Anti-inflammatory Effect of the Water-Soluble Portion of Porcine Placental Extract in Lipopolysaccharide-Stimulated RAW264.7 Murine Macrophage Cells.

Porcine placental extract (PPE) is used as a nonprescription drug for analeptics and in health foods and cosmetics in Japan, Korea and China. It was reported that PPE has anti-oxidative and anti-inflammatory activities; however, the mechanisms and the responsible molecules involved in these activities are still unclear. Here, we investigated how enzymatically prepared PPE affects proinflammatory factors such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in a cultured macrophage cell line, RAW264.7, when co-stimulated with lipopolysaccharide (LPS). Enhanced production of IL-1ß, IL-6 and TNF-α by LPS was significantly reduced by the addition of PPE and these effects were dose dependent. Nitric oxide (NO) production induced in cultured macrophages by LPS was also inhibited by PPE. Real-time PCR after the reverse transcription of total RNAs isolated from cells treated with PPE revealed that the mRNA expressions of IL-1ß, IL-6, TNFα, and NO synthase (NOS)-2 were reduced. The necessary concentration of PPE prepared by enzymatic digestion to mediate anti-inflammatory effects compared with the reported value of that extracted by phosphate buffered saline without digestion was proportional to the amount of extracted materials from the same amount of placenta (about 10-fold). This suggests that the molecules responsible for the anti-inflammatory activity exists in the placenta and can be extracted by phosphate buffered saline, and thus might survive enzymatic digestion.

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 constituents from Tabebuia avellanedae.

Five novel compounds were isolated from the water extract of Tabebuia avellanedae, and their structures were established by analysis of NMR spectroscopy and mass spectrometry. Compounds 1-5 at 25µM showed strong inhibitory activity on the inflammatory cytokine, tumor-necrosis factor-α and interleukin-1ß production in cultured human myeloma THP-1 cells co-stimulated with lipopolysaccharide without any significant cytotoxicity, and their anti-allergic and antioxidant activities were evaluated.

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.

Frequency of myeloid dendritic cells can predict the efficacy of Wilms' tumor 1 peptide vaccination.

BACKGROUND: The object of this study was to investigate the clinical predictive capability of peripheral myeloid dendritic cells (DCs) in Wilms' tumor 1 (WT1) vaccine therapy for patients with gynaecological cancer. PATIENTS AND METHODS: Six patients with WT1/human leukocyte antigen (HLA)-A*2402-positive gynaecological cancer were included in this study. The patients received intradermal injections of a modified 9-mer WT1 peptide every week for 12 weeks. Peripheral blood samples were obtained at 0, 4, 8 and 12 weeks after the initial vaccination. Circulating DCs were detected by flow cytometry. RESULTS: The frequencies of CD14(+)CD16(+)CD33(+)CD85(+) myeloid DCs were significantly higher in the therapeutically effective group than in therapeutically inert group (p<0.05). CONCLUSION: These results suggested that myeloid DCs, which should be associated with inducing cytotoxic T-cells, provided additional prognostic information in the use of cancer peptide vaccine.

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell-to-cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)-ß1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc-1) cells. TGF-ß1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E-cadherin from cell-to-cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT-specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG-ß1-treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF-ß1 and may contribute to EMT.

Anti-inflammatory and anti-melanogenic steroidal saponin glycosides from Fenugreek (Trigonella foenum-graecum L.) seeds.

Fenugreek seed ( Trigonella foenum-graecum L.) is used as an herbal medicine for treating metabolic and nutritive dysfunctions. To determine if this plant has other beneficial effects, we tested the inhibitory activities of a methanol (MeOH) extract of fenugreek seed on the production of inflammatory cytokines and melanin synthesis in cultured cell lines in vitro. The MeOH extract inhibited the production of phorbol-12-myristate-13-acetate-induced inflammatory cytokines such as tumor necrosis factor (TNF)-α in cultured THP-1 cells, and also restrained the intracellular synthesis of melanin in murine melanoma B16F1 cells. We isolated three active constituents from fenugreek seed extracts. These were identified as the steroidal saponins 26- O-ß-D-glucopyranosyl-(25 R)-furost-5(6)-en-3 ß,22 ß,26-triol-3- O-α-L-rhamno-pyranosyl-(1'' → 2')-O-[ß-D-glucopyranosyl-(1''' → 6')- O]-ß-D-glucopyranoside 1, minutoside B 2, and pseudoprotodioscin 3. Compounds 1 and 2 strongly suppressed the production of inflammatory cytokines, whereas 3 showed a weaker suppressing effect. Melanogenesis in B16F1 cells was significantly suppressed by 1 and 3, and weakly suppressed by 2. All three compounds showed moderate cytotoxicities. These results indicate that fenugreek extract and its active constituents could protect against skin damage.

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.

Bioactive monoterpene glycosides conjugated with gallic acid from the leaves of Eucalyptus globulus.

