Fargesin, a component of Flos Magnoliae, stimulates glucose uptake in L6 myotubes.

Flos Magnoliae (FM) is a commonly used Chinese medicinal herb for symptomatic relief of allergic rhinitis, sinusitis and headache. Although several FM species have been used as substitutes or adulterants for clinical use, possible differences in their pharmacological actions have not been reported. To confirm the effects of FM on skeletal muscle glucose metabolism, we tested the effects of several compounds isolated from FM on glucose uptake by L6 myotubes. We found that fargesin, a component of FM, dose-dependently stimulated glucose consumption in L6 myotubes, which was accompanied by enhanced glucose transporter (GLUT)-4 translocation to the cell surface. Fargesin-stimulated glucose uptake was blocked by wortmannin, a phosphatidylinositol-3 kinase (PI3 K) inhibitor. Fargesin stimulated Akt phosphorylation, a key component in the insulin signaling pathway, which was completely inhibited by wortmannin. Here, we demonstrated that fargesin, a bioactive component of Flos Magnoliae, increases basal glucose uptake and GLUT4 translocation in L6 myotubes by activating the PI3 K-Akt pathway.

Honokiol and magnolol stimulate glucose uptake by activating PI3K-dependent Akt in L6 myotubes.

Honokiol and magnolol, ingredients of Magnolia officinalis, which is used in traditional Chinese and Japanese medicines, have been reported to have antioxidant, anticancer, and antiangiogenic effects. Effects of these compounds on glucose metabolism in adipocytes have also been reported. However, their effects on skeletal muscle glucose uptake and the underlying molecular mechanisms are still unknown. Here, we investigated the direct effects and signaling pathways activated by honokiol and magnolol in skeletal muscle cells using L6 myotubes. We found that honokiol and magnolol dose-dependently acutely stimulated glucose uptake without synergistic effects of combined administration in L6 myotubes. Treatment with honokiol and magnolol also stimulated glucose transporter-4 translocation to the cell surface. Honokiol- and magnolol-stimulated glucose uptake was blocked by the phosphatidylinositol-3 kinase inhibitor, wortmannin. Both honokiol and magnolol stimulated Akt phosphorylation, a key element in the insulin signaling pathway, which was completely inhibited by wortmannin. These results suggest that honokiol and magnolol might have beneficial effects on glucose metabolism by activating the insulin signaling pathway.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 µM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Osteogenic activity of diphenyl ether-type cyclic diarylheptanoids derived from Acer nikoense.

Osteogenic activity of six diarylheptanoids, acerogenin A (1), (R)-acerogenin B (2), aceroside I (3), aceroside B(1) (4), aceroside III (5) and (-)-centrolobol (6) and two phenolic compounds; (+)-rhododendrol (7) and (+)-cathechin (8), isolated from the stem bark of Acer nikoense (Nikko maple) was evaluated using alkaline phosphatase (ALP) activity as a marker for early osteoblast differentiation. We found that the diphenyl ether-type cyclic diarylheptanoids 1-5 promoted ALP activity in mouse preosteoblastic MC3T3-E1 cells without affecting cell proliferation, but linear-type diarylheptanoid 6 and phenolic compounds 7 and 8 did not. Diphenyl ether-type cyclic diarylheptanoids 1-4 also increased protein production of osteocalcin, a late stage maker for osteoblast differentiation, and induced osteoblastic mineralization. Structure-activity relationships of these compounds demonstrated that the stimulative efficacy of aglycones was higher than that of its glycosides. Taken together, diphenyl ether-type cyclic diarylheptanoids promote early- and late-stage osteoblastogenesis, which may open the possibility for the development of novel osteogenic agents.

Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes.

Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 µM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

Effects of a Citrus depressa Hayata (shiikuwasa) extract on obesity in high-fat diet-induced obese mice.

Citrus depressa Hayata (commonly known as shiikuwasa) is cultivated in the northern areas of Okinawa, Japan, and used as a juice. In this study, we examined the anti-obesity effects and mechanism of action of shiikuwasa peel extract (SE) using high-fat diet (HFD)-induced obese mice. Mice were fed a low-fat diet (LFD), HFD or HFD containing 1% or 1.5% (w/w) SE (HFD+1 SE and HFD+1.5 SE, respectively) for 5 weeks. The body weight gain and white adipose tissue weight were significantly decreased in the HFD+1.5 SE group compared with the HFD group. The plasma triglyceride and leptin levels were also significantly reduced in the HFD+1.5 SE group compared with the HFD group. Histological examinations showed that the sizes of the adipocytes were significantly smaller in the HFD+1.5 SE group than in the HFD group. The HFD+1.5 SE group also showed significantly lower mRNA levels of lipogenesis-related genes, such as activating protein 2, stearoyl-CoA desaturase 1, acetyl-CoA-carboxylase 1, fatty acid transport protein and diacylglycerol acyltransferase 1, than the HFD group. These results suggest that the anti-obesity effects of SE may be elicited by regulating the expressions of lipogenesis-related genes in white adipose tissue.

Harmine, a ß-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo.

Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive bone resorption is involved in the pathogenesis of bone-related disorders such as osteoporosis, arthritis and periodontitis. To obtain new antiresorptive agents, we searched for natural compounds that can inhibit osteoclast differentiation and function. We found that harmine, a ß-carboline alkaloid, inhibited multinucleated osteoclast formation induced by receptor activator of nuclear factor-κB ligand (RANKL) in RAW264.7 cells. Similar results were obtained in cultures of bone marrow macrophages supplemented with macrophage colony-stimulating factor and RANKL, as well as in cocultures of bone marrow cells and osteoblastic UAMS-32 cells in the presence of vitamin D(3) and prostaglandin E(2). Furthermore, harmine prevented RANKL-induced bone resorption in both cell and bone tissue cultures. Treatment with harmine (10 mg/kg/day) also prevented bone loss in ovariectomized osteoporosis model mice. Structure-activity relationship studies showed that the C3-C4 double bond and 7-methoxy group of harmine are important for its inhibitory activity on osteoclast differentiation. In mechanistic studies, we found that harmine inhibited the RANKL-induced expression of c-Fos and subsequent expression of nuclear factor of activated T cells (NFAT) c1, which is a master regulator of osteoclastogenesis. However, harmine did not affect early signaling molecules such as ERK, p38 MAPK and IκBα. These results indicate that harmine inhibits osteoclast formation via downregulation of c-Fos and NFATc1 induced by RANKL and represses bone resorption. These novel findings may be useful for the treatment of bone-destructive diseases.

Palmatine attenuates osteoclast differentiation and function through inhibition of receptor activator of nuclear factor-κb ligand expression in osteoblast cells.

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.

Honokiol inhibits osteoclast differentiation and function in vitro.

Honokiol, a neolignan, is a physiologically active component of kouboku (Magnolia obovata), a herb used in traditional Chinese medicine. This study investigated the effects of honokiol on the differentiation and function of osteoclasts induced by receptor activator of nuclear factor-kappaB ligand (RANKL). Honokiol markedly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP-positive multinucleated cells in both bone marrow-derived monocytes and RAW264 cells. In experiments to elucidate its mechanism of action, honokiol was found to suppress RANKL-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The RANKL-induced expressions of c-Fos and nuclear factor of activated T cells-c1 (NFATc1), which are crucial transcriptional factors for osteoclastogenesis, were also reduced by treatment with honokiol. Furthermore, honokiol induced disruption of the actin rings in mature osteoclasts (mOCs) without affecting the cell viability and suppressed osteoclastic pit formation on dentin slices. Taken together, these results suggest that honokiol inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK), decreasing the expressions of c-Fos and NFATc1, and attenuates bone resorption by disrupting the actin rings in mOCs. Therefore, honokiol could prove useful for the treatment of bone diseases associated with excessive bone resorption.

Identification of two biologically crucial hydroxyl groups of (-)-epigallocatechin gallate in osteoclast culture.

(-)-Epigallocatechin gallate (EGCG) induces cell death of osteoclasts in an Fe(2+)- and H(2)O(2)-dependent manner. In the present study, we further explore the cytotoxic mechanism of EGCG using four EGCG analogues. Molecules methylated at position 4' in the B ring (EGCG-4'-O-Me) or at position 4'' in the D-ring (EGCG-4''-O-Me) showed markedly decreased cytotoxicity to osteoclasts, indicating that hydroxyl groups at these two positions of EGCG are crucial for inducing cell death of osteoclasts. EGCG-4'-O-Me also showed the lowest Fe(3+)-reducing activity among five EGCGs. The Fe(3+)-reducing activity of EGCG was enhanced under conditions whereby protonated EGCG levels were increased, indicating that the protonated status of EGCG was involved in the Fe(3+)-reducing activity. The hydroxyl group at position 4'' in the D-ring was shown by quantum chemical calculation to be preferentially deprotonated among all of the hydroxyl groups in EGCGs. It was also shown that the highest occupied molecular orbital (HOMO) was localized to the B-ring of EGCGs, except for EGCG-4'-O-Me. We report here that the HOMO on the B-ring plays crucial roles in both the Fe(3+)-reducing activity of EGCG and the cytotoxicity of EGCG to osteoclasts, while deprotonation of the hydroxyl group at position 4'' in the D-ring plays a supplementary role.

Bioactive guaianolides from siyekucai (Ixeris chinensis).

Two new guaianolides, named chinensiolides D (5) and E (6), were isolated from Ixeris chinensis Nakai, and their structures were determined to be 10alpha-hydroxy-3-oxoguaia-11(13)-eno-12,6alpha-lactone (5) and 10alpha-hydroxy-3beta-O-[2,6-di(p-hydroxyphenylacetyl)-beta-glucopylanosyl]guaia-4(15),11(13)-dieno-12,6alpha-lactone (6). The first isolation of (11S)-10alpha-hydroxy-3-oxoguaia-4-eno-12,6alpha-lactone (4) from natural sources and its characterization are also reported. Chinenciolide E (6) showed significant growth inhibitory activity toward VA-13 malignant lung tumor cells (IC50 = 0.72 microM).

