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Aim: The intent of the study was to assess the bone conserving effect of Butea monosperma total extract (BTE) prepared from the stem-bark, in ovariectomy (Ovx)-induced bone loss in Sprague Dawley (SD) rats. Methods: Sprague-Dawley rats were ovariectomized (Ovx) and BTE treatment with three different doses (100mg, 250mg and 500mg/kg body weight) commenced a day after Ovx and continued for 12 weeks. Skeletal parameters including trabecular and cortical bone microarchitecture, bone mineral density, bone mineral content, biomechanical strength, bone turnover markers (serum procollagen 1 N-terminal propeptide/P1NP and cross-linked C-telopeptide of type I collagen/CTX-1), new bone formation rate (periosteal bone formation rate and mineral apposition rate), and expression of osteogenic genes were assessed. Estrogenicity of BTE was assessed by studying uterus at the end of the treatments. Results: Ovx rats treated with BTE prevented the alterations in trabecular and cortical microarchitecture, maintained bone mineral density, bone mineral content, and increased bone biomechanical strength in comparison with Ovx rats. Treatment of BTE also enhanced new bone formation rate, increased the serum bone formation marker (P1NP), decreased the serum bone resorption marker (CTX-1), and upregulated the osteogenic genes in bones. BTE was devoid of estrogen-like effect in uterus. Conclusion: BTE protects against Ovx-induced bone loss by inhibiting bone resorption and increasing new bone formation. The most effective bone conserving dose of BTE was 250mg/kg. This study demonstrates the potential of BTE in preventing/delaying osteoporosis development in postmenopausal women. [ABSTRACT FROM AUTHOR]

The bone regeneration and healing effect of formononetin was evaluated in a cortical bone defect model that predominantly heals by intramembranous ossification. For this study, female Balb/c mice were ovariectomised (OVx) and a drill-hole injury was generated in the midfemoral bones of all animals. Treatment with formononetin commenced the day after and continued for 21 d. Parathyroid hormone (PTH1–34) was used as a reference standard. Animals were killed at days 10 and 21. Femur bones were collected at the injury site for histomorphometry studies using microcomputed tomography (μCT) and confocal microscopy. RNA and protein were harvested from the region surrounding the drill-hole injury. For immunohistochemistry, 5 µm sections of decalcified femur bone adjoining the drill-hole site were cut. μCT analysis showed that formononetin promoted bone healing at days 10 and 21 and the healing effect observed was significantly better than in Ovx mice and equal to PTH treatment in many aspects. Formononetin also significantly enhanced bone regeneration as assessed by calcein-labelling studies. In addition, formononetin enhanced the expression of osteogenic markers at the injury site in a manner similar to PTH. Formononetin treatment also led to predominant runt-related transcription factor 2 and osteocalcin localisation at the injury site. These results support the potential of formononetin to be a bone-healing agent and are suggestive of its promising role in the fracture-repair process. [ABSTRACT FROM AUTHOR]

Balance between adipocyte and osteoblast differentiation is the key link of disease progression in obesity and osteoporosis. We have previously reported that formononetin (FNT), an isoflavone extracted from Butea monosperma, stimulates osteoblast formation and protects against postmenopausal bone loss. The inverse relationship between osteoblasts and adipocytes prompted us to analyse the effect of FNT on adipogenesis and in vivo bone loss, triggered by high-fat diet (HFD)-induced obesity. The anti-obesity effect and mechanism of action of FNT was determined in 3T3-L1 cells and HFD-induced obese male mice. Our findings show that FNT suppresses the adipogenic differentiation of 3T3-L1 fibroblasts, through down-regulation of key adipogenic markers such as PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα) and sterol regulatory element-binding protein (SREBP) and inhibits intracellular TAG accumulation. Increased intracellular reactive oxygen species levels and AMP-activated protein kinase (AMPK) activation accompanied by stabilisation of β-catenin were attributed to the anti-adipogenic action of FNT. In vivo, 12 weeks of FNT treatment inhibited the development of obesity in mice by attenuating HFD-induced body weight gain and visceral fat accumulation. The anti-obesity effect of FNT results from increased energy expenditure. FNT also protects against HFD-induced dyslipidaemia and rescues deterioration of trabecular bone volume by increasing bone formation and decreasing bone resorbtion caused by HFD. FNT’s rescuing action against obesity-induced osteoporosis commenced at the level of progenitors, as bone marrow progenitor cells, obtained from the HFD mice group supplemented with FNT, showed increased osteogenic and decreased adipogenic potentials. Our findings suggest that FNT inhibits adipogenesis through AMPK/β-catenin signal transduction pathways and protects against HFD-induced obesity and bone loss. [ABSTRACT FROM PUBLISHER]

