Five compounds were separated from Sambucus chinensis and identified as maslinic acid(1), 12alpha, 13-dihydroxyolean-3-oxo-28-oic acid(2), 13-hydroxyolean-3-oxo-28-oic acid (3), 3-oxo oleanolic acid (4), corosolic acid (5). Of them,compound 3 was a new compound, and compounds 1, 2, 4, and 5 were seperated from this plant for the first time.
Oleanolic Acid ; analysis ; Sambucus ; chemistry ; Triterpenes ; analysis

To generate active recombinant human beta-secreatase (BACE1) for studying its interaction with its inhibitors, we constructed two recombinant plasmids, pPIC9K-MetBACE22 (bearing pro-bace1 gene) and pPIC9K-MetBACE46 (bearing bace1 gene). These two plasmids were then transformed into Pichia pastoris GS115 by electroporation to obtain the recombinant strains 9k-B22 and 9k-B46. After induction in buffered methanol complex medium, we found the supernatant activity of 9k-B22 significantly higher than that of 9k-B46. The culture filtrate of 9k-B22 was concentrated, and then purified by HisTrap affinity column. The purified proteins, showing good BACE1 protease activity, were found to be a mixture of glycoproteins because they can be stained by periodic acid-Schiff reagent. After this mixture was treated with Endo H(f) (a recombinant protein of endoglycosidase H), we found two new adjacent bands around 50 kDa on SDS-PAGE. These two bands were cut and subjected to peptide mass fingerprint analysis, and identified as proBACE1 and BACE1 proteins. Enzyme assays revealed that the activities of both BACE1 proteins in glycosylated and deglycosylated form were lower than that of commercial BACE1 (expressed in HEK-293), inferring glycosylation and the type of glycosylation are crucial to the activity. However, we found no apparent difference in the inhibition of those all above three enzyme forms by one known BACE1 inhibitor. This observation demonstrated that the glycosylation of BACE1 by Pichia pastoris does not affect its interaction with this inhibitor. After optimization of culture conditions, the production of BACE1 in Pichia pastoris was enhanced to about 1 mg/L. This work enables us to further investigate the interaction of BACE1 and its inhibitors, and assists in discovering and optimizing BACE1 inhibitors as anti-Alzheimer's disease agents.
Amyloid Precursor Protein Secretases ; biosynthesis ; genetics ; Aspartic Acid Endopeptidases ; biosynthesis ; genetics ; Electroporation ; Humans ; Pichia ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Tubulin has been shown to be an effective target for the development of cytotoxic agents against prostate cancer. Previously, we reported that Lx2-32c is an anti-tubulin agent with high binding affinity to tubulin. In this study, we investigated the potential of Lx2-32c to act as an effective cytotoxic agent in the treatment of prostate cancer. MTT assays showed that Lx2-32c was cytotoxic to all tested prostate cancer cell lines. The Lx2-32c-treated cells typically exhibited a rounded morphology associated with the onset of apoptosis, as evidenced by immunocytochemical staining. Human prostate cancer cell lines treated with Lx2-32c arrest in the G2/M phase of the cell cycle and the treatment is associated with an increased ratio of cells in the sub-G0/G1 phase as determined by flow cytometry. Furthermore, expression of the cleaved form of poly (ADP-ribose) polymerase in prostate cancer cell lines treated with Lx2-32c was shown by Western blotting assay. Xenograft implants of LNCaP and PC3-derived tumors in nude mice showed that Lx2-32c treatment significant inhibited tumor growth with effects equivalent to those of docetaxel. These findings demonstrate the potential of Lx2-32c as a candidate antitumor agent for the treatment of prostate cancer.