Preventive effects of withaferin A isolated from the leaves of an Indian medicinal plant Withania somnifera (L.): comparisons with 17-ß-estradiol and alendronate.

OBJECTIVE: Bone protective effects of withaferin A (WFA) from leaves of Withania somnifera (L.) were evaluated in preventive model of Balb/c mice with 17 ß-estradiol (E2) and alendronate (ALD). METHODS: Adult female Balb/c mice, 7 to 9 wk, were bilaterally ovariectomized (OVx) to mimic the state of E2 deficiency. Immediately after surgery mice were administrated WFA at doses of 1, 5, 10 mg/kg/d while other two OVx groups received ALD or E2 for 2 mo. Sham and OVx groups with vehicle and no treatment served as controls. RESULTS: WFA administration increased new bone formation, as well as improving microarchitecture and biomechanical strength of the bones. It prevented bone loss by reducing expression of osteoclastic genes tartrate resistant acid phosphatase (TRAP) and receptor activator of nuclear factor κ B (RANK). Increase in bone turnover marker, osteocalcin (OCN) and inflammatory cytokine tumor necrosis factor-alpha (TNF-α) because of ovariectomy were reduced with WFA treatment, with effects comparable to E2 administration. Histomorphometric analysis of uterus shows that WFA was not fraught with estrogenic or antiestrogenic effects. At cellular level, WFA promoted differentiation of bone marrow cells (BMCs) and increased mineralization by inducing expression of osteogenic genes. WFA has bone protective potential as its treatment prevents bone loss that is comparable to ALD and E2. CONCLUSIONS: It is surmised that WFA in preclinical setting is effective in preserving bone loss by both inhibition of resorption and stimulation of new bone formation before onset of osteoporosis with no uterine hyperplasia.

Ovariectomized Rats with Established Osteopenia have Diminished Mesenchymal Stem Cells in the Bone Marrow and Impaired Homing, Osteoinduction and Bone Regeneration at the Fracture Site.

We investigated deleterious changes that take place in mesenchymal stem cells (MSC) and its fracture healing competence in ovariectomy (Ovx)-induced osteopenia. MSC from bone marrow (BM) of ovary intact (control) and Ovx rats was isolated. (99m)Tc-HMPAO (Technitium hexamethylpropylene amine oxime) labeled MSC was systemically transplanted to rats and fracture tropism assessed by SPECT/CT. PKH26 labeled MSC (PKH26-MSC) was bound in scaffold and applied to fracture site (drill-hole in femur metaphysis). Osteoinduction was quantified by calcein binding and microcomputed tomography. Estrogen receptor (ER) antagonist, fulvestrant was used to determine ER dependence of osteo-induction by MSC. BM-MSC number was strikingly reduced and doubling time increased in Ovx rats compared to control. SPECT/CT showed reduced localization of (99m)Tc-HMPAO labeled MSC to the fracture site, 3 h post-transplantation in Ovx rats as compared with controls. Post-transplantation, Ovx MSC labeled with PKH26 (Ovx PKH26-MSC) localized less to fracture site than control PKH26-MSC. Transplantation of either control or Ovx MSC enhanced calcein binding and bone volume at the callus of control rats over placebo group however Ovx MSC had lower efficacy than control MSC. Fulvestrant blocked osteoinduction by control MSC. When scaffold bound MSC was applied to fracture, osteoinduction by Ovx PKH26-MSC was less than control PKH26-MSC. In Ovx rats, control MSC/E2 treatment but not Ovx MSC showed osteoinduction. Regenerated bone was irregularly deposited in Ovx MSC group. In conclusion, Ovx is associated with diminished BM-MSC number and its growth, and Ovx MSC displays impaired engraftment to fracture and osteoinduction besides disordered bone regeneration.

Osteogenic efficacy enhancement of kaempferol through an engineered layer-by-layer matrix: a study in ovariectomized rats.

