Abnish K. Gautam

Methoxylated isoflavones, cajanin and isoformononetin, have non‐estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling

Following a lead obtained from stem‐bark extract of Butea monosperma, two structurally related methoxyisoflavones; cajanin and isoformononetin were studied for their effects in osteoblasts. Cajanin had strong mitogenic as well as differentiation‐promoting effects on osteoblasts that involved subsequent activation of MEK‐Erk and Akt pathways. On the other hand, isoformononetin exhibited potent anti‐apoptotic effect in addition to promoting osteoblast differentiation that involved parallel activation of MEK‐Erk and Akt pathways. Unlike genistein or daidzein, none of these two compounds appear to act via estrogen receptors in osteoblast. Once daily oral (by gavage) treatment for 30 consecutive days was given to recently weaned female Sprague–Dawley rats with each of these compounds at 10.0 mg kg−1 day−1 dose. Cajanin increased bone mineral density (BMD) at all skeletal sites studied, bone biomechanical strength, mineral apposition rate (MAR) and bone formation rate (BFR), compared with control. BMD levels at various anatomic positions were also increased with isoformononetin compared with control however, its effect was less potent than cajanin. Isoformononetin had no effect on the parameters of bone biomechanical strength although it enhanced MAR and BFR compared with control. Isoformononetin had very mild uterotrophic effect, whereas cajanin was devoid of any such effect. Our data suggest that cajanin is more potent than isoformononetin in accelerating peak bone mass achievement. To the best of our knowledge, this work represents the first attempt to elucidate structure‐activity relationship between the two methoxylated isoflavones regarding their effects in osteoblasts and bone formation. J. Cell. Biochem. 108: 388–399, 2009.

Differential effects of formononetin and cladrin on osteoblast function, peak bone mass achievement and bioavailability in rats

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.

Medicarpin inhibits osteoclastogenesis and has nonestrogenic bone conserving effect in ovariectomized mice

Medicarpin, a pterocarpan class of naturally occurring benzopyran furanobenzene compound was synthesized in gram scale to investigate its effects on murine bone cells and in ovariectomized (OVx) mice. Medicarpin, at as low as 10(-10)M suppressed osteoclastogenesis in bone marrow cells (BMCs). Medicarpin-induced apoptosis of mature osteoclasts isolated from long bones. Effects of medicarpin in osteoclasts appear to be independent of estrogen receptor (ER) activation as ICI 180,782 failed to abrogate its effects on osteoclasts. In calvarial osteoblasts, medicarpin (10(-10)M) blocked nuclear factor kappaB (NF-kappaB) signaling assessed by tumor necrosis factor alpha (TNFalpha)-stimulated nuclear translocation of p65 subunit of NF-kappaB. Medicarpin also inhibited the expression of TNFalpha in mouse calvarial osteoblasts. This effect was ER dependent as ICI 180,782 reversed the suppressive effect of medicarpin on TNFalpha mRNA levels in osteoblasts. In addition, like 17beta-estradiol, presence of medicarpin inhibited TNFalpha-induced upregulation of interleukin-1, and -6 mRNA levels in osteoblasts. In co-cultures consisting of calvarial osteoblasts and BMCs, presence of medicarpin increased osteoprotegerin (OPG)/receptor activator of NF-kappaB ligand (RANKL) ratio and reduced mRNA levels of osteoclast markers including tartrate-resistant acid phosphatase and RANK. OVx mice administered medicarpin (10.0mgkg(-1)day(-1)) orally for 30days had reduced formation of osteoclasts but increased formation of osteoprogenitor cells in BMCs compared with OVx+vehicle group. Medicarpin treatment to OVx mice maintained parameters of trabecular microarchitecure. Medicarpin exhibited no uterine estrogenicity. Our findings point towards direct and indirect inhibitory effects of medicarpin on osteoclastogenesis in vitro that contribute to its bone sparing effect in OVx mice.

Total extract and standardized fraction from the stem bark of Butea monosperma have osteoprotective action: evidence for the nonestrogenic osteogenic effect of the standardized fraction.

