9-Demethoxy-medicarpin: A potential bone health supplement for the management of protein deficiency-induced bone loss in growing rats.

Human skeleton requires an adequate supply of many different nutritional factors for optimal growth and development. The role of nutrition in bone growth has piqued interest in recent years, especially in relation to maximizing peak bone mass and reducing the risk of osteoporosis. Protein deficiency-induced bone loss was induced in female growing rats. All experimental rodent diets were prepared as per recommendations for growing animals. 9-Demethoxy-medicarpin (DMM) treatment was given to growing Sprague Dawley (SD) rats at 1 mg and 10 mg dose orally for 30 days. Bones were collected for bone mineral density (BMD). Bone marrow cells were isolated from femur for calcium nodule formation. Serum samples were collected for biochemical parameters. We found that DMM treatment speeds up the recovery of musculoskeletal weakness by replenishing nutrients in proven rodent model. DMM supplementation for four weeks showed significantly increased vertebral, femur and tibial BMD compared with the untreated PD group. Albumin levels were significantly enhanced in treatment groups, in which 10 mg dose imparted a better effect. We conclude that DMM treatment led to increased BMD and biochemical parameters in protein deficient condition in growing rats and has potential as a bone growth supplement.

Maintenance of increased bone mass after PTH withdrawal by sequential medicarpin treatment via augmentation of cAMP-PKA pathway.

Osteoporosis is a metabolic bone disorder associated with impaired bone microarchitecture leading to fragility fractures. Long-term usage of parathyroid hormone (PTH) enhances bone resorption and leads to osteosarcoma in rats which limits its exposure to maximum 2 years in human. Notably, the anabolic effects of PTH do not endure in the absence of sustained administration. Studies in our lab identified osteogenic and antiresorptive activity in medicarpin, a phytoestrogen belonging to the pterocarpan class. Considering dual-acting property of medicarpin and limitations of PTH therapy, we envisaged that medicarpin sequential treatment after PTH withdrawal could serve as promising therapeutic approach for osteoporosis treatment. As PTH exerts its bone anabolic effect by increasing osteoblast survival, our study aims to determine whether medicarpin amplifies this effect of PTH. Our results show that PTH withdrawal led to reduced bone mineral density and bone parameters, while sequential treatment of medicarpin after PTH withdrawal significantly enhanced these parameters. Remarkably, these effects were more pronounced than 8-week PTH treatment. Sequential therapy also significantly increased P1NP levels and decreased CTX levels and TRAP positive cells compared to PTH 8W group where CTX levels were quite high due to bone resorptive action of PTH. Protein expression studies revealed that medicarpin along with PTH betters the antiapoptotic potential compared to PTH alone, through augmentation of cyclic adenosine monophosphate-PKA-CREB pathway. These results proclaim that medicarpin sequential treatment prevented the reduction in bone accrual and strength accompanying PTH withdrawal and also aided in antiapoptotic role of PTH. The study points toward the potential use of medicarpin as a replacement therapeutic option postdiscontinuation of PTH.

3-PEP promotes bone regeneration by up regulating BCL-2 expression via ERK phosphorylation.

Abstract: Bone healing and regeneration is a complex process that recapitulates embryonic skeletal development and is delayed in diseases like osteoporosis. Bone healing therapies like recombinant bone morphogenetic-2 protein (rhBMP-2) and parathyroid hormone (PTH), an approved bone anabolic therapy reduces fracture risks but are fraught with high cost and several side effects. Thus, there is an unmet need for cost-effective bone healing agents. In this study, we have synthesized 3-piperidinylethoxypterocarpan (3-PEP) which is a hybrid of bone supplement ipriflavone and anti-resorptive drug raloxifene and evaluated its bone regeneration and healing potential. Prior to studies in animal models, the potency of 3-PEP was confirmed in calvarial osteoblast cells. Bromodeoxy uridine cell proliferation and cell viability assay revealed that 3-PEP at 100 pM concentration increased the proliferation and survival of osteoblasts simultaneously inhibiting the apoptosis by involving activation of BCL-2 by phosphorylation at Ser70 site through MEK-ERK pathway. In vivo studies were conducted in estrogen-deficient ovariectomized Balb/c mice and drill hole injury was generated in the mid diaphysis of the femur in all the animals. Treatment with 3-PEP commenced the next day onward and terminated at 7 and 15 days. Micro-CT analysis and calcein labeling of newly generated bone at the drill hole injury site showed that 3-PEP promotes bone healing and new bone formation at a dose of 5 mg/kg at the injury site. These data were also corroborated in non-ovariectomized Balb/c mice cortical defect model. Owing to the side effects associated with rhBMP-2 and PTH, along with the expenses involved, our study proposes an alternative therapeutic option for bone healing.

Medicarpin confers powdery mildew resistance in Medicago truncatula and activates the salicylic acid signalling pathway.

