Osteoporosis is one of the prevalent diseases in ageing populations. Due to side effects of many chemotherapeutic agents, there is always a need to search for herbal products to treat the disorder. Punica granatum (PG) represent a potent fruit-bearing medicinal herb which exerted valuable anti-osteoporotic activities. The present study was carried out to validate the in vitro osteogenic effects of the PG seed extract in primary calvarial osteoblast cultures harvested from neonatal rats. The ethanolic extract of PG was subjected to evaluate cell proliferation, regeneration, mineralization and formation of collagen matrix using MTT, alkaline phosphatase, Alizarin Red-S staining and Sirius Red dye, respectively. Cell cycle progression and osteogenic gene Runx2 expression were carried out by flow cytometry and real time PCR, respectively. Exposure of different concentrations (10-100 μg/ml) of the extract on osteoblastic cells showed characteristic morphological changes and increment in cell number. A significant growth in cell proliferation, ALP activity, collagen contents and matrix mineralization of osteoblasts in a dose dependent manner (p < 0.05), suggested that PG has a stimulatory effect on osteoblastic bone formation or potential activity against osteoporosis. In addition, PG extract also enhanced DNA content in S phase of cell cycle and Runx2 gene expression level in osteoblasts. The data clearly indicated that PG promoting bone cell proliferation and differentiation in primary osteoblasts might be due to elevating the osteogenic gene Runx2 expression. The present study provides an evidence for PG could be a promising herbal medicinal candidate that able to develop drugs for osteoporosis.

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Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts

Bone undergoes continuous remodeling through bone formation and resorption, and maintaining the balance for skeletal rigidity. Bone resorption and loss are generally attributed to osteoclasts. Differentiation of osteoclasts is regulated by receptor activator of nuclear factor NF-kB ligand (RANKL), a member of tumor necrosis factor family. When the balance is disturbed, pathological bone abnormality ensues. Through the screening of traditional Korean medicinal plants, the effective molecules for inhibition and stimulation of RANKL-induced osteoclast differentiation in mouse bone marrow macrophages were identified. Among 222 methanol extracts, of medicinal plants, 10 samples exhibited ability to induce osteoclast differentiation. These include Dryobalanops aromatica, Euphoria longana, Lithospermum erythrorhizon, Prunus mume, Prunus nakaii, and Polygonatum odoratum. In contrast, Ailanthus altissima, Curcuma longa, Solanum nigrum, Taraxacum platycarpa, Trichosanthes kirilowii, and Daphne genkwa showed inhibitory effects in RANKL-induced osteoclast differentiation.

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Screening of Korean medicinal plants for possible osteoclastogenesis effects in vitro

Exposure to ultraviolet (UV) radiation induces various pathological changes, such as thickened skin and wrinkle formation. In particular, UVB irradiation increases matrix metalloproteinase (MMP)-1 production and collagen degradation, leading to premature aging, termed photoaging. The azuki bean (Vigna angularis; VA) has been widely used as a food product as well as a traditional medicine. However, its activity needs additional study to confirm its functional application in foods and cosmetics for protecting skin. In this study, hot-water extract from VA (VAE) and its active component, rutin, were investigated to determine their antiphotoaging effects. VAE was found to have antioxidant activity. In UVB-exposed normal human dermal fibroblasts cells with VAE and rutin treatments, MMP-1 production was significantly suppressed (90% and 47%, respectively). The effects of both topical and oral administration of VAE were tested in UVB-irradiated hairless mice. VAE suppressed wrinkle formation and skin thickness by promoting elastin, procollagen type I, and TGF-β1 expression (118%, 156%, and 136%, respectively) and by diminishing MMP-1 production. These results suggest that VAE may be effective for preventing skin photoaging accelerated by UVB radiation.

Vigna angularis Water Extracts Protect Against Ultraviolet B-Exposed Skin Aging In Vitro and In Vivo

Objectives The seed of Phaseolus calcaratus Roxburgh (PHCR) has traditionally been used as a herbal medicine, considered to have anti-inflammatory potential. Here we examined the ability of PHCR seed extract to inhibit inflammatory responses of macrophages to bacterial toxin and the mechanism involved.

Methods  In the present study, we prepared four fractions from an ethanol extract of PHCR seed and investigated their effects on the production of nitric oxide and cytokines, and the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells.

