Hexane Fraction of Turbo brunneus Inhibits Intermediates of RANK-RANKL Signaling Pathway and Prevent Ovariectomy Induced Bone Loss.

Osteoporosis is a "silent disease" characterized by fragile and impaired bone quality. Bone fracture results in increased mortality and poor quality of life in aged people particularly in postmenopausal women. Bone is maintained through the delicate balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The imbalance is caused most often by overly active osteoclasts due to estrogen deficiency. Natural products have long been used to prevent and treat osteoporosis since they have fewer side effects. The marine environment is a potential source of biologically and structurally novel biomolecules with promising biological activities but is less explored for the treatment of bone-related diseases. The present study aims to evaluate the antiosteoporotic effect of Hexane fraction of Turbo brunneus methanolic extract (HxTME) and to investigate its role in RANK-RANKL signaling pathway using in vitro osteoclasts cultures and in vivo ovariectomized (OVX) Swiss mice model. The present study demonstrated that the HxTME significantly inhibited RANKL induced osteoclast differentiation and maturation in vitro. HxTME completely downregulated the mRNA expression of key transcription factors such as NFATc1, c-FOS, and osteoclasts related genes involved in osteoclastogenesis. In vivo studies also depicted the effectiveness of HxTME in ovariectomized mice by preserving bone microarchitecture, mineral content, and inhibiting bone loss in treated mice as analyzed by Histomorphometry, MicroCT, and Raman spectroscopy. Oral administration of HxTME fraction resulted in the decreased percentage of F4/80+, CD11b+, and CD4+ RANKL+ T cells in OVX mice whereas pro-osteoclastic cytokine, IL6 was markedly reduced upon treatment with HxTME. On stimulation with PMA/Io and PHA, a significant decrease in proliferative response in the splenocytes of HxTME treated OVX mice was observed. Fatty acid profiling revealed that HxTME is rich in ω3 and ω6 polyunsaturated fatty acids (PUFAs), which have high nutraceutical properties and are known to play important role in growth, development and maintenance of health. Therefore, HxTME may be a good source of nutraceutical in the treatment of bone-related diseases particularly in postmenopausal osteoporosis and may be pursued as a potential candidate for treatment and management of osteoporosis.

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities.

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histological investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histo-logical investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Methanol Extract of Euchelus asper Prevents Bone Resorption in Ovariectomised Mice Model.

Marine molluscs are widely distributed throughout the world and many bioactive compounds exhibiting antiviral, antitumor, antileukemic, and antibacterial activity have been reported worldwide. The present study was designed to investigate the beneficial effect of methanol extract of Euchelus asper (EAME) on estrogen deficiency induced osteoporosis in ovariectomised mice model. Forty-two female Swiss albino mice were randomly assigned into Sham operated (Sham) group and six ovariectomised (OVX) subgroups such as OVX with vehicle (OVX); OVX with estradiol (2 mg/kg/day); OVX with EAME of graded doses (25, 50, 100, and 200 mg/kg/day). Bone turnover markers like serum alkaline phosphatase (ALP), serum acid phosphatase (ACP), serum calcium, and histological investigations of tibia and uterus were analysed. Metaphyseal DNA content of the femur bone was also studied. Antiosteoclastogenic activity of EAME was examined. Administration of EAME was able to reduce the increased bone turnover markers in the ovariectomised mice. Histomorphometric analysis revealed an increase in bone trabeculation and restoration of trabecular separation by EAME treatment. Metaphyseal DNA content of the femur of the OVX mice was increased by EAME administration. EAME also showed a potent antiosteoclastogenic behaviour. Thus, the present study reveals that EAME was able to successfully reduce the estrogen deficiency induced bone loss.

Turbo methanol extract inhibits bone resorption through regulation of T cell function.

