Eleven monoterpenes including seven new chemical structures or new natural products covering two pairs of scalemic enantiomers, together with four known analogues, were isolated from an aqueous extract of the Angelica sinensis root head (Guitou) by separation techniques of column chromatography over macroporous adsorbent resin, MCI resin, silica gel, Sephadex LH-20, and Toyopearl HW-40C, together with preparative thin-layer chromatography as well as reversed phase and chiral HPLC. Their structures were determined by spectroscopic data analysis, combined with theoretic calculation of electronic circular dichroism (ECD) spectra and single crystal X-ray diffraction. The new structures or new natural products named (+)-/(-)-angelinones A and B [(+)-/(-)-1 and (+)-/(-)-2], angelinones C and D (3 and 4), and angelinol A (5), respectively, while the known analogues were 6β, 9-dihydroxy-(+)-α-pinene (6), 1, 1, 5-trimethyl-2-hydroxymethyl-cyclohexa-2, 5-dien-4-one (7), jasminol E (8), and (+)-trans-sobrerol (9). All the isolates were reported in this plant for the first time, except for the previously reported 6 from an ethanol extract of the aerial parts of A. sinensis, of which the structure was confirmed by X-ray crystallography in this study.

Two new ursane triterpenoids along with twelve known compounds were isolated from 80% ethanol extract of Agastache rugosa (Fisch. et. Mey.) O. Kuntze by using silica gel column, MCI column, ODS column and HPLC. The structures of the new compounds were identified as 2α, 3α-dihydroxy-24-nor-urs-4(23), 12(13)-dien-28-oic acid (1) and 2α, 3α-dihydroxy-24-nor-urs-4(23), 12(13), 20(30) -trien-28-oic acid (2) by HR-ESI-MS, NMR and ECD spectral data, named agasursacid A and agasursacid B. In addition, compounds 3, 4, 6, 8 showed anti-coxsackievirus B3 (CVB3) activities with a IC₅₀ as 4.77, 1.59, 11.11 and 25.87 μmol·L^-1, resepectively.

An UHPLC-Q-exactive orbitrap MS method for the simultaneous determination of 19 chemical components in Qilong Zhuang'er oral liquid was established and the quality differences between different batches of samples was compared by chemometric analysis to provide a basis for the quality evaluation of the preparation. The contents of allantoin, L-proline, pyroglutamic acid, hordenine, adenosine, L-phenylalanine, guanosine, L-tryptophan, caffeic acid, calycosin-7-glucoside, verbascoside, isoacteoside, ononin, calycosin, 3-hydroxy-9, 10-dimethoxyptercarpan, formononetin, atractylenolide Ⅲ, atractylenolide Ⅱ and astragaloside A were analyzed by cluster heat map, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) using Hiplot platform and Markerlynx^XS software to comprehensively evaluate the quality difference of different batches of Qilong Zhuang'er oral liquid. The 19 chemical compounds showed good linearity in their respective concentration ranges (r ≥ 0.999). The RSD of precision, repeatability and stability (24 h) tests were all less than 1.94%. The average recovery was 97.24%-102.75% (RSD < 2.74%, n = 6). The 10 batches of samples were divided into two categories by cluster heat map and PCA analysis. 3-Hydroxy-9, 10-dimethoxyptercarpan, atractylenolide Ⅲ, calycosin, atractylenolide Ⅱ, formononetin, allantoin and caffeic acid were identified as differential markers by PLS-DA. The established multi component quantitative method of Qilong Zhuang'er oral liquid combined with chemometric analysis can provide reference for the quality evaluation of the preparation.

Antibody-drug conjugate (ADC) has the characteristics of low toxicity and high efficiency, and plays an important role in cancer treatment. However, due to the complexity of its structure, it brings difficulties in pharmacokinetic (PK) bioanalysis. This study established an analytical method for the detection of ADC (RC108) in cynomolgus monkey plasma by ligand-binding assay (LBA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which was used to analyze and quantify the total antibody, bound antibody and free drug in cynomolgus monkey plasma. Based on the LBA method, rabbit anti-RC108 Fab and mouse anti-MMAE (monomethyl auristatin E) mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents, respectively. The samples to be tested were added, and then the detection reagents were added in turn. Goat anti-human IgG (H+L)-HRP, chromogenic solution tetramethylbenzidine (TMB), H₂SO₄ terminate the reaction, read data at 450 nm/630 nm wavelength of microplate reader; LC-MS/MS analysis method quantifies MMAE concentration, and refer to relevant regulations for methodological validation. The analytical method for quantifying total antibody, bound antibody and free drug of RC108 drug obtained good accuracy and precision, and the selectivity, dilution linearity, hook effect, parallelism and stability were verified. Meet the requirements of biological analysis. Finally, a bioanalytical method for the determination of the concentration of the test substance RC108 (total antibody, conjugated antibody, free MMAE) in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method. Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

