Kunal Sharan

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.

Daidzein Prevents the Increase in CD4+CD28null T Cells and B Lymphopoesis in Ovariectomized Mice: A Key Mechanism for Anti-Osteoclastogenic Effect

Estrogen deficiency leads to an upregulation of TNF-α producing T cells and B-lymphopoesis which augments osteoclastogenesis. Estrogen deficiency also increases the population of premature senescent CD4+CD28null T cells which secrete a higher amount of TNF-α thus leading to enhanced osteoclastogenesis. Isoflavonoids like daidzein and genistein are found mostly in soybeans, legumes, and peas. These share structural similarity with 17β-stradiol (E2) and have osteoprotective role. This study explores the effect of daidzein (Daid) on the proliferation of TNF-α producing T cells, premature senescent T cells and B cell lymphopoesis under estrogen deficient conditions. For this study adult Balb/c mice were treated with Daid at 10 mg/kg body weight dose by oral gavage daily post ovariectomy (Ovx). After six weeks animals were autopsied and bone marrow and spleen cells were collected for FACS analysis. Blood serum was collected for ELISA. It was observed that Ovx mice treated with Daid for six weeks show reduction in Ovx induced expansion of CD4+ T cells in bone marrow and spleen when analysed by flow cytometry. Estrogen deficiency led to increased prevalence of TNF-α secreting CD4+CD28null T cells, however, treatment with Daid increased the percentage of CD4+CD28+ T cells. Co-culture of CD4+CD28null T cells and bone marrow resulted in enhanced osteoclastogenesis as evident by increased tartarate resistant acid phosphatase (TRAP) expression, an osteoclast marker. However, treatment with Daid resulted in reduced osteoclastogenesis in CD4+CD28null T cells and bone marrow cell co-culture. Daid also regulated B lymphopoesis and decreased mRNA levels of RANKL in B220+ cells. Taken together, we propose that one of the mechanisms by which Daid prevents bone loss is by reversing the detrimental immune changes as a result of estrogen deficiency.

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.

[6]-Gingerol induces bone loss in ovary intact adult mice and augments osteoclast function via the transient receptor potential vanilloid 1 channel.

SCOPE [6]-Gingerol, a major constituent of ginger, is considered to have several health beneficial effects. The effect of 6-gingerol on bone cells and skeleton of mice was investigated. METHODS AND RESULTS The effects of 6-gingerol on mouse bone marrow macrophages and osteoblasts were studied. 6-Gingerol-stimulated osteoclast differentiation of bone marrow macrophages but had no effect on osteoblasts. Capsazepine, an inhibitor of TRPV1 (transient receptor potential vanilloid 1) channel, attenuated the pro-osteoclastogenic effect of 6-gingerol or capsaicin (an agonist of TRPV1). Similar to capsaicin, 6-gingerol stimulated Ca(2) + influx in osteoclasts. The effect of daily feeding of 6-gingerol for 5 wk on the skeleton of adult female Balb/cByJ mice was investigated. Mice treated with capsaicin and ovariectomized (OVx) mice served as controls for osteopenia. 6-Gingerol caused increase in trabecular osteoclast number, microarchitectural erosion at all trabecular sites and loss of vertebral stiffness, and these effects were comparable to capsaicin or OVx group. Osteoclast-specific serum and gene markers of 6-gingerol-treated mice were higher than the OVx group. Bone formation was unaffected by 6-gingerol. CONCLUSION Daily feeding of 6-gingerol to skeletally mature female mice caused trabecular osteopenia, and the mechanism appeared to be activation of osteoclast formation via the TRPV1 channel.

Identification of Novel 2-((1-(Benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid Analogues as BMP-2 Stimulators

The synthesis and SAR studies of 10 new chemical entities (NCEs) that have shown BMP-2 stimulation and osteoblast differentiation are reported. Among these, 2-((1-(benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid (11) was the most effective while its analogue 13 also showed good activity in inducing osteoblast BMP-2 production. Compound 11 induced osteoblast differentiation in vitro, and this effect was abrogated by a physiological BMP-2 inhibitor, noggin. It also exhibited dose dependent increase in nascent bone formation (2.16- and 3.12-fold more than the control at 1 and 5 mg/kg dose, respectively) at the fracture site in rats. At the maximum osteogenic concentration, compound 11 significantly inhibited osteoblastic proteosomal activity. This compound was safe, as it had no effect on BMP synthesis in cardiovascular tissue.