UDP glycosyltransferase (UGT) is a terminal modifying enzyme for the formation of flavonoid glycosides. In this study, we obtained two glycosyltransferase genes, CtUGT25 and CtUGT18, which are closely related to the synthesis of safflower flavonoids, through the Pierce correlation analysis of the expression of glycosyltransferase genes in the safflower corolla transcriptome database at different developmental stages with the contents of the major constituents of safflower metabolome database, and bioinformatically analyzed the gene and protein sequences of the two genes. Expression pattern analysis revealed that CtUGT25 was mainly expressed in the corolla, with the highest expression on day 3 of flowering stage; CtUGT18 was mainly expressed in the root, with the highest expression on day 1 of flowering stage. Functional validation was verified in safflower by Agrobacterium-mediated pollen-tube pathway transgenesis method, demonstrating that CtUGT25 promoted the accumulation of kaempferol-3-O-glucoside and hydroxysafflor yellow A (HSYA), and CtUGT18 promoted the accumulation of kaempferol-3-O-glucoside and orientin, both of which may be the glycosyl-modifying enzymes for the synthesis of safflower flavonoids. Meanwhile, in vitro experiments demonstrated the catalytic activity of CtUGT25 protein on naringenin, quercetin, apigenin, kaempferol, luteoin, 2-hydroxynaringenin and galangin. This study serves as reference for future advancements in regulating the quality of safflower using molecular biotechnology, particularly focuses on the industrial production of safflower exclusive component HSYA. Additionally, it offers valuable insights for researching related genes in other plants.

Palmatine, the main effective ingredient of Fibraurea recisa, is a typical berberine isoquinoline alkaloid with extensive anti-inflammatory and antibacterial activities. In this work, the studies of metabolomics and transcriptomics were utilized to detect differentially expressed genes (DEGs) that are significantly associated with the synthesis of palmatine. In addition, eight of these DEGs were verified by quantitative real-time PCR (qRT-PCR). A total of 106 alkaloids were detected in the metabolomics study, including 23 isoquinoline alkaloids. Palmatine ranked in the top ten of differential metabolites in the group of root vs leaf, and its relative content in root was about 47.5 times higher than that in leaf. In the transcriptomics study, a total of 188 genes were annotated to the pathway of isoquinoline alkaloid biosynthesis. Among them, there were 36 DEGs were significantly different. In the comparison group of root and leaf, a total of 33 DEGs were significantly different, and 30 DEGs were annotated on the biosynthetic pathway of palmatine. Finally, the results of the correlation analysis between metabolomics and transcriptomics showed that the expression patterns of four gene sequences were screened to be significantly correlated with palmatine. The results of qRT-PCR experiments showed that the expression trends of eight DEGs were consistent with the results of transcriptomic. This study not only enriched the omics data of F. recisa, but also established the foundation for the study of the synthetic biology of palmatine. It further provided a reference for the analysis of the key enzyme genes on the biosynthetic pathway of other isoquinoline alkaloids.

Protein disulfide isomerase A6 (PDIA6) is closely related to inflammation and endoplasmic reticulum stress. To obtain the glycosyl derivatives of benzophenone polyphenols targeting PDIA6 with strong anti-inflammatory effects, twenty-five target glycosyl derivatives were synthesized by Friedel-Crafts acylation and deacetylation reaction, starting from the substituted benzophenone and α-bromoacetyl saccharide, and their interactions with PDIA6 were quantitatively investigated by bio-layer interferometry (BLI) technique. Their in vitro anti-inflammatory properties were also evaluated. The results showed that target compounds 4b, 10b, 17b, 18b, and 25b not only exhibit high affinity with PDIA6, but also present strong anti-inflammatory abilities. Above results suggest that this class of compounds can affect the signaling pathways related to inflammation by directly acting on PDIA6. In particular, such compounds exhibit the strong inhibitory effects on IL-1β and IL-6 release, suggesting the potential development prospect in the treatment of inflammatory diseases.

