A novel extraction method enhanced the osteogenic and anti-osteoporosis effect of tea extract without any hepatotoxicity in ovariectomized rats.

Tea (Camellia sinensis) has several reported health benefits, including that on bone health attributed to catechins of which the most abundant is epigallocatechin-3-gallate (EGCG). However, several preclinical and clinical studies raise safety concerns about EGCG in tea extract causing acute liver failure. Tea also contains kaempferol, albeit scanty, and it has hepatoprotective and osteogenic effects. Here, we utilized a novel extraction procedure of acid hydrolysis to enhance the osteogenic effect of tea extract while reducing its hepatotoxicity. The resultant extract (USKECSE) has a ~40-fold increase in kaempferol and a 2.5-fold reduction in EGCG content compared with the hydroethanolic extract (USCSE). In a female Sprague Dawley (SD) rat femur osteotomy model, USKECSE (100 mg/kg) but not USCSE promoted bone regeneration. In a rat postmenopausal osteoporosis model induced by bilateral ovariectomy (OVX), USKECSE through an osteogenic mechanism maintained bone mass, strength, and microarchitecture to the levels of ovary-intact rats with no hepatotoxic effect. After a single oral dose (100 mg/kg) of USKECSE to adult rats, kaempferol was detectable for 48 hours, suggesting its significant absorption and distribution in plasma. Peak kaempferol concentration in plasma (Cmax) was 483 ng/ml (2 µM), and at this concentration, kaempferol induces osteoblast differentiation. USKECSE had no genotoxicity, and its safety index assessed by preclinical toxicity studies, including safety pharmacology, was >20-fold. Taken together, we report a novel extraction process that enhanced the osteogenicity and concomitantly reduced hepatotoxicity of tea extract with significant kaempferol bioavailability and a favorable systemic safety profile. Based on these data, we propose assessing the USKECSE effect for postmenopausal osteoporosis treatment.

Oral Administration of Isovitexin, a Naturally Occurring Apigenin Derivative Showed Osteoanabolic Effect in Ovariectomized Mice: A Comparative Study with Teriparatide.

Isovitexin (apigenin-6C-glucopyranose) is found in several food items and medicinal plants. Recently, we showed that isovitexin stimulated osteoblast differentiation through mitochondrial biogenesis and respiration that required adiponectin receptors (AdipoRs). Here, we studied whether oral isovitexin has a bone anabolic effect in vivo. At first, using a femur osteotomy model in adult mice, we compared the bone regenerative effect of isovitexin and apigenin. Whereas isovitexin-stimulated bone formation at the osteotomy site at 2.5 mg/kg and 5 mg/kg dose, apigenin had no effect. Subsequently, we tested the effect of isovitexin (5 mg/kg) in ovariectomized (OVX) osteopenic mice and observed that it restored bone mass and architecture of trabecular bones (femur metaphysis and fifth lumbar vertebra/L5) and cortical bones (femur diaphysis). Isovitexin completely restored bone strength at L5 (compressive strength) and femur (bending strength) in OVX mice. The bone anabolic effect of isovitexin was demonstrated by the increased surface referent bone formation parameters, increased expression of osteogenic genes (Runx2, bone morphogenetic protein-2 and type 1 collagen) in bones, and increased serum procollagen type 1N-terminal propeptide in OVX mice and these were on a par with teriparatide. Isovitexin inhibited bone and serum sclerostin as well as the serum type I collagen cross-linked C-telopeptide in OVX mice. Isovitexin has an oral bioavailability of 14.58%. Taken together, our data show that isovitexin had a significant oral bioavailability that translated to osteoanabolic effect equivalent to teriparatide and inhibited bone resorption, which implied a durable effect over teriparatide.

Diosmin, a citrus fruit-derived phlebotonic bioflavonoid protects rats from chronic kidney disease-induced loss of bone mass and strength without deteriorating the renal function.