Marine organisms have bioactive potential which has tremendous pharmaceutical promise. Emerging evidence highlights the importance of the interplay between bone and the immune system of which T lymphocytes and their product act as key regulators of bone resorption. In the present investigation we have analyzed the anti-osteoporotic effect of turbo methanol extract (TME) in the reversal of bone resoprtion. Forty-two female Swiss albino mice were used and randomly assigned into sham-operated group (sham) and six ovariectomized (OVX) subgroups, i.e. OVX with vehicle (OVX) that received daily oral administration of water ad libitum; OVX with estradiol (2mg/kg/day); and OVX with different doses of TME i.e. TME 100mg/kg, TME 50mg/kg, TME 25mg/kg and TME 12.5mg/kg. Oral administration of TME or estradiol started on the second week after ovariectomy for a period of 4weeks. We observed that the administration of TME increased the trabeculation in tibia and reduced the atrophy in the uterus. TME significantly decreased the serum alkaline phosphatase (ALP) and acid phosphatase (ACP) activity in OVX mice. Micro CT analysis revealed that the TME administration preserved the bone volume, connectivity density, trabecular number, trabecular thickness and trabecular separation in OVX mice. Bone mineralization was measured in different groups of mice by Raman spectroscopy. Reversal of bone resorption was observed in TME treated group of mice. To further investigate the mechanism of action of TME, we analyzed the T lymphocyte proliferation and profiles of cytokine TNFα and sRANKL in TME treated ovariectomized mice. Decrease in the elevation of T cell subsets was observed after the supplementation with TME. The extract significantly lowered the T cell proliferation responses to mitogens, phorbol 12-myristate 13-acetate (PMA) and ionomycin (Io) and phytohemagglutinin (PHA). A marked reduction in TNFα and sRANKL secretion in serum and TNFα in cell free supernatants of activated T lymphocytes was observed upon TME administration. TME could significantly inhibit the in vitro osteoclastogenesis and the bone resorption observed using artificial calcium coated slides. Collectively, these results indicate that TME has the potential to inhibit bone resorption and may prove to be a potential candidate for the development of an anti-osteoporosis drug.

Antiangiogenic, antimicrobial, and cytotoxic potential of sponge-associated bacteria.

The bacteria associated with marine invertebrates are a rich source of bioactive metabolites. In the present study bacteria associated with the sponge Suberites domuncula and its primmorphs (3-dimensional aggregates containing proliferating cells) were isolated and cultured. These bacteria were extracted, and the extracts were assayed for antiangiogenic, hemolytic, antimicrobial, and cytotoxic activities. Our studies revealed that extract obtained from the bacterium (PB2) isolated from sponge primmorphs is a potent angiogenesis inhibitor. In the chick chorio-allantoic membrane (CAM) assay, it showed 50% activity at 5 microg ml(-1) and 100% activity at 10 and 20 microg ml(-1) concentrations. Extracts obtained from 5 bacterial strains isolated from sponge and its primmorphs showed hemolytic activity. The sponge-associated bacteria belonging to the alpha subdivision of Proteobacteria and the primmorph-associated bacterium identified as a possible novel Pseudomonas sp. displayed remarkable antimicrobial activity. It is important to note that these bacterial extracts were strongly active against multidrug-resistant clinical strains such as Staphylococcus aureus and Staphylococcus epidermidis, isolated from hospital patients. The bacterial extracts having antimicrobial activity also showed cytotoxicity against HeLa and PC12 cells. In summary, this investigation explores the importance of sponge-associated bacteria as a valuable resource for the discovery of novel bioactive molecules.

One step flow-through adsorptive purification of tubulin from tissue homogenate.

Tubulin, a potential target for anti-cancer drugs, has been purified in one step and obtained as flow-through fraction directly from an extract of a mammalian brain tissue by adsorption chromatography on H-CELBEADS, an indigenously developed rigid, superporous cross-linked cellulose based weakly hydrophobic adsorbent. The fibrous polymerized tubulin mass passed through the H-CELBEADS bed while the associated proteins were separated by adsorption. The final tubulin preparation was obtained free from other proteins as seen on SDS-PAGE. Purified tubulin was obtained in a yield of about 29 mg/100 g brain, and its bioactivity, evaluated through its ability to bind colchicine, was found to be preserved.

