Increasingly natural products particularly flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. This study has reviewed previous studies on the two better known flavonoids, genistein and icariin, their structures, functions, action mechanisms, relative potency, and potential application in regulating bone remodeling and preventing bone loss. Genistein, an isoflavone abundant in soy, has dual functions on bone cells, able to inhibit bone resorption activity of osteoclasts and stimulate osteogenic differentiation and maturation of bone marrow stromal progenitor cells (BMSCs) and osteoblasts. Genistein is an estrogen receptor (ER)-selective binding phytoestrogen, with a greater affinity to ERβ. Genistein inhibits tyrosine kinases and inhibits DNA topoisomerases I and II, and may act as an antioxidant. Genistein enhances osteoblastic differentiation and maturation by activation of ER, p38MAPK-Runx2, and NO/cGMP pathways, and it inhibits osteoclast formation and bone resorption through inducing osteoclastogenic inhibitor osteoprotegerin (OPG) and blocking NF-κB signaling. Icariin, a prenylated flavonol glycoside isolated from Epimedium herb, stimulates osteogenic differentiation of BMSCs and inhibits bone resorption activity of osteoclasts. Icariin, whose metabolites include icariside I, icariside II, icaritin, and desmethylicaritin, has no estrogenic activity. However, icariin is more potent than genistein in promoting osteogenic differentiation and maturation of osteoblasts. The existence of a prenyl group on C-8 of icariin molecular structure has been suggested to be the reason why icariin is more potent than genistein in osteogenic activity. Thus, the prenylflavonoids may represent a class of flavonoids with a higher osteogenic activity.

Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling.

Traditional Chinese medicine formulas for the treatment of osteoporosis: Implication for antiosteoporotic drug discovery

This paper reviews the latest understanding of biological and pharmacological properties of osthole (7-methoxy-8-(3-methyl-2-butenyl)-2H-1-benzopyran-2-one), a natural product found in several medicinal plants such as Cnidium monnieri and Angelica pubescens. In vitro and in vivo experimental results have revealed that osthole demonstrates multiple pharmacological actions including neuroprotective, osteogenic, immunomodulatory, anticancer, hepatoprotective, cardiovascular protective, and antimicrobial activities. In addition, pharmacokinetic studies showed osthole uptake and utilization are fast and efficient in body. Moreover, the mechanisms of multiple pharmacological activities of osthole are very likely related to the modulatory effect on cyclic adenosine monophosphate (cAMP) and cyclic adenosine monophosphate (cGMP) level, though some mechanisms remain unclear. This review aims to summarize the pharmacological properties of osthole and give an overview of the underlying mechanisms, which showcase its potential as a multitarget alternative medicine.

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Osthole: A Review on Its Bioactivities, Pharmacological Properties, and Potential as Alternative Medicine.

Background
Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed tumors worldwide and is known to be resistant to conventional chemotherapy New therapeutic strategies are urgently needed for treating HCC Osthole, a natural coumarin derivative, has been shown to have anti-tumor activity However, the effects of osthole on HCC have not yet been reported

Growth Inhibition and Apoptosis Induced by Osthole, A Natural Coumarin, in Hepatocellular Carcinoma

Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K–AKT–eNOS–NO–cGMP–PKG☆

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats Osthol was supplemented into cultured medium at 10–7, 10–6, 10–5 and 10–4 mol/l, respectively No stimulating effect was found on cell proliferation, but 10–5 mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 152, 274 and 20 times higher, respectively, than in the control cells Results of ALP histochemical staining and mineralized bone nodule assays both showed that the number and area achieved in osthol-treated cells were 153-fold higher than in control cells The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated The increase in mRNA expression was 194, 174, 168, 183 and 231 times, respectively, higher compared to the control Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen p38MAPK protein and collagen in osthol-treated cells were 142 and 158 times higher in osthol-treated cells compared to the control The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs

Osthol, a coumarin isolated from common cnidium fruit, enhances the differentiation and maturation of osteoblasts in vitro.