Kidney Disease Improving Global Outcomes (KDIGO) 2017 Clinical Practice Guideline has recommended treatment decisions for patients with chronic kidney disease (CKD) with osteoporosis and/or high risk of fracture. Bisphosphonates, the first-line anti-osteoporosis drugs have the concern of worsening kidney functions. Moreover, despite impaired bone formation in CKD patients, teriparatide, the formation-stimulating drug is not recommended. Thus, there is an urgent need for safe and effective treatment of osteoporosis in CKD patients. Here, in CKD rats, we tested the osteoprotective effect of diosmin, a citrus-derived bioflavonoid used as a phlebotonic in chronic venous insufficiency and has a renoprotective effect. CKD was developed by 5/6th nephrectomy and diosmin at the human equivalent dose (100 mg kg-1) did not advance renal failure but reduced blood pressure to the level of sham control. Fibroblast growth factor-23 and parathyroid hormone were increased in CKD and diosmin suppressed both. CKD reduced bone mass and deteriorated the microarchitecture of trabecular bones, and diosmin maintained both to control levels. Bone formation and strength were impaired in the CKD and diosmin maintained these levels to control levels. Nanoindentation of bone showed that diosmin significantly increased tissue hardness over the control. Diosmetin, the metabolic surrogate of diosmin had comparable pharmacokinetic profiles between the control and CKD groups. Furthermore, diosmetin (50 mg kg-1) protected against CKD-induced bone loss. These data suggest that diosmin and its metabolic surrogate, diosmetin protect against CKD-induced osteopenia. Since diosmin has no renal adverse effect and protected bone mass and strength in CKD rats, we propose assessing its anti-osteoporosis effect in CKD patients.

Moderate/subclinical calcium deficiency attenuates trabecular mass, microarchitecture and bone growth in growing rats.

Adequate dietary calcium (Ca) intake is essential for bone accretion, peak bone mass (PBM) attainment, bone quality and strength during the mammalian growth period. Severe Ca deficiency during growing age results in secondary hyperparathyroidism (SHPT) and poor bone quality and strength. However, the impact of moderate Ca deficiency during rats early growth period on bone health and the reversibility with supplementing calcium later in adult life remains unclear. Female Sprague-Dawley (SD) rats (postnatal 28th day, P28) were initiated either with a moderate calcium-deficient diet (MCD, 0.25% w/w Ca) or a control diet (0.8% w/w Ca, control group) till P70. Thereafter, MCD rats were continued either with MCD diet or supplemented with calcium diet (0.8% w/w Ca, calcium supplemented group, CaS) till P150. Another group (control rats) were fed 0.8% w/w Ca containing diet from P28 till P150. MCD group, as compared to the control group, had significantly reduced serum ionized Ca and procollagen type 1 N-terminal propeptide (P1NP) at P70 while no significant change was observed in serum corrected Ca, inorganic phosphate (P), alkaline phosphatase (ALP), 25-hydroxy vitamin D [25(OH)D], intact parathyroid hormone (iPTH), and urinary C-terminal telopeptide of collagen 1 (CTX-1), Ca, and P. Femoral and tibial metaphysis in MCD rats had significantly reduced linear growth, cortical and trabecular volumetric BMD (vBMD), trabecular microarchitecture (BV/TV%, trabecular thickness, separation and number, structural model index and connectivity density), cortical thickness, and bone stiffness despite the absence of secondary hyperparathyroidism (SHPT). Continued MCD at P70-P150 results in persistence of compromised bone strength while calcium supplementation (CaS group) improved all the parameters related to bone strength and microarchitecture. Our results indicate that uncorrected moderate/subclinical calcium deficiency in growing rats can result in poor bone quality and strength despite the absence of SHPT. This finding could have relevance in children with poor calcium intake in childhood and adolescence.

Selective dietary polyphenols induce differentiation of human osteoblasts by adiponectin receptor 1-mediated reprogramming of mitochondrial energy metabolism.

Anabolic therapies for osteoporosis including dietary polyphenols promote osteoblast function by influencing its energy metabolism. Among the dietary polyphenols, the beneficial skeletal effects of genistein (an isoflavone), kaempferol (a flavone), resveratrol (RES, a stilbenoid) and epigallocatechin gallate (EGCG, a catechin) have been reported in preclinical studies. We studied the action mechanism of these nutraceuticals on osteoblast bioenergetics. All stimulated differentiation of human fetal osteoblasts (hFOB). However, only EGCG and RES stimulated mitochondrial parameters including basal and maximum respiration, spare respiratory capacity and ATP production (a measure of the activity of electron transport chain/ETC). Increases in these parameters were due to increased mitochondrial biogenesis and consequent upregulation of several mitochondrial proteins including those involved in ETC. Rotenone blocked the osteogenic effect of EGCG and RES suggesting the mediatory action of mitochondria. Both compounds rapidly activated AMPK, and dorsomorphin (an AMPK inhibitor) abolished ATP production stimulated by these compounds. Moreover, EGCG and RES upregulated the mitochondrial biogenesis factor, PGC-1α which is downstream of AMPK activation, and silencing PGC-1α blocked their stimulatory effects on ATP production and hFOB differentiation. Adiponectin receptor 1 (AdipoR1) is an upstream regulator of PGC-1α, and both compounds increased the expression of AdipoR1 but not AdipoR2. Silencing AdipoR1 blocked the upregulation of EGCG/RES-induced PGC-1α and hFOB differentiation. In rat calvarium, both compounds increased AdipoR1, PGC-1α, and RunX2 (the osteoblast transcription factor) with a concomitant increase in mitochondrial copy number and ATP levels. We conclude that EGCG and RES display osteogenic effects by reprogramming osteoblastic bioenergetics by acting as the AdipoR1 agonists.