AIM: A layer-by-layer matrix (LBL) comprising kaempferol (LBL-KEM) was prepared for improved osteogenic action. MATERIALS & METHODS: The LBL-KEM consisted of alternate layers of sodium alginate and protamine sulfate, which were sequentially deposited on the preformed kaempferol (KEM)-loaded CaCO3 core (CaCO3-KEM) by LBL self-assembly. The LBL matrix developed was evaluated for layer growth by ζ-potential and size alterations after self-assembly of each layer. Its physicochemical properties and intestinal absorption pattern were characterized and its pharmacokinetic behavior, mineralization of bone marrow cells, bone mineral density, bone strength, microcrack formation and estrogenicity were evaluated after oral administration. RESULTS: The entrapment efficiency of KEM was 94 ± 2% and the cumulative %KEM released from LBL-KEM was 19.2 and 63.5% at pH 1.4 and 7.4, respectively, after 24 h. Stepwise polyelectrolyte assembly onto initially positively charged particles (+21.2 mV) resulted in alterations between -28.5 and +10.9 mV. A final ζ-potential of -8.9 mV was obtained after terminal surface modification with sodium deoxycholate. Fluorescein isothiocyanate-labeled LBL matrix was diffused into the basolateral lacteal region upon oral administration to rats. The area under the KEM serum concentration curve following oral administration of LBL-KEM to rats was 2479 ± 682 ng·h/ml, nearly twofold higher than free KEM. The concentration-time profile in bone marrow indicated improved penetration and retention of KEM on administration of LBL-KEM. Treatment with LBL-KEM restored bone mineralization, bone mineral density, microcrack formation and empty osteocyte lacunae density in ovariectomized (OVx) rats, which was significantly (p < 0.05) improved in femoral diaphysea, tibial head and vertebrae compared with free KEM treatment. Administration of LBL-KEM to growing female rats for 4 weeks resulted in no estrogenicity when compared with OVx rats. CONCLUSION: The data suggests that LBL matrix enhanced drug delivery, improved pharmacokinetics and maintained better bone quality under OVx conditions.

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: BMC's 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.

In vivo efficacy studies of layer-by-layer nano-matrix bearing kaempferol for the conditions of osteoporosis: a study in ovariectomized rat model.

A prototype formulation based on layer-by-layer (LbL) nano-matrix was developed to increase bioavailability of kaempferol with improved retention in bone marrow to achieve enhanced bone formation. The layer-by-layer nano-matrix was prepared by sequential adsorption of biocompatible polyelectrolytes over the preformed kaempferol-loaded CaCO(3) template. The system was pharmaceutically characterized and evaluated for osteogenic activity in ovariectomized (OVx) rats. Data have been compared to the standard osteogenic agent parathyroid hormone (PTH). Single oral dose of kaempferol loaded LbL nano-matrix formulation increased bioavailability significantly compared to unformulated kaempferol. Three months of Formulated kaempferol administration to osteopenic rats increased plasma and bone marrow Kaempferol levels by 2.8- and 1.75-fold, respectively, compared to free Kaempferol. Formulated Kaempferol increased bone marrow osteoprogenitor cells, osteogenic genes in femur, bone formation rate, and improved trabecular micro-architecture. Withdrawal of Formulated kaempferol-in OVx rats resulted in the maintenance of bone micro-architecture up to 30days, whereas micro-architectural deterioration was readily observed in OVx rats treated with unformulated kaempferol-within 15days of withdrawal. The developed novel formulation has enhanced anabolic effect in osteopenic rats through increased stimulatory effect in osteoblasts. Treatment post-withdrawal sustenance of formulated kaempferol could become a strategy to enhance bioavailability of flavanoids.

A standardized phytopreparation from an Indian medicinal plant (Dalbergia sissoo) has antiresorptive and bone-forming effects on a postmenopausal osteoporosis model of rat.

OBJECTIVE: The aim of this study was to evaluate the skeletal effects of an extract made from the leaves and pods of Dalbergia sissoo (butanol-soluble standardized fraction [BSSF]) on ovariectomized rats, a model for postmenopausal osteopenia. METHODS: Adult Sprague-Dawley rats were ovariectomized and administered BSSF (50 and 100 mg/kg per day) or 17ß-estradiol orally for 12 weeks. The sham-operated group and the ovariectomy + vehicle group served as controls. Bone microarchitecture, bone turnover markers (serum osteocalcin and C-telopeptide fragment of collagen type I), biomechanical strength, new bone formation (based on mineral apposition rate and bone formation rate), and skeletal expressions of osteogenic and resorptive gene markers were studied. Uterine histomorphometry was used to assess estrogenicity. Bioactive marker compounds in BSSF were analyzed by high-performance liquid chromatography. One-way analysis of variance was used to test the significance of effects. RESULTS: In comparison with ovariectomized rats treated with vehicle, BSSF treatment in ovariectomized rats resulted in an improved trabecular microarchitecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and type I collagen) and expression of skeletal osteoclastogenic genes, and increased new bone formation and expression of osteogenic genes in the femur. Overall, the osteoprotective effects of BSSF were comparable to those of 17ß-estradiol. BSSF did not exhibit uterine estrogenicity. Analysis of marker compounds revealed the presence of osteogenic methoxyisoflavones, including caviunin 7-O-[ß-D-apiofuranosyl-(1→6)-ß-D-glucopyranoside] (a novel compound), biochanin A, and pratensin. CONCLUSIONS: Oral doses of BSSF in the preclinical setting are effective in preventing estrogen deficiency-induced bone loss by dual action: inhibition of bone resorption and stimulation of new bone formation.