Abstract: Phytochemical investigation from the stem bark of Butea monosperma, led to the isolation and identification of three new compounds named buteaspermin A (1), buteaspermin B (2) and buteaspermanol (3), along with 19 known compounds. The structure of compounds 1–22 were established on the basis of their spectroscopic data. The isolated compounds 2–17 were evaluated using neonatal (1–3 day old) rat calvaria derived primary osteoblast cultures. Five of these compounds 7, 10–13 showed promising osteogenic activity, attributed to increased osteoblast proliferation, differentiation and mineralization as evidenced by marked increase in expression of alkaline phosphatase, an early phase differentiation marker, and alizarin Red S staining of osteoblasts cultured for 48h and von Kossa silver staining of nodules formed 15days after culture with these compounds. Quantification of mineralization by optical density measurement of Alizarin Red S extracted from stained osteoblasts cultured for 7days in presence of these compounds showed significant (P <0.05, vs corresponding vehicle control group) increase in mineralization. On the basis of biological results, structure–activity relationships are discussed. [Copyright &y& Elsevier]

Glucocorticoid-induced osteoporosis (GIOP) is a common clinical consequence that arises due to the extensive usage of glucocorticoids. Cladrin (Clad), a methoxylated isoflavone has been reported to have a bone protecting effect by enhancing osteoblast proliferation and differentiation. However, its consequences on GIOP are not reported yet. This study investigates whether Clad protects against the deleterious effects of Dexamethasone (Dex) on osteoblast and bone. Mice calvarial osteoblasts were treated with Clad and then exposed to Dex to study the effect on osteoblast differentiation, proliferation, and survival. Further, GIOP mice were treated with Clad (5 and 10 mg/kg) doses along with reference standard alendronate (ALN 3 mg/kg) for evaluation of bone protecting effect of Clad. We analyzed bone and vertebral microarchitecture, mechanical strength, and biochemical parameters. We observed that Clad at 10 nM concentration mitigated Dex-induced cytotoxicity and defend osteoblasts against apoptosis. Subsequent results demonstrate that Clad suppressed apoptosis of osteoblast in the presence of Dex by enhancing autophagy in a way that was reliant on the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway. Furthermore, micro-CT scanning, eco MRI results, and serum CTX levels revealed that 12 weeks of Clad treatment prevented bone loss and preserved trabecular bone mass in GIOP animals. We also observed that Clad treated osteoblasts had a lower rate of apoptosis and a greater LC3-II/LC3-I ratio than the Dex group. Our findings show that Clad can protect osteoblasts against glucocorticoids by inducing autophagy via the AMPK/mTOR pathway. [Display omitted] • Glucocorticoid-induced osteoporosis arises due to the extensive usage of glucocorticoids. • Cladrin shows a protective effect on osteoblast cells following exposure to Dexamethasone (DEX) via autophagy activation. • Cladrin-mediated autophagy induction by Dex is mediated by targeting of AMPK/mTOR pathway. • Animal studies show that Cladrin inhibits bone resorption in the GIOP mice modelby autophagy enhancement. [ABSTRACT FROM AUTHOR]

