A phase II randomized, double-blind, placebo-controlled study of Nuvastatic (C50SEW505OESA), a standardized rosmarinic acid-rich polymolecular botanical extract formulation to reduce cancer-related fatigue in patients with solid tumors.

AIM: We evaluated the efficacy and safety of Nuvastatic™ (C5OSEW5050ESA) in improving cancer-related fatigue (CRF) among cancer patients. METHODS: This multicenter randomized double-blind placebo-controlled phase 2 trial included 110 solid malignant tumor patients (stage II-IV) undergoing chemotherapy. They were randomly selected and provided oral Nuvastatic™ 1000 mg (N = 56) or placebo (N = 54) thrice daily for 9 weeks. The primary outcomes were fatigue (Brief Fatigue Inventory (BFI)) and Visual Analog Scale for Fatigue (VAS-F)) scores measured before and after intervention at baseline and weeks 3, 6, and 9. The secondary outcomes were mean group difference in the vitality subscale of the Medical Outcome Scale Short Form-36 (SF-36) and urinary F2-isoprostane concentration (an oxidative stress biomarker), Eastern Cooperative Oncology Group scores, adverse events, and biochemical and hematologic parameters. Analysis was performed by intention-to-treat (ITT). Primary and secondary outcomes were assessed by two-way repeated-measures analysis of variance (mixed ANOVA). RESULTS: The Nuvastatic™ group exhibited an overall decreased fatigue score compared with the placebo group. Compared with the placebo group, the Nuvastatic™ group significantly reduced BFI-fatigue (BFI fatigue score, F (1.4, 147) = 16.554, p < 0.001, partial η2 = 0.333). The Nuvastatic™ group significantly reduced VAS-F fatigue (F (2, 210) = 9.534, p < 0.001, partial η2 = 0.083), improved quality of life (QoL) (F (1.2, 127.48) = 34.07, p < 0.001, partial η2 = 0.243), and lowered urinary F2-IsoP concentrations (mean difference (95% CI) = 55.57 (24.84, 86.30)), t (55) = 3.624, p < 0.001, Cohen's d (95% CI) = 0.48 (0.20, 0.75)). Reported adverse events were vomiting (0.9%), fever (5.4%), and headache (2.7%). CONCLUSION: Nuvastatic™ is potentially an effective adjuvant for CRF management in solid tumor patients and worthy of further investigation in larger trials. TRIAL REGISTRATION: ClinicalTrial.gov ID: NCT04546607. Study registration date (first submitted): 11-05-2020.

Rosmarinic acid mitigates intestinal inflammation and oxidative stress in bullfrogs (Lithobates catesbeiana) fed high soybean meal diets.

High proportions of soybean meal in aquafeed have been confirmed to induce various intestinal pathologies. This study aims to investigate the regulatory effects of rosmarinic acid (RA), an antioxidant with anti-inflammatory and antimicrobial properties, when added to high soybean meal feeds in different doses, (0, 0.5, 1, and 4 g/kg). During the 56-day feeding trial, results indicated that, compared to the control group without RA (0 g/kg), the 1 g/kg and 4 g/kg RA groups increased bullfrog survival rates and total weight gain while reducing feed coefficient. Additionally, these doses markedly suppressed the expression of key intestinal inflammatory markers (tlr5, myd88, tnfα, il1ß, cxcl8, cxcl12) and the activity and content of intestinal antioxidants (CAT, MDA, GSH, GPX). Concurrently, RA significantly downregulated the transcription levels of antioxidant-related genes (cat, gpx5, cyba, cybb, mgst, gclc, gsta, gstp), suggesting RA's potential to alleviate intestinal inflammation and oxidative stress induced by high soybean meal and to help downregulate and restore normal expression of antioxidant enzyme genes. However, the 0.5 g/kg RA group did not show a significant improvement in survival rates; instead, it upregulated the transcription of some antioxidant genes (cat, gpx5, cyba, cybb), revealing the complexity and dose-dependency of RA's antioxidant action. Furthermore, RA supplementation significantly reshaped the intestinal microbial community structure and relative abundance in bullfrogs, particularly affecting the genera Hafnia, Phascolarctobacterium, and Lactococcus. Notably, high doses of RA (1 g/kg, 4 g/kg) were able to downregulate pathways associated with the enrichment of gut microbiota in diseases such as Parkinson's, Staphylococcus aureus infection, and Systemic lupus erythematosus, suggesting its potential in anti-inflammatory action and health maintenance to prevent potential diseases.

