Protective effect of Pterospermum rubiginosum bark extract on bone mineral density and bone remodelling in estrogen deficient ovariectomized Sprague-Dawley (SD) rats.

Osteoporosis is a common metabolic old age disorder characterised by low bone mass content (BMC) and mineral density (BMD) with micro-architectural deterioration of the extracellular matrix, further increasing bone fragility risk. Several traditional remedies, including plant extracts and herbal formulations, are used worldwide by local healers to improve the overall bone health and metabolism as an excellent osteoregenerative agent. Pteropsermum rubiginosum is an underexplored medicinal plant used by tribal peoples of Western Ghats, India, to treat bone fractures and associated inflammation. The proposed study evaluates the elemental profiling and phytochemical characterisation of P. rubiginosum methanolic bark extract (PRME), along with detailed In vitro and In vivo biological investigation in MG-63 cells and Sprague-Dawley (SD) rats. AAS and ICP-MS analysis showed the presence of calcium, phosphorus, and magnesium and exceptional levels of strontium, chromium, and zinc in PRME. The NMR characterisation revealed the presence of vanillic acid, Ergost-4-ene-3-one and catechin. The molecular docking studies revealed the target pockets of isolated compounds and various marker proteins in the bone remodelling cycle. In vitro studies showed a significant hike in ALP and calcium content, along with upregulated mRNA expression of the ALP and COL1, which confirmed the osteoinductive activity of PRME in human osteoblast-like MG-63 cells. The in vivo evaluation in ovariectomised (OVX) rats showed remarkable recovery in ALP, collagen and osteocalcin protein after 3 months of PRME treatment. DEXA scanning reports in OVX rats supported the above in vitro and in vivo results, significantly enhancing the BMD and BMC. The results suggest that PRME can induce osteogenic activity and enhance bone formation with an excellent osteoprotective effect against bone loss in OVX animals due to estrogen deficiency. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03942-7.

Targeting the Role of PRME in Regulating Bone Remodelling During Postmenopausal Osteoporosis.

Kariavattom Campus Postmenopausal osteoporosis (PMO) is an old age disorder associated with estrogen deficiency, which reduces bone mass and makes bones more prone to fracture. The present study was proposed to evaluate the invivo osteogenic efficiency of Pterospermum rubiginosum methanolic bark extract (PRME) in the PMO model. Molecular docking studies on transcription factor NFATC1 showed excellent interactions with phytochemical ligands with the lowest binding energies. Female Sprague Dawley (SD) rats (n=24) were divided into four groups, (n=6 each) sham control (Group I) and osteoporotic control (Group II) groups treated with saline, PRME (50 mg/kg/day) and alendronate (10 mg/kg/day) treated with Group III and Group IV (n=6) respectively. The serum tartrate-resistant acid phosphatase 5b and cathepsin-K also exhibited a significant rise after PRME treatment 12.33±2.30 mU/ml and 427.68±46.97 pg/ml, respectively. DEXA results exhibited a remarkable increase in total bone mineral content and density values in PRME-treated animals (0.175±0.002 g/cm2) and (7.95±0.23 g) when compared to osteoporotic control (0.163±0.004 g/cm2) and (6.83±0.34 g). Long-term toxicity study revealed that PRME is non-toxic, up to 100 mg/kg bodyweight for 6 months. Our findings suggest PRME protects osteoporotic SD rats from PMO damage resulting from estrogen deficiency by regulating bone remodelling markers and upregulating BMD indices.

An integrated approach to the structural characterization, long-term toxicological and anti-inflammatory evaluation of Pterospermum rubiginosum bark extract.