Abstract Ethnopharmacological relevance Cassia occidentalis L., a synonym of Senna occidentalis (belongs to Caesalpiniaceae family) is an annual plant. Pursuing a lead from a folk practice prevalent since the late nineteenth century in Andhra Pradesh, a Southern state of India, of use of Cassia occidentalis leaf and stem for treating patients with fracture and bone diseases, we have not only confirmed its fracture healing activity but also demonstrated efficacy in preventing glucocorticoid-induced osteoporosis (GIO), the commonest form of medication-induced bone loss caused chiefly due to impairment of bone formation. Aim of the study In the present work, the effects of extract and fraction of leaf and stem of Cassia occidentalis was investigated in fracture healing and GIO models of rat. The study also aimed to identify osteogenic compounds from this plant. Materials and methods Ethanolic extracts from leaf and stem of Cassia occidentalis were prepared and their efficacy tested in rat femur osteotomy (fracture healing) model. Subsequently, a butanolic fraction was prepared and osteogenic efficacy compared with the ethanolic extract, and upon finding the former to be more potent, its osteogenic effect was studied in details in GIO model. Chemical finger-printing and isolation of ten pure compounds were done to assess their osteogenic effect in rat primary osteoblast cultures. Results Ethanolic extract of stem was more effective than the leaf extract in enhancing bone regeneration at the site of osteotomy. Further, butanolic fraction of the ethanolic extract of stem was more effective than the later in bone regeneration at the femur osteotomy site and in preventing bone loss in GIO model. The mechanism of skeletal preservation involved stimulation of new bone formation and inhibition of bone resorption. As many as six osteogenic compounds were isolated out of which apigenin-6 C -glucopyranoside was most effective in vitro. Conclusion Our study found that a standardized extract of an ethanolic extract and its butanolic fraction from the stem of Cassia occidentalis has osteogenic as well as anti-resorptive effects, resulting in the protection against glucocorticoid-induced bone loss. Our results contribute towards validation of the traditional use of Cassia occidentalis in fracture healing and also suggest its beneficial use in GIO for which clinical trials are warranted. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/β-catenin pathway assessed by phosphorylation of GSK-3β. Over-expressing of constitutively active GSK-3β (caGSK-3β) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3β over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation. [ABSTRACT FROM AUTHOR]