A study on the antioxidant activity of rosmarinic acid against carbon tetrachloride-induced liver toxicity in adult male albino rats / Estudio sobre la actividad antioxidante del ácido rosmarínico contra la toxicidad hepática inducida por tetracloruro de carbono en ratas albinas macho adultas

SUMMARY: Carbon tetrachloride (CCl4) is a manufactured chemical and does not occur naturally in the environment. CCl4 is a clear liquid that evaporates very easily. It has a sweet odor. CCl4 is toxic to the mammalian liver and is hepatocarcinogenic in both rats and mice. Rosemary (Rosmarinus Officinalis) is commonly used as a spice and flavoring agent in food processing. It is known for its antioxidant properties. The present study aims to investigate the antioxidant activity of rosmarinic acid (RA) on CCl4-induced liver toxicity in adult male albino rats. Forty adult male albino rats were divided into 4 groups with 10 rats in each group. Group I (control group). Group II animals received RA at a dose of 50 mg/kg/day by oral gavage for 4 weeks. Group III animals received CCl4 intraperitoneally at a dose of 3ml/kg twice weekly for 4 weeks. Group IV animals received CCl4 Plus RA. At the end of the experiment, liver specimens are processed for histological, immunohistochemical, EM and biochemical studies. Administration of RA deceased the elevated serum liver enzymes (AST, ALT, and ALP), elevated MDA level and immunoexpression of the proapoptotic protein (Bax) induced by CCl4. It increased reduced glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and immunoexpression of the antiapoptotic protein (Bcl2). It also improved the histological and ultrastructural changes induced by CCl4. It appears that Rosmarinic acid has protective effects against CCl4-induced hepatotoxicity as indicated by biochemical, histological, immunohistochemical and ultrastructural results.

RESUMEN: El tetracloruro de carbono (CCl4) es un producto químico fabricado y no se encuentra de forma natural en el medio ambiente. CCl4 es un líquido transparente que se evapora fácilmente; tiene un olor dulce. CCl4 es tóxico para el hígado de los mamíferos y es hepatocarcinogénico tanto en ratas como en ratones. El romero (Rosmarinus officinalis) se usa comúnmente como condimento y agente aromatizante en el procesamiento de alimentos. Es conocido por sus propiedades antioxidantes. El presente estudio tuvo como objetivo investigar la actividad antioxidante del ácido rosmarínico (RA) sobre la toxicidad hepática inducida por CCl4 en ratas albinas macho adultas. Se dividieron cuarenta ratas albinas macho adultas en 4 grupos con 10 ratas en cada grupo. Grupo I (grupo control). Los animales del grupo II recibieron AR a una dosis de 50 mg / kg / día por sonda oral durante 4 semanas. Los animales del grupo III recibieron CCl4 por vía intraperitoneal a una dosis de 3 ml / kg dos veces por semana durante 4 semanas. Los animales del grupo IV recibieron CCl4 Plus RA. Al final del experimento, las muestras de hígado se procesaron para estudios histológicos, inmunohistoquímicos, EM y bioquímicos. La administración de AR eliminó las enzimas hepáticas séricas elevadas (AST, ALT y ALP), el nivel elevado de MDA y la inmunoexpresión de la proteína proapoptótica (Bax) inducida por CCl4. Aumentó el glutatión reducido (GSH), glutatión peroxidasa (GSH-Px), la superóxido dismutasa (SOD) y la inmunoexpresión de la proteína antiapoptótica (Bcl2). También mejoró los cambios histológicos y ultraestructurales inducidos por CCl4. El ácido rosmarínico puede tener efectos protectores contra la hepatotoxicidad inducida por CCl4, tal como lo indican los resultados bioquímicos, histológicos, inmunohistoquímicos y ultraestructurales.

NLRP3 inflammasome in rosmarinic acid-afforded attenuation of acute kidney injury in mice.