Influence of antiangiogenic fraction from Diogenes avarus (Heller) on fertility and implantation in mice.

The methanol extract isolated from hermit crab, D. avarus degenerated ovarion and uterine tissues in cyclic and pregnant mice, treated before and after the implantation. Immunohistochemical staining using CD31 and Factor VIII specific to endothelial cells showed reduction in microvessel density. The hormonal assay showed decrease in the progesterone secretion in all experimental mice.

Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system.

Sponges (Porifera), represent the phylogenetically oldest metazoan phylum still extant today. Recently, molecular biological studies provided compelling evidence that these animals share basic receptor/ligand systems, especially those involved in bodyplan formation and in immune recognition, with the higher metazoan phyla. An in vitro cell/organ-like culture system, the primmorphs, has been established that consists of proliferating and differentiating cells, but no canals of the aquiferous system. We show that after the transfer of primmorphs from the demosponge Suberites domuncula to a homologous matrix (galectin), canal-like structures are formed in these 3D-cell aggregates. In parallel with the formation of these structures a gene is expressed whose deduced protein falls into the CD36/LIMPII receptor family. The receptor was cloned and found to be strongly expressed after adhesion to the galectin matrix. This process was suppressed if primmorphs were co-incubated with a homologous polypeptide containing the CSVTCG domain, as found in thrombospondin-1 (and related) molecules of vertebrates. In situ hybridization studies revealed that the S. domuncula CD36/LIMPII receptor is localized in the pinacocytes that surround the canals of the sponge. Furthermore, a secondary metabolite from a sponge-associated bacterium was isolated and characterized, the 2-methylthio-1,4-naphthoquinone (MTN). MTN causes inhibition of cell proliferation of vertebrate tumor cells at concentrations of >80 ng/ml. However, doses of only 2 ng are required to potently inhibit angiogenesis in the chick chorio-allantoic membrane assay. At concentrations of 10 ng/ml this compound was also found to suppress the expression of the S. domuncula CD36/LIMPII; this result is a first indication that this secondary metabolite has a conserved functional activity: the suppression of the formation of the circulation system, from sponges to vertebrates.

Fatty acids derived from a marine crustacean Diogenes avarus (Heller) and their antiangiogenic activity.

An organic extract from a marine crustacean D. avarus was examined for antiangiogenic activity by using the chick embryo chorioallantoic membrane (CAM) assay. The methanol extract (HCM) inhibited angiogenesis in a dose dependent manner. The extract was further fractionated by bioactivity-guided separation to purify the active fractions successively. This resulted in three fractions HCM1, HCM2 and HCM3. The 50% inhibition shown by HCM was 600 ng/disc, HCM1 was 100 ng/disc and of HCM3 was 2.7ng/disc. HCM3 which was separated by column chromatography and showed single spot on TLC was analysed by GLC and showed the presence of saturated and unsaturated fatty acids such as lauric, myristic, palmitic, stearic, oleic, linoleic. The antiangiogenic activity of the fatty acids obtained from a marine crustacean is reported for the first time.

In vivo and in vitro evaluation for immunomodulatory activity of three marine animal extracts with reference to phagocytosis.

The whole body ether extracts of a marine prawn Nematopaleamon tenuipes and two gastropods viz. Euchelus asper and Hemifusus pugilinus, obtained by Soxhlet extraction and cold percolation were tested for their effects on phagocytosis by in vitro (slide method) and by in vivo (carbon clearance) methods. Extract of E. asper exhibits immunostimulatory activity in vitro and immunosuppressant activity in vivo. The in vitro test for N. tenuipes and H. pugilinus shows biphasic activity, but the former shows immunostimulatory while the latter shows immunosuppresant activity in vivo test.