Abstract: Extracts rich in antioxidants, such as rosemary extracts, are currently obtained by extraction of the plant material using hydro-alcoholic mixtures with high ethanol content. As this ratio is generally chosen by default and scarcely optimized, we intended to investigate the impact of the hydro-alcoholic composition on extract characteristics such as extraction yield, composition profile in selected compounds, and antioxidant/reducing activity such as Folin-Ciocalteu, DPPH, and ORAC. A theoretical determination of rosmarinic (RA) and carnosic (CA) acid solubilities in ethanol:water mixtures was also performed using COSMO-RS and was confronted to experiments. While the best solubilizing solvent (100% ethanol) was also the best extracting solvent for CA, it was not the case with RA since pure ethanol appeared as a poor solvent compared to 30% ethanol which was optimal. Finally, the best antioxidant activities were obtained with 30% ethanol.

Abstract: This study evaluated the protective effects of two rosemary components, rosmarinic acid (RA) and carnosic acid (CA), against hypoglycemia, hyperlipidemia, oxidative stress and an imbalanced gut microbiota architecture in diabetic rats. Treatment with RA and CA (30 mg kg-1) decreased the levels of fasting plasma glucose (23.7%, 15.6%), total cholesterol (30.4%, 14.1%) and triglyceride (65.7%, 47.8%) at 15 weeks. RA and CA also exhibited an anti-oxidative and anti-glycative effect by lowering the formation of malondialdehyde and advanced glycation end products. In addition, they showed protective effects against tissue damage and inflammation in the abdominal aorta, based on microscopic observations and the analysis of protein expression. Finally, the prebiotic effects of RA and CA on gut microbiota were demonstrated by increasing the population of diabetes-resistant bacteria and decreasing the amounts of diabetes-sensitive bacteria. Overall, RA showed a stronger protective effect than CA in mitigating diabetic symptoms in rats.

Abstract: Carnosic acid (CA) is a phenolic diterpene obtained from Rosmarinus officinalis L. and has demonstrated cytoprotective properties in several experimental models. CA exerts antioxidant effects by upregulating the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which controls the expression of antioxidant and phase II detoxification enzymes. Heme oxygenase-1 (HO-1) expression is modulated by Nrf2 and has been demonstrated as part of the mechanism underlying the CA-induced cytoprotection. Nonetheless, it remains to be studied whether and how HO-1 would mediate CA-elicited anti-inflammatory effects. Therefore, we have investigated here whether and how CA would prevent paraquat (PQ)-induced inflammation-related alterations in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were pretreated for 12 h with CA at 1 μM before exposure to PQ for further 24 h. CA suppressed the PQ-induced alterations on the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) through a mechanism involving the activation of the Nrf2/HO-1 axis. Furthermore, we observed a crosstalk between the Nrf2/HO-1 signaling pathway and the activation of the nuclear factor-κB (NF-κB) transcription factor, since administration of ZnPP IX (specific inhibitor of HO-1) or Nrf2 knockdown using small interfering RNA (siRNA) abolished the anti-inflammatory effects induced by CA. Moreover, administration of SN50 (specific inhibitor of NF-κB) inhibited the PQ-induced inflammation-related effects in SH-SY5Y cells. Therefore, CA exerted anti-inflammatory effects in SH-SY5Y cells through an Nrf2/HO-1 axis-dependent manner associated with downregulation of NF-κB.

Abstract: Carnosic acid (CA), a major polyphenolic diterpene present in Rosmarinus officinalis, has been reported to have multiple functions, including antitumor activity. The MTT assay, BrdU incorporation, wound healing, and colony formation were used to detect melanoma B16F10 cell growth and proliferation. Flow cytometry was used for cell cycle detection. p21 and p27 expression was detected by Western blotting. B16F10 cell xenograft model was established, and treated with CA, carmustine (BCNU), or lomustine (CCNU). The present study found that CA exhibits significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggers cell cycle arrest at G0/G1 phase, and enhances p21 expression. Additionally, CA can enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibits tumor growth, and reduces the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo The present study study concluded that CA may be safe and useful as a novel chemotherapeutic agent.