Cisplatin (CP) is a well-known anticancer drug used to effectively treat various kinds of solid tumors. CP causes acute kidney injury (AKI) and unfortunately, there is no therapeutic approach in hand to prevent AKI. Several signaling pathways are responsible for inducing AKI which leads to inflammation in proximal convoluted tubule cells in the kidney. Furthermore, the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome is involved in the CP-induced AKI. In this study, we investigated therapeutic effects of rosmarinic acid (RA) against inflammation-induced AKI. RA was orally administered at the dose of 100 mg/kg for two consecutive days after 24 h of a single injection of CP at the dose of 20 mg/kg administered intraperitoneally in Swiss albino male mice. Treatment of RA inhibited the activation of NLRP3 signaling pathway by blocking the activated caspase-1 and downstream signal molecules such as IL-1ß and IL18. CP activated HMGB1-TLR4/MyD88 axis was also found to be downregulated with the RA treatment. Activation of nuclear factor-κB and elevated protein expression of cyclooxygenase-2 (COX-2) were also found to be downregulated in RA-treated animals. Alteration of early tubular injury biomarker, kidney injury molecule-1 (KIM-1), was found to be subsided in RA-treated mice. RA has been earlier reported for antioxidant and anti-inflammatory properties. Our findings show that blocking a critical step of inflammasome signaling pathway by RA treatment can be a novel and beneficial approach to prevent the CP-induced AKI.

Rosmarinic acid ameliorates acetaminophen-induced acute liver injury in mice via RACK1/TNF-α mediated antioxidant effect.

CONTEXT: Rosmarinic acid (RA) dose-dependently ameliorates acetaminophen (APAP) induced hepatotoxicity in rats. However, whether RA hepatoprotective effect is by regulating RACK1 and its downstream signals is still unclear. OBJECTIVE: This study explores the RA protective effect on APAP-induced ALI and its mechanism. MATERIALS AND METHODS: Sixty Kunming mice 6-8 weeks old were randomly separated into six groups (n = 10) and pre-treated with normal saline, ammonium glycyrrhetate (AG) or RA (10, 20 or 40 mg/kg i.p./day) for two consecutive weeks. Then, APAP (300 mg/kg, i.g.) was administrated to induce ALI, except for the control. Serum alanine/aspartate aminotransferases (ALT and AST), malondialdehyde (MDA), superoxide dismutase (SOD) and histopathology were used to authenticate RA effect. The liver RACK1 and TNF-α were measured by western blot. RESULTS: Compared with the APAP group, different dosages RA significantly decreased ALT (52.09 ± 7.98, 55.13 ± 10.19, 65.08 ± 27.61 U/L, p < 0.05), AST (114.78 ± 19.87, 115.29 ± 31.91, 101.78 ± 21.85 U/L, p < 0.05), MDA (2.37 ± 0.87, 2.13 ± 0.87, 1.86 ± 0.39 nmol/mg, p < 0.01) and increased SOD (306.178 ± 90.80, 459.21 ± 58.54, 444.01 ± 78.09 U/mg, p < 0.05). With increasing doses of RA, RACK1 and TNF-α expression decreased. Moreover, the RACK1 and TNF-α levels were positively correlated with MDA (r = 0.8453 and r = 0.9391, p < 0.01). DISCUSSION AND CONCLUSIONS: Our findings support RA as a hepatoprotective agent to improve APAP-induced ALI and the antioxidant effect mediated through RACK1/TNF-α pathway.

Effectiveness and Anticancer Activity of a Novel Phenolic Compound from Garcinia porrecta Against the MCF-7 Breast Cancer Cell Line in vitro and in silico.

BACKGROUND: Cancer is a leading cause of death worldwide, with breast cancer being the most common invasive cancer type in women. Several therapeutic strategies have been explored to reduce the mortality rates of breast cancer. Chemotherapy is the most commonly used systemic treatment, but associated with numerous side-effects. Development of anticancer agents with high efficacy and minimal negative effects is therefore an important focus of research. Natural materials provide an excellent source of bioactive compounds. For instance, Garcinia porrecta from the Clusiaceae family has multiple pharmacological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral, anti-HIV, antidepressant, and anticancer properties. PURPOSE: The main objective of this study was to investigate the potential anticancer effects of compounds extracted from the bark of G. porrecta. MATERIALS AND METHODS: Our experiments were divided into three steps: (1) chromatographic isolation of compounds using various separation techniques, such as extraction, separation and purification, (2) characterization via infrared (IR), nuclear magnetic resonance (NMR) and mass spectroscopy, and (3) evaluation of anticancer activity in vitro (MTT assay) and in silico (via analysis of molecular docking against caspase-9, tumor necrosis factor alpha (TNF-α), estrogen receptor alpha (ER-α), and human epidermal growth factor receptor 2 (HER-2)). RESULTS: Depsidone (1) and benzophenone (2) from the ethyl acetate extract of bark of G. porrecta were identified as bioactive components. Examination of the activities of these compounds against MCF-7 cells revealed an IC50 value of 119.3 µg/mL for benzophenone, whereas IC50 for depsidone could not be estimated. Benzophenone activity was lower than that of the positive control doxorubicin (6.9 µg/mL). Depsidone showed the highest binding affinity for HER-2 (-9.2 kcal.mol-1) and benzophenone for ER-α (-8.0 kcal.mol-1). CONCLUSION: Benzophenone displays potency as an anticancer agent through blocking ER-α.

