Intramyocardial cell-based therapy with Lomecel-B during bidirectional cavopulmonary anastomosis for hypoplastic left heart syndrome: the ELPIS phase I trial.

Aims: Hypoplastic left heart syndrome (HLHS) survival relies on surgical reconstruction of the right ventricle (RV) to provide systemic circulation. This substantially increases the RV load, wall stress, maladaptive remodelling, and dysfunction, which in turn increases the risk of death or transplantation. Methods and results: We conducted a phase 1 open-label multicentre trial to assess the safety and feasibility of Lomecel-B as an adjunct to second-stage HLHS surgical palliation. Lomecel-B, an investigational cell therapy consisting of allogeneic medicinal signalling cells (MSCs), was delivered via intramyocardial injections. The primary endpoint was safety, and measures of RV function for potential efficacy were obtained. Ten patients were treated. None experienced major adverse cardiac events. All were alive and transplant-free at 1-year post-treatment, and experienced growth comparable to healthy historical data. Cardiac magnetic resonance imaging (CMR) suggested improved tricuspid regurgitant fraction (TR RF) via qualitative rater assessment, and via significant quantitative improvements from baseline at 6 and 12 months post-treatment (P < 0.05). Global longitudinal strain (GLS) and RV ejection fraction (EF) showed no declines. To understand potential mechanisms of action, circulating exosomes from intramyocardially transplanted MSCs were examined. Computational modelling identified 54 MSC-specific exosome ribonucleic acids (RNAs) corresponding to changes in TR RF, including miR-215-3p, miR-374b-3p, and RNAs related to cell metabolism and MAPK signalling. Conclusion: Intramyocardially delivered Lomecel-B appears safe in HLHS patients and may favourably affect RV performance. Circulating exosomes of transplanted MSC-specific provide novel insight into bioactivity. Conduct of a controlled phase trial is warranted and is underway.Trial registration number NCT03525418.

The Role of ERK1/2 Pathway in the Pathophysiology of Alzheimer's Disease: An Overview and Update on New Developments.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. Several findings suggest that correcting the dysregulated signaling pathways may offer a potential therapeutic approach in this disease. Extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase family, plays a major role in regulation of cell proliferation, autophagy process, and protein synthesis. The available literature suggests dysregulated ERK1/2 in AD patients with potential implications in the multifaceted underlying pathologies of AD, including amyloid-ß plaque formation, tau phosphorylation, and neuroinflammation. In this regard, in the current review, we aim to summarize the reports on the potential roles of ERK1/2 in AD pathophysiology.

The PI3K/AKT signaling pathway in cancer: Molecular mechanisms and possible therapeutic interventions.

The human body consists of countless cells with the possibility of excessive and uncontrolled proliferation under certain dysregulated circumstances that could cause abnormal states such as cancer. Phosphatidylinositol 3 kinase (PI3K) and its downstream target, Protein kinase B or AKT, play a critical role in cell survival, proliferation, differentiation, migration, and metabolism. Research studies have examined the aberrant expression of signaling molecules that regulate PI3K/AKT pathway with the purpose of target discovery. The present review aims to recapitulate the relationship between the PI3K/AKT signaling pathway and factors contributing to initiation and development of various cancers. In addition, therapeutic interventions regulating this signaling pathway have been summarized.

Col4a3-/- Mice on Balb/C Background Have Less Severe Cardiorespiratory Phenotype and SGLT2 Over-Expression Compared to 129x1/SvJ and C57Bl/6 Backgrounds.