Powdery mildew (PM) caused by the obligate biotrophic fungal pathogen Erysiphe pisi is an economically important disease of legumes. Legumes are rich in isoflavonoids, a class of secondary metabolites whose role in PM resistance is ambiguous. Here we show that the pterocarpan medicarpin accumulates at fungal infection sites, as analysed by fluorescein-tagged medicarpin, and provides penetration and post-penetration resistance against E. pisi in Medicago truncatula in part through the activation of the salicylic acid (SA) signalling pathway. Comparative gene expression and metabolite analyses revealed an early induction of isoflavonoid biosynthesis and accumulation of the defence phytohormones SA and jasmonic acid (JA) in the highly resistant M. truncatula genotype A17 but not in moderately susceptible R108 in response to PM infection. Pretreatment of R108 leaves with medicarpin increased SA levels, SA-associated gene expression, and accumulation of hydrogen peroxide at PM infection sites, and reduced fungal penetration and colony formation. Strong parallels in the levels of medicarpin and SA, but not JA, were observed on medicarpin/SA treatment pre- or post-PM infection. Collectively, our results suggest that medicarpin and SA may act in concert to restrict E. pisi growth, providing new insights into the metabolic and signalling pathways required for PM resistance in legumes.

Medicarpin prevents arthritis in post-menopausal conditions by arresting the expansion of TH17 cells and pro-inflammatory cytokines.

Autoimmune diseases are characterized by alteration in balance of various cytokines. Rheumatoid arthritis is a well-known inflammatory disease leading to destruction of cartilage at knee and hands. Collagen-induced arthritis (CIA) is a common autoimmune model for rheumatoid arthritis study. Here, we have investigated the therapeutic role of medicarpin, a natural pterocarpan with known anti-osteoclastogenic activities, in postmenopausal polyarthritis model of DBA/1J mice. For this, mice were ovariectomized and CIA was induced in OVx animals with primary immunization. After 21 days, booster dose was injected in Ovariectomy (OVx) mice to develop postmenopausal poly-arthritis mice model. Medicarpin treatment in mice at dose of 10.0 mg/kg/body wt was started after 21 days of primary immunization for one month of time period every day orally. We found that medicarpin prevented alteration of TH-17/Treg ratio in CIA model leading to reduced osteoclastogenesis. Micro Computed Tomography (Micro-CT) analysis demonstrated that medicarpin prevents cartilage erosion in joints and restores loss of trabeculae parameters in distal tibia. Treatment with medicarpin also prevented alteration of various cytokines level by down-regulating various pro-inflammatory cytokines like TNF-α, IL-6 and IL-17A, while up-regulating anti-inflammatory cytokine IL-10 in CIA model of mice. Biological marker of arthritis is cartilage oligomeric matrix protein (COMP). COMP level was up-regulated in CIA induced mice while treatment with medicarpin significantly restored the serum level of COMP compared with untreated groups. Cartilage staining by Safranin-O also indicates that cartilage destruction in joints of CIA mice was prevented by medicarpin treatment. From this study, we can conclude that medicarpin is effective in preventing arthritis in post-menopausal conditions.

Bioavailability, tissue distribution and excretion studies of a potential anti-osteoporotic agent, medicarpin, in female rats using validated LC-MS/MS method.

Medicarpin, one of the active constituents isolated from the extract of Butea monosperma, has been shown to have various pharmacological activities including potent anti-osteoporotic properties. The aim of this study was to investigate the oral pharmacokinetics, tissue distribution and excretion of medicarpin following single oral dose administration in female rats. Oral pharmacokinetics was explored at 5 and 20 mg/kg while tissue distribution, urinary and fecal excretion were studied following 20 mg/kg oral dose. Medicarpin was quantified in rat plasma, urine, feces and tissue samples using a validated LC-MS/MS method following reverse-phase HPLC separation on RP18 column (4.6 mm × 50 mm, 5.0 µm) using methanol and 10 mM ammonium acetate (pH 4.0) as mobile phase in the ratio of 80:20 (v/v) at a flow rate of 0.8 mL/min. The oral bioavailability of medicarpin was found to be low with low systemic levels. The concentration in tissues was significantly higher than plasma. Highest tissue concentrations were found in the liver followed by bone marrow. Urinary and fecal excretion of medicarpin was < 1 %. In conclusion, medicarpin was found to be highly distributed in body tissues and minimally excreted via urine or feces.

White Light Induced E/Z-Photoisomerization of Diphenylamine-Tethered Fluorescent Stilbene Derivatives: Synthesis, Photophysical, and Electrochemical Investigation.