Chemotherapeutic efficacy of ascofuranone in Trypanosoma vivax-infected mice without glycerol.

Ascofuranone, an antibiotic isolated from Ascochyta visiae, showed trypanocidal activity in Trypanosoma vivax-infected mice. A single dose of 50 mg/kg ascofuranone effectively cured the mice without the help of glycerol. Repeated administrations of this drug further enhanced its chemotherapeutic effect. After two, three, and four consecutive days treatment, the doses needed to cure the infection decreased to 25, 12, and 6 mg/kg, so that the total doses administered were 50, 36 and 24 mg/kg, respectively. Ascofuranone (50 mg/kg) also had a prophylactic effect against T. vivax infection within the first two days after administration. This prophylactic activity diminished to 80% by day 3 and completely disappeared four days after administration. Of particular interest in this study was that ascofuranone had trypanocidal activity in T. vivax-infected mice in the absence of glycerol, whereas co-administration of glycerol or repeated administrations of this drug are needed for Trypanosoma brucei brucei infection. Our present results strongly suggest that ascofuranone is also an effective tool in chemotherapy against African trypanosomiasis in domestic animals.

Direct evidence for cyanide-insensitive quinol oxidase (alternative oxidase) in apicomplexan parasite Cryptosporidium parvum: phylogenetic and therapeutic implications.

Cryptosporidium parvum is a parasitic protozoan that causes the diarrheal disease cryptosporidiosis, for which no satisfactory chemotherapy is currently available. Although the presence of mitochondria in this parasite has been suggested, its respiratory system is poorly understood due to difficulties in performing biochemical analyses. In order to better understand the respiratory chain of C. parvum, we surveyed its genomic DNA database in GenBank and identified a partial sequence encoding cyanide-insensitive alternative oxidase (AOX). Based on this sequence, we cloned C. parvum AOX (CpAOX) cDNA from the phylum apicomplexa for the first time. The deduced amino acid sequence (335 a.a.) of CpAOX contains diiron coordination motifs (-E-, -EXXH-) that are conserved among AOXs. Phylogenetic analysis suggested that CpAOX is a mitochondrial-type AOX, possibly derived from mitochondrial endosymbiont gene transfer. The recombinant enzyme expressed in Escherichia coli showed quinol oxidase activity. This activity was insensitive to cyanide and highly sensitive to ascofuranone, a specific inhibitor of trypanosome AOX.

The efficacy of ascofuranone in a consecutive treatment on Trypanosoma brucei brucei in mice.

Consecutive administration of ascofuranone without glycerol was found to have therapeutic efficacy against Trypanosoma brucei brucei infection in mice. A suspension of ascofuranone (25-100 mg/kg) was administrated intraperitoneally every 24 h for 1-4 consecutive days to trypanosome-infected mice and efficacy was compared with oral treatment. With intraperitoneal administration, all mice treated with 100 mg/kg ascofuranone for 4 consecutive days were cured. On contrary, with oral treatment a higher dose of ascofuranone (400 mg/kg) was needed for 8 consecutive days to cure the mice. With intraperitoneal treatment, parasitemia was strongly suppressed, with almost all long slender bloodstream forms of the parasite changed to short stumpy forms by day 3 and the parasites have been eliminated 4 days after the start of treatment. These ascofuranone-induced short stumpy forms were morphologically analogous to the stumpy forms 2 days after peak parasitemia of pleomorphic clone of T. b. brucei GUTat 3.1. However, the properties of ubiquinol oxidase activity, which is the target of ascofuranone, in mitochondria isolated from before and after treatment, were almost same. The enzymatic activities of ubiquinol oxidase were only decreased to approximately 30% within a day after treatment, and then kept at nearly the same level. In the present study, we have improved regimen for administration of ascofuranone without glycerol, and demonstrated that consecutively administrated ascofuranone showed trypanocidal effects in T. b. brucei infected mice. Our present results strongly suggest that consecutive administration of ascofuranone may be an effective chemotherapy for African trypanosomiasis.

Safflower polysaccharides activate the transcription factor NF-kappa B via Toll-like receptor 4 and induce cytokine production by macrophages.

Two active polysaccharide fractions (SF1 and SF2) purified from dried safflower petals (Carthamus tinctorius L.) stimulated the synthesis of various cytokines by peritoneal macrophages. In a number of cell types, SF1 and SF2 induced a rapid degradation of IkappaB alpha essential for the activation of the transcription factor NF-kappaB. Toll-like receptor 4 (TLR4), but not TLR2, was expressed in all cell lines that responded to SF1 and SF2. Enforced expression of TLR4 and MD-2 rendered responsiveness to SF1 and SF2. Moreover, these safflower polysaccharides failed to induce the production of TNF-alpha and NO by peritoneal macrophages prepared from C3H/HeJ mice that have a point mutation in the Tlr4 gene. Thus, these observations clearly indicate that safflower polysaccharides activate the NF-kappaB signaling pathway via TLR4.