Abstract: The discovery of new therapeutic drugs with the ability of preventing inflammation and joint destruction with less adverse effects is urgently needed for rheumatoid arthritis (RA). Carnosic acid (CA), a major phenolic compound isolated from the leaves of Rosemary (Rosmarinus officinalis L.), has been reported to have antioxidative and antimicrobial properties. However, its effects on RA have not been elucidated. Here, we investigated the effects of CA on osteoclasts and fibroblast-like synoviocytes in vitro and on collagen-induced arthritis (CIA) in Wistar rats in vivo. Our in vitro and in vivo studies showed that CA suppressed the expression of pro-inflammatory cytokines including TNFɑ, IL-1β, IL-6, IL-8, IL-17 and MMP-3, and downregulated the production of RANKL. More importantly, we observed that CA inhibited osteoclastogenesis and bone resorption in vitro and exerted therapeutic protection against joint destruction in vivo. Further biochemical analysis demonstrated that CA suppressed RANKL-induced activations of NF-κB and MAPKs (JNK and p38) leading to the downregulation of NFATc1. Taken together, our findings provide the convincing evidence that rosemary derived CA is a promising natural compound for the treatment of RA.

Abstract: Drastic macrophages activation triggered by exogenous infection or endogenous stresses is thought to be implicated in the pathogenesis of various inflammatory diseases. Carnosic acid (CA), a natural phenolic diterpene extracted from Salvia officinalis plant, has been reported to possess anti-inflammatory activity. However, its role in macrophages activation as well as potential molecular mechanism is largely unexplored. In the current study, we sought to elucidate the anti-inflammatory property of CA using an integrated approach based on unbiased proteomics and bioinformatics analysis. CA significantly inhibited the robust increase of nitric oxide and TNF-α, downregulated COX2 protein expression, and lowered the transcriptional level of inflammatory genes including Nos2, Tnfα, Cox2, and Mcp1 in LPS-stimulated RAW264.7 cells, a murine model of peritoneal macrophage cell line. The LC-MS/MS-based shotgun proteomics analysis showed CA negatively regulated 217 LPS-elicited proteins which were involved in multiple inflammatory processes including MAPK, nuclear factor (NF)-κB, and FoxO signaling pathways. A further molecular biology analysis revealed that CA effectually inactivated IKKβ/IκB-α/NF-κB, ERK/JNK/p38 MAPKs, and FoxO1/3 signaling pathways. Collectively, our findings demonstrated the role of CA in regulating inflammation response and provide some insights into the proteomics-guided pharmacological mechanism study of natural products.

Abstract: The present studies have been executed to explore the protective mechanism of carnosic acid (CA) against NaAsO2-induced hepatic injury. CA exhibited a concentration dependent (1–4 μM) increase in cell viability against NaAsO2 (12 μM) in murine hepatocytes. NaAsO2 treatment significantly enhanced the ROS-mediated oxidative stress in the hepatic cells both in in vitro and in vivo systems. Significant activation of MAPK, NF-κB, p53, and intrinsic and extrinsic apoptotic signaling was observed in NaAsO2-exposed hepatic cells. CA could significantly counteract with redox stress and ROS-mediated signaling and thereby attenuated NaAsO2-mediated hepatotoxicity. NaAsO2 (10 mg/kg) treatment caused significant increment in the As bioaccumulation, cytosolic ATP level, DNA fragmentation, and oxidation in the liver of experimental mice ( ). The serum biochemical and haematological parameters were significantly altered in the NaAsO2-exposed mice ( ). Simultaneous treatment with CA (10 and 20 mg/kg) could significantly reinstate the NaAsO2-mediated toxicological effects in the liver. Molecular docking and dynamics predicted the possible interaction patterns and the stability of interactions between CA and signal proteins. ADME prediction anticipated the drug-likeness characteristics of CA. Hence, there would be an option to employ CA as a new therapeutic agent against As-mediated toxic manifestations in future.