Rosmarinic acid attenuates acrylamide induced apoptosis of BRL-3A cells by inhibiting oxidative stress and endoplasmic reticulum stress.

Acrylamide (AA) is a common endogenous contaminant in food, with a complex toxicity mechanism. The study on liver damage to experimental animals caused by AA has aroused a great attention. Rosmarinic acid (RosA) as a natural antioxidant shows excellent protective effects against AA-induced hepatotoxicity, but the potential mechanism is still unclear. In the current study, the protective effect of RosA on BRL-3A cell damage induced by AA was explored. RosA increased the activity of SOD and GSH, reduced the content of ROS and MDA, and significantly reduced the oxidative stress (OS) damage of BRL-3A cells induced by AA. RosA pretreatment inhibited the MAPK signaling pathway activated by AA, and down-regulated the phosphorylation of JNK, ERK and p38. RosA pretreatment also reduced the production of calcium ions caused by AA. In addition, the key proteins p-IRE1α, XBP-1s, TRAF2 of the IRE1 pathway, and the expression of endoplasmic reticulum stress (ERS) characteristic proteins GRP78, p-ASK1, Caspase-12 and CHOP were also down-regulated by RosA. NAC blocked the activation of the MAPK signaling pathway and inhibited the ERS pathway. RosA reduced the rate of apoptosis and down-regulated the expression of Bax/Bcl-2 and Caspase-3, thereby inhibiting AA-induced apoptosis. In conclusion, RosA reduced the OS and ERS induced by AA in BRL-3A cells, thereby inhibiting cell apoptosis, and it could be used as a potential protective agent against AA toxicity.

Preparation and In Vivo Evaluation of Rosmarinic Acid-Loaded Transethosomes After Percutaneous Application on a Psoriasis Animal Model.

The topical use of rosmarinic acid (RA) in skin inflammatory pathologies is restricted due to its poor water solubility, poor permeability, and chemical instability. In this study, RA-loaded transethosomes-in-Carbopol® formulations have been developed to evaluate its anti-inflammatory activity on imiquimod-induced psoriasis-like skin inflammation in mice. In vitro release profiles demonstrated sustained behavior due to the retentive action of gel and the entrapment of RA into the vesicles. However, the low viscosity of the combined formulation increased the drug release rate. Animal evaluation of anti-inflammatory activity demonstrated that transethosomes-in-gel containing dexamethasone (Dex-TE-Gel), as positive control, showed effect in all the pro-inflammatory parameters evaluated, evidencing that these drug-loaded nanocarriers have been effectively reached the site of action. In addition, transethosomes-in-gel containing RA (RA-TE-Gel) formulations produced a great reduction in the punch edema (P < 0.001) and in TNF-α and IL-6 (P < 0.05). However, non-significant differences were obtained for IL-1ß, IL17, and MPO. Despite the protecting effect of Carbopol® and transethosomes on oxidation index and antioxidant activity of RA over the 7 days of treatment, however, a degradation process of this antioxidant to caffeic acid may be the cause of these in vivo results. We have also checked that the pH existing into the intercellular space of damaged cells (pH 6.8) may be affecting. Therefore, our results suggest that RA-TE-Gel could act as an effective RA formulation for skin delivery; further studies will help to understand the loss of activity at the cellular level.

Chitosan-coated rosmarinic acid nanoemulsion nasal administration protects against LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats.

