, and 46.7%, respectively (P < 0.05, 0.01). Furthermore, the time to reach peak concentration (t_max) for both drugs in mitochondria was earlier in the combination group, and the mean residence time (MRT_0—t) of CTD in the endoplasmic reticulum was significantly prolonged (P < 0.01), indicating a spatiotemporally synergistic drug delivery optimization. Conclusions The combination of CTD and BA could synergistically optimize the distribution of drugs in key subcellular structures such as nucleus, mitochondria, endoplasmic reticulum and lysosomes, and may enhance the anti-hepatocellular carcinoma effect by inducing DNA damage, accelerating mitochondria-mediated apoptosis and promoting endoplasmic reticulum stress, which provides a theoretical basis for the design of traditional Chinese medicine combinations based on organelle targeting.

Objective To analyze the correlation between the content changes of characteristic components and its antithrombotic effect in different proportions of the Danshen (Salviae Miltiorrhizae Radix et Rhizoma, SMRR)-Honghua (Carthami Flos, CF) herb pair, and optimize the compatibility ratio of SMRR and CF for promoting blood circulation and removing blood stasis. Methods Fingerprint profiles and quantitative determination methods for characteristic components were established for SMRR-CF herb pair at different ratios (1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1). The HPLC combined with a comprehensive scoring method was used to determine the optimal extraction process of the SMRR-CF herb pair. The anti-thrombotic activity of the SMRR-CF extracts at different ratios was measured using zebrafish as a model organism. The principal component analysis (PCA) method was used to integrate the content of characteristic components and the intensity of antithrombotic activity, and the optimal ratio of SMRR and CF was comprehensively evaluated from two aspects of composition and efficacy. Results The fingerprint profiles of the SMRR-CF herb pair in nine different ratios collectively identified 17 common peaks, among which nine characteristic components were characterized. These include hydroxysafflor yellow A (peak 2), luteoloside (peak 3), kaempferol-3-O-rutinoside (peak 4), quercitrin (peak 5), salvianolic acid B (peak 8), dihydrotanshinone I (peak 12), cryptotanshinone (peak 14), tanshinone I (peak 15), and tanshinone II_A (peak 17). The optimal extraction process was determined to be maceration with 10 times the amount of 70% ethanol for 1 h by reflux. The content determination showed that the characteristic components changed significantly after the compatibility of SMRR and CF compared with the single herb. When the SMRR-CF ratio was 1:5, decoction yielded the highest concentrations of salvianolic acids and tanshinones derived from SMRR. Conversely, ratios of 3:1, 4:1 and 5:1 produced the greatest amounts of hydroxysafflor yellow A, luteoloside and kaempferol-3-O-rutinoside from CF, indicating that co-decoction facilitates the extraction of bioactive constituents from both herbs. Pharmacodynamic evaluation revealed that the 3:1 ratio exhibited the strongest antithrombotic activity, achieving a thrombosis inhibition rate of 43.7%. Principal-component analysis of the “composition-effect” dataset ranked the comprehensive indices (F-values) as 3:1 > 4:1 > 5:1 > 2:1 > 1:1 > 1:5 > 1:4 > 1:2 > 1:3, confirming that the 3:1 ratio represents the optimal compatibility of the SMRR-CF pair. Conclusion In this study, chemical profiling and in vivo pharmacodynamic evaluation were integrated to elucidate the quantitative relationship between the dynamic changes of characteristic constituents and the antithrombotic efficacy of the SMRR-CF herb pair across different compatibility ratios. An optimal ratio was identified through a “composition-effect”-based comprehensive assessment. These findings not only establish a foundation for subsequent dose-effect investigations of this classic combination, but also offer a rational reference for determining prescription design in latent prescriptions.

Herbal textual research, through systematic collation and analysis of historical texts, clarifies the nomenclature and historical applications of medicinal materials, and serves as a key factor in ensuring their quality and clinical efficacy. This study primarily conducted a comprehensive textual investigation into the nomenclature, botanical origin, efficacy, harvesting and collection, processing and geo-authentic producing areas of Chaihu (Bupleuri Radix) as recorded in historical herbal literature. Simultaneously, it systematically reviewed modern ecological research on the quality formation of geo-authentic Bupleuri Radix. The study identified three major shifts in the documented efficacy of Bupleuri Radix, ultimately establishing its current recognized functions of “dispelling pathogenic factors to relieve fever, soothing the liver to alleviate depression, and elevating yang qi” through three major revolutions. Furthermore, the geo-authentic producing areas of Bupleuri Radix have remained relatively stable historically, with their distribution centered on the adjoining region of Shaanxi and Henan provinces and gradually expanding outward along the watersheds of the Yellow River and the Wei River. Concurrently, modern research, from the perspective of interactions between ecological factors and microorganisms, elucidates the “stress effect” and the multidimensional regulatory network underlying the quality formation of Bupleuri Radix, thereby providing a theoretical basis for understanding the causes of its geo-authenticity. This study interprets the scientific connotation of dao-di herbs through the cross-verification of “traditional textual research” and “scientific inquiry”. It identifies ecological factors—ranging from water, heat, and light to microorganisms—as the core elements driving the formation ofthe geo-authentic quality of Bupleuri Radix. This provides a theoretical foundation for enhancing the quality of Bupleuri Radix, promoting in-depth resource development, and guiding rational clinical application.