ETHNOPHARMACOLOGICAL RELEVANCE OF STUDY: Pterospermum rubiginosum is an evergreen plant in Western Ghats, India, used by traditional tribal healers due to its excellent biological potential for treating inflammation and pain relief procedures. The bark extract is also consumed to relieve the inflammatory changes at the bone fractured site. The traditional medicinal plant in India have to be characterized for its diverse phytochemical moieties, its interactive multiple target sites, and to reveal the hidden molecular mechanism behind the biological potency. AIM OF THE STUDY: The study focussed on plant material characterization, computational analysis (prediction study), toxicological screening (In vivo), and anti-inflammatory evaluation of P. rubiginosum methanolic bark extracts (PRME) in LPS-induced RAW 264.7 cells. MATERIALS AND METHODS: The pure compound isolation of PRME and their biological interactions were used to predict the bioactive components, molecular targets, and molecular pathways of PRME in inhibiting inflammatory mediators. The anti-inflammatory effects of PRME extract were evaluated in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage cell model. The toxicity evaluation of PRME was performed in healthy 30 Sprague-Dawley experimental rats, were randomly divided into five groups for toxicological evaluation for 90 days. The tissue levels of oxidative stress and organ toxicity markers were measured using the ELISA method. Nuclear magnetic resonance spectroscopy (NMR) was performed to characterize the bioactive molecules. RESULTS: Structural characterization revealed the presence of vanillic acid, 4-O-methyl gallic acid, E-resveratrol, gallocatechin, 4'-O-methyl gallocatechin, and catechin. Molecular docking of NF-kB exhibited significant interactions with vanillic acid and 4-O-methyl gallic acid with binding energy -351.159 Kcal/Mol and -326.5505 Kcal/Mol, respectively. The PRME-treated animals showed an increase in total GPx and antioxidant levels (SOD and catalase). Histopathological examination revealed no variation in the liver, renal and splenic tissue's cellular pattern. PRME inhibited the pro-inflammatory parameters (IL-1ß, IL-6, and TNF-α) in LPS-induced RAW 264.7 cells. The protein level of TNF-α and NF-kB protein expression study brought out a notable reduction and exhibited a good correlation with the gene expression study. CONCLUSION: The current study establishes the therapeutic potential of PRME as an effective inhibitory agent against LPS-activated RAW 264.7 cells induced inflammatory mediators. Long-term toxicity evaluation on SD rats confirmed the non-toxic nature of PRME up to 250mg/body weight for 3 months.

Nanohydroxyapatite incorporated photocrosslinked gelatin methacryloyl/poly(ethylene glycol)diacrylate hydrogel for bone tissue engineering.

The development of novel strategies that aim to augment the regenerative potential of bone is critical for devising better treatment options for bone defects or injuries. Facilitation of bone repair and regeneration utilizing composite hydrogels that simulates bone matrix is emerging as a viable approach in bone tissue engineering. The present study aimed to develop nanohydroxyapatite-incorporated gelatin methacryloyl (GelMA)/poly(ethylene glycol) diacrylate (PEGDA) hydrogel (GMPH hydrogel). A facile blending and photocrosslinking approach was employed to incorporate nanohydroxyapatite into the inter-crosslinked polymeric hydrogel network to obtain an ECM mimicking matrix for assisting bone tissue regeneration. Chemical characterization of GelMA and the GMPH hydrogel was carried out using FTIR and 1H NMR. Physical properties of GMPH, such as gelation, swelling and degradation ratios, and internal morphology, signified the suitability of GMPH hydrogel for tissue engineering. Cell viability assay demonstrated a healthy proliferation of MG63 osteoblast cells in GMPH hydrogel extracted growth medium, indicating the hydrogel's cytocompatibility and suitability for bone tissue engineering. Our study documented the fabrication of a novel GelMA/PEGDA-nanohydroxyapatite hydrogel that possesses ideal physicochemical and biological properties for bone tissue engineering.

Alcohol induced NLRP3 inflammasome activation in the brain of rats is attenuated by ATRA supplementation.

Alcohol abuse affects several neurological pathways and causes significant alterations in the brain. Abstention from alcohol is an effective intervention against alcohol related diseases. But the recovery of the damaged cells to normal presents a major problem in those who have stopped alcohol consumption. Hence therapeutic interventions are needed. Our previous studies have shown that all trans retinoic acid (ATRA) is effective in reducing alcohol induced neuro toxicity. Chronic alcohol administration up-regulates and activates the NLRP3 inflammasome leading to caspase-1 activation and IL-1ß production causing neuroinflammation. Hence, we investigated whether ATRA has any impact on NLRP3 inflammasomes activation. Rats were divided into two groups and were maintained for 90 days as control and ethanol group (4 â€‹g/kg body weight). After 90 days, ethanol administration was stopped and animals in the control group were divided into control and control â€‹+ â€‹ATRA (100 â€‹µg/kg body weight per day) groups; those in the ethanol group as ethanol abstention and ATRA (100 â€‹µg/kg body weight per day) and maintained for 30 days. Administration of ATRA reduced reactive oxygen species and endotoxins which were elevated in alcoholic rats. There was also reduction in the expression of NLRP3 inflammasome and caspase 1. Our results suggested ATRA down regulated NLRP3 activation with concomitant decrease in the release of caspase -1 and production of IL1ß. However, all these parameters were higher in abstention in comparison with ATRA supplemented group. In short therapeutic intervention with ATRA regressed alcohol induced inflammasome activation better than abstention.