Alport syndrome (AS) is a hereditary renal disorder with no etiological therapy. In the preclinical Col4a3-/- model of AS, disease progression and severity vary depending on mouse strain. The sodium-glucose cotransporter 2 (SGLT2) is emerging as an attractive therapeutic target in cardiac/renal pathologies, but its application to AS remains untested. This study investigates cardiorespiratory function and SGLT2 renal expression in Col4a3-/- mice from three different genetic backgrounds, 129x1/SvJ, C57Bl/6 and Balb/C. male Col4a3-/- 129x1/SvJ mice displayed alterations consistent with heart failure with preserved ejection fraction (HFpEF). Female, but not male, C57Bl/6 and Balb/C Col4a3-/- mice exhibited mild changes in systolic and diastolic function of the heart by echocardiography. Male C57Bl/6 Col4a3-/- mice presented systolic dysfunction by invasive hemodynamic analysis. All strains except Balb/C males demonstrated alterations in respiratory function. SGLT2 expression was significantly increased in AS compared to WT mice from all strains. However, cardiorespiratory abnormalities and SGLT2 over-expression were significantly less in AS Balb/C mice compared to the other two strains. Systolic blood pressure was significantly elevated only in mutant 129x1/SvJ mice. The results provide further evidence for strain-dependent cardiorespiratory and hypertensive phenotype variations in mouse AS models, corroborated by renal SGLT2 expression, and support ongoing initiatives to develop SGLT2 inhibitors for the treatment of AS.

Metformin in Alzheimer's disease: An overview of potential mechanisms, preclinical and clinical findings.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. The association between AD and other diseases such as diabetes is well-studied. In parallel, potential disease-modifying effects of therapeutic agents used for diabetes have been investigated in the context AD of. Metformin is a biguanide and the most commonly prescribed medication for type 2 diabetes Due to its pleiotropic properties, metformin's potential disease-modifying effects are widely studied on different pathophysiological plyers of AD such as amyloid-ß (Aß) production and clearance, tau phosphorylation, and neuroinflammation, in relevant in vitro and in vivo models. In this review, we summarize the relevant scientific literature on the effects of metformin on various aspects of AD pathophysiology.

MicroRNAs in the pathophysiology of Alzheimer's disease and Parkinson's disease: an overview.

Neurodegenerative diseases are characterized by a progressive loss of neurons of the central nervous system (CNS) and serve as a major cause of morbidity, mortality and functional dependence especially among the elderly. Despite extensive research and development efforts, the success rate of clinical pipelines has been very limited. However, microRNAs (miRs) have been proved to be of crucial importance in regulating intracellular pathways for various pathologic conditions including those of a neurodegenerative nature. There is ample evidence of altered levels of various miRs in clinical samples of Alzheimer's disease and Parkinson's disease patients with potentially major clinical implications. In the current review, we aim to summarize the relevant literature on the role of miRs in the pathophysiology of Alzheimer's disease (AD) and Parkinson's disease (PD) as the two globally predominant neurodegenerative conditions.

Carvedilol and exercise combination therapy improves systolic but not diastolic function and reduces plasma osteopontin in Col4a3-/- Alport mice.

There are currently no Food and Drug Administration-approved treatments for heart failure with preserved ejection fraction (HFpEF). Here we compared the effects of exercise with and without α/ß-adrenergic blockade with carvedilol in Col4a3-/- Alport mice, a model of the phenogroup 3 subclass of HFpEF with underlying renal dysfunction. Alport mice were assigned to the following groups: no treatment control (n = 29), carvedilol (n = 11), voluntary exercise (n = 9), and combination carvedilol and exercise (n = 8). Cardiac function was assessed by echocardiography after 4-wk treatments. Running activity of Alport mice was similar to wild types at 1 mo of age but markedly reduced at 2 mo (1.3 ± 0.40 vs. 4.5 ± 1.02 km/day, P < 0.05). There was a nonsignificant trend for increased running activity at 2 mo by carvedilol in the combination treatment group. Combination treatments conferred increased body weight of Col4a3-/- mice (22.0 ± 1.18 vs. 17.8 ± 0.29 g in untreated mice, P < 0.01), suggesting improved physiology, and heart rates declined by similar increments in all carvedilol-treatment groups. The combination treatment improved systolic parameters; stroke volume (30.5 ± 1.99 vs. 17.8 ± 0.77 µL, P < 0.0001) as well as ejection fraction and global longitudinal strain compared with controls. Myocardial performance index was normalized by all interventions (P < 0.0001). Elevated osteopontin plasma levels in control Alport mice were significantly lowered only by combination treatment, and renal function of the Alport group assessed by urine albumin creatinine ratio was significantly improved by all treatments. The results support synergistic roles for exercise and carvedilol to augment cardiac systolic function of Alport mice with moderately improved renal functions but no change in diastole.NEW & NOTEWORTHY In an Alport mouse model of heart failure with preserved ejection fraction (HFpEF), exercise and carvedilol synergistically improved systolic function without affecting diastole. Carvedilol alone or in combination with exercise also improved kidney function. Molecular analyses indicate that the observed improvements in cardiorenal functions were mediated at least in part by effects on serum osteopontin and related inflammatory cytokine cascades. The work presents new potential therapeutic targets and approaches for HFpEF.