Key findings  The fractions inhibited LPS-induced nitric oxide production and cyclooxygenase-2 (COX-2) expression in the cells. The ethyl acetate fraction at 100 µg/ml almost completely suppressed NO production, iNOS and COX-2 expression, and TNF-α and IL-6 secretion in cells stimulated with LPS. The fraction also inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and p38 in LPS-stimulated cells with the attendant suppression of IκBα nuclear translocation and nuclear factor (NF)-κB activation. Furthermore, PHCR seed extracts contained a large number of phenolic compounds having antioxidant potentials against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and hydroxyl radicals. We identified catechin-7-O-β-d-glucopyranoside as one of the active compounds responsible for the biological activity of PHCR seed extract.

Conclusions  These results suggest for the first time that ethanol extracts from PHCR seed have anti-inflammatory potential on LPS-stimulated macrophages through the down-regulation of ERK/p38- and NF-κB-mediated signalling pathways.

Anti-inflammatory potential of Phaseolus calcaratus Roxburgh, a oriental medicine, on LPS-stimulated RAW 264.7 macrophages

In this study, the possible molecular mechanisms involved in the estrogen-like activities of ethanol extracts from two common Chinese legumes, i.e. Adzuki bean (Phaseolus angularis Wight.) and Lima bean (Phaseolus lunatus L.) were investigated. Reverse transcription-polymerase chain reaction (RT-PCR) measurement and western blot analysis were conducted to detect the functional mRNA and protein expressions, while immunofluorescence was used to determine the estrogen receptor (ER) intracellular distribution in the MCF-7/BOS cell line. The results indicated that (1) both mRNA and protein levels of progesterone receptor (PR) in the cell line increased after 48 h treatment with the extracts compared with those in the control; (2) Adzuki and Lima bean extracts might alter the ER intracellular distributions in two different manners: for the Adzuki bean, the ER appeared in both cytoplasm and nucleus, while for the Lima bean, it exhibited the same pattern as that of estradiol in which the ER almost disappeared in the nucleus and mainly occurred in the cytoplasm. Above all, the results showed a novel pattern of ER intracellular distribution in the MCF-7/BOS cell line upon exposure to Adzuki bean extract, suggesting that different pathways were involved in the estrogen-like activities of the ethanol extracts of Adzuki bean and Lima bean.

Effects of ethanol extracts from Adzuki bean (Phaseolus angularis Wight.) and Lima bean (Phaseolus lunatus L.) on estrogen and progesterone receptor phenotypes of MCF-7/BOS cells.

Ethanol extracts of several Chinese legumes were prepared and their estrogen-like activities were examined using the modified MCF-7 cell proliferation assay method (E-SCREEN assay). The possible mechanisms were addressed using cell cycle analysis and reverse transcription-polymerase chain reaction (RT-PCR) measurement in which a pure estrogen receptor antagonist ICI182,780 was employed as the tool agent. The legumes selected in this study were Mung bean (Vigna radiata (L.) Wilczek), Adzuki bean (Phaseolus angularis Wight.), Black soybean (Glycine max (L.) Merr.), Lima bean (Phaseolus lunatus L.), Broad bean (Vicia faba L.), Pea (Pisum sativum L.), Common bean I (Phaseolus vulgaris L.), and Common bean II (Phaseolus vulgaris var. humilis Alief), respectively. The results showed that ethanol extracts of Adzuki bean (100  μg/ml), Black soybean (1–200 μg/ml) and Lima bean (100 μg/ml), could strongly stimulated the proliferation of MCF-7 cells (P<0.01 vs. control), of which the estrogen-like effects of the one from Adzuki bean and Lima bean were the firstly reported. Cell cycle analysis revealed that the proliferation stimulatory effects of ethanol extracts of Adzuki bean, Black soybean and Lima bean were associated with a significant increase in the number of MCF-7 cells in S phase and in the G2/M population, similar to those of 17β-estradiol (E2). Using RT-PCR measurement, it was found that ethanol extracts of Adzuki bean, Black soybean and Lima bean could induce pS2 transcription in MCF-7 cells. Moreover, these reactions could be significantly inhibited by the specific estrogen receptor antagonist ICI182,780 (P<0.01 vs. the same concentration of compound without treatment of ICI182,780), suggesting that ethanol extracts of Adzuki bean, Black soybean and Lima bean exhibited estrogen-like activities through the estrogen response pathway by interacting with the estrogen receptor.

Qing-wei Zhao

Papers

Effects of ethanol extracts from Adzuki bean (Phaseolus angularis Wight.) and Lima bean (Phaseolus lunatus L.) on estrogen and progesterone receptor phenotypes of MCF-7/BOS cells.

Estrogen-like effects of ethanol extracts from several Chinese legumes on MCF-7 cell