Rosmarinic acid (RA) lipid-nanotechnology-based delivery systems associate with mucoadhesive biopolymers for nasal administration has arisen as a new promising neuroprotective therapy for neurodegenerative disorders (ND). We have previously demonstrated the glioprotective effect of chitosan-coated RA nanoemulsions (RA CNE) against lipopolysaccharide (LPS)-induced damage in rat astrocyte primary culture. Here, we further investigate the protective effect of RA CNE nasal administration on LPS-induced memory deficit, neuroinflammation, and oxidative stress in Wistar rats, since these in vivo studies were crucial to understand the impact of developed delivery systems in the RA neuroprotective effects. The animals were treated through nasal route with RA CNE (2 mg·mL-1), free RA (2 mg·mL-1), blank CNE, and saline (control and LPS groups) administrations (n.a., 100 µL per nostril) twice a day (7 a.m./7 p.m.) for six days. On the sixth day, the animals received the last treatments and LPS was intraperitoneally (i.p.) administrated (250 µg·kg-1). Overall results, proved for the first time that the RA CNE nasal administration elicits a neuroprotective effect against LPS-induced damage, which was associated with increased 1.6 times recognition index, decreased 5.0 and 1.9 times in GFAP+ cell count and CD11b expression, respectively, as well as increased 1.7 times SH in cerebellum and decreased 3.9 times TBARS levels in cerebral cortex in comparison with LPS group. RA CNE treatment also facilitates RA bioavailability in the brain, confirmed by RA quantification. Free RA also demonstrates a protective effect in some studied parameters, although no RA was quantified in the brain.

Rosmarinic Acid Protects Mice from Concanavalin A-Induced Hepatic Injury through AMPK Signaling.

Rosmarinic acid (RA) is extensively utilized in herbal medicine in China. The AMP-activated protein kinase (AMPK) signaling can be activated by RA and inhibited by the synthetic, reversible AMP-competitive inhibitor, Compound C (CC). The objective of this study was to investigate the role of AMPK signaling involving the protective effects of RA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) in mice. BALB/c mice were treated with RA, with or without CC, followed by the pretreatment with Con A. Analysis of serum aminotransferases and cytokines were conducted and liver tissue histology was performed to evaluate hepatic injury. Cytokine levels in serum and hepatic tissue were respectively measured by enzyme-linked immunoassay (ELISA) and used quantitative (q)PCR. Levels of phosphorylated acetyl CoA carboxylase in the liver, representing AMPK activation, were detected by Western blotting. Compared with the Con A group, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in RA group (100 and 150 mg/kg/d) were significantly reduced. RA also reduced hepatocyte swelling, cell death, and infiltration of leukocytes in the liver of Con A-treated mice. Serum levels of cytokines, such as interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-1ß (IL-1ß), were reduced by RA pretreatment, while the levels of serum interleukin-10 (IL-10), an anti-inflammatory cytokine, was elevated. These protective effects were reversed by treatment with CC. RA treatment reduced the hepatic damage via the activation of AMPK in the mice of Con A-induced. So RA acts as a potential part in the therapy of autoimmune hepatitis.

Rosmarinic acid prevents gastric ulcers via sulfhydryl groups reinforcement, antioxidant and immunomodulatory effects.

Rosmarinic acid (RA) is a secondary metabolite present in several plant species that has already demonstrated antioxidant, antiallergic, anticancer, antimicrobial, neuroprotective, and hepatoprotective effects experimentally. Due to the promising pharmacological properties found previously, this study aimed to assess the oral acute toxicity and the gastroprotective effect of RA using animal models. Acute toxicity was assessed according to OECD guide 423. Ethanol, stress, NSAIDs, and pylorus ligature-induced gastric ulcer models were used to investigate antiulcer properties. The related mechanisms of action were also evaluated from ethanol-induced gastric lesions protocol. RA (300 and 2000 mg/kg) showed no changes in behavioral, water and food intake, body and organs weight parameters with LD50 set around 2500 mg/kg. RA presented gastroprotective activity in all assessed doses (25, 50, 100, and 200 mg/kg) using different animal models. Besides, it was observed that this effect is not related to the modulation of gastric juice parameters (pH, volume, and [H+]), the participation of nitric oxide, mucus, and prostaglandins. However, increased sulfhydryl groups, GSH and IL-10 levels as well as reduced of proinflammatory cytokine (TNF-α and IL-1ß) levels were found for RA-treated groups. RA presents low acute toxicity and gastroprotective activity, preventing ulcer formation via cytoprotective, antioxidant, and anti-inflammatory mechanisms. Graphical abstract.