The structural modifications of pregnenolone have been described via the introduction of heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized derivatives, four compounds showed significantly increased ALP activity. Among all four active compounds, the novel compound 3a has shown significant bone matrix mineralization and mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1 pM concentration. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Mistletoe extracts (decoctions) are used traditionally in eastern Nigeria for the management of bone pain, post menopausal syndrome and diabetes amongst several other ailments. While scientific evidence supporting its folkloric use as an antidiabetic agent has been documented, the age-long practice of its use in treatment of post menopausal syndrome has not been scientifically validated. Postmenopausal osteoporosis accounts for one of the prevalent disease conditions in aging population globally. This situation is exacerbated by the lack of osteogenic therapy. In search for plants of Nigerian origin with osteogenic potential, we evaluated eastern Nigerian mistletoe, having ethnotraditional claims of anti-diabetic, anti-hypertensive and anti-cancer activities as well as preventive effect in various post-menopausal syndromes. Materials and Methods: Methanolic extracts of mistletoe leaves harvested from three host tress – Kola acuminata (KM), Citrus spp (CM) and Garcinia kola (GKM) – were evaluated for osteoblast viability and osteogenic activities using primary rat calvaria culture. Lupeol (1) was isolated from the stem bark of Bombax ciba and its congener, dihydoxylupeol palmitate (2) in addition to three other compounds; 3-methoxy quercetin (3), 3,4,5-trimethoxy gallate (4), and friedelin (5) were isolated from the leaves of mistletoes species. Following their chemical characterization, the compounds were evaluated for osteogenic potential using validated models including alkaline phosphatase (ALP) assay, mineralization assay and expression of osteogenic genes – bone morphogenetic protein-2 (BMP2) and osteoblast transcription factor (RUNX2) – in primary calvarial cultures harvested from neonatal rats. Uterine estrogenicity of the extracts was tested in adult female Sprague Dawley rats. Results: Methanol extracts of mistletoe from three hosts exhibited increase in ALP activity (a marker of osteoblast differentiation) at lower concentrations (0.2–0.8μg/ml) and either no or inhibitory effect at higher concentrations (1.6 and 3.2μg/ml). None of the extract had cytotoxicity to osteoblasts at the concentrations tested. Five compounds viz. 1 from Bombax ciba, and 2–5 were isolated from the mistletoe leaves. Out of these, 5 exhibited significant loss of osteoblast viability and hence it was not considered further. All four compounds exhibited stimulatory effects on osteoblast differentiation as assessed by ALP assay and determination of osteogenic gene expression. Compound 2 was relatively more potent than its precursor, compound 1 in stimulating BMP2 upregulation. KM did not show uterine estrogenicity. Conclusion: Methanolic extracts from the three mistletoes species possess in vitro osteogenic activity, and from these extracts three new classes of compounds have been found to promote osteoblast differentiation in vitro. In light of these findings, we propose that mistletoe species may be developed as safer alternative(s) in the management of diseases where lack of bone formation is the pathology. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: The whole plant or some part of Peperomia pellucida (L.) HBK is used in some parts of Cameroon as a treatment for fracture healing. Aim of the study: To evaluate the effect of ethanolic extracts of Peperomia pellucida (L.), a Cameroonian medicinal plant on bone regeneration following bone and marrow injury, and determine the mode of action. Materials and methods: Ethanol extract of Peperomia pellucida was administered at 100 and 200mg/kg doses orally to adult female Sprague-Dawley rats having a drill hole injury (0.8mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of calcein labeling at the drill hole site was performed to evaluate bone regeneration. 3-D microarchitecture of drill hole site was analyzed by micorocomputed tomography. Osteogenic effects of the extract were evaluated by assessing mineralized nodule formation of bone marrow stromal cells and expression of osteogenic genes (mRNA level of type-1 collagen, bone morphogenetic protein-2 and osteocalcin genes) in the femur. Results: Ethanol extract from Peperomia Pellucida (L.) dose-dependently induced bone regeneration at the fracture site. At 200mg/kg dose, the extract significantly increased mineral deposition compared to controls. The extract also improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. In addition, the extract increased the formation of mineralized nodules from the bone marrow stromal cells. Furthermore, the extract induced the expression of osteogenic genes in the femur including type 1 collagen, osteocalcin and BMP-2, compared to control. Conclusion: Ethanolic extract of P. pellucid (L.) accelerates fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying its traditional use. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Elephantopus mollis, Spilanthes africana, Urena lobata, Momordica multiflora, Asystasia gangetica and Brillantaisia ovariensis are used in Cameroonian traditional medicine for the treatment of bone diseases and fracture repair. The aim of this study was to evaluate the effect of ethanolic extracts of six Cameroonian medicinal plants on bone regeneration following bone and marrow injury. Materials and methods: Ethanol extract of Cameroonian medicinal plants were administered (each extract at 250, 500 and 750mg/kg doses) orally to adult female Sprague–Dawley rats having a drill hole injury (0.8mm) in the femur diaphysis. Vehicle (gum-acacia in distilled water) was given to the control group. After 12 days of treatment, animals were euthanized and femur bones collected. Confocal microscopy of fractured bone was performed to evaluate bone regeneration (calcein labeling). Only active plant extracts were used for further experiments. Thus, callus was analyzed by microcomputed tomography. Osteogenic effects of the extracts were evaluated by assessing mineralized nodules formation of bone marrow stromal cells and osteoblast recruitment at drill hole site by immunohistochemistry. Results: Ethanolic extract of the leaves and twigs of Elephantopus mollis (EM) and whole plant of Spilanthes africana (SA) dose-dependently stimulated bone regeneration at the drill hole site. EM at 250 and 750mg/kg doses and SA at 750mg/kg dose significantly increased mineral deposition compared to controls. Both extracts at 500 and 750mg/kg doses improved microarchitecture of the regenerating bone evident from increased bone volume fraction, trabecular thickness, trabecular number, and decreased trabecular separation and structure model index. EM and SA extracts increased the formation of mineralized nodules from the bone marrow stromal cells. In addition, EM and SA extracts increased osteoblast recruitment at the drill hole site evident from increased Runx-2 positive cells following their treatments compared to control. Conclusion: Ethanolic extracts of EM and SA accelerate fracture repair in rats via stimulatory effects on osteoblast differentiation and mineralization, thereby justifying their traditional use. [Copyright &y& Elsevier]

Abstract: Dietary isoflavones including genistein and daidzein have been shown to have favorable bone conserving effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of medicarpin (Med); a phytoalexin that is structurally related to isoflavones and is found in dietary legumes. Med stimulated osteoblast differentiation and mineralization at as low as 10−10 M. Studies with signal transduction inhibitors demonstrated involvement of a p38 mitogen activated protein kinase-ER-bone morphogenic protein-2 pathway in mediating Med action in osteoblasts. Co-activator interaction studies demonstrated that Med acted as an estrogen receptor (ER) agonist; however, in contrast to 17β-estradiol, Med had no uterine estrogenicity and blocked proliferation of MCF-7 cells. Med increased protein levels of ERβ in osteoblasts. Selective knockdown of ERα and ERβ in osteoblasts established that osteogenic action of Med is ERβ-dependent. Female Sprague–Dawley (weaning) rats were administered Med at 1.0- and 10.0 mg.kg−1 doses by gavage for 30 days along with vehicle control. Med treatment resulted in increased formation of osteoporgenitor cells in the bone marrow and osteoid formation (mineralization surface, mineral apposition/bone formation rates) compared with vehicle group. In addition, Med increased cortical thickness and bone biomechanical strength. In pharmacokinetic studies, Med exhibited oral bioavailability of 22.34% and did not produce equol. Together, our results demonstrate Med stimulates osteoblast differentiation likely via ERβ, promotes achievement of peak bone mass, and is devoid of uterine estrogenicity. In addition, given its excellent oral bioavailability, Med can be potential osteogenic agent. [Copyright &y& Elsevier]