Objective To explore the mechanism by which Astragalus polysaccharides (APS) improves the consumption of adipose tissue in cancer cachexia (CC) using lipidomics and transcriptomics. Methods A random number table was used to divide 50 male mice into control group, model group, APS low-, medium-and high-dose (200, 400, 800 mg/kg) groups, with 10 mice in each group. CC model was constructed using abdominal sc CT-26 colorectal cancer cells. The treatment group was given 0.2 mL APS solution by ig daily, while the control group and model group were given an equal volume of physiological saline by ig for four consecutive weeks. The diameter of epididymal adipocytes was evaluated using hematoxylin-eosin (HE) staining; ELISA method was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclic adenosine monophosphate (cAMP) in epididymal adipose tissue and level of free fatty acids (FFA) in plasma. After effectiveness and significance verification, transcriptome and lipidomics sequencing were performed on control group, model group and APS high-dose group to screen differentially expressed genes (DEGs) and differentially expressed lipids (DELs), and enrichment analysis was performed; Western blotting was used to verify the expressions of relevant proteins. Results High-dose APS significantly alleviated inflammatory infiltration in epididymal adipose tissue and reversed adipocyte atrophy (P < 0.01), significantly reduced the levels of TNF-α, IL-6, cAMP in adipose tissue and level of FFA in plasma (P < 0.01, 0.001), indicating that high-dose APS inhibits inflammation and lipolysis reactions in adipocytes. The lipidomics results showed that high-dose APS intervention reduced the expression levels of 66 DELs, including ceramides and their modifications, triacylglycerols, etc. Metabolic pathway enrichment analysis showed that these DELs mainly involved glycerophospholipid metabolism and sphingolipid metabolism pathways. The transcriptomic results showed that the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) pathway was repeatedly enriched in DEGs, indicating that this pathway may be the core pathway for APS to function. In addition, the expressions of 18 DEGs was significantly down-regulated by APS, and the gene interaction network diagram showed that these genes had complex interactions with Akt. Western blotting results confirmed that APS intervention reversed the expression levels of Tribbles homologous protein 3 (TRIB3), phosphorylated Akt (p-Akt), phosphodiesterase 3B (PDE3B), phosphorylated protein kinase A (p-PKA), phosphorylated hormone sensitive lipase (p-HSL) and adipose triglyceride lipase (ATGL) (P < 0.05, 0.01, 0.001). Conclusion APS may alleviate inflammation in adipose tissue, reduce the synthesis of sphingolipids such as ceramides, regulate TRIB3-Akt axis, restore insulin resistance to lipolysis, promote lipid signaling, and improve the consumption of CC adipose tissue.

Objective To investigate the effect of Dendrobium officinale polysaccharide (DOP) on prediabetes (PDM) and explore its mechanism in improving PDMs through the gut short-chain fatty acids (SCFAs)-G protein-coupled receptor 43 (GPR43)-glucagon-like peptide 1 (GLP1) pathway. Methods SD Rats were randomly divided into control group, model group and DOP low-, high-dose (0.1, 0.2 g/kg) groups, with 10 rats in each group. Except for the control group, the remaining rats were fed a high-sugar and high-fat diet for six weeks to establish a PDM model. During the modeling period, the corresponding drugs were administered intravenously, and the control group and model group were given distilled water of equal volume. Changes in general physiological indicators, glucose/lipid levels, glucose regulation function, intestinal/pancreatic histopathology, fecal SCFAs contents, expressions of GPR43 and GLP1 and its receptor in intestinal/pancreatic were analyzed. Results Compared with model group, DOP significantly increased tail microcirculatory blood flow in PDM rats (P < 0.01) and decreased anal temperature (P < 0.01), significantly reduced levels of total cholesterol and low-density lipoprotein cholesterol in serum (P < 0.05, 0.01), significantly decreased the oral glucose tolerance test results, fasting blood glucose, 2 h postprandial glucose, homeostasis model assessment-insulin resistance index, and C-peptide levels in PDM rats (P < 0.05, 0.01), increased the insulin sensitivity index (P < 0.05), and alleviated the pathological damage of pancreas, ileum and colon. Gas chromatography results showed that DOP administration could significantly increase the levels of acetic acid, butyric acid, isovaleric acid and SCFAs in feces (P < 0.05, 0.01). ELISA, Western blotting and qRT-PCR results showed that DOP administration could significantly increase the plasma GLP1 level, as well as the mRNA and protein expressions of GPR43 and GLP1 receptor in ileum and pancreas (P < 0.05, 0.01). Conclusion DOP could improve PDM, and its effect may be related to enhancing gut-derived SCFA production, up-regulating GPR43 expressions in intestine and pancreas, subsequent promoting GLP1 secretion, restoring β-cell function and improving glucose regulation.