Effects of a Hot-Water Extract of Allium hookeri Roots on Bone Formation in Human Osteoblast-Like MG-63 Cells In Vitro and in Rats In Vivo.

Allium hookeri is a wild herb found mainly in the Himalayas, growing at altitudes of 1400-4200 m. A. hookeri is widely consumed as a vegetable and herbal medicine in Asia, but its effects on bone health have not been reported previously. This study investigated the effects of a hot-water extract of A. hookeri roots on bone formation. The hot-water extract significantly increased the proliferation of in vitro human osteoblast-like MG-63 cells and the stimulatory effects on osteoblast differentiation were noticeably greater for the hot-water extract than for daidzein (a positive control), as reflected by alkaline phosphatase activity, collagen content, and mineral deposition. Expression of the bone-remodeling marker osteocalcin production and bone microstructural parameters were significantly improved in Sprague-Dawley rats in vivo after oral treatment with the hot-water extract compared with their control (saline-administered) counterparts. The chemical compounds of the hot-water extract were characterized by liquid chromatography-mass spectrometry, and alliin, sinapic acid, and ferulic acid, which exert beneficial effects on bone health, were identified. These findings indicate that A. hookeri can be used as a natural resource for increasing bone formation. This is the first report of the anabolic effects of A. hookeri extracts on bone formation in vitro and in vivo.

Effects of Glucosinolates from Turnip (Brassica rapa L.) Root on Bone Formation by Human Osteoblast-Like MG-63 Cells and in Normal Young Rats.

Turnip (Brassica rapa L.) root ethanol extract (TRE) was prepared, and its chemical constituents were characterized by ultra-performance liquid chromatography and mass spectrometry. Thirteen glucosinolates (GSLs) were identified, comprising eight aliphatic, four indolic, and one aromatic compounds. The effects of these GSLs on bone formation were investigated in vitro by incubating human osteoblast-like MG-63 cells with TRE and then analyzing their viability, alkaline phosphatase (ALP) activity, collagen content, and mineralization and in vivo by administering TRE orally to normal young rats (500 mg/kg/day) and assessing subsequent changes in serum osteocalcin and bone microstructure in these animals. No TRE-related toxicity was found, and the levels of cell viability, ALP activity, collagen synthesis, and mineralization were significantly increased relative to the negative control. In particular, stimulatory effects on the differentiation of MG-63 cells were strongly enhanced as compared with a positive control (daidzein). Serum osteocalcin was also significantly increased, and some important bone microstructural parameters were improved in TRE-administered rats compared with their saline-administered counterparts. GSLs therefore appear to have a stimulatory effect on bone formation in both MG-63 cells and normal young rats. This is the first report on the usefulness of turnip root and its GSL compounds for bone formation.

Effects of Watercress Containing Rutin and Rutin Alone on the Proliferation and Osteogenic Differentiation of Human Osteoblast-like MG-63 Cells.

Most known osteoporosis medicines are effective for bone resorption, and so there is an increasing demand for medicines that stimulate bone formation. Watercress (N. officinale R. Br.) is widely used as a salad green and herbal remedy. This study analyzed a watercress extract using ultra-performance liquid chromatography/mass spectrometry, and identified a rutin as one of its major constituents. Osteogenic-related assays were used to compare the effects of watercress containing rutin (WCR) and rutin alone on the proliferation and differentiation of human osteoblast-like MG-63 cells. The reported data are expressed as percentages relative to the control value (medium alone; assigned as 100%). WCR increased cell proliferation to 125.0±4.0% (mean±SD), as assessed using a cell viability assay, and increased the activity of alkaline phosphatase, an early differentiation marker, to 222.3±33.8%. In addition, WCR increased the expression of collagen type I, another early differentiation marker, to 149.2±2.8%, and increased the degree of mineralization, a marker of the late process of differentiation, to 122.9±3.9%. Rutin alone also increased the activity of ALP (to 154.4±12.2%), the expression of collagen type I (to 126.6±6.2%), and the degree of mineralization (to 112.3±5.0%). Daidzein, which is reported to stimulate bone formation, was used as a positive control; the effects of WCR on proliferation and differentiation were significantly greater than those of daidzein. These results indicate that WCR and rutin can both induce bone formation via the differentiation of MG-63 cells. This is the first study demonstrating the effectiveness of either WCR or rutin as an osteoblast stimulant.

Cleavage of the terminal N-acetylglucosamine of egg-white ovalbumin N-glycans significantly reduces IgE production and Th2 cytokine secretion.

Ovalbumin (OA) is one of the most abundant of the glycoprotein allergens, and induces a T-helper type 2 immune response that results in an IgE-mediated hypersensitivity. In this study, the terminal carbohydrates of N-glycans from intact OA were cleaved with the exoglycosidases galactosidase, mannosidase, and N-acetylglucosaminidase to generate degalactosylated-OA, demannosylated-OA, and de-N-acetylglucosaminylated-OA, respectively, in order to evaluate their role in allergenicity. The exoglycosidase digestion procedure did not result in either degradation or contamination of the three deglycosylated sample, and the digestion efficiency was confirmed by comparing the results of glycan analysis of the three exoglycosidase-treated OAs with that of glycans of intact OA. Mice were immunized with either intact or exoglycosidase-treated OAs, and their respective allergic reactions were compared. IgE production in the de-N-acetylglucosaminylated-OA group was reduced to 58.8% of that in the intact OA group. In addition, the production levels of the cytokines interleukin-4 and interleukin-5 were significantly reduced in the de-N-acetylglucosaminylated-OA group to 53.4% and 45.8% of the levels in the intact OA group, respectively. However, there were almost no changes (or only slight reductions) in the degalactosylated-OA and demannosylated-OA groups, respectively. These results indicate that cleavage of the terminal carbohydrate, and particularly N-acetylglucosamine, reduces the allergenicity of OA. This is the first report of the effect of cleavage of the terminal carbohydrate on glycoprotein allergenicity.

Binding Specificity of Philyra pisum Lectin to Pathogen-Associated Molecular Patterns, and Its Secondary Structure.

We recently reported a Philyra pisum lectin (PPL) that exerts mitogenic effects on human lymphocytes, and its molecular characterization. The present study provides a more detailed characterization of PPL based on the results from a monosaccharide analysis indicating that PPL is a glycoprotein, and circular dichroism spectra revealing its estimated α-helix, ß-sheet, ß-turn, and random coil contents to be 14.0%, 39.6%, 15.8%, and 30.6%, respectively. These contents are quite similar to those of deglycosylated PPL, indicating that glycans do not affect its intact structure. The binding properties to different pathogen-associated molecular patterns were investigated with hemagglutination inhibition assays using lipoteichoic acid from Gram-positive bacteria, lipopolysaccharide from Gram-negative bacteria, and both mannan and ß-1,3-glucan from fungi. PPL binds to lipoteichoic acids and mannan, but not to lipopolysaccharides or ß-1,3-glucan. PPL exerted no significant antiproliferative effects against human breast or bladder cancer cells. These results indicate that PPL is a glycoprotein with a lipoteichoic acid or mannan-binding specificity and which contains low and high proportions of α-helix and ß-structures, respectively. These properties are inherent to the innate immune system of P. pisum and indicate that PPL could be involved in signal transmission into Gram-positive bacteria or fungi.