In this study, inspired by biomimetic mineralization process, we have developed imidazole-zeolite framework (ZIF-8) conjugated VNP20009, and chemotherapy drug doxorubicin hydrochloride (DOX) was encapsulated to obtain ZD@VNP. The morphology and the combination of ZIF-8 and VNP was characterized by transmission electron microscopy and laser confocal microscopy. Fluorescence spectrophotometry was used to examine the encapsulation rate and in vitro release rate of DOX. CCK-8 and FDA/PI cell viability staining experiments were used to evaluate the ability of ZD@VNP to inhibit cell proliferation. Melanoma mouse model was established to investigate the effect of ZD@VNP to inhibit tumor growth. It was shown that ZIF-8 was evenly bounded to the surface of VNP20009, and laser confocal microscopy results also confirm the combination of ZIF-8 with VNP in ZD@VNP. The encapsulation rate of DOX in ZD@VNP was calculated to be 85.7% ± 3.7%, and the release of DOX under the buffer at pH 6.0 was significantly higher than that of pH 7.4. ZD@VNP treatment resulted in a greater inhibitory effect on B16F10 cell proliferation compared to DOX treatment. Animal experiment results showed that compared with VNP+DOX, ZD@VNP treatment can significantly inhibit the growth of B16F10 tumors in C57BL/6 mice and prolonged survival (all animal experiments were approved by the Institutional Animal Care and Ethics Committee Guangdong Medical University, No. GDMU-2023-2518). In summary, ZD@VNP was prepared through a facile, one-step method, which can significantly enhance the proliferation inhibitory effect of DOX to inhibit tumor growth and prolong survival. Our results demonstrate that ZD@VNP has great application prospects in the field of drug delivery.

Xiaoke formula (XKF) is a classic formula for the treatment of insulin resistance (IR), but there is still unclear on bioactive equivalent combinatorial components (BECC) of XKF. In this study, based on the previous research of our team, three components, berberine, astragaloside Ⅳ and chlorogenic acid, were selected as the BECC of XKF, and their efficacy and mechanism were investigated. A high-fat diet-induced IR mouse model was used to detect blood glucose, insulin sensitivity, lipid metabolism, immune & inflammatory factors, etc., and staining of pathology sections was used to detect histopathological changes. Network pharmacology was used to predict the potential targets and signaling pathways of XKF and its BECC, and the results of the network were verified by Western blot. The animal welfare and experimental procedures followed the regulations of the Laboratory Animal Ethics Committee of Beijing MDKN Biotech Company (MDKN-2023-019). The results showed that BECC, which was composed of berberine, astragaloside Ⅳ and chlorogenic acid in the ratio of the original formula of XKF, was comparable to XKF in improving the glycemia, insulin sensitivity, histopathological damage, dyslipidemia, and immuno-inflammation in IR mice. The results of network pharmacology and Western blot suggested that the BECC of XKF and XKF might alleviate IR by promoting the activation of hepatic phosphatidylinositol 3-kinase (PI3K), phosphorylation of protein kinase B (AKT), and inhibiting the expression of glucose-6-phosphate phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1), the key limiting enzymes of hepatic gluconeogenesis. The above results suggest that berberine, astragaloside Ⅳ and chlorogenic acid can be used as the potential BECC of XKF to improve IR, and can regulate lipid metabolism, immuno-inflammation, and promote hepatic PI3K/AKT signaling to inhibit hepatic gluconeogenesis, regulate glucose homeostasis, and improve IR in mice.

Proteolysis targeting chimera (PROTAC) is a drug discovery strategy using ubiquitin proteasome system (UPS) to degrade the target protein. Unlike traditional small molecule drugs utilizing occupancy-driven pharmacology as the mode of action (MOA) to regulate protein activity, PROTACs function through forming stable target protein-PROTAC-E3 ubiquitin ligase ternary complex and use ubiquitin proteasome system to degrade the target protein. However, only a few E3 ubiquitin ligases have been used in PROTAC drug design now, and the space of target proteins that PROTAC can target needs to be further expanded. On the other hand, the complicated system of ternary crystal structures is difficult to capture and identify, computational simulation provides modeling of PROTAC-mediated ternary complex formation with effective approaches. In view of this, this review describes the recent progress of bioinformatics on expanding the landscape of E3 ubiquitin ligases and target proteins, and summarizes the methods of computation simulation in modeling PROTAC ternary complex. Finally, the trend of development about PROTAC is prospected.

The compounds were isolated and purified by silica gel, MCI, Sephadex LH-20 and semi-preparative high performance liquid chromatography. The structures of the compounds were determined by NMR and MS spectroscopic data. Twenty monomer compounds were isolated from the ethyl acetate extract of Lindera reflexa root from Hunan province, and identified as: (1″S, 3″S, 6″R)-2′, 4′, 6′-trihydroxy-3′-[1″-(3″-hydroxy)-p-menthanyl]-chalcone (1), sumadain D (2), quercetin (3), catechin (4), epicatechin (5), N-cis-feruloyltyramine (6), N-trans-feruloyltyramine (7), N-trans-feruloyl-3-methoxytyramine (8), flavifloramides B (9), northalifoline (10), isolariciresinol (11), syringaresinol (12), pinoresinol (13), medioresinol (14), dehydroconiferyl alcohol (15), 4-methoxyl-denudaquinol (16), miliusanal (17), syringic acid (18), abscisic acid (19), (E)-cinnamyl-(E)-cinnamate (20). Compound 1 is a new compound, and compounds 2‒20 have been isolated from Lindera reflexa for the first time. The results showed that compounds 1, 2, 3 and 16 could significantly reduce the survival ability of MGC-803 cells with IC₅₀ values of 5.58, 23.41, 25.72 and 20.96 μmol·L^-1, respectively. Compounds 5 and 20 can reduce the survival ability of MGC-803 cells with IC₅₀ values of 98.83 and 89.26 μmol·L^-1, respectively.