OBJECTIVE To investigate the effects of icariin and it's main metabolites-icariside II on the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). METHODS rBMSCs were cultured by adherence screening method, icariin and icariside II were supplemented into the culture at 5 x 10(-5) mol/L respectively. The osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-F(ALp), osteocalcin secretion, calcium deposition and mineralized bone modulus were compared among the icariin-supplemented group, icariside II and the control. The gene expressions of bFGF, IGF-1, Osterix and Runx-2 were examined by RT-Real Time PCR. RESULTS Both icariside II and icariin significantly improved ALP activity, CFU-F(ALP) amount, osteocalcin secretion, calcium deposition and mineralized modulus. Besides, they enhanced the gene expressions of bFGF, IGF-1, Osterix and Runx-2. Icariside II was obviously stronger than icariin at the above activities. CONCLUSION Icariside II is stronger than icariin at enhancing the osteogenic differentiation of rBMSCs, suggesting that icariin can be administered via oral and it's metabolites are the effective constitutes for antiosteoporosis activity.

Comparative study on the osteogenic differentiation of rat bone marrow stromal cells effected by icariin and icariside II

Objective To investigated the effects of isopsoralen on bone marrow stromal stem cells (BMSCs) differentiate and proliferate in vitro. Method The stratum of mononuclear cells were separated and collected from the rat bone marrow sample by the all bone marrow cell culture methods. The cells were cultured in DMEM contained 10% fetal bovine serum. The culture medium was changed after three days. Nine days later, cells were treatment by isopsoralen with the concentration 1 x 10(-5), 1 x 10(-4), 1 x10(-6), 1 x 10(-7) mol x L(-1). MTT method was used for the proliferated analyzing. Under the induced condition, the alkaline phosphatase (ALP) activity, calcium salt sediment yield and osteocalcin were measured at the 4, 8, 12, 16 d. At the fifteenth day, histochemistry dyeing for calcified tubercle and ALP was proceeded. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix and Runx-2 were investigated by Real Time PCR. Result The BMSCs proliferation refrained by isopsoralen with dose dependent. But it significantly enhanced osteogenesis, which was represented by the promotion of the ALP activity, calcium salt sediment yield, osteocalcin, and calcified tubercle amount. Besides, it also enhanced the mRNA level of bFGF, IGF-1, Osterix and Runx-2. Conclusion The isopsoralen with the concentration 1 x 10(-5) mol x L(-1) can promote BMSCs differentiation to osteoblasts. It demonstrated the isopsoralen can prevent antiosteoporotic, which is an active part of the traditional Chinese medicine psoralea corylifolia.

Effects of isopsoralen on bone marrow stromal stem cells differentiate and proliferate in vitro

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.

Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro

Objective To investigate the effects of icariin on the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). Method rBMSCs were cultured by adherence screening method. Icariin was supplemented into the culture at 1 x 10(-5) mol x L(-1). The osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-F(ALP) and mineralized bone modulus were compared between the icariin-supplemented group and the control group. Total RNA was isolated and the gene expression of bFGF, IGF-1, Osterix(OSX) and Runx-2 was investigated by RT Real-time PCR. Result Icariin significantly improved ALP activity, CFU-F(ALP) amounts and mineralized modulus. It also can enhance the mRNA level of bFGF, IGF-1, Osterix and Runx-2. Conclusion Icariin enhances the osteogenic differentiation of rBMSCs significantly, which suggested that icariin has the potentiality to be a new drug of anti-osteoporosis or fracture healing.

Ke-Ming Chen

Papers

Osthol, a coumarin isolated from common cnidium fruit, enhances the differentiation and maturation of osteoblasts in vitro.

Comparative study on the osteogenic differentiation of rat bone marrow stromal cells effected by icariin and icariside II

Effects of isopsoralen on bone marrow stromal stem cells differentiate and proliferate in vitro

Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro

Icariin promotes osteogenic differentiation of rat bone marrow stromal cells in vitro