ß2-Adrenergic receptor agonism as a therapeutic strategy for kidney disease.

Approximately 14% of the general population suffer from chronic kidney disease that can lead to acute kidney injury (AKI), a condition with up to 50% mortality for which there is no effective treatment. Hypertension, diabetes, and cardiovascular disease are the main comorbidities, and more than 660,000 Americans have kidney failure. ß2-Adrenergic receptors (ß2ARs) have been extensively studied in association with lung and cardiovascular disease, but with limited scope in kidney and renal diseases. ß2ARs are expressed in multiple parts of the kidney including proximal and distal convoluted tubules, glomeruli, and podocytes. Classical and noncanonical ß2AR signaling pathways interface with other intracellular mechanisms in the kidney to regulate important cellular functions including renal blood flow, electrolyte balance and salt handling, and tubular function that in turn exert control over critical physiology and pathology such as blood pressure and inflammatory responses. Nephroprotection through activation of ß2ARs has surfaced as a promising field of investigation; however, there is limited data on the pharmacology and potential side effects of renal ß2AR modulation. Here, we provide updates on some of the major areas of preclinical kidney research involving ß2AR signaling that have advanced to describe molecular pathways and identify potential drug targets some of which are currently under clinical development for the treatment of kidney-related diseases.

Osteopontin and LDLR Are Upregulated in Hearts of Sudden Cardiac Death Victims With Heart Failure With Preserved Ejection Fraction and Diabetes Mellitus.

Background: Diabetes mellitus (DM) is associated with increased risk of sudden cardiac death (SCD), particularly in patients with heart failure with preserved ejection fraction (HFpEF). However, there are no known biomarkers in the population with DM and HFpEF to predict SCD risk. Objectives: This study was designed to test the hypothesis that osteopontin (OPN) and some proteins previously correlated with OPN, low-density lipoprotein receptor (LDLR), dynamin 2 (DNM2), fibronectin-1 (FN1), and 2-oxoglutarate dehydrogenase-like (OGDHL), are potential risk markers for SCD, and may reflect modifiable molecular pathways in patients with DM and HFpEF. Methods: Heart tissues were obtained at autopsy from 9 SCD victims with DM and HFpEF and 10 age and gender-matched accidental death control subjects from a Finnish SCD registry and analyzed for the expression of OPN and correlated proteins, including LDLR, DNM2, FN1, and OGDHL by immunohistochemistry. Results: We observed a significant upregulation in the expression of OPN, LDLR, and FN1, and a marked downregulation of DNM2 in heart tissues of SCD victims with DM and HFpEF as compared to control subjects (p < 0.01). Conclusions: The dysregulated protein expression of OPN, LDLR, FN1, and DNM2 in patients with DM and HFpEF who experienced SCD provides novel potential modifiable molecular pathways that may be implicated in the pathogenesis of SCD in these patients. Since secreted OPN and soluble LDLR can be measured in plasma, these results support the value of further prospective studies to assess the predictive value of these plasma biomarkers and to determine whether tuning expression levels of OPN and LDLR alters SCD risk in patients with DM and HFpEF.

Effects of Klotho supplementation on hyperoxia-induced renal injury in a rodent model of postnatal nephrogenesis.

