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.

In silico, anti-inflammatory and acute toxicological evaluation of an indigenous medicinal plant Pterospermum rubiginosum using Sprague-Dawley rats.

BACKGROUND: Pterospermum rubiginosum has been traditionally used by the tribal inhabitants of Southern India for treating bone fractures and as a local anti-inflammatory agent; however, experimental evidence to support this traditional usage is lacking. The present study aimed to investigate the phytochemical characterization, in silico and in vitro anti-inflammatory evaluation, followed by in vivo toxicological screening of P. rubiginosum methanolic bark extract (PRME). RESULTS: The LCMS evaluation revealed the presence of 80 significant peaks; nearly 50 molecules were identified using the LCMS database. In silico analysis showed notable interactions with inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6). In vitro gene expression study supported the docking results with significant down-regulation of iNOS, IL-6, and IL-10. PRME was administered orally to the SD rats and was found to be non-toxic up to 1000 mg/kg body weight for 14 days. The antioxidant enzymes catalase and sodium dismutase exhibited an increased value in PRME-administered groups, possibly due to the diverse phytochemical combinations in bark extract. CONCLUSIONS: PRME administration significantly downregulated the gene expression of inflammatory markers, such as iNOS, IL-6, and IL-10. The molecular docking analysis of iNOS and IL-6 supports the in vitro study. In vivo toxicological study of PRME in SD rats was found to be non-toxic up to a concentration of 1000 mg/kg body weight for 14 days.

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.

Modulation of neutrophil (dys)function by Ayurvedic herbs and its potential influence on SARS-CoV-2 infection.

For centuries, traditional medicines of Ayurveda have been in use to manage infectious and non-infectious diseases. The key embodiment of traditional medicines is the holistic system of approach in the management of human diseases. SARS-CoV-2 (COVID-19) infection is an ongoing pandemic, which has emerged as the major health threat worldwide and is causing significant stress, morbidity and mortality. Studies from the individuals with SARS-CoV-2 infection have shown significant immune dysregulation and cytokine overproduction. Neutrophilia and neutrophil to lymphocyte ratio has been correlated to poor outcome due to the disease. Neutrophils, component of innate immune system, upon stimulation expel DNA along with histones and granular proteins to form extracellular traps (NETs). Although, these DNA lattices possess beneficial activity in trapping and eliminating pathogens, NETs may also cause adverse effects by inducing immunothrombosis and tissue damage in diseases including Type 2 Diabetes and atherosclerosis. Tissues of SARS-CoV-2 infected subjects showed microthrombi with neutrophil-platelet infiltration and serum showed elevated NETs components, suggesting large involvement and uncontrolled activation of neutrophils leading to pathogenesis and associated organ damage. Hence, traditional Ayurvedic herbs exhibiting anti-inflammatory and antioxidant properties may act in a manner that might prove beneficial in targeting over-functioning of neutrophils and there by promoting normal immune homeostasis. In the present manuscript, we have reviewed and discussed pathological importance of NETs formation in SARS-CoV-2 infections and discuss how various Ayurvedic herbs can be explored to modulate neutrophil function and inhibit NETs formation in the context of a) anti-microbial activity to enhance neutrophil function, b) immunomodulatory effects to maintain neutrophil mediated immune homeostasis and c) to inhibit NETs mediated thrombosis.