Synthesis and Structure-Activity Relationship of Salvinal Derivatives as Potent Microtubule Inhibitors.

Salvinal is a natural lignan isolated from the roots of Salvia mitorrhiza Bunge (Danshen). Previous studies have demonstrated its anti-proliferative activity in both drug-sensitive and -resistant cancer cell lines, with IC50 values ranging from 4-17 µM. In this study, a series of salvinal derivatives was synthesized and evaluated for the structure-activity relationship. Among the twenty-four salvinal derivatives, six compounds showed better anticancer activity than salvinal. Compound 25 displayed excellent anticancer activity, with IC50 values of 0.13-0.14 µM against KB, KB-Vin10 (overexpress MDR/Pgp), and KB-7D (overexpress MRP) human carcinoma cell lines. Based on our in vitro microtubule depolymerization assay, compound 25 showed depolymerization activity in a dose-dependent manner. Our findings indicate that compound 25 is a promising anticancer agent with depolymerization activity that has potential for the management of malignance.

Isolation and identification of alpha-CEHC sulfate in rat urine and an improved method for the determination of conjugated alpha-CEHC.

2,5,7,8-Tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC), the water-soluble metabolite of alpha-tocopherol (alpha-TOH) with a shortened side chain but an intact hydroxychroman structure, has been identified in human urine and are thought to be produced in significant amount at excess intake of alpha-TOH. In previous studies, CEHCs in biological specimens were measured by HPLC, GC-MS or LC-MS, preceded by a hydrolysis procedure using either enzyme or methanolic HCl. In an attempt to analyze alpha-CEHC in rat urine accordingly, we observed that enzyme hydrolysis was relatively inefficient in releasing alpha-CEHC compared to high concentrations of HCl. The HCl releasable alpha-CEHC conjugate was isolated and chemically identified as 6-O-sulfated alpha-CEHC (alpha-CEHC sulfate). Using the synthetic alpha-CEHC sulfate standard, it was found that sulfatase could not hydrolyze to a significant extent. On the other hand, pretreatment with HCl at 60 degrees C in the presence of ascorbate, followed by a one-step ether extraction, not only hydrolyzed the sulfate conjugate completely but also extracted alpha-CEHC with high recovery. The inclusion of ascorbate minimized the conversion of alpha-CEHC to alpha-tocopheronolactone in the HCl pretreatment. A complete procedure for the quantitative analysis of alpha-CEHC including HCl hydrolysis, ether extraction and reverse phase isocratic HPLC-ECD was thus established. In conclusion, alpha-CEHC sulfate was isolated and identified as the HCl-releasable conjugate of alpha-CEHC in rat urine. A rapid and sensitive method with high reproducibility for the determination of free, conjugated and total alpha-CEHC is then established.

Inhibitory activity against tobacco mosaic virus (TMV) replication of pinoresinol and syringaresinol lignans and their glycosides from the root of Rhus javanica var. roxburghiana.

Four new diepoxylignan glycosides, pinoresinol-4'-O-[6' '-O-(E)-feruloyl]-beta-D-glucopyranoside (1), pinoresinol-4'-O-[4' ',6' '-O-(E)-diferuloyl]-beta-D-glucopyranoside (2), pinoresinol-4'-O-[3' ',6' '-O-(E)-diferuloyl]-beta-D-glucopyranoside (3), and syringaresinol- 4'-O-[4' ',6' '-O-(E)-diferuloyl]-beta-D-glucopyranoside (4), together with three known compounds, pinoresinol (5), syringaresinol (6), and pinoresinol-4'-O-beta-D-glucopyranoside (7), were isolated from the n-butanol extract of Rhus javanica var. roxburghiana, and their structures were established using various spectroscopic techniques. Three glycosides (2-4) of the lignans showed moderate inhibition of multiplication of the tobacco mosaic virus.

Rhusemialins A-C, new cyclolignan esters from the roots of Rhus javanica var. roxburghiana.

Three new cyclolignan esters and seven known compounds including two cyclolignan isolariciresinol, lynoiresinol, and five aromatic compounds methyl ferulate, vanillin, 4-hydroxy-3,5-dimethoxybenaldehyde, 4-methoxygallic acid, gallic acid were isolated from n-butanol extract of Rhus semialata (Anacardiaceae). The structural elucidations of rhusemialins A (1), B (2), and C (3) were based on FAB-MS, 1D and 2D NMR spectra.