Formononetin reverses established osteopenia in adult ovariectomized rats.

OBJECTIVE: Formononetin (Formo) prevents ovariectomy (Ovx)-induced bone loss in rats. However, there are no reports on the curative effects of Formo. The objective of this study was to investigate the ability of Formo in restoring trabecular microarchitecture and promoting new bone formation in osteopenic rats. METHODS: Adult Sprague-Dawley rats were ovariectomized and left for 90 days for osteopenia to develop. After 90 days, Formo (10.0 mg kg d) was given orally for the next 12 weeks to Ovx rats in a therapeutic protocol. Sham-operated, Ovx + vehicle, and Ovx + parathyroid hormone (PTH) groups served as controls. Trabecular microarchitecture, osteoid formation, bone turnover/resorption markers, and bone osteoprotegerin-to-receptor activator for nuclear κB ligand ratio were studied. One-way analysis of variance was used to test significance of effects. RESULTS: Formo treatment significantly restored the lost trabecular microarchitecture in the femurs and tibia of osteopenic Ovx rats and promoted new bone formation. Formo was devoid of any uterine estrogenicity. Serum levels of type I collagen N-terminal propeptide, which is a reliable marker of bone formation, were increased in Ovx rats treated with Formo compared with Ovx + vehicle group, and the levels were comparable with those in the sham group. Formo prevented the Ovx-induced increase in bone turnover markers, including serum osteocalcin and urinary type I collagen degradation product. Furthermore, Formo-treated Ovx rats had an increased bone osteoprotegerin-to-receptor activator for nuclear κB ligand ratio compared with the Ovx + vehicle group. CONCLUSIONS: Daily oral administration of Formo for 12 weeks has a substantial anabolic effect, thus raising the possibility of its use in postmenopausal osteoporosis.

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.

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 d) and ASF (100 mg kg d) 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.

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-beta-d-glucopyranosyl-(2S,3S)-(+)-3',4',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 d) was given orally to ovariectomized (OVx) rats (180-200 g) for 12 weeks. Sham operated + vehicle, ovariectomy + 17beta-estradiol (2.5 microg kg d), 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 day 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) at approximately 1 hour, indicating its slow absorption. Its absolute bioavailability was found to be 1.04% with a plasma elimination half-life of approximately 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.

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 + 17beta-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.

Effects of Egb 761 on bone mineral density, bone microstructure, and osteoblast function: Possible roles of quercetin and kaempferol.

The effects of standardized and concentrated extract of Ginkgo biloba, Egb 761, were studied on estrogen deficiency-induced bone loss in ovariectomized (OVx) rats rendered osteopenic. Upon osteopenia development, Egb 761 was administered at a dose of 100mgkg(?1)day(?1) by oral gavage to OVx rats whereas control group received vehicle. Following 5 weeks of treatment, the OVx+Egb 761 group (n=12) of rats exhibited significantly higher whole body BMD and lower bone turnover markers (serum alkaline phosphatase and osteocalcin) than OVx rats that were given vehicle (n=12). BMD levels in excised bones were also found to be higher in both trabecular (most robustly in lumbar vertebrae) and cortical bones of OVx+Egb 761 compared with OVx+vehicle group. Egb 761 did not exhibit estrogen agonistic activity at the uterine level. Microcomputed tomography demonstrated that OVx+Egb 761 group had better bone microarchitectural parameters compared with OVx+vehicle group. Moreover, OVx+Egb 761 group had higher femoral mRNA levels of osterix, type I collagen and osteocalcin compared with OVx+vehicle group. Determination of levels of three flavonoids of Egb 761, that are known to have bone conserving property, in serum and bone marrow suggests that kaempferol and quercetin, and not rutin, likely mediate the beneficial actions observed with Egb 761 treatment. These results show for the first time that oral administration of Egb 761 restores bone mass in aged OVx rats.