Lactobacillus plantarum GKM3 and Lactobacillus paracasei GKS6 Supplementation Ameliorates Bone Loss in Ovariectomized Mice by Promoting Osteoblast Differentiation and Inhibiting Osteoclast Formation.

Osteoporosis, an imbalance in the bone-forming process mediated by osteoblasts and the bone-resorbing function mediated by osteoclasts, is a bone degenerative disease prevalent among the aged population. Due to deleterious side effects of currently available medications, probiotics as a potential treatment of osteoporosis is an appealing approach. Hence, this study aims to evaluate the beneficial effects of two novel Lactobacilli strain probiotics on bone health in ovariectomized (OVX) induced osteoporotic mice model and its underlying mechanisms. Forty-five 9-week-old Institute of Cancer Research (ICR) mice underwent either a sham-operation (n = 9) or OVX (n = 36). Four days after the operation, OVX mice were further divided into four groups and received either saline alone, Lactobacillus plantarum GKM3, Lactobacillus paracasei GKS6 or alendronate per day for 28 days. After sacrifice by decapitation, right distal femur diaphysis was imaged via micro-computed tomography (MCT) and parameters including bone volume/tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and bone mineral density (BMD) were measured. Moreover, GKM3 and GKS6 on RANKL-induced osteoclast formation and osteoblast differentiation using in vitro cultures were also investigated. The results showed that both probiotics strains inhibited osteoporosis in the OVX mice model, with L. paracasei GKS6 outperforming L. plantarum GKM3. Besides this, both GKS6 and GKM3 promoted osteoblast differentiation and inhibited RANKL-induced osteoclast differentiation via the Bone Morphogenetic Proteins (BMP) and RANKL pathways, respectively. These findings suggested that both strains of Lactobacilli may be pursued as potential candidates for the treatment and management of osteoporosis, particularly in postmenopausal osteoporosis.

Biovalorization of soybean residue (okara) via fermentation with Ganoderma lucidum and Lentinus edodes to attain products with high anti-osteoporotic effects.

Okara, despite being a soybean processing by-product, still holds many nutrients. Thus, considerable attention has been recently paid to its reuse. In this study, solid-state fermentation was performed using Ganoderma lucidum and Lentinus edodes. Antioxidant activity and bioactive compound levels in G. lucidum-fermented okara (GLFO) and L. edodes-fermented okara (LEFO) were assayed. Antiosteoporosis bioactivity was evaluated using an animal model. The results demonstrated that solid-state fermentation significantly improved the antioxidant activity and bioactive compound levels. Furthermore, GLFO and LEFO increased trabecular bone volume, although only the GLFO-treated group exhibited significantly improved trabecular separation compared with the bilateral ovariectomy-treated control group. GLFO-related outcomes were superior to those of LEFO. The results demonstrate that okara products are effective for treating postmenopausal osteoporosis in humans.

Subchronic and Genetic Safety Assessment of a New Medicinal Dendrobium Species: Dendrobium Taiseed Tosnobile in Rats.

Dendrobium Taiseed Tosnobile is a new species of herba dendrobii (Shi-Hu) that was developed by crossbreeding D. tosaense and D. nobile. Its pharmacological activity and active component have been reported, but its subchronic toxicity and genetic safety have not yet been investigated. This study assessed the 90-day oral toxicity and genetic safety of the aqueous extracts of D. Taiseed Tosnobile (DTTE) in male and female Sprague-Dawley (SD) rats. Eighty rats were divided into four groups, each consisting of ten male and ten female rats. DTTE was given orally to rats at 800, 1600, or 2400 mg/kg for 90 consecutive days, and distilled water was used for the control group. Genotoxicity studies were performed using a bacterial reverse mutation assay and in vivo mammalian cell micronucleus test in ICR mice and analyzed using flow cytometry. Throughout the study period, no abnormal changes were observed in clinical signs and body weight or on ophthalmological examinations. Additionally, no significant differences were found in urinalysis, hematology, and serum biochemistry parameters between the treatment and control groups. Necropsy and histopathological examination indicated no treatment-related changes. Based on results, the no-observed-adverse-effect level of DTTE is greater than 2400 mg/kg in SD rats.

Type II arabinogalactan from Anoectochilus formosanus induced dendritic cell maturation through TLR2 and TLR4.

BACKGROUND: Polysaccharides, considered as immunomodulators with the capacity to activate immunity against microbial pathogens and tumors, have been employed for their dietary and medical benefits. PURPOSE: This study investigated the immunomodulatory effect of polysaccharide such as type II arabinogalactan from Anoectochilus formosanus (AGAF) on dendritic cell (DC) maturation and the underlying molecular mechanisms. METHODS AND RESULTS: Exposing DCs to AGAF induces cell maturation, which is characterized by the upregulation of CD86, CD83, CD80, CD40, and MHC class I and class II expression through flow cytometry analysis and morphological change without cytotoxicity. In addition, AGAF-triggered DC2.4 cells were involved in priming T-cell activation in vitro and in vivo. Transfection of toll-like receptor (TLR) 2 proteins and TLR4 siRNA suppressed DC maturation, suggesting that AGAF induced DC maturation through TLR2 and TLR4. CONCLUSION: These findings indicate that AGAF may be a potentially effective immunomodulator in stimulating DC maturation.