Sulfated polysaccharides from the edible marine algae Padina tetrastromatica attenuates isoproterenol-induced oxidative damage via activation of PI3K/Akt/Nrf2 signaling pathway - An in vitro and in vivo approach.

The reactive oxygen species (ROS) induced oxidative stress is an inevitable factor for the pathogenesis of cardiovascular diseases. The edible marine algae-derived sulfated polysaccharides gained special attention as novel bioactive compounds having potential pharmacological activities. The present study evaluated in vitro and in vivo cardioprotective properties of sulfated polysaccharides from the edible brown marine algae Padina tetrastromatica (PSPS) against isoproterenol (ISO) induced cardiac damage. The cardioprotective properties of PSPS were first evaluated in H9c2 cardiac myoblasts and the results were confirmed by in vivo studies conducted in male Sprague-Dawley rats. The biochemical parameters, histopathological analysis, mRNA expressions, and ELISA studies indicated that PSPS significantly decreased (p < 0.05) the cardiac damage induced by ISO by reducing lipid peroxidation and improving antioxidant status, both in vitro and in vivo, via modulating PI3k/Akt/Nrf2 signaling pathway. The histopathological evidence further reinforced our findings and highlighted the promising cardioprotective activities offered by PSPS.

All trans retinoic acid modulates TNF-α and CYP2E1 pathways and enhances regression of ethanol-induced fibrosis markers in hepatocytes and HSCs in abstaining rodent model.

Context: Our previous studies showed that all trans retinoic acid (ATRA) ameliorates alcohol-induced toxicity. Hence, we evaluated the efficacy of ATRA and abstention in the regression of alcohol-induced hepatotoxicity. Materials and methods: After ethanol administration to rats for 90 days, the regression of alcohol-induced toxicity was studied by supplementing ATRA at a dose of 100 µg/kg body weight for 30 days. It was also compared with animals in abstention. Results and discussion: Ethanol administration enhanced oxidative stress, activated HSCs and increased collagen deposition. All these alterations were reversed to a certain extent by ATRA supplementation. Conclusions: ATRA had better efficacy than just abstention in reducing ethanol-induced toxicity. The mechanism might be downregulation of CYP2E1, leading to reduced oxidative stress in the hepatocytes and thus impeding NFκB activation, cytokine production, activation of HSC and resulting in the reduction of inflammation and remodelling of fibrosis by modulating MMP and TIMP.

Isolation, Purification and Characterisation of a D-galactose and N-acetyl-D-galactosamine Specific Lectin from Marine Sponge Fasciospongia cavernosa.

BACKGROUND: Marine sponges, belonging to the phylum Porifera, are gaining more attention by researchers and industrial sectors from all over the world due to their ability to produce a variety of bioactive secondary metabolites that have many applications including drug discovery. Marine sponges are a promising source of bioactive lectins, which are structurally diverse, many of them in the form of glycoproteins. OBJECTIVE: To purify and characterize lectin from a marine sponge Fasciospongia cavernosa and to study its antibacterial activity. METHOD: Lectin from a marine sponge Fasciospongia cavernosa was purified by guar gum affinity chromatography and checked for its biophysical and antibacterial properties. The lectin was subjected to evaluation for inhibition of microbial growth against bacteria by aggregation test. The activity of FCL against the biofilms formed by P. aeruginosa was also carried out. Biofilm is defined as the undesirable accumulation of microorganisms on artificial surfaces immersed in a common matrix. The effect of FCL on biofilm-forming gram negative bacteria P. aeruginosa was tested by crystal violet assay. RESULTS: This lectin, named FCL, has a molecular weight of 80 KDa approximately, was found to agglutinate human ABO, rat, rabbit and chicken erythrocytes. The hemagglutinating activity of FCL was reduced by demetallisation with E.D.T.A and regained by the addition of Ca2+, Mg2+, Mn2+, Ba2+ and Fe2+, which shows the metal dependency of the lectin. The hemagglutinating activity by the lectin was inhibited by D-galactose and N-acetyl-D-galactosamine. The lectin was stable over a range of pH from 2 to 10.5, and up to a temperature 70°C for 20 min. FCL agglutinated B. subtilis, S. aureus and P. aeruginosa and was able to reduce biofilm mass formed by P. aeruginosa. Thus, the marine sponge F. cavernosa lectin, FCL could be used as an antibacterial agent. FCL significantly reduced the biomass of bacterial biofilm tested. Biofilm mass of P. aeruginosa, K. pneumoniae and E. coli were decreased in a dose dependent manner. CONCLUSION: A novel lectin was isolated and purified from marine sponge F. cavernosa. FCL, a galactose-binding lectin displayed considerable antimicrobial activity in vitro, particularly against gram-positive bacteria and also exhibited a strong antibiofilm activity.

