Gaurav Swarnkar

Kaempferol has osteogenic effect in ovariectomized adult Sprague–Dawley rats

Kaempferol (K), a flavonol, is known to have anti-osteoclastogenic effect. We here show that K, from 0.2 to 5.0 microM, increased mineralized nodules in rat primary osteoblasts. K also significantly attenuated adipocyte formation from bone marrow cells (BMCs). A single oral dose of 1 mg/kg body weight of K in Sprague-Dawley (180-200 g) rats resulted in a peak serum level of 2.04+/-0.8 nM in 30 min (Tmax), suggesting its rapid absorption. The Cmax of K in bone marrow was 0.684 nM after 90 min. Rats were ovariectomized (OVx) along with sham-operated rats and left for 4 weeks. Daily oral administration of K (5 mg/kg body weight) was then started to one group of OVx rats, and continued for 10 weeks. K levels were found to be 0.311 and 0.838 nM at the end of 4 and 10 weeks, respectively. K exhibited no estrogenicity at the uterine level. The K-treated group exhibited significantly higher bone mineral density (BMD) in the trabecular regions (femur neck, proximal tibia and vertebrae) and lower serum ALP (bone turnover marker) compared with the OVx rats. The compressive energy of the vertebrae was significantly higher in the OVx+K-treated group compared with the OVx group. K treatment of OVx rats resulted in the increase in osteoprogenitor cells as well as inhibition of adipocyte differentiation from BMCs compared with the OVx group. Together we show that K is non-estrogenic in vivo and exerts bone anabolic activity with attendant inhibition of bone marrow adipogenesis.

Role of Phytochemicals in the Prevention of Menopausal Bone Loss: Evidence from In Vitro and In Vivo, Human Interventional and Pharmacokinetic Studies

Substantial body of data generated from cultured bone cells and rat models of osteoporosis supports a significant bone-conserving effect of phytochemicals. Flavonoids including isoflavones, stilbenes and lignans with variable efficacy have shown promising therapeutic application in osteoporosis. Majority of the phytochemicals assessed for their effects on bone cells revealed multiple beneficial actions such as promoting osteoblast functions, and inhibiting osteoclast and adipocyte functions. A variety of molecular targets mediate multiple effects of phytochemicals in bone cells. In vivo, quite a few phytochemicals have been found to afford bone-sparing effect and in some cases even bone restoring effect. However, important pharmacokinetic and bioavailaibility studies associated with these phytochemicals are mostly lacking. As a result, translating these findings to the clinic has been challenging, and so far only a few clinical studies have attempted to evaluate the effect of phytochemicals in menopausal osteoporosis. Clinical studies so far performed are with dietary supplements rather than pure phytochemicals. Clinical trials with pure molecules necessitate preclinical regulatory and safety studies that are not available with the phytochemicals except ipriflavone with bone-conserving properties. Ipriflavone is the only marketed anti-osteoporosis agent that was obtained following a lead from natural substance. As phytochemicals have multiple beneficial influences on bone cells, making analogues of the most potent molecule for developing synthetic series with rational drug design approach could pay rich dividends in menopausal osteoporosis therapy.

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.

Co-expression of Arabidopsis transcription factor, AtMYB12, and soybean isoflavone synthase, GmIFS1, genes in tobacco leads to enhanced biosynthesis of isoflavones and flavonols resulting in osteoprotective activity.

Isoflavones, a group of flavonoids, restricted almost exclusively to family Leguminosae are known to exhibit anticancerous and anti-osteoporotic activities in animal systems and have been a target for metabolic engineering in commonly consumed food crops. Earlier efforts based on the expression of legume isoflavone synthase (IFS) genes in nonlegume plant species led to the limited success in terms of isoflavone content in transgenic tissue due to the limitation of substrate for IFS enzyme. In this work to overcome this limitation, the activation of multiple genes of flavonoid pathway using Arabidopsis transcription factor AtMYB12 has been carried out. We developed transgenic tobacco lines constitutively co-expressing AtMYB12 and GmIFS1 (soybean IFS) genes or independently and carried out their phytochemical and molecular analyses. The leaves of co-expressing transgenic lines were found to have elevated flavonol content along with the accumulation of substantial amount of genistein glycoconjugates being at the highest levels that could be engineered in tobacco leaves till date. Oestrogen-deficient (ovariectomized, Ovx) mice fed with leaf extract from transgenic plant co-expressing AtMYB12 and GmIFS1 but not wild-type extract exhibited significant conservation of trabecular microarchitecture, reduced osteoclast number and expression of osteoclastogenic genes, higher total serum antioxidant levels and increased uterine oestrogenicity compared with Ovx mice treated with vehicle (control). The skeletal effect of the transgenic extract was comparable to oestrogen-treated Ovx mice. Together, our results establish an efficient strategy for successful pathway engineering of isoflavones and other flavonoids in crop plants and provide a direct evidence of improved osteoprotective effect of transgenic plant extract.