Two monoterpene glycosides, conjugated with gallic acid [globulusin A (1) and B (2)], together with four known compounds, cypellocarpin A (3), eucaglobulin (4), cuniloside (5) and (1S, 2S, 4R)-trans-2-hydroxy-1,8-cineole beta-d-glucopyranoside (6), were isolated from hot-water extracts of the leaves of Eucalyptus globulus. The structures of compounds 1 and 2 were determined by 1D, 2D NMR and MS spectroscopic analyses. The absolute stereochemistry of 1 was determined by correlating the spectroscopic data with those of synthetic compound 6 with a known configuration. Globulusin A (1) and B (2), cypellocarpin A (3) and eucaglobulin (4), scavenged DPPH free radicals and globulusin A (1) showed a higher antioxidant activity than the other tested compounds, with an IC50 of 3.8microM. Globulusin A (1) and eucaglobulin (4) concentration-dependently suppressed inflammatory cytokine production, tumor-necrosis factor-alpha and interleukin-1beta in cultured human myeloma THP-1 cells co-stimulated with phorbol myristate acetate. These compounds also inhibited melanogenesis in cultured murine melanoma B16F1 cells, without any significant cytotoxicity. These results suggested that globulusin A (1) and eucaglobulin (4), which were isolated as antioxidants from E. globulus, also had anti-inflammatory as well as anti-melanogenesis activity.

Cytotoxic fatty acid from Pleurocybella porrigens.

The new conjugated ketonic fatty acid, porrigenic acid (1), was isolated as a cytotoxic constituent of Pleurocybella porrigens. The structure of 1 was elucidated using spectroscopic methods including 1D and 2D NMR and MS. The absolute stereochemistry of 1 was determined by application of the exciton chirality method. Compound 1 exhibited cytotoxic activity against myeloma THP-1 cells, but did not show any significant toxicity against B16F1 melanoma. This is the first report of the isolation and structural elucidation of the new cytotoxic constituent porrigenic acid (1) from the edible mushroom P. porrigens.

Aqueous extract of peanut skin and its main constituent procyanidin A1 suppress serum IgE and IgG1 levels in mice-immunized with ovalbumin.

In this study, we investigated the effects of an aqueous extract of peanut (Arachis hypogaea L.) seed skin (PSE) and its main constituent procyanidin A1 (PA) on the allergic response to allergen ovalbumin (OVA) in a mouse model. Mice immunized interaperitoneally with OVA dramatically increased anti-OVA IgE and total IgG1 levels in serum compared with non-treated control mice. Oral injection of PSE at doses ranging from 10 to 100 mg/kg/d (for 21 consecutive days) decreased anti-OVA IgE and IgG1 levels 21 d after OVA-immunization. OVA-induced increments in spleen weight and peripheral white blood cell count were also suppressed by this PSE administration. Polyphenol-enriched fractions from apple (30 mg/kg) and grape seed (30 mg/kg) also decreased anti-OVA IgE level but did not affect total IgG1 levels. Oral injection of PA (1 to 10 mg/kg/d) purified from PSE resulted in a suppression of IgE and total IgG1 levels in serum. An increment of serum interleukin-4 level in mice that were immunized with OVA was reduced by all tested samples, whereas PSE and PA were the only compounds that could reverse the reduced interferon-gamma level by OVA. These findings suggest that intake of PSE or its main active constituent PA may prevent an allergic reaction by inhibiting immunoglobulin synthesis, and the mechanism of this action of PSE and PA is in part due to their regulation of T helper cytokine production.

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.

Water-soluble extracts from Angelica acutiloba Kitagawa enhance hematopoiesis by activating immature erythroid cells in mice with 5-fluorouracil-induced anemia.

The extract from the root of Angelica acutiloba Kitagawa (AR), which is used as herbal medicine in Japan, has been reported to be clinically effective for postmenstrual blood loss and erythropoietin (EPO)-resistant anemia in chronic renal failure, although the pharmacological mechanisms underlying its clinical efficacy are unknown. We prepared an animal model of anemia by bolus injection of 5-fluorouracil (5FU) at 150 mg/kg to mice (8- to 12-week-old female C57BL/6J), and then administered orally the water-soluble fraction of AR to the anemic mice for 10 days. After confirming the anti-anemic effect of the water-soluble fraction of AR (AR-3) containing polysaccharides, we examined the effects of AR-3 on immature erythroid cell activity, EPO production, and plasma cytokine levels. AR-3 administration at 50 mg/kg activated erythroid progenitor cells in bone marrow on day 10, increased the percentage of peripheral reticulocytes in red blood cells on day 15, and led to the recovery of red blood cell count to a value that was almost equal to the basal level on day 20. Although EPO production, which was determined by examining EPO mRNA expression in kidney and liver, remained unaltered by AR-3 administration, this treatment significantly lowered plasma interferon-gamma level, which may suppress the activity of erythroid progenitor cells. These results suggest that the polysaccharides in AR promote hematopoiesis by activating immature erythroid cells, in part, by suppressing cytokine secretion. Since the hematopoietic effect was achieved by high-dose AR-3, identification of specific polysaccharides is still required for the development of a novel medicine for anemia caused by a malignancy or chemotherapy.

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.