BACKGROUND: Hyperoxia (HO) causes kidney injury in preterm infants; however, whether these effects are modifiable is unknown. We hypothesized that administration of exogenous soluble Klotho, a kidney-derived antioxidant, would attenuate HO-induced kidney injury during postnatal nephrogenesis in rats. METHODS: Sprague Dawley neonatal rats assigned to normoxia (21% O2) or HO (85% O2) groups from postnatal day (P) 1 to 21 were randomly assigned to receive alternate day intraperitoneal injections of recombinant Klotho or placebo for 3 weeks. They were recovered in normoxia for an additional 3 weeks and sacrificed at 6 weeks. Renal artery resistance and pulsatility indices, tubular injury scores, glomerular area, and renal antioxidant capacity were assessed. RESULTS: Rodents exposed to HO during postnatal nephrogenesis had reduced kidney Klotho expression, glomerulomegaly, and higher tubular injury scores. Exogenous Klotho administration improved renal perfusion as indicated by decreases in both resistance and pulsatility indices and increased antioxidant enzyme expression. CONCLUSIONS: HO exposure during postnatal nephrogenesis in rodents results in a decline in kidney Klotho expression, decreased renal perfusion, enlarged glomerular size, and tubular injury. The exogenous administration of Klotho attenuated HO-induced kidney injury and augmented antioxidant capacity.

Neonatal hyperoxia exposure induces aortic biomechanical alterations and cardiac dysfunction in juvenile rats.

Supplemental oxygen (O2 ) therapy in preterm infants impairs lung development, but the impact of O2 on long-term systemic vascular structure and function has not been well-explored. The present study tested the hypothesis that neonatal O2 therapy induces long-term structural and functional alterations in the systemic vasculature, resulting in vascular stiffness observed in children and young adults born preterm. Newborn Sprague-Dawley rats were exposed to normoxia (21% O2 ) or hyperoxia (85% O2 ) for 1 and 3 weeks. A subgroup exposed to 3 weeks hyperoxia was recovered in normoxia for an additional 3 weeks. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound and pressure myography. Aorta remodeling was assessed by collagen deposition and expression. Left ventricular (LV) function was assessed by echocardiography. We found that neonatal hyperoxia exposure increased vascular stiffness at 3 weeks, which persisted after normoxic recovery at 6 weeks of age. These findings were accompanied by increased PWV, aortic remodeling, and altered LV function as evidenced by decreased ejection fraction, cardiac output, and stroke volume. Importantly, these functional changes were associated with increased collagen deposition in the aorta. Together, these findings demonstrate that neonatal hyperoxia induces early and sustained biomechanical alterations in the systemic vasculature and impairs LV function. Early identification of preterm infants who are at risk of developing systemic vascular dysfunction will be crucial in developing targeted prevention strategies that may improve the long-term cardiovascular outcomes in this vulnerable population.

Osteopontin Promotes Left Ventricular Diastolic Dysfunction Through a Mitochondrial Pathway.

BACKGROUND: Patients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction, and kidney failure in Col4a3-/- mice, a model of human Alport syndrome. OBJECTIVES: The purpose of this study was to identify potential OPN targets in biopsies of HF patients, healthy control subjects, and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), and to characterize the cardiac phenotype of Col4a3-/- mice, relate this to HFpEF, and investigate possible causative roles for OPN in driving the cardiomyopathy. METHODS: OGDHL mRNA and protein were quantified in myocardial samples from patients with HFpEF, heart failure with reduced ejection fraction, and donor control subjects. OGDHL expression was quantified in hiPS-CMs treated with or without anti-OPN antibody. Cardiac parameters were evaluated in Col4a3-/- mice with and without global OPN knockout or AAV9-mediated delivery of 2-oxoglutarate dehydrogenase-like (Ogdhl) to the heart. RESULTS: OGDHL mRNA and protein displayed abnormal abundances in cardiac biopsies of HFpEF (n = 17) compared with donor control subjects (n = 12; p < 0.01) or heart failure with reduced ejection fraction patients (n = 12; p < 0.05). Blockade of OPN in hiPS-CMs conferred increased OGDHL expression. Col4a3-/- mice demonstrated cardiomyopathy with similarities to HFpEF, including diastolic dysfunction, cardiac hypertrophy and fibrosis, pulmonary edema, and impaired mitochondrial function. The cardiomyopathy was ameliorated by Opn-/- coincident with improved renal function and increased expression of Ogdhl. Heart-specific overexpression of Ogdhl in Col4a3-/- mice also improved cardiac function and cardiomyocyte energy state. CONCLUSIONS: Col4a3-/- mice present a model of HFpEF secondary to CKD wherein OPN and OGDHL are intermediates, and possibly therapeutic targets.