A facile synthesis and detailed photophysical investigation of E/Z-isomerization of fluorescent diphenylamine tethered stilbene derivatives (DPASs) under white light exposure have been carried out to understand the effect on fluorescence, electrochemical properties, and photostability under various activation/deactivation pathways. In solution state, in the dark, the E-isomer of DPASs (6a-d) exhibited high fluorescence quantum yields (Φfl ≈ 53% to 60% in DMSO). However, on white light exposure, 1H NMR and HPLC studies revealed that pure E-isomer of the DPAS 6a (∼9.5 mM) started converting into its Z-form by photoisomerization until it reaches to nearly equilibrium. At low concentrations (∼10 µM), the absorption band of the pure E-isomer in the range of 350-450 nm gradually decreased to adopt Z-conformation 6a' until a photostationary state was reached. The structure of the E-isomer 6a was unequivocally confirmed by X-ray diffraction analysis. The synthesized DPAS compounds 6a-d possessed positive solvatochromic properties, two photon absorption properties, and good thermal stability. The electrochemical investigations using DPASs showed reversible oxidation resulting in formation of a stable radical cation. Owing to useful photophysical, electrochemical and thermal properties, these DPAS derivatives are suitable for their application in biomedical imaging as well as in fabrication of electroluminescent materials.

3-Piperidylethoxypterocarpan: A potential bone anabolic agent that improves bone quality and restores trabecular micro-architecture in ovariectomized osteopenic rats.

A series of new 6H-benzofuro[3, 2-c]chromenes (BFC, pterocarpans) with structure-activity relationships were investigated for their potential use in osteoporosis treatment. One of the BFCs 3-piperidylethoxypterocarpan 20 promotes osteoblast differentiation and mineralization at a dose as low as 1 pM via activation of ER/P38MAPK/BMP-2 pathway. When evaluated for in-vivo osteogenic activity in female Sprague-Dawley rats, BFC 20 increased bone mineral density and new bone formation, compared with control at 1.0 and 10.0 mg/kg/body weight by oral gavage for 30 days. The compound was devoid of any uterotrophic effect and led to the new bone formation in adult ovariectomized osteopenic rats. BFC 20 compound also inhibited bone resorption by reducing Ovx induced increase in urinary CTx, thus exhibiting both bone anabolic and anti-catabolic action. Finally, BFC 20 treatment to Ovx rats led to improved trabecular microarchitectural restoration and exhibited therapeutic potential as a dual acting anti-osteoporotic agent for the management of osteoporosis.

Identification of GRP78 as a molecular target of medicarpin in osteoblast cells by proteomics.

Osteogenic activity was identified in medicarpin (Med), a natural pterocarpan. Further, it was decided to study the differentially regulated protein expression during osteoblast differentiation in the presence of Med. Using 2D proteomic approach, we found that Med treatment to osteoblasts significantly downregulated GRP78, an ER chaperone with anti-apoptotic properties which also controls the activation of unfolded protein response signaling, a pro-survival strategy for normal ER functioning. However, severe stress leads to triggering of apoptotic responses and signaling switches to pro-apoptotic. In order to elucidate the effect of Med downregulation of GRP78, osteoblasts were transfected with SiGRP78 or SiGRP78+ Med or Med alone. It was seen that mRNA and protein levels of ER stress markers like GRP78, ATF-4, and CHOP were decreased in all the three groups with maximum reduction in SiGRP78+ Med group. Med targets GRP78 by inhibiting mitochondrial-mediated apoptosis which is evident by reduced levels of cytochrome c, caspase-3, Bax/BCL2 ratio, and enhanced expression of survivin. Finally, Annexin-PI staining of apoptotic cells revealed that MED inhibition of GRP78 leads to reduced osteoblast apoptosis and increased osteoblast survival. Altogether, our data show that Med inhibits ER stress-induced apoptosis and promotes osteoblast cell survival by targeting GRP78.

Medicarpin, a Natural Pterocarpan, Heals Cortical Bone Defect by Activation of Notch and Wnt Canonical Signaling Pathways.

We evaluated the bone regeneration and healing effect of Medicarpin (med) in cortical bone defect model that heals by intramembranous ossification. For the study, female Sprague-Dawley rats were ovariectomized and rendered osteopenic. A drill hole injury was generated in mid femoral bones of all the animals. Med treatment was commenced the day after and continued for 15 days. PTH was taken as a reference standard. Fifteen days post-treatment, animals were sacrificed. Bones were collected for histomorphometry studies at the injury site by micro-computed tomography (µCT) and confocal microscopy. RNA and protein was harvested from newly generated bone. For immunohistochemistry, 5µm sections of decalcified femur bone adjoining the drill hole site were cut. By µCT analysis and calcein labeling of newly generated bone it was found that med promotes bone healing and new bone formation at the injury site and was comparable to PTH in many aspects. Med treatment led to increase in the Runx-2 and osteocalcin signals indicating expansion of osteoprogenitors at the injury site as evaluated by qPCR and immunohistochemical localization. It was observed that med promoted bone regeneration by activating canonical Wnt and notch signaling pathway. This was evident by increased transcript and protein levels of Wnt and notch signaling components in the defect region. Finally, we confirmed that med treatment leads to elevated bone healing in pre-osteoblasts by co localization of beta catenin with osteoblast marker alkaline phosphatase. In conclusion, med treatment promotes new bone regeneration and healing at the injury site by activating Wnt/canonical and notch signaling pathways. This study also forms a strong case for evaluation of med in delayed union and non-union fracture cases.