Improvement of antioxidant activity of oregano (Origanum vulgare L.) with an oral pharmaceutical form.

Aging-related diseases can be triggered by multiple factors such as oxidative stress. Oxidative stress is an imbalance between free radicals and antioxidants, so today, compounds capable of reducing or neutralizing free radicals are being studied for a therapeutic use. Origanum vulgare L. is a traditional medicinal plant used for a wide number of health problems due to its antimicrobial, carminative and antioxidant activities. However, when administered orally, gastrointestinal digestion can modify some of therapeutical properties. To avoid this, two different solid oral formulations have been designed for an O. vulgare extract evaluating their antioxidant behaviours in vitro and in vivo after a simulation of gastrointestinal digestion. The results showed that the divided powder has a lower antioxidant activity both in vitro and in vivo than the encapsulated extract. The quantitative difference of polyphenols found on HPLC-DAD (especially luteolin, apigenin and caffeic acid) may explain the differences in pharmacological activity. Thus, we propose that the best form to administrate O. vulgare extracts to maintain the antioxidant properties is the encapsulated form, that is, two capsules of 250 mg of a hydroalcoholic extract of O. vulgare with a minimum of 33 % of rosmarinic acid as a daily dose.

Rosmarinic acid exhibits broad anti-enterovirus A71 activity by inhibiting the interaction between the five-fold axis of capsid VP1 and cognate sulfated receptors.

Enterovirus A71 (EV-A71), a positive-stranded RNA virus of the Picornaviridae family, may cause neurological complications or fatality in children. We examined specific factors responsible for this virulence using a chemical genetics approach. Known compounds from an anti-EV-A71 herbal medicine, Salvia miltiorrhiza (Danshen), were screened for anti-EV-A71. We identified a natural product, rosmarinic acid (RA), as a potential inhibitor of EV-A71 by cell-based antiviral assay and in vivo mouse model. Results also show that RA may affect the early stage of viral infection and may target viral particles directly, thereby interfering with virus-P-selectin glycoprotein ligand-1 (PSGL1) and virus-heparan sulfate interactions without abolishing the interaction between the virus and scavenger receptor B2 (SCARB2). Sequencing of the plaque-purified RA-resistant viruses revealed a N104K mutation in the five-fold axis of the structural protein VP1, which contains positively charged amino acids reportedly associated with virus-PSGL1 and virus-heparan sulfate interactions via electrostatic attraction. The plasmid-derived recombinant virus harbouring this mutation was confirmed to be refractory to RA inhibition. Receptor pull-down showed that this non-positively charged VP1-N104 is critical for virus binding to heparan sulfate. As the VP1-N104 residue is conserved among different EV-A71 strains, RA may be useful for inhibiting EV-A71 infection, even for emergent virus variants. Our study provides insight into the molecular mechanism of virus-host interactions and identifies a promising new class of inhibitors based on its antiviral activity and broad spectrum effects against a range of EV-A71.

A Polyphenolic Complex Attenuates Inflammatory Response and Blood- Brain Barrier Disruption.

BACKGROUND: Cerebral ischemia causes a strong inflammatory response. Neumentix is a dietary supplement containing 14.9% rosmarinic acid and 29.9% total phenolic content, which has been proved to be beneficial against inflammatory response. Therefore, Neumentix's effect on anti-inflammatory and blood brain barrier (BBB) disruption in transient middle cerebral artery occlusion (tMCAO) model mice is investigated in this study. METHODS: After the pretreatment of vehicle or Neumentix 134 mg/kg/d, intraperitoneal injection (i.p.) (containing rosmarinic acid 20 mg/kg/d) for 14 days, mice were subjected to tMCAO for 60 min and kept receiving vehicle or Neumentix daily 5 days afterward. RESULTS: Neumentix treatment ameliorated neurobehavioral impairment in the corner test (5d after tMCAO, **P<0.01), reduced infarct volume (#P<0.05), suppressed expression of ionized calciumbinding adapter molecule-1 (Iba-1), tumor necrosis factor alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) (###P<0.001), and improved the integrity of BBB (§P<0.05) at 5 days after tMCAO. CONCLUSION: The present study provided an evidence of Neumentix's anti-inflammatory and neuroprotection effect against BBB disruption on experimental tMCAO model mice, suggesting that Neumentix could be a potential therapeutic agent for stroke.

