Suppression of melanin synthesis by the phenolic constituents of sappanwood (Caesalpinia sappan).

Sappanwood (Caesalpinia sappan Linn.) is used as an herbal medicine. It is sometimes used to treat skin damage or as a facial cleanser. In the present study, the methanol (MeOH) extract of sappanwood was found to inhibit melanin synthesis in cultured human melanoma HMV-II cells stimulated with forskolin, and six active compounds (1-5 and 7) were isolated from the extract along with a non-active compound (6). Compounds 2-7 were identified as sappanchalcone (2), 3'-deoxy-4-O-methylsappanol (3), brazilein, (4), brazilin (5), sappanol (6), and 4-O-methylsappanol (7). Compound 1 was a new compound, and its structure was determined to be (6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c]chromene-3,6a,10,11-tetrol by spectroscopic analyses. Among the six active compounds, brazilin (5) (EC50: 3.0 ± 0.5 µM) and 4-O-methylsappanol (7) (EC50: 4.6 ± 0.7 µM) strongly suppressed melanin synthesis in HMV-II cells. Bioactive compounds showed moderate cytotoxicities against HMV-II cells with IC50 values of 83.1 ± 4.0 µM (for 2), 72.0 µM ± 2.4 (for 3), 33.8 ± 1.1 µM (for 4), 18.4 ± 0.8 µM (for 5), and 20.2 ± 0.8 (for 7), respectively. Brazilin (5) selectively suppressed the expression of mRNAs for tyrosinase-related protein (TYRP) 2 and tyrosinase but did not influence the expression of TYRP1. These results suggest that brazilin (5) is a new class of melanin inhibitor and that sappanwood could be used as a cosmetic material.

Anti-inflammatory and anti-melanogenic proanthocyanidin oligomers from peanut skin.

Peanut skin (Arachis hypogaea L., Fabaceae) is an abundant source for polyphenols, such as proanthocyanidin oligomers. To determine whether proanthocyanidin has beneficial effects on skin, we tested for inhibitory activity of proanthocyanidins isolated from peanut skin on inflammatory cytokine production and melanin synthesis in cultured cell lines. Administration of peanut skin extract (PSE, 200 µg/mL) decreased melanogenesis in cultured human melanoma HMV-II co-stimulated with phorbol-12-myristate-13-acetate. It also decreased production of inflammatory cytokines (PSE at 100 µg/mL), tumor necrosis factor-α and interleukin-6, in cultured human monocytic THP-1 cells in response to lipopolysaccharide. We isolated ten known proanthocyanidins and one new proanthocyanidin trimer from the PSE. The structure of the new compound (5) was determined by 1D- and 2D-NMR and mass spectrometry analyses, and was determined as epicatechin-(2ß→O→7,4ß→6)-epicatechin-(4ß→6)-epicatechin. The other known proanthocyanidins were identified as proanthocyanidin monomers (1), dimers (6-9), trimers (3-5) and tetramers (2, 10, 11). They showed suppressive activities against melanogenesis and cytokine production at concentrations ranging from 0.1-10 µg/mL. Among the tested compounds, suppressive activities of proanthocyanidin dimers or trimers in two assay systems were stronger than those obtained with monomer or tetramers. These data indicate that proanthocyanidin oligomers from peanut skin have the potential to reduce dermatological conditions such as inflammation and melanogenesis.

Oral Administration of Ren-Shen-Yang-Rong-Tang 'Ninjin'yoeito' Protects Against Hematotoxicity and Induces Immature Erythroid Progenitor Cells in 5-Fluorouracil-induced Anemia.

The purpose of this study was to investigate the efficacy of four different Japanese and Chinese herbal prescriptions, Ren-Shen-Yang-Rong-Tang (Ninjin'yoeito, NYT), Chai-Hu-Gui-Zhi-Gan-Jiang-Tang (Saikokeishikankyoto, SKKT), Si-Jun-Zi-Tang (Shikunshito, SKT) and Si-Wu-Tang (Shimotsuto, SMT), which are traditionally used for anemia and fatigue, against hematotoxicity in mice treated with 5-fluorouracil (5-FU). NYT 1-100 mg kg(-1) day(-1) injected orally for 7 consecutive days before and after 5-FU injection significantly suppressed reductions in red blood cell, white blood cell and platelet counts in peripheral blood, and accelerated their recovery. Administration of SKKT also produced a slight but significant improvement in 5-FU-induced erythrocytopenia, whereas SMT and SKT could not prevent anemia. Oral injection of NYT also inhibited 5-FU-induced decreases in peripheral reticulocyte and bone marrow cell counts on day 10, and markedly hastened their recovery on day 20, in a dose-dependent manner. Erythroid progenitor colonies, such as colony forming units-erythroid and burst forming units-erythroid, formed by marrow cells from mice treated with 5-FU were significantly increased by oral administration of NYT. These findings suggest that NYT has the potential to protect against hematotoxicity, and also has hematopoietic activity, through stimulation of immature erythroid progenitor cell differentiation.

The liquid culture filtrates of entomogenous fungus Paecilomyces tenuipes and its glycoprotein constituent protects against anemia in mice treated with 5-fluorouracil.

