Flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanin synthase (ANS) are members of the 2-oxoglutarate-dependent oxygenase (2-ODD) superfamily, which play important functions in plant flavonoid biosynthesis. In this study, 16 2-ODD genes involved in flavonoid biosynthesis pathway were screened and identified from safflower genome database using bioinformatics method. The length of the proteins encoded by these genes ranged from 283 (CtFLS2) to 442 aa (CtANS6), and the molecular weight ranged from 32 062.05 (CtFLS2) to 48 245.49 Da (CtANS6). The proteins encoded by these genes were hydrophilic. Phylogeny divided it into three subfamilies, two conserved residues, H-x-D-xn-H and R-x-S, were found in this gene family using conserved motifs analysis, the analysis of cis-acting elements in the upstream 2 000 bp region showed that these genes might be regulated by environmental factors such as light, temperature and plant hormones such as salicylic acid, methyl jasmonate and so on. The members of the safflower 2-ODD gene family involved in flavonoid biosynthesis pathway were analyzed by qRT-PCR, the result showed that the expression patterns of these members were different in diverse flowering period and leaf. This study can lay a foundation for further exploring the function of 2-ODD genes involved in flavonoid biosynthesis pathway in safflower.

The metabonomics method was used to explore the processing and synergistic mechanism of epimedium fried with suet oil in warming the kidney and enhancing yang. The kidney-yang deficiency rat model was established by injection of hydrocortisone. Then the UPLC-Q-TOF-MS (ultra-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry) metabolomics method was combined with multivariate statistical analysis methods and univariate statistical analysis to screen and identify kidney-yang deficiency potential biomarkers in plasma and urine samples. Finally the metabolic regulation mechanism of suet oil group, the epimedium raw product group, the epimedium heating product group, and the epimedium fried with suet oil group improved kidney-yang deficiency was analyzed. The results showed that the plasma and urine metabolism of rats with kidney-yang deficiency induced by hydrocortisone showed obvious trajectory changes. 15 biomarkers related to kidney-yang deficiency were identified in plasma and urine, involving 5 metabolic pathways, namely glycerophospholipid metabolism, sphingolipid metabolism, sulfur metabolism, glyoxylate acid and dicarboxylate metabolism, and cysteine and methionine metabolism. The metabolic pathway of epimedium fried with suet oil warming kidney and promoting yang involved glycerophospholipid metabolism, cysteine and methionine metabolism, and the two processing factors of epimedium fried with suet oil "heating" and "suet oil" enhanced its function of warming the kidney and promoting yang by regulating glycerophospholipid metabolism, cysteine and methionine metabolism, respectively. In this way, the processing and synergistic mechanism of epimedium fried with suet oil was clarified. The animal experiments involved in this article comply with ethical standards and have been approved by the Animal Ethics Committee of Jiangsu Provincial Academy of Chinese Medicine (approval number: AEWC-20200702-119).

Twenty-five candidate MLO genes of Lonicera japonica were obtained by whole genome sequencing. Bioinformatics analysis showed that the number of amino acids in the protein family ranged from 137 to 846, the theoretical isoelectric point ranged from 5.02 to 9.50, and it was rich in basic amino acids. One protein did not contain transmembrane domain, and the other proteins ranged from 3 to 10. The results of subcellular localization showed that 21 proteins were located on the cell membrane and 1 protein was located on the chloroplast. Phylogenetic trees were constructed from 133 MLO proteins of Lonicera japonica, Triticum aestivum, Arabidopsis thaliana, Solanum lycopersicum, Nicotiana tabacum, Nicotiana sylvestris and Nicotiana tomentosiformis. The results indicated that MLO family proteins of Lonicera japonica could be divided into five subgroups. Tissue specific analysis showed that the expression of MLO genes in Lonicera japonica had obvious tissue specificity. Among them, 8 genes were highly expressed in leaves, 2 genes in stems and 2 genes in flowers. Four genes were significantly upregulated after inoculation with powdery mildew, among which MLO14 increased the most, which was more than 2 000 times higher than that of the control. In this study, the composition and expression of the MLO gene related to the occurrence of powdery mildew of Lonicera japonica were preliminarily analyzed, which laid a foundation for the further use of MLO as a target gene to develop new germplasm resistant to powdery mildew of Lonicera japonica.

Compared with systemic administration such as oral delivery or injection, inhaled medicines directly locate in the respiratory tract to exert therapeutic effects, offering obvious advantages in the treatment of respiratory diseases. Marketed inhaled medicines are yet difficult to meet the clinical demands, and there are considerable challenges in the discovery and development of novel inhaled medicines due to the lack of experiences- and property-based rules for inhaled compounds. Personalized modification of candidate drugs through prodrug technology to meet the requirements of inhalation therapy is the current alternative approach for inhaled drug development. In this review, we intend to summary the applications of prodrug technology in the research of inhaled medicines over the past 20 years. These studies have shown that esterified prodrugs and macromolecule conjugates could effectively prolong lung retention; mannose modification or acid-sensitive bond connection can achieve targeted drug release in alveolar macrophages; personalized modified prodrugs can obtain suitable physicochemical properties for pulmonary delivery and reduce drug toxicity. In general, the application of prodrug technology can modify the physicochemical and biopharmaceutical properties of drugs and may promote the discovery and development of novel inhaled medicines.