Dual functional liposomes carrying antioxidants against tau hyperphosphorylation and apoptosis of neurons.

Quercetin (QU) and rosmarinic acid (RA) were loaded in phosphatidic acid-liposomes (QU/RA-PA-liposomes) with surface apolipoprotein E (ApoE) using a process of thin-film hydration, followed by covalent crosslinking to activate biological pathways for penetrating the blood-brain barrier (BBB) and redeeming the neuronal apoptosis from attack of ß-amyloid 1-42 (Aß1-42) and neurofibrillary tangles. The conjugation of liposomes with PA improved the activity of QU and RA against neurotoxicity of Aß1-42. The fluorescent images of brain capillaries revealed that surface modification with ApoE improved the permeation ability of QU/RA-PA-ApoE-liposomes across the BBB. In addition, the highest therapeutic efficacy was obtained in the case of QU/RA-PA-ApoE-liposomes, compared to other QU/RA formulations studied using in vivo Aß1-42-insulted rats mimicking Alzheimer's disease (AD). The cellular and molecular evidence from AD rats included the decrease in Aß1-42 plaque formation and interleukin-6 secretion, increase in the neuronal count in Nissl staining, and reduction in the expression of phosphorylated extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, p38 kinase and tau protein at serine 202 as well as caspase-3. The use of PA-ApoE-liposomes as a dual targeting formulation enhances the QU and RA ability to infiltrate the BBB, docks Aß1-42 plaques and can be a potent approach to rescue degenerated neurons from AD.

Rosmarinic acid, the active component of Salvia miltiorrhizae, improves gliquidone transport by regulating the expression and function of P-gp and BCRP in Caco-2 cells.

Salvia miltiorrhiza (Danshen) is typically used in the treatment of diabetic complications and is often co-prescribed with gliquidone in China. However, whether danshen affects the absorption of gliquidone has not been elucidated. In this study, the effects of an aqueous extract of danshen (danshen injection, DSI) and its primary compounds (danshensu, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B) on gliquidone transport across Caco-2 monolayer cells was investigated. DSI enhanced the transport of gliquidone in Caco-2 cell monolayers from the apical (AP) to basolateral (BL) sides and from the BL to AP sides. Rosmarinic acid (RA) also significantly increased the Papp (AP-BL) value for gliquidone transport. Verapamil (a P-gp inhibitor) and Ko143 (a BCRP inhibitor) inhibited the BL-AP transport of gliquidone and promoted the AP-BL transport of gliquidone, whereas MK571 (an MRP1 inhibitor), probenecid (an MRP2 inhibitor), and benzbromarone (an MRP3 inhibitor) had no effect on gliquidone transport. RA also enhanced the intracellular accumulation of Rho123 and Hoechst 33342. The expression of P-gp and BCRP was significantly downregulated, and P-gp ATPase activity was promoted by RA in a dose-dependent manner. These results indicate that an aqueous extract of danshen can increase the transport of gliquidone in Caco-2 cell monolayers and that RA may be the primary compound associated with this activity, which is in agreement with RA simultaneously suppressing the function and expression of P-gp and BCRP.

The cytotoxic concentration of rosmarinic acid increases MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells.

Rosmarinic acid (RA) is a natural polyphenolic compound derived from many common herbal plants. Although it is known that RA has many important biological activities, its effect on proteasome inhibitor-induced changes in cancer treatment or its effects on any experimental proteasome inhibition model is unknown. The aim of the study was to investigate the effect of RA on MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells. HepG2 cells were treated with 10, 100, and 1000 µM RA in the presence of MG132 for 24 h; 10 and 100 µM RA did not affect but 1000 µM RA decreased cell viability in HepG2 cells. MG132 caused a significant decrease in cell viability and phosphorylation of mammalian target of rapamycin and a significant increase in levels of polyubiquitinated protein, microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II), heat shock protein 70 (HSP70), binding immunoglobulin protein (BiP), activating transcription factor 4 (ATF4), protein carbonyl, and cleaved poly(adenosine diphosphate-ribose) polymerase 1 (PARP1); 10 and 100 µM RA did not significantly change these effects of MG132 in HepG2 cells; 1000 µM RA caused a significant decrease in cell viability and a significant increase in polyubiquitinated protein, LC3B-II, HSP70, BiP, ATF4, protein carbonyl, and cleaved PARP1 levels in MG132-treated cells. Our study showed that only 1000 µM RA increased MG132-induced cytotoxicity, proteasome inhibition, autophagy, cellular stresses, and apoptosis in HepG2 cells. According to our results, cytotoxic concentration of RA can potentiate the effects of MG132 in hepatocellular carcinoma treatment.

