Jawed A. Siddiqui

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.

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.

Phytochemicals for Breast Cancer Therapy: Current Status and Future Implications

Breast cancer is one of the most common malignancies among women, representing nearly 30% of newly diagnosed cancers every year. Till date, various therapeutic interventions, including surgery, chemotherapy, hormonal therapy, and radiotherapy are available and are known to cause a significant decline in the overall mortality rate. However, therapeutic resistance, recurrence and lack of treatment in metastasis are the major challenges that need to be addressed. Increasing evidence suggests the presence of cancer stem cells (CSCs) in heterogeneous population of breast tumors capable of selfrenewal and differentiation and is considered to be responsible for drug resistance and recurrence. Therefore, compound that can target both differentiated cancer cells, as well as CSCs, may provide a better treatment strategy. Due to safe nature of dietary agents and health products, investigators are introducing them into clinical trials in place of chemotherapeutic agents.This current review focuses on phytochemicals, mainly flavonoids that are in use for breast cancer therapy in preclinical phase. As phytochemicals have several advantages in breast cancer and cancer stem cells, new synthetic series for breast cancer therapy from analogues of most potent natural molecule can be developed via rational drug design approach.

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.

Evaluation of Cameroonian plants towards experimental bone regeneration

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.

Design and synthesis of 1,3-biarylsulfanyl derivatives as new anti-breast cancer agents

A new series of 1,3-biarylsulfanyl derivatives (homodibenzyl core motif) have been designed and synthesized as new estrogen receptor ligands by chopping benzothiophene core of raloxifene to engender seco-raloxifene scaffold. All the synthesized compounds were screened for anti-proliferative, anti-osteoporotic, and anti-implantation activity. Compounds (35, 36) having basic amino anti-estrogenic side chain were exhibiting potential anti-proliferative activity in MCF-7, MDA-MB-231 and ishikawa cell lines. Some of the synthesized compounds having homodibenzyl motif (5, 8, 10) have shown moderate anti-osteoporotic activity.