Fractionation and identification of 9c, 11t, 13t-conjugated linolenic acid as an activator of PPARalpha in bitter gourd (Momordica charantia L.).

Bitter gourd (Momordica charantia L.) is a common vegetable in Asia that has been used in traditional medicine for the treatment of Diabetes. PPARs are ligand-dependent transcription factors that belong to the steroid hormone nuclear receptor family and control lipid and glucose homeostasis in the body. We previously reported that the ethyl acetate (EA) extract of bitter gourd activated peroxisome proliferator receptors (PPARs) alpha and gamma. To identify the active compound that activated PPARalpha, wild bitter gourd EA extract was partitioned between n-hexane and 90% methanol/10% H(2)O, and the n-hexane soluble fraction was further separated by silica gel column chromatography and finally by preparative HPLC. A transactivation assay employing a clone of CHOK1 cells stably transfected with a (UAS)(4)-tk-alkaline phosphatase reporter and a chimeric receptor of GAL4-rPPARalpha LBD was used to track the active component. Based on Mass, NMR, and IR spectroscopy, 9cis, 11trans, 13trans-conjugated linolenic acid (9c, 11t, 13t-CLN) was identified as a PPARalpha activator in wild bitter gourd. The isolated 9c, 11t, 13t-CLN rich fraction also significantly induced acyl CoA oxidase (ACO) activity in a peroxisome proliferator-responsive murine hepatoma cell line, H4IIEC3, implying that 9c, 11t, 13t-CLN was able to act on a natural PPARalpha signaling pathway as well. The content of 9c, 11t, 13t-CLN was estimated to be about 7.1 g/kg of our dried wild bitter gourd sample. The concentration of 9c, 11t, 13t-CLN and activation activity in the hydrolyzed EA extract of the seeds was higher than that of the flesh. The potential health benefits of 9c, 11t, 13t-CLN through the PPARalpha regulated mechanism are worthy to be further characterized in in vivo studies.

Salvinal, a novel microtubule inhibitor isolated from Salvia miltiorrhizae Bunge (Danshen), with antimitotic activity in multidrug-sensitive and -resistant human tumor cells.

Aqueous extracts of Salvia miltiorrhizae Bunge have been extensively used in the treatment of cardiovascular disorders and cancer in Asia. Recently, a compound, 5-(3-hydroxypropyl)-7-methoxy-2-(3'-methoxy-4'-hydroxyphenyl)-3-benzo[b]furancarbaldehyde (salvinal), isolated from this plant showed inhibitory activity against tumor cell growth and induced apoptosis in human cancer cells. In the present study, we investigated the cytotoxic effect and mechanisms of action of salvinal in human cancer cell lines. Salvinal caused inhibition of cell growth (IC50 range, 4-17 microM) in a variety of human cancer cell lines. Flow cytometry analysis showed that salvinal treatment resulted in a concentration-dependent accumulation of cells in the G(2)/M phase. We observed, using Hoechst 33258 dye staining, that salvinal blocked the cell cycle in mitosis. In vitro and in vivo examinations showed that salvinal inhibited tubulin polymerization in a concentration-dependent manner. Immunocytochemical studies demonstrated that salvinal treatment caused the changes of cellular microtubule network, similar to the effect of colchicine. In addition, salvinal treatment resulted in upregulation of cyclin B1 levels, activation of Cdc2 kinase, and Cdc25c phosphorylation. Furthermore, elevation of levels of MPM-2 phosphoepitopes in salvinal-treated cells in a concentration-dependent manner was also observed. Similar to the effect of other antitubulin agent, hyperphosphorylation of Bcl-2, induction of DNA fragmentation and activation of caspase-3 activity occurred in salvinal-treated cells. In particular, salvinal exhibited similar inhibitory activity against parental KB, P-glycoprotein-overexpressing KB vin10 and KB taxol-50 cells, and multidrug resistance-associated protein (MRP)-expressing etoposide-resistant KB 7D cells. Taken together, our data demonstrate that salvinal inhibits tubulin polymerization, arrests cell cycle at mitosis, and induces apoptosis. Notably, Salvinal is a poor substrate for transport by P-glycoprotein and MRP. Salvinal may be useful in the treatment of human cancers, particularly in patients with drug resistance.