A nutraceutical composition containing diosmin and hesperidin has osteogenic and anti-resorptive effects and expands the anabolic window of teriparatide.

A combination of diosmin and hesperidin (9:1 ratio) is marketed as a dietary supplement/nutraceutical for cardiovascular health. We studied the skeletal effect of this combination (90% diosmin and 10% hesperidin, henceforth named as DH). We showed that a) in rats with femur osteotomy, DH stimulated callus bone regeneration, b) in growing rats, DH promoted peak bone mass achievement and c) in OVX rats rendered osteopenic, DH completely restored femur trabecular bones and strength along with the increases in surface referent bone formation and serum osteogenic marker. Furthermore, DH suppressed bone resorption in OVX rats as well as in OVX rats treated with teriparatide (human parathyroid hormone 1-34) but did not affect the osteoanabolic effect of teriparatide. These data suggested that DH could prolong the anabolic window of teriparatide. To understand the mechanism of DH action, we performed pharmacokinetic studies and observed that upon its oral administration the only circulating metabolites was diosmetin (the aglycone form of diosmin) while none of the two input flavanones were detectable. Accordingly, subsequent experiments with diosmetin revealed that it was a selective estrogen receptor-ß agonist that stimulated osteoblast differentiation and suppressed sclerostin the anti-osteoblastogenic Wnt antagonist. Taken together, our study defined a positive skeletal effect of DH.

Extract and fraction of Cassia occidentalis L. (a synonym of Senna occidentalis) have osteogenic effect and prevent glucocorticoid-induced osteopenia.

ETHNOPHARMACOLOGICAL RELEVANCE: Cassia occidentalis L., a synonym of Senna occidentalis (belongs to Caesalpiniaceae family) is an annual plant. Pursuing a lead from a folk practice prevalent since the late nineteenth century in Andhra Pradesh, a Southern state of India, of use of Cassia occidentalis leaf and stem for treating patients with fracture and bone diseases, we have not only confirmed its fracture healing activity but also demonstrated efficacy in preventing glucocorticoid-induced osteoporosis (GIO), the commonest form of medication-induced bone loss caused chiefly due to impairment of bone formation. AIM OF THE STUDY: In the present work, the effects of extract and fraction of leaf and stem of Cassia occidentalis was investigated in fracture healing and GIO models of rat. The study also aimed to identify osteogenic compounds from this plant. MATERIALS AND METHODS: Ethanolic extracts from leaf and stem of Cassia occidentalis were prepared and their efficacy tested in rat femur osteotomy (fracture healing) model. Subsequently, a butanolic fraction was prepared and osteogenic efficacy compared with the ethanolic extract, and upon finding the former to be more potent, its osteogenic effect was studied in details in GIO model. Chemical finger-printing and isolation of ten pure compounds were done to assess their osteogenic effect in rat primary osteoblast cultures. RESULTS: Ethanolic extract of stem was more effective than the leaf extract in enhancing bone regeneration at the site of osteotomy. Further, butanolic fraction of the ethanolic extract of stem was more effective than the later in bone regeneration at the femur osteotomy site and in preventing bone loss in GIO model. The mechanism of skeletal preservation involved stimulation of new bone formation and inhibition of bone resorption. As many as six osteogenic compounds were isolated out of which apigenin-6C-glucopyranoside was most effective in vitro. CONCLUSION: Our study found that a standardized extract of an ethanolic extract and its butanolic fraction from the stem of Cassia occidentalis has osteogenic as well as anti-resorptive effects, resulting in the protection against glucocorticoid-induced bone loss. Our results contribute towards validation of the traditional use of Cassia occidentalis in fracture healing and also suggest its beneficial use in GIO for which clinical trials are warranted.

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/ß-catenin signaling.

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/ß-catenin pathway assessed by phosphorylation of GSK-3ß. Over-expressing of constitutively active GSK-3ß (caGSK-3ß) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3ß over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats.

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.