Characterization and immunomodulatory activity of polysaccharides derived from Dendrobium tosaense.

Dendrobium tosaense is a medicinal Dendrobium species widely used in traditional medicine. This study demonstrated some structural characterizations and immunomodulatory activity of the water-soluble polysaccharides derived from the stem of D. tosaense (DTP). DTP was fractioned using DEAE-650 M anion-exchange gel filtration chromatography, producing one neutral polysaccharide fraction (DTP-N), which was investigated for its structural characteristics, using HPAEC-PAD, HP-SEC, GC-MS, and NMR spectroscopy. DTP and DTP-N consisted of galactose, glucose, and mannose in ratios of 1:9.1:150.7 and 1:12.2:262.5, respectively. DTP-N comprised (1 → 4)Man as its main backbone, and its average molecular weight was 220 kDa. We also investigated the immunomodulatory effects of DTP administered orally to BALB/c mice for 3 weeks. DTP substantially boosted the population of splenic natural killer (NK) cells, NK cytotoxicity, macrophage phagocytosis, and cytokine induction in splenocytes. This is the first study to demonstrate the structural characteristics of an active polysaccharide derived from D. tosaense and its immunopharmacological effects in vivo.

Structurally characterized arabinogalactan from Anoectochilus formosanus as an immuno-modulator against CT26 colon cancer in BALB/c mice.

In this study, the innate immuno-modulatory effects and anti-cancer action of arabinogalactan (AG), a derivative of a well-known orchid, Anoectochilus formosanus, were investigated. The innate immuno-modulatory effects of AG were determined in vitro using RAW 264.7 cells for microarray analysis, and in vivo using BALB/c mice administrated with AG at 5 and 15 mg/kg intra-peritoneally for 3 weeks. The anti-cancer activity of AG was evaluated by CT26 colon cancer-bearing BALB/c mice. The microarray analysis was performed to evaluate the innate immunity and demonstrated that AG significantly induced the expression of cytokines, chemokines, and co-stimulatory receptors, such as IL-1α, CXCL2, and CD69. An intraperitoneal injection of AG in mice increased the spleen weight, but not the body weight. The treatment of mitogen, LPS significantly stimulated splenocyte proliferation in AG treated groups. The AG treatment also promoted splenocyte cytotoxicity against YAC-1 cells and increased the percentage of CD3(+)CD8(+) cytotoxic T cells in innate immunity test. Our experiments revealed that AG significantly decreased both tumour size and tumour weight. Besides, AG increased the percentage of DC, CD3(+)CD8(+) T cells, CD49b(+)CD3(-) NK cells among splenocytes, and cytotoxicity activity in tumour-bearing mice. In addition, the immunohistochemistry of the tumour demonstrated that the AG treatments increased the tumour-filtrating NK and cytotoxic T-cell. These results demonstrated that AG, a polysaccharide derived from a plant source, has potent innate immuno-modulatory and anti-cancer activity. AG may therefore be used for cancer immunotherapy.

A Type II Arabinogalactan from Anoectochilus formosanus for G-CSF Production in Macrophages and Leukopenia Improvement in CT26-Bearing Mice Treated with 5-Fluorouracil.

Anoectochilus formosanus is an herb well known in Asian countries. The polysaccharide isolated from A. formosanus consists of type II arabinogalactan (AGAF), with branched 3,6-Gal as the major moiety. In this study, AGAF was examined for the granulocyte colony-stimulating factor (G-CSF) production and related protein expression in RAW 264.7 murine macrophages. The signaling pathway of G-CSF production involves AGAF and mitogen-activated protein kinases (MAPKs) inhibitors and pattern-recognition receptor antibodies. AGAF was evaluated to ease the leukopenia in CT26-colon-cancer-bearing mice treated with 5-fluorouracil (5-FU). The results of this study showed that AGAF was a stimulant for Toll-like receptor 2 and Dectin-1 and that it induced G-CSF production, through p38 and ERK MAPK, and NF- κ B pathways. In vivo examination showed that the oral administration of AGAF mitigated the side effects of leukopenia caused by 5-FU in colon-cancer-bearing mice. In conclusion, the botanic type II AGAF in this study was a potent G-CSF inducer in vivo and in vitro.

The prebiotic effect of Anoectochilus formosanus and its consequences on bone health.