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells.

Mitochondrial dysfunction in the renal tubular epithelial cells (TECs) can lead to renal fibrosis, a major cause of chronic kidney disease (CKD). Therefore, assessing mitochondrial function in primary TECs may provide valuable insight into the bioenergetic status of the cells, providing insight into the pathophysiology of CKD. While there are a number of complex protocols available for the isolation and purification of proximal tubules in different species, the field lacks a cost-effective method optimized for tubular cell isolation without the need for purification. Here, we provide an isolation protocol that allows for studies focusing on both primary mouse proximal and distal renal TECs. In addition to cost-effective reagents and minimal animal procedures required in this protocol, the isolated cells maintain high energy levels after isolation and can be sub-cultured up to four passages, allowing for continuous studies. Furthermore, using a high throughput extracellular flux analyzer, we assess the mitochondrial respiration directly in the isolated TECs in a 96-well plate for which we provide recommendations for the optimization of cell density and compound concentration. These observations suggest that this protocol can be used for renal tubular ex vivo studies with a consistent, well-standardized production of renal TECs. This protocol may have broader future applications to study mitochondrial dysfunction associated with renal disorders for drug discovery or drug characterization purposes.

Osteopontin deficiency ameliorates Alport pathology by preventing tubular metabolic deficits.

Alport syndrome is a rare hereditary renal disorder with no etiologic therapy. We found that osteopontin (OPN) is highly expressed in the renal tubules of the Alport mouse and plays a causative pathological role. OPN genetic deletion ameliorated albuminuria, hypertension, tubulointerstitial proliferation, renal apoptosis, and hearing and visual deficits in the Alport mouse. In Alport renal tubules we found extensive cholesterol accumulation and increased protein expression of dynamin-3 (DNM3) and LDL receptor (LDLR) in addition to dysmorphic mitochondria with defective bioenergetics. Increased pathological cholesterol influx was confirmed by a remarkably increased uptake of injected DiI-LDL cholesterol by Alport renal tubules, and by the improved lifespan of the Alport mice when crossed with the Ldlr-/- mice with defective cholesterol influx. Moreover, OPN-deficient Alport mice demonstrated significant reduction of DNM3 and LDLR expression. In human renal epithelial cells, overexpressing DNM3 resulted in elevated LDLR protein expression and defective mitochondrial respiration. Our results suggest a potentially new pathway in Alport pathology where tubular OPN causes DNM3- and LDLR-mediated enhanced cholesterol influx and impaired mitochondrial respiration.

An in vitro ethnopharmacological study on Prangos ferulacea: a wound healing agent.

Introduction: Traditionally Prangos ferulacea root is being used as an effective wound healing agent especially for pus-filled wounds both in human and stocks in the western north of Iran. Regarding the subject we decided to study P. ferulacea roots essential oil (PFE) for its antimicrobial and wound healing activities. Methods: The in vitro wound healing activity of PFE was evaluated in the mouse fibroblast cell line L929 using MTT assay of cell viability and cytotoxicity indices. Scratch assay as an in vitro model of wound healing assay was also conducted in this study. Moreover, the type I collagen content was used as an indicator of progress in wound healing process using Sircol collagen assay. Besides, PFE was subjected to GC/MS to identify the chemical constituents, and antimicrobical property was also evaluated against S. aureus, S. epidermidis, E. coli, P. aeruginosa,S. paratyphi and C. albicans using agar dilution method. Results: GC/MS analysis showed that the monoterpene hydrocarbones dominated in PFE, amounting to a total percentage of 95.1% with the major constituents: ß-Phellandrene (32.1%), m-Tolualdehyde (26.2%), and δ-3-carene (25.8%). PFE inhibited the growth of S. aureus and P. aeruginusa with the MIC value of 20 µg/mL. In addition, at the second day of treatment, PFE at concentrations of 4 and 16 µg/mL significantly (P<0.001) enhanced the migration rate of L929 cells by 87.05±2.4 and 63.5±0.08 %, respectively. Moreover, the collagen production by L929 cells was increased greatly (P<0.001). Conclusion: It is proposed that the excellent antimicrobial activity along with the significant increase of migration rate and collagen production by fibroblast cells might be associated with the high content and synergistic effect of the monoterpens, corroborating the traditional usage of this plant as a wound healing agent.

