Hot water extract of adzuki (Vigna angularis) suppresses antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice.

The hot water extract of adzuki (HWEA), which is produced as a byproduct in the adzuki bean boiling process, has anti-tumor, antioxidative, and anti-diabetic activities. In this study, we fractionated HWEA to 4 fractions using stepwise gradient column chromatography with water and ethanol, and demonstrated the effects of each fraction on antigen (Ag)-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. The 40% ethanol eluate extract (EtEx.40) showed the strongest inhibition level of these fractions. To reveal the inhibitory mechanisms underlying degranulation by EtEx.40, we investigated intracellular reactive oxygen species (ROS) production, intracellular free Ca²âº concentration ([Ca²âº]i), and early intracellular signaling pathways. Treatment with EtEx.40 markedly inactivated Lyn following Ag stimulation, resulting in the suppressions of intracellular elevation of [Ca²âº]i and production of ROS. To identify the active compound in EtEx.40, we isolated 7 flavonoids from EtEx.40 and calculated their inhibition levels on Ag-stimulated degranulation. These flavonoids inhibited degranulation by about 25-60%. We further examined the in vivo effects of HWEA or EtEx.40 using a passive cutaneous anaphylaxis (PCA) reaction. Both extracts strongly suppressed the PCA reaction. These findings suggest that HWEA and/or EtEx.40 are beneficial for alleviating type I allergic symptoms.

Achyranthoside H methyl ester, a novel oleanolic acid saponin derivative from Achyranthes fauriei roots, induces apoptosis in human breast cancer MCF-7 and MDA-MB-453 cells via a caspase activation pathway.

Achyranthoside H methyl ester (AH-Me) is an oleanolic acid saponin derivative isolated from the roots of Achyranthes fauriei through diazomethane treatment. AH-Me exhibited significant cytotoxicity against human breast cancer MCF-7 and MDA-MB-453 cells, with respective ID(50) values of 4.0 and 6.5 muM: in the MTT assay. AH-Me is a unique saponin containing three methoxycarbonyl groups in the sugar moiety linked to the C-3 position of oleanolic acid. The demethylation of these methoxycarbonyl groups by alkaline hydrolysis caused a marked reduction of the cytotoxicity of AH-Me, suggesting that the methoxycarbonyl groups of AH-Me are key groups for the acquisition of cytotoxicity against human cancer cells. The staining of cancer cells with 4',6'-diamidino-2-phenylindole (DAPI) showed that the population of cells with altered nuclear morphology, for example chromatin condensation and fragmentation, increased markedly after AH-Me treatment. Exposure of MCF-7 and MDA-MB-453 cells to AH-Me resulted in a dose-dependent and time-dependent increase in the sub-G1 population, and in the cleavage of poly-ADP-ribose polymerase (PARP) followed by the formation of an 89 kD peptide. Pretreatment of the cells with the pan-caspase inhibitor z-VAD-fmk abolished the cleavage of PARP by AH-Me treatment and suppressed the antiproliferative effect of AH-Me on tumor cell growth. These results together led to the suggestion that AH-Me induces apoptosis via the caspase activation pathway in human breast cancer cells, and apoptosis is the major mode of the cytotoxic effect triggered by AH-Me.

Two new glucuronide saponins, Achyranthosides G and H, from Achyranthes fauriei root.

Two oleanolic acid saponins named achyranthosides G (1) and H (2) were newly isolated from Achyranthes fauriei root as methyl esters in addition to methyl esters of achyranthosides A - F and five oleanolic acid glucuronides (chikusetsusaponins IVa, V, 28-deglucosyl chikusetsusaponin V, pseudoginsenoside RT(1), and oleanolic acid 3-O-beta-D-glucuronopyranoside) as well as oleanolic acid 28-O-beta-D-glucopyranoside, beta-ecdysterone, and polypodine B. Their structures were characterized as follows on the basis of the chemical and spectroscopic evidences.

Specific inhibition of hypoxia-inducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids.

Screening using a reporter under the control of the hypoxia-response element (HRE) identified several flavonoids and homoisoflavonoids that inhibit the activation of HRE under hypoxic conditions. Among various compounds, isorhamnetin, luteolin, quercetin, and methyl ophiopogonanone B (MOB) were effective at 3 to 9 microg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by MOB in HepG2 cells, but the effective doses were 10 to 20 microg/ml. MOB caused destabilization of hypoxia-inducible factor (HIF)-1alpha, as revealed by Western blotting, that was dependent on proteasome activity and the tumor suppressor, p53. The tubular formation and migration of human umbilical vein endothelial cells was also inhibited by MOB. MOB is expected to act as an inhibitor of angiogenesis.

Cytotoxic and radical scavenging activity of blended herbal extracts.

Chinese medicines have been applied to a variety of diseases producing various favorable effects, possibly due to the interactions between individual components. Establishment of an evaluation method for such interactions may facilitate the production of new natural medicines. We investigated here the interaction of the hot water extract of Aconiti Tuber (one of the most prominent Chinese medicines) and that of Scutellariae Radix, Coptidis Rhizoma, Glycyrrhizae Radix, Atractylodesi, Lanceae Rhizoma or Poria, by measuring the superoxide anion (O2-), hydroxyl radical (OH) and nitric oxide (NO) scavenging activity, using ESR spectroscopy. We found that a 1:1 mixture of the hot water extract of one herb and that of another herb (referred to as a combined formula) showed a higher radical scavenging activity and cytotoxic activity than the hot water extract of a 1:1 mixture of two herbs (referred to as a blended formula). Both formulae showed higher cytotoxic activity against human oral tumor cell lines than against normal cells. These data further confirm the medicinal usefulness of combinations of empirical Chinese medicines.

Sophoranone, extracted from a traditional Chinese medicine Shan Dou Gen, induces apoptosis in human leukemia U937 cells via formation of reactive oxygen species and opening of mitochondrial permeability transition pores.

Screening of various natural products in a search for novel inducers of apoptosis in human leukemia cells led us to identify the strong apoptosis-inducing activity in a fraction extracted with methanol from the roots of Sophora subprostrata Chun et T. Chen. We purified the compound that induced apoptosis in human leukemia cells and identified it as sophoranone. Sophoranone inhibited cell growth and induced apoptosis in various lines of cells from human solid tumors, with 50% inhibition of growth of human stomach cancer MKN7 cells at 1.2 +/- 0.3 microM. The growth-inhibitory and apoptosis-inducing activities of sophoranone for leukemia U937 cells were very much stronger than those of other flavonoids, such as daidzein, genistein and quercetin. At the early stages of treatment of U937 cells with sophoranone, reactive oxygen species were formed, mitochondrial permeability pores were opened and cytochrome c was released from mitochondria. Cytochrome c was also released upon treatment of isolated mitochondria with sophoranone. Inhibitors of complexes III and IV, but not complexes I and II, of the mitochondrial respiratory chain prevented the release of cytochrome c from isolated mitochondria by sophoranone, as well as the induction of apoptosis in U937 cells in response to sophoranone. Our results indicate that sophoranone might be a unique apoptosis-inducing anticancer agent that targets mitochondria.