A naturally occurring naringenin derivative exerts potent bone anabolic effects by mimicking oestrogen action on osteoblasts

BACKGROUND AND PURPOSE Naringenin and its derivatives have been assessed in bone health for their oestrogen‐‘like’ effects but low bioavailability impedes clinical potential. This study was aimed at finding a potent form of naringenin with osteogenic action.

A novel flavonoid isolated from the steam-bark of Ulmus wallichiana planchon stimulates osteoblast function and inhibits osteoclast and adipocyte differentiation.

(2S,3S)-Aromadendrin-6-C-β-d-glucopyranoside (AG) is a novel flavonol isolated from the extract of Ulmus wallichiana (Himalayan Elm). Extract of U. wallichiana is used as a traditional medicine for rapid fracture repair in India. We characterized the mechanism of action of AG in mouse bone cells by investigating its effect on the precursors of osteoblasts, osteoclasts and adipocytes. At nanomolar concentrations, AG increased differentiation of preosteoblasts obtained from neonatal mouse calvaria. The gene expression of osteogenic markers, including runt-related transcription factor 2 (Runx-2), bone morphogenetic protein-2 (BMP-2), type I collagen and osteocalcin were elevated in the preosteoblasts. The extracellular matrix mineralization was higher in preosteoblast and bone marrow cells when AG was present in the medium. Furthermore, AG protected the differentiated osteoblasts from serum deprivation-induced apoptosis, and increased the expression of the anti-osteoclastogenic cytokine, osteoprotegerin. It inhibited osteoclast differentiation of bone marrow precursor cells to osteoclasts in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and monocyte/macrophage-colony stimulating factor (M-CSF). Additionally, in 3T3-L1 preadipocytes, AG decreased the expression of genes involved in adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element binding protein (SREBP) and CCAAT/enhancer-binding protein alpha (CEBP/α), and induced apoptosis of differentiated adipocytes. Induction of adipogenic differentiation was also inhibited in the presence of AG. AG exhibited no estrogenic/antiestrogenic effect. Together, our data show that AG has potent osteogenic, anti-osteoclastogenic and anti-adipogenic effects, which may translate to a better skeletal outcome in postmenopausal osteoporosis.

Extract and fraction from Ulmus wallichiana Planchon promote peak bone achievement and have a nonestrogenic osteoprotective effect.

Objective: This study aimed to determine the skeletal effects of total ethanolic extract (TEE) and its butanolic fraction (BF) from the stem-bark of Ulmus wallichiana, which is rich in C-glycosylated flavonoids, in growing rats (for peak bone [PB] achievement) and in ovariectomized (OVx) rats (for menopausal bone loss). Methods: TEE (750 mg kg−1 d−1) and BF (50 mg kg−1 d−1) were given orally for 10 weeks to weaning female Sprague-Dawley rats and for 12 weeks to adult OVx rats of the same strain, respectively. In studies with OVx rats, sham operated + vehicle, OVx + 17&bgr;-estradiol, and OVx + vehicle groups served as various controls. Bone mineral density (BMD), biomechanical strength, bone histology, formations of osteoprogenitor cells, osteoid formation, and bone turnover/resorption markers were studied. Bioactive marker compounds in TEE and BF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test significance of effects. Results: In growing rats, both TEE and BF increased BMD, bone strength, and bone formation rate, suggesting higher PB achievement. OVx rats treated with either TEE or BF exhibited increased BMD at various anatomical positions and improved bone strength and trabecular architecture compared with the OVx + vehicle group. Serum osteocalcin and urinary type 1 collagen degradation product levels in OVx rats treated with either TEE or BF were significantly lower than those of the OVx + vehicle group. Neither TEE nor BF exhibited uterine estrogenicity. Analysis of marker compounds revealed significant enrichment of two bioactive markers in BF over TEE. Conclusions: Derived from U wallichiana, BF at much a lower dose than TEE was effective in PB achievement and prevention of OVx-induced bone loss.

Quercetin-6-C-β-D-glucopyranoside isolated from Ulmus wallichiana planchon is more potent than quercetin in inhibiting osteoclastogenesis and mitigating ovariectomy-induced bone loss in rats.