Objective: The aim of this study was to determine the skeletal effects of Butea total extract (BTE) and its acetone soluble fraction (ASF) from Butea monosperma, which is rich in methoxyisoflavones, in ovariectomized (OVx) rats, a model for postmenopausal bone loss. Methods: BTE (1.0 g kg−1 d−1) and ASF (100 mg kg−1 d−1) were given orally for 12 weeks to adult OVx rats. The sham-operated and ovariectomy + vehicle groups served as controls. Bone mineral density, osteoid formation (mineral apposition rate and bone formation rate), bone microarchitecture, and bone turnover/resorption markers were studied. Phytoestrogens in rats given BTE and ASF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. Results: OVx rats treated with either BTE or ASF exhibited increased bone mineral density in trabecular bones and improved trabecular microarchitecture compared with the ovariectomy + vehicle group. ASF treatment was more efficient than BTE treatment in maintaining trabecular microarchitecture. Serum osteocalcin and urinary type 1 collagen levels in OVx rats treated with either BTE or ASF were significantly lower than those of the ovariectomy + vehicle group. ASF treatment led to increased mineral apposition rate and bone formation rate compared with ovariectomy + vehicle, whereas BTE had no such effect. In the uterotropic assay, BTE was mildly estrogenic in adult OVx rats. In immature rats, BTE exhibited both estrogenicity and antiestrogenicity. ASF had neither uterine estrogenicity nor antiestrogenicity. Analysis of phytoestrogens revealed significant enrichment of cladrin, isoformononetin, and medicarpin in ASF over BTE. Conclusions: Derived from B monosperma, ASF at a 10-fold lower dose than that of BTE was effective in preventing OVx-induced bone loss and stimulated new-bone formation.

Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass achievement in rats: evidence for estrogen receptor β-mediated osteogenic action of medicarpin.

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.

Anti-osteoporotic constituents from Indian medicinal plants

The objective of this study was to determine the in vitro osteogenic activities of selected medicinal plants used traditionally in India. The compounds isolated from three plants viz. Allophylus serratus, Cissus quadrangularis and Vitex negundo were evaluated for their in vitro osteogenic activities. Primary cultures of osteoblasts were used to determine the effects of these components on osteoblast functions. Five of the fourteen compounds isolated led to increase in osteoblast differentiation and mineralization. These findings lend support to the use of Allophylus serratus, Cissus quadrangularis and Vitex negundo in traditional medicine.

Identification of kaempferol-regulated proteins in rat calvarial osteoblasts during mineralization by proteomics.

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.

Synthetic analogs of daidzein, having more potent osteoblast stimulating effect.

A series of didzein derivatives were synthesized and assessed for stimulation of osteoblast differentiation using primary cultures of rat calvarial osteoblasts. Data suggested that three synthetic analogs, 1c, 3a and 3c were several folds more potent than daidzein in stimulating differentiation and mineralization of osteoblasts. Further, these three compounds did not show any estrogen agonistic activity, however had mild estrogen antagonistic effect. Out of the three compounds, 3c was found to maximally increase the mineralization of bone marrow osteoprogenitor cells. Compound 3c also robustly increased the mRNA levels of osteogenic genes including bone morphogenetic protein-2 and osteocalcin in osteoblasts. Unlike daidzein, 3c did not inhibit osteoclastogenesis. Collectively, we demonstrate osteogenic activity of daidzein analogs at significantly lower concentrations than daidzein.

9-Demethoxy-medicarpin promotes peak bone mass achievement and has bone conserving effect in ovariectomized mice: Positively regulates osteoblast functions and suppresses osteoclastogenesis

We report a new bone anabolic and anti-catabolic pterocarpan 9-demethoxy-medicarpin (DMM) for the management of postmenopausal osteoporosis. DMM promoted osteoblast functions via activation of P38MAPK/BMP-2 pathway and suppressed osteoclastogenesis in bone marrow cells (BMCs). In calvarial osteoblasts, DMM blocked nuclear factor kappaB (NFκB) signaling and inhibited the mRNA levels of pro-inflammatory cytokines. DMM treatment led to increased OPG (osteoprotegrin) and decreased transcript levels of TRAP (tartarate resistant acid phosphatase), RANK (receptor activator of NFκB) and RANKL (RANK ligand) in osteoblast-osteoclast co-cultures. Immature female SD rats administered with DMM exhibited increased bone mineral density, bone biomechanical strength, new bone formation and cortical bone parameters. Ovx mice administered with DMM led to significant restoration of trabecular microarchitecture and had reduced formation of osteoclasts and increased formation of osteoprogenitor cells in BMCs. DMM exhibited no uterine estrogenicity. Overall, these results demonstrate the therapeutic potential of DMM for the management of postmenopausal osteoporosis.