All Trans Retinoic Acid Attenuates Markers of Neuroinflammation in Rat Brain by Modulation of SIRT1 and NFκB.

Alcohol abuse affects several neurological pathways and causes significant alterations in the brain. Abstention from alcohol causes only a marginal decrease in oxidative stress and neuro inflammation. Our previous studies had shown that an active metabolite of vitamin A, all trans retinoic acid (ATRA), ameliorates alcohol induced toxicity. Hence in the present study we investigated whether ATRA regressed alcohol induced neuroinflammation. We focused on the role of silent mating type information regulation 2 homolog 1(SIRT1) and nuclear factor kappa-B (NFκB). Animals were administered with ethanol at a daily dose of (4 g/kg body weight) for 90 days. On the 91st day ethanol administration was stopped and animals were divided into ethanol abstention (A) and ATRA supplementation group (ATRA + A) (100 µg/kg body weight) and maintained for 30 days. Ethanol exposure increased markers of oxidative stress, inflammation and the activities of alcohol and acetaldehyde dehydrogenases and reduced the expression of SIRT1 in the whole brain.The ethanol induced altered expressions of NFκB and SIRT1 were modulated by supplementation of ATRA. Abstention also reduced toxicity, but to a lower extent in comparison with supplementation of ATRA. Our results seemed to suggest that ATRA regressed the mediators of ethanol induced neuroinflammation by reducing oxidative stress and by regulating the expression of NFκB and SIRT1. The ameliorative potential of ATRA was much higher than abstention.

Molecular mechanisms of the antiglycative and cardioprotective activities of Psidium guajava leaves in the rat diabetic myocardium.

CONTEXT: Antiglycative potential of Psidium guajava L. (Myrtaceae) leaves has been established. However, the molecular basis of its antiglycative potential remains unknown. OBJECTIVE: The ethyl acetate fraction of P. guajava leaves (PGEt) was evaluated to determine the cardioprotective effect and its mechanism of action compared to quercetin. MATERIALS AND METHODS: After the induction of diabetes by streptozotocin (55 mg/kg body weight), PGEt and quercetin (50 mg/kg body weight) was administered for 60 days. Rats were grouped as follows: Group C: Control, Group D: Diabetic, Group D + E: Diabetic rats treated with PGEt, Group D + Q: Diabetic rats treated with quercetin. The antiglycative potential was evaluated by assaying glycosylated haemoglobin, serum fructosamine and advanced glycation end product levels. The differential receptor for advanced glycation end products and nuclear factor kappa B (NFκB) protein levels was determined by western blot and the transcript level changes of connective tissue growth factor (CTGF), brain natriuretic peptide (BNP) and TGF-ß1 in heart tissue were assessed by RT-PCR analysis. RESULTS: Glycated haemoglobin and serum fructosamine levels were found to be enhanced in diabetic rats when compared with control. Administration of PGEt significantly reduced the glycated haemoglobin and fructosamine levels to a larger extent than quercetin treated diabetic rats. PGEt reduced the translocation of NFκB from cytosol to nucleus when compared with diabetic rats. Expression of TGF-ß1, CTGF and BNP was downregulated in PGEt treated groups compared with diabetic controls. DISCUSSION AND CONCLUSION: Administration of PGEt ameliorated diabetes associated changes in the myocardium to a greater extent than quercetin.

