Structure Elucidation of a Novel Polysaccharide Isolated from Euonymus fortunei and Establishing Its Antioxidant and Anticancer Properties.

Euonymusfortunei polysaccharides (EFPs) have not been extensively investigated yet in terms of their extraction and biological activity. The orthogonal experimental design was employed in this study to evaluate the optimum yield of EFPs. A maximum yield of 2.63 ± 0.23% was attained using material-liquid ratios of 60 mL/g, extraction temperature of 80°C, ultrasonic power of 144 W, and extraction time of 75 mins. The polysaccharide content reached 53.47 ± 0.31% when deproteinized thrice. An analysis of monosaccharide composition revealed that these polysaccharides consist of Gal, Glc, Man, Fuc, and Rha with a molar ratio of 7.14 ∶ 23.99 ∶ 6.29 ∶ 6.55 ∶ 1.00, respectively, in EFPs. Subsequently, the in vitro scavenging capacities of 2,2-diphenylpicrylhydrazyl (DPPH) and ·OH and superoxide anion radicals, along with the reducing power of EFPs, were studied. Results revealed that EFPs have higher antioxidant activity, particularly ·OH scavenging, as well as reducing power, as compared to Astragalus polysaccharides (ASPs) and Lycium barbarum polysaccharides (LBPs). The Cell Counting Kit-8 (CCK-8) method was used to evaluate the effects of different concentrations of polysaccharides on SKOV3 cell proliferation, and the results revealed their inhibition at concentrations in the range of 200-800 µg/mL. In addition, findings from flow cytometry further confirmed that EFPs blocked the cell cycle at G0/G1 and S phases and induced SKOV3 cell apoptosis. In a word, EFPs could be exploited and used further based on the experimental results from this study.

Positive Selection of Squalene Synthase in Cucurbitaceae Plants.

Triterpenoid saponins are secondary metabolites synthesized through isoprenoid pathways in plants. Cucurbitaceae represent an important plant family in which many species contain cucurbitacins as secondary metabolites synthesized through isoprenoid and triterpenoid pathways. Squalene synthase (SQS) is required for the biosynthesis of isoprenoids, but the forces driving the evolution of SQS remain undetermined. In this study, 10 SQS cDNA sequences cloned from 10 species of Cucurbitaceae and 49 sequences of SQS downloaded from GenBank and UniProt databases were analyzed in a phylogenetic framework to identify the evolutionary forces for functional divergence. Through phylogenetic construction and positive selection analysis, we found that SQS sequences are under positive selection. The sites of positive selection map to functional and transmembrane domains. 180L, 189S, 194S, 196S, 265I, 289P, 389P, 390T, 407S, 408A, 410R, and 414N were identified as sites of positive selection that are important during terpenoid synthesis and map to transmembrane domains. 196S and 407S are phosphorylated and influence SQS catalysis and triterpenoid accumulation. These results reveal that positive selection is an important evolutionary force for SQS in plants. This provides new information into the molecular evolution of SQS within the Cucurbitaceae family.

Molecular identification of Uncaria (Gouteng) through DNA barcoding.

BACKGROUND: While DNA barcoding is an important technology for the authentication of the botanical origins of Chinese medicines, the suitable markers for DNA barcoding of the genus Uncaria have not been reported yet. This study aims to determine suitable markers for DNA barcoding of the genus Uncaria (Gouteng). METHODS: Genomic DNA was extracted from the freshly dried leaves of Uncaria plants by a Bioteke's Plant Genomic DNA Extraction Kit. Five candidate DNA barcode sites (ITS2, rbcL, psbA-trnH, ITS, and matK) were amplified by PCR with established primers. The purified PCR products were bidirectionally sequenced with appropriate amplification primers in an ABI-PRISM3730 instrument. The candidate DNA barcodes of 257 accessions of Uncaria in GenBank were aligned by ClustalW. Sequence assembly and consensus sequence generation were performed with CodonCode Aligner 3.7.1. The identification efficiency of the candidate DNA barcodes was evaluated with BLAST and nearest distance methods. The interspecific divergence and intraspecific variation were assessed by the Kimura 2-Parameter model. Genetic distances were computed with Molecular Evolutionary Genetics Analysis 6.0. RESULTS: The accessions of the five candidate DNA barcodes from 11 of 12 species of Uncaria in China and four species from other countries were included in the analysis, while 54 of total accessions were submitted to GenBank. In a comparison of the interspecific genetic distances of the five candidate barcodes, psbA-trnH exhibited the highest interspecific divergence based on interspecific distance, theta prime, and minimum interspecific distance, followed by ITS2. The distribution of the interspecific distance of ITS2 and psbA-trnH was higher than the corresponding intraspecific distance. Additionally, psbA-trnH showed 95.9 % identification efficiency by both the BLAST and nearest distance methods regardless of species or genus level. ITS2 exhibited 92.2 % identification efficiency by the nearest distance method, but 87 % by the BLAST method. CONCLUSION: While psbA-trnH and ITS2 (used alone) were applicable barcodes for species authentication of Uncaria, psbA-trnH was a more suitable barcode for authentication of Uncaria macrophylla.

IPP5, a novel inhibitor of protein phosphatase 1, suppresses tumor growth and progression of cervical carcinoma cells by inducing G2/M arrest.

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here, we report the characterization of a novel human bone marrow stromal cell (BMSC)-derived protein called protein phosphatase 1 inhibitor 5 (IPP5), which was obtained by large-scale random sequencing of a human BMSC cDNA library. The human IPP5 cDNA encodes a protein of 116 amino acid residues, which shares high homology with human protein phosphatase 1 inhibitor-1 (PPI-1). The effect of IPP5 on tumor growth and the underlying molecular mechanisms were investigated by overexpression of IPP5 in HeLa cells, a human cervical carcinoma cell line. Our results demonstrated that overexpression of the active mutant IPP5 inhibited the growth of HeLa cells both in vitro and in vivo. Biochemical analysis demonstrated that active mutant IPP5-mediated G2/M arrest of HeLa cells involved regulation of cyclin A1, cyclin B1, CDK1, p21, and p53, as well as increased inhibition of ERK activation. Furthermore, overexpression of the active mutant IPP5 leads to the formation of dikaryons following the failure of cytokinesis. Therefore, IPP5 might be a potential growth inhibitor for human tumor cells, especially for cervical carcinoma cells, and it could contribute to the development of new therapeutic strategies for human cervical cancer treatment.