As an edible eukaryotic microorganism, Saccharomyces cerevisiae has the characteristics of high safety, rapid proliferation, low cost, easy transformation, etc. It has been widely used to produce vaccines, antibodies, insulin, etc. Up to now, yeast components, such as cell wall and yeast microcapsules, have been widely used in the treatment of tumors, inflammatory virus infection, post-traumatic osteoarthritis and other diseases. Among them, the components of yeast cell membrane are relatively simple and stable, which are easy to be extracted on a large scale. Therefore, yeast cell membrane material was used to construct yeast membrane vesicle nanosystem, and its biomedical application was preliminarily explored. In this study, Saccharomyces cerevisiae membrane vesicle (SMV) was prepared by co-extrusion method, and the particle size and surface potential of SMV, drug loading and release characteristics, stability, cell safety, and in vitro therapeutic effect were investigated. The results showed that the average particle size of SMV was 185.1 nm. Curcumin and silica nanoparticles were effectively encapsulated by co-incubation and ultrasonic methods, and the characteristics of cell membrane proteins were maintained. Moreover, SMV had good stability and biocompatibility. In addition, SMV could be effectively uptaken by macrophages RAW 264.7, and curcumin loaded SMV could effectively eliminate reactive oxygen species (ROS). In conclusion, the yeast plasma membrane vesicles prepared in this study could effectively deliver curcumin drugs and encapsulate nanoparticles, and could be effectively absorbed by macrophages and effectively eliminate ROS, providing new ideas and new methods for biomedical applications of yeast membrane materials.

We constructed and optimized the plasmid DNA (pDNA) Opt-S encoding the gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, using poly (lactic-co-glycolic acid) copolymer (PLGA) as a delivery carrier for pDNA. PLGA-pDNA NPs were loaded by nanoprecipitation and its properties in vitro were preliminary evaluated. The results showed that the prepared PLGA-pDNA NPs were regular morphology, clear edges, with an average particle size of (184.2 ± 2.4) nm, polydisperse index (PDI) of 0.093 ± 0.013, zeta potential of (-68.10 ± 0.36) mV, and encapsulation rate of (98.92 ± 0.22)%. The PLGA-pDNA NPs were stable at -20 ℃ for 7 months and could protect pDNA against nuclease degradation. And they also exhibited sustained release of pDNA in vitro. The PLGA-pDNA NPs have low cytotoxicity and high safety. In addition, in vitro transfection experiments showed that the SARS-CoV-2 S gene could enter cells and be expressed. These results indicate that PLGA-pDNA NPs non-viral gene vector have simple preparation process and good performance, which are expected to provide a new idea for the research and development of SARS-CoV-2 vaccine.

Long-acting analgesia is a common clinical treatment method after surgery. The slow-release injection with long-acting analgesia has the advantages of less medication frequency and stable effect. In this study, the analgesic drug lappaconitine hydrobromide lyotropic liquid crystal injection was prepared, and its sustained release mechanism, drug release and pharmacodynamic characteristics were evaluated. The results of polarizing microscope and freeze-transmission electron microscope showed that the lyotropic liquid crystal injection of the liquid crystal precursor preparation of lappaconitine hydrobromide could be obtained by the combination of glycerol monooleate (GMO) and soybean lecithin (SPC) in different proportions. The results of dissolution study in vitro showed that the drug release rate of different forms of liquid crystal preparations was layered liquid crystal > cubic liquid crystal > hexagonal liquid crystal. The mathematical model fitting results of the release data showed that the external release of layered liquid crystal, cubic liquid crystal and hexagonal liquid crystal conforms to the Ritger-Peppas model, and the release mechanism was Fick diffusion. The results of pharmacodynamics study in vivo showed that the analgesic effect of lappaconitine hydrobromide lyotropic liquid crystal injection lasted for 3 days, and there was no abnormality in the incision and local tissue, showing good safety and tolerance. The study on drug release and elimination process of the in vivo gel repository showed that lappaconitine hydrobromide could be completely released from the lyotropic liquid crystal 3 days after administration, and the sustained-release materials could be gradually eliminated locally. All animal experiments were approved by the Experimental Animal Ethics Committee of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (No. 2021-08-GY-61) and the experiments were conducted in accordance with the relevant guiding principles and regulations. The lyotropic liquid crystal injection of lappaconitine hydrobromide prepared in this study presented a solution state at room temperature, and underwent phase transition in contact with the body fluid at the administration site, formed a drug depot and exerted a slow drug release effect. This preparation can reduce systemic toxicity, prolong the duration of analgesia, reduce the number of administrations, improve the compliance of postoperative patients, and provide a reference for the design of long-term sustained release analgesic preparations.