Anti-aging formulation of rosmarinic acid-loaded ethosomes and liposomes.

The study was aimed to evaluate the effectiveness of rosmarinic acid (RA) loaded ethosomes (ETHs) and liposomes (LPs) when subjected to the transdermal application. RA-loaded ETHs and LPs were prepared, optimised, and characterised. The ex vivo permeation studies of formulations using mouse abdominal skin were performed. Antioxidant activities and the inhibitory effects of formulations on collagenase and elastase enzymes were measured. Optimised ethosomal formulation (F3) was showed nanometric size range (138 ± 1.11 nm) and greatest entrapment (55 ± 1.80%), was selected for further transdermal permeation studies. Skin permeation profile of the nanoformulations analysed by HPLC revealed an enhanced permeation of ETHs. Transdermal flux of ETHs was found to be higher than RA solution and LPs. Enzyme inhibitions of ETHs were the significant difference found between ETHs and LPs (p < 0.05). ETHs were found to be more effective and successful than LPs. Results suggest that ETHs are more effective than LPs for transdermal delivery of RA.

Development, physico-chemical characterization and in-vitro studies of hydrogels containing rosmarinic acid-loaded nanoemulsion for topical application.

OBJECTIVES: Natural sources with antioxidant activity, such as rosmarinic acid (RA), have been considered as an interesting approach for the development of new anti-ageing skin products. In this context, this study aimed to develop hydrogels containing RA-loaded nanoemulsions and evaluate the effect of the addition of Tween® 80 (a nonionic cosurfactant) in formulations intended to be used for topical application. METHODS: Physico-chemical characterization, in-vitro release and skin retention/permeation from hydrogels of RA-loaded nanoemulsions (containing or not Tween® 80) were evaluated. The RA-loaded nanoemulsion safety profiles were also investigated in keratinocytes (HaCaT cells). KEY FINDINGS: It was observed that all formulations presented adequate physico-chemical characterization for topical application. Furthermore, the results also demonstrated that the presence of Tween® 80 decreased the droplet size and polydispersity index of nanoemulsions and hydrogels. An extended RA release was noted for the hydrogels. However, when comparing the hydrogels, a positive effect of the presence of Tween® 80 on RA retention/permeation in total skin was seen. The RA-loaded nanoemulsion safety profiles demonstrated a good tolerability (3.125-100 µm) in HaCaT cells. CONCLUSIONS: The overall results demonstrated that the formulations developed in this study can be considered as a suitable carrier for RA in a topical application targeting new anti-ageing skin care products.

Rosmarinic acid attenuates inflammatory responses through inhibiting HMGB1/TLR4/NF-κB signaling pathway in a mouse model of Parkinson's disease.

Inflammation contributes to the pathological processes in patients and animal models of PD. Rosmarinic acid (RA) has been demonstrated to protect neurons in PD models. The present study aimed to evaluate the anti-inflammatory effect of RA on PD and reveal possible pharmacological mechanisms. 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) was injected to mice to establish PD model in vivo. BV-2 cells were exposed to 1-methyl-4-phenylpyridinium (MPP+) and α-synuclein to establish PD model in vitro. Results showed that treatment with RA dose-dependently improved motor function of PD mice, increased the number of tyrosine hydroxylase-positive cells, reduced production of pro-inflammatory cytokines, and inhibited microglia activation in ventral midbrain. In cell study, RA also decreased MPP+ or α-synuclein-induced secretion of pro-inflammatory cytokines. Furthermore, RA treatment downregulated the expression levels of HMGB1, TLR4 and Myd88 and inhibited NF-κB nuclear expression both in PD animal and cell models. These findings indicated that RA could attenuate inflammatory responses through suppressing HMGB1/TLR4/NF-κB signaling pathway, which may contribute to its anti-PD activity.