Beneficial effects of Psidium guajava leaf extract on diabetic myocardium.

Non enzymatic glycosylation (glycation) between reducing sugar and protein results in the formation of advanced glycation end products (AGEs), which is believed to play an important role in diabetes associated cardiovascular complications. Thus agents that inhibit the formation of AGEs are believed to have therapeutic potential against diabetic complications. In the present study we evaluated the antiglycative potential of ethyl acetate fraction of Psidium guajava leaves (PGEt) by administering the extract into streptozotocin induced diabetic rats. Daily administration of the extract for a period of one month significantly decreased the blood glucose, glycated hemoglobin and fructosamine levels in a dose dependent manner. Evaluation of the toxicity markers like SGOT and SGPT revealed the non toxic nature of the extract. Apart from this we evaluated the presence of cardiac isoform of liver alpha 2 macroglobulin, which is a major protein associated with earlier stages of cardiac hypertrophy. SDS-PAGE analysis showed that the level of this protein decreased significantly in extract treated groups compared to diabetic control. These findings support that the administration of PGEt extract may be beneficial for preventing cardiovascular complications associated with diabetes.

Role of cardiac isoform of alpha-2 macroglobulin in diabetic myocardium.

Earlier studies from one of the investigator's laboratory have demonstrated the presence of a high molecular weight protein (182 kDa) in the blood serum of laboratory animals subjected to pressure-induced cardiac hypertrophy and suggested that this protein may be involved in the development of cardiac hypertrophy. Studies have shown that this protein is also involved in earlier stages of cardiac complications associated with diabetes, but the role of this protein in diabetic heart is less understood. So we aimed to check whether this protein is having any protective role in diabetic heart. The protein was purified from serum of rats induced with cardiac hypertrophy and the purified protein was injected through tail vein of diabetic rats for further studies. The results of various antioxidant enzymes and the TBARS levels have indicated the antioxidant activity of this protein. Real-time PCR analysis of gene expression revealed the upregulation of certain muscle-specific genes like ß-MHC, MLC-2, and skeletal α actin in diabetic group and also in presence of 182-kDa protein. The results further showed a down regulation of genes such as cardiac α-actin and α- MHC implicating the role of this protein in the development of cardiac hypertrophy in diabetes. Increased cardiac hypertrophy as revealed by the expression of various genes and improved antioxidant potential in presence of 182 kDa protein in diabetes at the earlier stages is beneficial for counteracting the myocardial damage associated with diabetes.

Antioxidant and antiglycative potential of ethyl acetate fraction of Psidium guajava leaf extract in streptozotocin-induced diabetic rats.

Hyperglycemia causes increased protein glycation and the formation of early glycation products and advanced glycation end products (AGEs) which are major factors responsible for the complications associated with diabetes. The aim of the present study was to investigate the antioxidant as well as antiglycative potential of ethyl acetate fraction of guava leaves. Oral administration of the extract at different doses showed a significant decrease in blood glucose level. It also showed an improved antioxidant potential as evidenced by decreased lipid peroxidation and a significant increase in the activity of various antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Glycated hemoglobin as well as fructosamine which are indicators of glycation was also reduced significantly in treated groups when compared to diabetic control. In vitro studies also support the antioxidant as well as antiglycative potential of guava leaves.

Modulatory effect of coffee on platelet function.

Blood platelets play a major role in cardiovascular disease (CVD) and thrombosis. Conflicting information exists regarding the effect of coffee consumption on the cardiovascular system. We have investigated whether the consumption of moderate amount of coffee affect platelet functions and primary hemostasis in vivo in normal and high fat diet fed rats. Coffee fed group showed significant (P < 0.05) decrease in mean platelet volume, platelet crit and platelet distribution width as compared to high fat diet (HFD) group. The concentration of malondialdehyde in platelets increased in atherosclerotic group indicates the increased thromboxane A2 (TXA2) production from membrane arachidonic acid and it was decreased in coffee treated group. Platelet aggregation studies with ADP, collagen, arachidonic acid and epinephrine showed significant (P < 0.05) decrease in aggregation in coffee fed group. Scanning electron microscopic studies revealed that platelet aggregation tendency increased in HFD group and was reduced in coffee fed group. These results indicate that coffee is active in inhibiting platelet aggregation, a critical step involved in thrombosis.