A high performance liquid chromatography (HPLC) method utilizing correction factors was established for the quantitative detection of related substances in flumazenil. Separation was achieved using an Agilent Pursuit XRs C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic elution of dilute phosphoric acid, methanol, and tetrahydrofuran as the mobile phases. Correction factors calculated from a standard curve method were applied to determine the impurity content. The quantification of impurities in flumazenil was conducted using both external standard and correction factor methods, followed by validation and comparison of the two. For the identification of degradation products, a forced degradation approach was employed to prepare a flumazenil degradation solution, and the resulting impurities were confirmed by LC-MS analysis. The separation of flumazenil and its impurities was found to be efficient. The limits of quantification for impurities A, B, D, and E were established at 0.169 9, 0.314 7, 0.143 9, and 0.270 8 ng, respectively, with the limits of detection at 0.055 8, 0.096 9, 0.048 8, and 0.089 0 ng. These impurities demonstrated a strong linear relationship across the concentration ranges of 0.034 9-7.847 0, 0.038 7-8.710 7, 0.034 6-7.794 1, and 0.032 4-7.292 8 µg·mL^-1, respectively (n = 7). The method achieved average recoveries between 98.25% and 99.42%, with an RSD of less than 2.0% (n = 9), indicating high accuracy. The external standard and correction factor methods were used to determine the related substances in flumazenil, and the results of the two methods were consistent. The established HPLC method is characterized by its high accuracy, sensitivity, and repeatability, and is suitable for determining related substances in flumazenil.

cAMP response element binding protein (CREB) is an eukaryotic intranuclear protein widely expressed in a variety of organs, and its activation increases the transcriptional activity of downstream genes and promotes the expression of related genes. The neuronal function of CREB is related to many intracellular processes, such as proliferation, differentiation, survival, long-term synaptic potentials, neurogenesis and neuronal plasticity. Increasing evidence has demonstrated that CREB plays an important role in the stroke development and therefore, it may serve as a potential target for stroke therapy. Since some herbal medicines as well as their active ingredients regulate the CREB signaling, this article will summarize the role of CREB signaling pathway in stroke pathophysiology. The research progress of traditional Chinese medicine and its active ingredients modulating CREB activity will also be discussed, with the aim of providing the basis and reference for the future research and development of natural medicines against stroke.

Celastrol and wilforlide A are the main active triterpenoids of the traditional Chinese medicine Lei Gong Teng, which have anti-tumour, anti-inflammatory and immunosuppressive activities, and are the material basis for the clinical efficacy of Lei Gong Teng-related Chinese medicinal preparations. By analysing the biosynthetic pathway of active ingredients, optimizing genetic elements and utilizing "cell factory" to produce triterpenoids heterologously will be an effective way to obtain from Tripterygium wilfordii in the future in a "low-cost and high-efficient" manner. CYP712Ks are the first cytochrome P450s involved in the skeleton modification of friedelane-type and oleanane-type triterpenoids in T. wilfordii, and they can catalyse the generation of polpunonic acid from friedelin and 3-epi-katonic acid from β-amyrin by carboxylation at C-29 position. In this study, four multifunctional TwCYP712K1/2/3/5 were used to clarify the catalytic function and substrate selection preference using in vivo functional characterization in Saccharomyces cerevisiae. The spatial structure of the protein-substrate binding was clarified through homology modeling and molecular docking, and then the differential amino acids in the active pocket of the protein were mutated to clarify the crucial amino acids determining the catalytic function and the selection of substrate structure. A total of 63 mutant elements were constructed, and the amino acid sites affecting the carboxylation function of TwCYP712Ks were analyzed. In particular, the key amino acids affecting the substrate selectivity of TwCYP712K2 towards oleanane-type and friedelane-type triterpenoids were revealed and the TwCYP712K2^F127I and TwCYP712K2^A227T mutants would result in a reversal of the product ratio. In conclusion, four proteins of TwCYP712Ks were semi-rationally designed by homologous protein alignment and mutual mutation to elucidate multiple amino acid sites determining the catalytic function of the proteins, and a series of activity-enhancing or altering mutants were obtained, which provide abundant catalytic elements for the biosynthesis of active triterpenoids from T. wilfordii, and the mechanism of carboxylation in the C-29 position was initially elucidated.