Atractylodes chinensis has important medicinal and economic values. In this study, the chloroplast genome sequences of four A. chinensis samples from different producing areas were sequenced using the Illumina platform. The specific DNA barcodes were screened and the germplasm resources of A. chinensis samples from different producing areas and the genetic diversity of the population were analyzed basing on the specific barcodes. The whole chloroplast genomes of the four A. chinensis samples had a typical cyclic tetrad structure, with 112 genes annotated. The comparative genomics results indicated that ccsA and trnC-GCA_petN were potential specific DNA barcodes for intraspecific identification of A. chinensis. Polymerase chain reaction (PCR) analysis of ccsA and trnC-GCA_petN was performed on 256 samples from 14 areas in 9 provinces, and the amplification efficiency was 100%. Sequence analysis showed that ccsA and trnC-GCA_petN had 11 and 22 variant positions, which could identify 16 and 22 haplotypes, respectively. The combined sequence analysis identified 39 haplotypes, named Hap1-Hap39, of which the most abundant and widely distributed genotype was Hap9. Haplotype diversity (H_d) = 0.896 and nucleotide diversity (Pi) = 0.002 22 indicated high genetic diversity at the species level in A. chinensis. The genetic distances of the haplotypes were 0.000 00-0.004 88, indicating that there were small genetic differences among the haplotypes. The results of phylogenetic tree analysis showed that 39 haplotypes had very close genetic relationship, and formed two obvious branches with other groups of the same genus except Atractylodes macrocephala. This study plays an important role in the identification of the origin of A. chinensis and the protection and breeding of germplasm resources.

The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism. Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids (TAs) content in A. belladonna. In this study, the rolB gene was overexpressed in A. belladonna plants to study the effect of rolB gene on the biosynthesis of TAs. The phenotype, TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A. belladonna were analyzed. The results showed that transgenic A. belladonna had developed root system, enlarged leaves, increased leaf fresh weight, deepened leaf color, enlarged flowers, changed flower shape, reduced pistil height and decreased pollen vitality. The content of TAs in the stems of transgenic A. belladonna was significantly higher than that of the control, and the contents of scopolamine, anisodamine, hyoscyamine can reach 2.11-2.91, 1.23-2.37 and 4.88-5.20 times of the control, respectively. Compared with the control group, the expressions of key enzymes putrescine N-methyltransferase (PMT), type Ⅲ polyketide synthase (PYKS), tropinone reductase I (TRI), aromatic amino acid aminotransferase 4 (ArAT4), UDP-glycosyltransferase 1 (UGT1) and hyoscyamine 6-β-hydroxylase (H6H) in the TAs biosynthesis pathway were up-regulated, and the expression of tropinone reductase Ⅱ (TRⅡ) as a metabolic shunting gene was down-regulated. The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A. belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway. This study laid a foundation for molecular breeding of A. belladonna with high-yield TAs content using rolB gene.

Fusarium oxysporum widely exists in farmland soil and is one of the main pathogenic fungi of root rot, which seriously affects the growth and development of plants and often causes serious losses of cash crops. In order to screen out natural compounds that inhibit the activity of Fusarium oxysporum more economically and efficiently, random forest, support vector machine and artificial neural network based on machine learning algorithms were constructed using the information of known inhibitory compounds in ChEMBL database in this study. And the antibacterial activity of the screened drugs was verified thereafter. The results showed that the prediction accuracy of the three models reached 77.58%, 83.03% and 81.21%, respectively. Based on the inhibition experiment, the best inhibition effect (MIC = 0.312 5 mg·mL^-1) of ononin was verified. The virtual screening method proposed in this study provides ideas for the development and creation of new pesticides derived from natural products, and the screened ononin is expected to be a potential lead compound for the development of novel inhibitors of Fusarium oxysporum.