The purpose of the present study was to investigate the efficacy of a liquid culture filtrates of the entomogenous fungus Paecilomyces tenuipes (PTCF) and its main active glycoprotein-enriched (PGF) fraction against hematotoxicity in mice treated with 5-fluorouracil (5-FU). Oral administration of PTCF (100 mg/kg/d) for 7 consecutive days after 5-FU injection significantly suppressed reductions in the red and white blood cell counts in peripheral blood, and accelerated their recoveries. From PTCF, glycoprotein-enriched fraction (PGF, >90% protein, approximately 15 kDa determined by SDS-PAGE) was separated as active ingredient that ameliorates 5-FU-induced anemia. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of trypsinized-PGF showed 11 fragment ion peaks. Effective recoveries of erythrocytopenia and leukocytopenia were observed when PGF was co-administered with murine recombinant erythropoietin (mrEPO; 5 U/mouse). Oral administration of PGF also inhibited 5-FU-induced decreases in peripheral reticulocyte and bone marrow cell counts on day 12, and markedly hastened their recoveries on day 20, in dose-dependent manners. Reductions in erythroid progenitor colonies, such as colony-forming units (CFU)-erythroid and burst-forming units-erythroid mix, formed by bone marrow cells from 5-FU-treated mice were markedly improved by oral administration of PGF with subcutaneous mrEPO. Oral administration of PGF also increased the myeloid lineage progenitor, CFU-granulocyte-macrophages, in cultured bone marrow cells. These findings suggest that PGF isolated from P. tenuipes has the potential to protect against 5-FU-inudced erythrocytopenia and leukopenia, especially in combination with mrEPO, and also has hematopoietic activity, through stimulation of immature erythroid as well as myeloid progenitor cell differentiation.

The liquid culture filtrates of Paecilomyces tenuipes (Peck) Samson (=Isaria japonica Yasuda) and Paecilomyces cicadae (Miquel) Samson (=Isaria sinclairii (Berk.) Llond) regulate Th1 and Th2 cytokine response in murine Peyer's patch cells in vitro and ex vivo.

The effects of liquid culture filtrates of medicinal entomogenous fungi, Paecilomyces tenuipes (Peck) Samson (=Isaria japonica Yasuda or Isaria tenuipes) (PTCF) and Paecilomyces cicadae (Miquel) Samson (=Isaria sinclairii (Berk.) Llond) (PCCF), on cytokine productions in cultured Peyer's patches (PP) from C57BL/6J mice were investigated in vitro and ex vivo. In an in vitro experiment, PTCF (100 and 10 microg/ml) enhanced the production of T helper 1 (Th1) cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, in cultured PP cells stimulated with 5 microg/ml concanavalin A (Con A) but did not influence on the production of T helper 2 (Th2) cytokines, IL-4 and IL-5. PTCF also enhanced the production of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-10 in the cultured PP cells. While, PCCF enhanced the production of IFN-gamma but did not alter the level of IL-2 in the PP cells. In an ex vivo experiment using PP cells removed from the mice after oral treatment of PTCF (10 and 100 mg/kg daily for 7 consecutive days), the production of IL-2 and IFN-gamma were increased in response to Con A. On the other hand, orally treated PCCF (10 mg/kg/day) suppressed IL-2 production but did not change the levels of IFN-gamma and IL-10 in the isolated PP cells. The flow cytometric analysis revealed that the population of CD3(+) cells in the PP cells slightly but significantly increased after oral administration of PCCF. Orally administered PTCF did not change the population of T (CD3(+)), B (CD19(+)), T cell subset (CD4(+)and CD8(+)) and Th1 (IFN-gamma(+)) and Th2 (IL-4(+)). From PTCF, the fraction rich in proteoglycans was separated as active fraction that stimulates Th1 immune response. These results indicate that the mode of action of PTCF and PCCF on mucosal immune response is different and this is contributed to their metabolites. Taken together, there is a possibility of PTCF and PCCF being therapeutic or preventive agents for immune diseases such as cancer, allergy and parasitic disease through activation of mucosal immune response.

Protective effect of (+)-catechin against 5-fluorouracil-induced myelosuppression in mice.

The object of this study was to investigate the efficacy of (+)-catechin, which was isolated from Actinidia arguta Planch (Actinidiaceae), as a bone marrow cell proliferation-promoting compound against the hematotoxicity of 5-fluorouracil (5-FU) in mice. Intraperitoneally injected (+)-catechin (1 and 10 mg/kg per day) accelerated the recovery of the number of white blood cells (WBC) and platelets (PLT) but did not affect the number of circulating red blood cells (RBC). (+)-Catechin also augmented the number of myelocytes and splenocytes. Dual color flow cytometric analysis revealed that (+)-catechin reversed the reduction of the population of leukocytes (CD11b+ monocytes, Gr-1+ granulocytes and CD3+ T and CD45RA+ B lymphocytes) in whole blood, spleen and bone marrow caused by 5-FU. (+)-Catechin (1 and 10 mg/kg per day) showed remarkable recovery of Gr-1+ cells in all three types of tissues and of CD11b+ cells in the bone marrow cells. These findings suggest that (+)-catechin selectively enhances the recovery of the population of granulocytes reduced by 5-FU in mice.

Isolation of (+)-catechin and (-)-epicatechin from Actinidia arguta as bone marrow cell proliferation promoting compounds.

The MeOH extract of stems of Actinidia arguta promoted proliferation of cultured bone marrow cells and stimulated formation of myeloid colonies from bone marrow cells. (+)-Catechin ( 1) and (-)-epicatechin ( 2) were isolated as active compounds from the MeOH extract. Compounds 1 and 2 stimulated the cell proliferation in a concentration-dependent manner in the range of 1 to 100 mg/mL. Compounds 1 and 2 also stimulated formation of myeloid colonies and enhanced the effect of interleukin-3 (IL-3) to increase the number of colony forming-units in culture (CFU-c). In an ex vivo experiment using a model mouse of decreasing bone marrow functions, orally administrated 1 (100 mg/kg/day) stimulated IL-3-induced CFU-c formation of the bone marrow cells.