Abstract: Phytochemical investigation of ethanol extracts of the Pterospermum acerifolium flowers led to the isolation and identification of two new flavones, 4′-(2-methoxy-4-(1,2,3-trihydroxypropyl) phenoxy luteolin (1) and 5,7,3′-trihydroxy-6-O-β-d-glucopyranosyl flavone (2), and one new lactone, 3,5-dihydroxyfuran-2(5H)-one (3) along with 14 known compounds (4–17). The structure of compounds 1–17 was established based on MS, 1D and 2D NMR, spectroscopic analysis. Eight of these compounds (1–6, 8 and 9) were assessed for osteogenic activity by using primary cultures of rat osteoblast. The compounds 1, 3 and 4 significantly stimulated osteoblast differentiation and mineralization as evident from a marked increase in expression of alkaline phosphatase and alizarin red-S staining of osteoblasts. [Copyright &y& Elsevier]

Abstract: Dietary soy isoflavones including genistein and daidzein have been shown to have favorable effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of cladrin and formononetin, two structurally related methoxydaidzeins found in soy food and other natural sources. Cladrin, at as low as 10 nM, maximally stimulated both osteoblast proliferation and differentiation by activating MEK-Erk pathway. On the other hand, formononetin maximally stimulated osteoblast differentiation at 100 nM that involved p38 MAPK pathway but had no effect on osteoblast proliferation. Unlike daidzein, these two compounds neither activated estrogen receptor in osteoblast nor had any effect on osteoclast differentiation. Daily oral administration of each of these compounds at 10.0 mg kg−1 day−1 dose to recently weaned female Sprague–Dawley rats for 30 consecutive days, increased bone mineral density at various anatomic positions studied. By dynamic histomorphometry of bone, we observed that rats treated with cladrin exhibited increased mineral apposition and bone formation rates compared with control, while formononetin had no effect. Cladrin had much better plasma bioavailability compared with formononetin. None of these compounds exhibited estrogen agonistic effect in uteri. Our data suggest that cladrin is more potent among the two in promoting parameters of peak bone mass achievement, which could be attributed to its stimulatory effect on osteoblast proliferation and better bioavailability. To the best of our knowledge, this is the first attempt to elucidate structure–activity relationship between the methoxylated forms of daidzein and their osteogenic effects. [Copyright &y& Elsevier]

Abstract: An efficient one step synthesis of new 2-hydroxymethylisoflavone is reported. A series of deoxybenzoin was subjected to cyclization with glyoxal in the presence of basic condition (KOH/EtOH) to afford the 2-hydroxymethyl isoflavone. The structures of compounds 5a–g were confirmed by NMR experiments including 1H, 13C, HMBC, HSQC and COSY. These compounds were assessed for stimulation of osteoblast function using primary culture of rat calvarial osteoblasts in vitro. Compounds 5a, 5d, 5f and 5g were potent in stimulating differentiation of osteoblasts as assessed by measuring alkaline phosphatase (ALP) activity. Besides, effect of these analogs was also seen on the transcript levels of osteogenic genes like Runx-2, osteocalcin and Bone morphogenetic protein-2 (BMP-2), involved in osteoblast differentiation and mineralization. Based on quantitative PCR data, compound 5f was found to be the potent followed by 5d. Compound 5f robustly increased the mRNA levels of Runx-2 (8.0 fold), BMP-2 (∼2 fold) and osteocalcin (∼2.0 fold) in osteoblasts. Collectively, we demonstrate osteogenic activity of the novel 2-hydroxymethyl isoflavones with 5f having the most potent activity. [Copyright &y& Elsevier]