Osteopontin RNA aptamer can prevent and reverse pressure overload-induced heart failure.

AIMS: Cardiac myocyte hypertrophy, the main compensatory response to chronic stress in the heart often progresses to a state of decompensation that can lead to heart failure. Osteopontin (OPN) is an effector for extracellular signalling that induces myocyte growth and fibrosis. Although increased OPN activity has been observed in stressed myocytes and fibroblasts, the detailed and long term effects of blocking OPN signalling on the heart remain poorly defined. Targeting cardiac OPN protein by an RNA aptamer may be beneficial for tuning down OPN pathologic signalling. We aimed to demonstrate the therapeutic effects of an OPN RNA aptamer on cardiac dysfunction. METHODS AND RESULTS: In vivo, we show that in a mouse model of pressure overload, treating at the time of surgeries with an OPN aptamer prevented cardiomyocyte hypertrophy and cardiac fibrosis, blocked OPN downstream signalling (PI3K and Akt phosphorylation), reduced expression of extracellular matrix (Lum, Col3a1, Fn1) and hypertrophy (Nppa, Nppb) genes, and prevented cardiac dysfunction. Treating at two months post-surgeries with the OPN aptamer reversed cardiac dysfunction and fibrosis and myocyte hypertrophy. While genetic homozygous deletion of OPN reduced myocardial wall thickness, surprisingly cardiac function and myocardial fibrosis, specifically collagen deposition and myofibroblast infiltration, were worse compared with wild type mice at three months of pressure overload. CONCLUSION: Taken together, these data demonstrate that tuning down cardiac OPN signalling by an OPN RNA aptamer is a novel and effective approach for preventing cardiac hypertrophy and fibrosis, improving cardiac function, and reversing pressure overload-induced heart failure.

Chromatographic Fingerprint Analysis of Marrubiin in Marrubium vulgare L. via HPTLC Technique.

PURPOSE: In the present study we aimed to quantify marrubiin, as the major active compound, in the aerial parts of Marrubium vulgare from Iran using a HPTLC-densitometry technique. METHODS: Quantitative determination of marrubiin in M. vulgare methanol extract was performed by HPTLC analysis via a fully automated TLC scanner. Later on, the in vitro antioxidant activity of the M. vulgare methanol extract was determined using 1,1-diphenyl-2-picryl-hydrazil (DPPH) free radical scavenging assay. Furthermore, total phenolics and flavonoids contents of the methanol extract were quantified, spectrophotometrically. RESULTS: The amount of marrubiin was calculated as 156 mg/g of M. vulgare extract. The antioxidant assay revealed a strong radical scavenging activity for the M. vulgare methanol extract with RC50 value of 8.24µg/mL. Total phenolics and flavonoids contents for M. vulgare were determined as 60.4 mg gallic acid equivalent and 12.05 mg quercetin equivalent per each gram of the extract, correspondingly. CONCLUSION: The presented fingerprint of marrubiin in M. vulgare extract developed by HPTLC densitometry afforded a detailed chemical profile, which might be useful in the identification as well as quality evaluation of herbal medications based on M. vulgare. Besides, the considerable antioxidant activity of M. vulgare was associated with the presence of marrubiin along with phenolics and flavonoids exerting a synergistic effect.