Objective: The aim of this study was to determine the skeletal effect of quercetin-6-C-&bgr;-d-glucopyranoside (QCG) isolated from the extract of Ulmus wallichiana and compare this effect with quercetin (Q) in a rat model of postmenopausal bone loss. Methods: Murine bone marrow cells were used to study the effect of QCG or Q on osteoclast differentiation. QCG or Q (1.0 and 5.0 mg kg−1 d−1 doses) was administered orally to ovarietomized (OVx) rats for 12 weeks. Sham-operated + vehicle and OVx + vehicle groups served as positive and negative controls, respectively. Bone mineral density, bone microarchitecture, biomechanical strength, bone turnover markers, and uterotrophic effect were studied. One-way analysis of variance was used to test significance of effects. Results: QCG at 1.0 nM significantly inhibited differentiation of multinucleated osteoclasts and expression of osteoclastogenic genes from bone marrow cells, whereas Q at 10.0 &mgr;M had comparable results. OVx rats treated with QCG exhibited significantly higher bone mass and better microarchitecture in trabecular and cortical bones compared with OVx + vehicle. QCG treatment of OVx rats had better functional impact than did Q-treated OVx rats, evident from increased bone biomechanical strength. Serum osteocalcin and urinary fragments of type 1 collagen were significantly lower in QCG-treated OVx rats compared with OVx + vehicle group. The protective effect of QCG under ovariectomy-induced bone loss setting was found to be significantly better than Q. Uterine histomorphometry parameters of OVx rats did not change with QCG treatment. Conclusions: QCG improves bone biomechanical quality more effectively than Q through positive modifications of bone mineral density and bone microarchitecture without a hyperplastic effect on the uterus.

A novel flavonoid, 6-C-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanone, isolated from Ulmus wallichiana Planchon mitigates ovariectomy-induced osteoporosis in rats.

Objective: The aim of this study was to determine the skeletal effect of 6-C-&bgr;-d-glucopyranosyl-(2S,3S)-(+)-3&vprime;,4&vprime;,5,7-tetrahydroxyflavanone (GTDF)/Ulmoside A, a new compound isolated from the extract of Ulmus wallichiana in a rat model of postmenopausal bone loss. Methods: GTDF (1.0 and 5.0 mg kg−1 d−1) was given orally to ovariectomized (OVx) rats (180-200 g) for 12 weeks. Sham operated + vehicle, ovariectomy + 17&bgr;-estradiol (2.5 &mgr;g kg−1 d−1), and ovariectomy + vehicle groups served as various controls. Bone mineral density (BMD), trabecular microarchitecture, bone biomechanical strength, levels of bone turnover/resorption markers, uterotropic effect, and plasma pharmacokinetics were studied. One-way analysis of variance was used to test significance of effects. Results: OVx rats treated with both doses of GTDF exhibited significantly higher BMD in the trabecular (distal femur, proximal tibia, and vertebrae) and cortical (femur shaft) regions compared with the ovariectomy + vehicle group. Micro-CT demonstrated that OVx rats treated with 5.0 mg kg−1 day−1 of GTDF had better bone microarchitectural parameters compared with the ovariectomy + vehicle group. Serum osteocalcin and urinary C-terminal teleopeptide of Type I collagen levels in OVx rats treated with GTDF (at both doses) were significantly lower than those in the ovariectomy + vehicle group. At neither of the two doses did GTDF exhibit uterine estrogenicity. A pharmacokinetic study revealed that GTDF achieved maximum plasma concentration (40.67 ng mL−1) at ∼1 hour, indicating its slow absorption. Its absolute bioavailability was found to be 1.04% with a plasma elimination half-life of ∼5 hours. Conclusions: GTDF, a novel compound isolated from U wallichiana extract, improves bone biomechanical quality through positive modifications of BMD and trabecular microarchitecture without a hyperplastic effect on the uterus.

EGb 761 promotes osteoblastogenesis, lowers bone marrow adipogenesis and atherosclerotic plaque formation.

AIM OF THE STUDY Our earlier study has demonstrated that EGb 761 (standardized extract of Ginkgo) has the bone sparing effect on the estrogen deficiency induced bone loss model. In the present study, we have addressed the question whether treatment of osteoporosis benefits arterial calcification or vice versa, because both adipocyte and osteoblast originate from the same mesenchymal cell of the bone marrow cell (BMC) population. MATERIALS AND METHODS Bone marrow cells were isolated to study the effect of EGb 761 on osteoblast and adipocytes. For in vivo effect hamsters were fed high fat diet and the effect of EGb 761 studied on atherosclerotic plaque formation and endothelial function. RESULTS BMCs undergoing induced osteogenic or adipogenic differentiations in the presence of EGb 761 show increase and decrease in mineralization and adipogenesis respectively. Osteogenic and adipogenic mRNAs, reveal lineage dependent expression patterns. Runx-2 (osteoblast transcription factor) showed a progressive increase, whereas PPAR-γ (adipogenic regulator) was attenuated, with same pattern of expression being for late osteogenic and adipogenic genes. EGb 761 led to increase in apoptotic cells and ROS, an important upstream signal. In vivo experiments in hamsters after induction with high cholesterol diet (HCD) show improvement in endothelial function by EGb 761 with lowering in total plasma cholesterol levels. EGb 761 led to vascular preservation of the aortic lumen with impairment of the endothelium dependent relaxation which was corroborated by micro-CT and histological sections of the thoracic region of the aorta. CONCLUSION From this data, it can be implied that EGb 761 controls bone loss, adiposity and lowers atherogenic risk factor after HCD induction.