The present study evaluated the prebiotic effect of a standardised aqueous extract of Anoectochilus formosanus (SAEAF) and its effects on osteoporosis in ovariectomised (OVX) rats. The OVX rats were randomly divided into five groups and orally treated with water, SAEAF (200 and 400 mg/kg daily) and inulin (400 mg/kg daily) for 12 weeks. The sham group was orally treated with water. The SAEAF treatment enhanced the number of faecal bifidobacteria in OVX rats. The results of a Ca-balance experiment showed that SAEAF increased apparent Ca absorption and retention. The OVX rats were killed after SAEAF treatment lasting 12 weeks. The SAEAF decreased the caecal pH values and increased the caecal wall weight, caecal mucosa calbindin-D9k mRNA expression, free-Ca concentration and levels of SCFA in the caecum. The mineral content, density and biomechanical strength of bones were lower in OVX rats than the sham group, but these bone losses were prevented by SAEAF administration. Microtomography scanning showed that the SAEAF-treated rats had higher trabecular bone volume than the OVX rats. These results suggest that SAEAF prevented bone loss associated with ovarian hormone deficiency in the rats.

Characterization and prebiotic activity of aqueous extract and indigestible polysaccharide from Anoectochilus formosanus.

Anoectochilus formosanus (Orchidaceae) is a folk medicine in Asia. This study investigated the in vivo and in vitro prebiotic effects of an aqueous extract of A. formosanus (SAEAF) and of an indigestible polysaccharide (AFP) isolated from SAEAF. Chemical analyses showed AFP was mainly composed of arabinogalactan type II (AG-II), with an average molecular weight of 29 kDa. Following 4 weeks of oral administration to rats, SAEAF exhibited prebiotic effects including a decrease in cecum pH and increases of calcium absorption and fecal bifidobacteria. Furthermore, through a bioactivity-guided separation strategy, AFP was proven to be a bifidogenic component in vitro fecal strains fermentation and in vivo administration to mice. In RT-PCR analysis of Bifidobacterium , AFP increased the expression of ABC transporter related to nutrient uptake. Thus, AFP, a polysaccharide from A. formosanus, was demonstrated to be a prebiotic that has a positive health effect on gut microbiota.

Kinsenoside, a high yielding constituent from Anoectochilus formosanus, inhibits carbon tetrachloride induced Kupffer cells mediated liver damage.

AIM: In the present study, we have evaluated the hepatoprotective ability of kinsenoside, a major component of Anoectochilus formosanus, in vitro and in vivo. MATERIALS AND METHODS: The inhibitory action of kinsenoside on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells and Kupffer cells were investigated. Mice hepatic injury was produced by CCl(4) twice a week for 3 weeks. Mice in the three CCl(4) group were treated daily with water and kinsenoside throughout the experimental period. RESULTS: In LPS-stimulated macrophage RAW 264.7 cells and Kupffer cells, kinsenoside inhibited nitric oxide (NO) production and also blocked LPS-induced inducible NO synthase expression. Furthermore, kinsenoside inhibited the NF-κB activation induced by LPS, and this is associated with the abrogation of IκBα degradation, with subsequent decreases in nuclear p65 and p50 protein levels. Moreover, the phosphorylations of p38, ERK and JNK in LPS-stimulated RAW 264.7 cells were suppressed by kinsenoside. In the in vivo study, kinsenoside significantly protected the liver from injury, by reducing the activities of plasma aminotransferase, and by improving the histological architecture of the liver. kinsenoside inhibited Kupffer cell activation by reducing the CD 14 mRNA and protein expressions. CONCLUSION: These results indicate that kinsenoside alleviates CCl(4)-induced liver injury, and this protection is probably due to the suppression of Kupffer cell activation.

A standardized aqueous extract of Anoectochilus formosanus ameliorated thioacetamide-induced liver fibrosis in mice: the role of Kupffer cells.

Anoectochilus formosanus is used in traditional folk medicine as an hepatoprotective agent. The purpose of this study was to investigate the effects of a standardized aqueous extract of A. formosanus (SAEAF) on thioacetamide (TAA)-induced liver fibrosis. An in vitro study showed that the inhibitive effect of kinsenoside, a major component of SAEAF, on tumor necrosis factor alpha (TNF-alpha) secretion from Kupffer cells might be derived at least partly from downregulation of LPS-receptor Toll-like receptor 4 (TLR4) signaling. Hepatic fibrosis was produced by TAA (200 mg/kg, i.p.) 3 times per week for 12 weeks. Mice in the three TAA groups were treated daily with distilled water and SAEAF (1.0, 0.2 g/kg) via gastrogavage throughout the experimental period. The mice that received the SAEAF treatment had significantly reduced plasma alanine aminotransferase activity, relative liver weights, and hepatic hydroxyproline contents. A histological examination also confirmed that SAEAF reduced the degree of fibrosis caused by TAA treatment. RT-PCR analysis showed that SAEAF treatment reduced mRNA expression of collagen (alpha1)(I), lipopolysaccharide-binding protein, CD14, TLR4, and TNF receptor 1. An immunohistochemical examination also indicated that SAEAF reduced the number of CD68-positive cells (macrophages). In conclusion, oral administration of SAEAF significantly reduced TAA-induced hepatic fibrosis in mice, probably through inhibition of hepatic Kupffer cell activation.