We established in vitro evaluation methods of the nebulization characteristics of budesonide suspension for inhalation and analyzed the influence factors. The delivery rate and total drug substance delivered (TDD) of two manufacturers were determined by using the breath simulator with different nebulizers. The aerodynamic particle size distribution was investigated by next generation impactor (NGI) and HPLC as well. The fine particle dose (FPD), the mass median aerodynamic diameter (MMAD), the delivery rate and TDD results of the same sample with different nebulizers were significantly different (P < 0.01), mainly due to the different design parameters of the nebulizers. The FPD of two samples were significantly different (P < 0.01) by the same nebulizers, probably due to differences in physical and chemical properties differences such as suspension particle size. The analysis method of nebulization characteristics provided in this paper can be used to select the nebulization device with appropriate delivery dose and aerodynamic particle size distribution (APSD) for different inhalation liquid preparations in clinical practice, and can also be used as the guidance for the selection of nebulization device and analysis method in the research and development of generic inhalation liquid preparations and quality consistency evaluation.

Hypoxia is one of the most significant characteristics of solid tumors. Hypoxia microenvironment can lead to the overexpression of hypoxia inducible factor-1α (HIF-1α). As the most critical transcription factor in the hypoxia response, HIF-1α activates downstream gene expression resulting in abnormal tumor cell proliferation, tumor angiogenesis, unusual energy metabolism, increased drug resistance, invasion, and metastasis. Down-regulation of HIF-1α expression is considered as a promising approach for the treatment of solid tumors, whereas the clinical efficacy of most existing HIF-1α inhibitors is restricted in low efficacy and high toxicity. Therefore, it is particularly important to develop powerful and safe novel drugs against the overexpression of HIF-1α. In recent years, numbers of studies have proved that a variety of chemical components of traditional Chinese medicine can directly or indirectly inhibit the activation of HIF-1α, which has a broad prospect in the fight against hypoxia-induced tumor progression. In this review, we summarized various anti-tumor active components of traditional Chinese medicines responsible for inhibiting the expression of HIF-1α in last ten years and analyzed the corresponding mechanism, with a view to further research as a reference.

Twenty-five quinolizidine alkaloids (including matrine-type 1-14, sparteine-type 15-17, cytisine-type 18-23, other types 24 and 25) were isolated from the roots and rhizomes of Sophora tonkinensis by various chromatographic methods. Their structures were elucidated by physicochemical properties, NMR and MS spectral data. Among them, 12-(1-acetoxyethyl)-cytisine (23) is a new alkaloid derivative, and compounds 13, 16, 17, 24 were isolated from the roots and rhizomes of S. tonkinensis for the first time. Compounds 1, 6, 19 and 20 showed potent inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages, with IC₅₀ values of 39.86 ±0.65, 23.66 ±0.37, 34.56 ±0.45, 47.68 ±0.58 μmol·L^-1, respectively.

Blocking the binding of programmed death 1 (PD-1) on the T cells and programmed death ligand 1 (PD-L1) on the tumor cells has become a hotspot in the field of tumor immunotherapy. Small-molecule checkpoint inhibitor targeting PD-1/PD-L1 axis is the new direction of tumor immunotherapy. In the present study, we investigated the anti-tumor role of hyperoside by regulating the PD-L1 level in non-small cell lung cancer (NSCLC). Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot, flow cytometry, and PD-1/PD-L1 interaction assays. The expression of mRNA level of PD-L1 was detected by real-time PCR. The cytotoxicity of activated human T cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment. The antitumor effect of hyperoside in vivo was examined by C57BL/6 mice bearing Lewis xenograft tumor. Western blot and flow cytometry assay showed that hyperoside significantly downregulated the abundance of PD-L1 in H1975 and HCC827 cells in dose- and time-dependent manner. PD-1/PD-L1 binding assay revealed that hyperoside reduced the binding of tumor cells to recombinant PD-1 protein. In addition, hyperoside decreased the abundance of c-Myc, a key transcriptional regulator of PD-L1, in H1975 and HCC827 cells. Cell impedance and crystal violet staining indicated that hyperoside enhanced the killing activity of co-cultured T cells toward tumor cells. Animal experiments (all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences) revealed that hyperoside treatment displayed significant suppression in the growth of Lewis tumor xenografts in C57BL/6 mice with an inhibition rate of 48.3% at 25 mg·kg^-1. Our results demonstrate that hyperoside exerts its anti-NSCLC activity by reducing the PD-L1 level. Our study provides an important material basis and scientific basis for developing hyperoside into a new small molecule drug for tumor immunotherapy.

Five compounds were isolated from an ethanol extract of Artemisia deversa Diels by solvent extraction, Diaion HP-20, CHP20/P120 MCI, Sephadex LH-20, silica gel and preparative high performance liquid chromatography. Their structures were elucidated by MS, NMR and X-ray as artemideversal (1), 7-hydroxy-6-methoxycoumarin (2), 6, 7-dimethoxycoumarin (3), caffeic acid (4) and 4', 5, 7-trihydroxy flavone (5). Compound 1 is a new eudesmane-type sesquiterpene. In vitro cytotoxic activities of the five compounds were explored by MTT testing with HepG2, A549, HeLa and MRC-5 cell lines. Results show that compounds 1 and 5 significantly inhibited cellular proliferation. The IC₅₀ of compound 1 in A549 and HepG2 cells was 8.36 and 16.51 μmol·L^-1; the IC₅₀ of compound 5 in A549 and HepG2 cells was 17.06 and 7.95 μmol·L^-1.

As one of the most critical post-translational modifications, glycosylation of therapeutic proteins has a profound impact on their safety, efficacy and consistent. However, glycosylation is not a template-driven process, therefore variability in the glycosylation pattern of a protein can arise. This makes challenges of glycan analysis and control. Here, we review the overall control strategy, basic requirements for standardized protocols and the novel technologies of glycosylation analysis to accelerate the development of therapeutic glycoproteins.