Abstract: Carnosic acid (CA; C20H28O4), a phenolic diterpene characterized as an ortho-dihydroquinone-type molecule, is a pro-electrophile agent that becomes an electrophile after reacting with free radicals. The electrophile generated from CA interacts with and activates the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, which is a major modulator of redox biology in mammalian cells. CA induces antioxidant and anti-inflammatory effects in several cell types, as observed in both in vitro and in vivo experimental models. In this context, CA has been viewed as a neuroprotective agent by activating signaling pathways associated with cell survival during stressful conditions. Indeed, CA exhibits the ability to promote mitochondrial protection in neural cells. Mitochondria are the main source of both ATP and reactive species in animal cells. Mitochondrial dysfunction plays a central role in the start and development of neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, among others. Therefore, the study of strategies aiming to reduce mitochondrial impairment in the case of neurodegeneration is of pharmacological interest. In the present review, it is described and discussed the effects of CA on brain mitochondria in experimental models of neural lesion. Based on the data discussed here, CA is a potential candidate to be listed as a neuroprotective agent by acting on the mitochondria of neural cells.

Abstract: Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Parkinson’s disease, are characterized by the progressive loss of neurons in specific regions of the brain and/or spinal cord. Neuronal cell loss typically occurs by either apoptotic or necrotic mechanisms. Oxidative stress and nitrosative stress, along with excitotoxicity and caspase activation, have all been implicated as major underlying causes of neuronal cell death. Diverse nutraceuticals (bioactive compounds found in common foods) have been shown to have neuroprotective effects in a variety of in vitro and in vivo disease models. In the current study, we compared the neuroprotective effects of two polyphenolic compounds, rosmarinic acid and carnosic acid, which are both found at substantial concentrations in the herb rosemary. The capacity of these compounds to rescue primary cultures of rat cerebellar granule neurons (CGNs) from a variety of stressors was investigated. Both polyphenols significantly reduced CGN death induced by the nitric oxide donor, sodium nitroprusside (nitrosative stress). Rosmarinic acid uniquely protected CGNs from glutamate-induced excitotoxicity, while only carnosic acid rescued CGNs from caspase-dependent apoptosis induced by removal of depolarizing extracellular potassium (5K apoptotic condition). Finally, we found that carnosic acid protects CGNs from 5K-induced apoptosis by activating a phosphatidylinositol 3-kinase (PI3K) pro-survival pathway. The shared and unique neuroprotective effects of these two compounds against diverse modes of neuronal cell death suggest that future preclinical studies should explore the potential complementary effects of these rosemary polyphenols on neurodegenerative disease progression.

Abstract: Fibrosis reflects a progression to liver cancer or cirrhosis of the liver. Recent studies have shown that high-mobility group box-1 (HMGB1) plays a major role in hepatic injury and fibrosis. Carnosic acid (CA), a compound extracted from rosemary, has been reported to alleviate alcoholic and non-alcoholic fatty liver injury. CA can also alleviate renal fibrosis. We hypothesized that CA might exert anti-liver fibrosis properties through an HMGB1-related pathway, and the results of the present study showed that CA treatment significantly protected against hepatic fibrosis in a bile duct ligation (BDL) rat model. CA reduced the liver expression of α-smooth muscle actin (α-SMA) and collagen 1 (Col-1). Importantly, we found that CA ameliorated the increase in HMGB1 and Toll-like receptor 4 (TLR4) caused by BDL, and inhibited NF-κB p65 nuclear translocation in fibrotic livers. In vitro, CA inhibited LX2 cell activation by inhibiting HMGB1/TLR4 signaling pathway. Furthermore, miR-29b-3p decreased HMGB1 expression, and a dual-luciferase assay validated these results. Moreover, CA down-regulated HMGB1 and inhibited LX2 cell activation, and these effects were significantly counteracted by antago-miR-29b-3p, indicating that the CA-mediated inhibition of HMGB1 expression might be miR-29b-3p dependent. Collectively, the results demonstrate that a miR-29b-3p/HMGB1/TLR4/NF-κB signaling pathway, which can be modulated by CA, is important in liver fibrosis, and indicate that CA might be a prospective therapeutic drug for liver fibrosis.

Abstract: Rosemary (Rosmarinus officinalis, L.) is an aromatic evergreen herb widely used around the globe as a spice/seasoning, flavoring agent and for medicinal applications. Rosemary extract derived from rosemary leaves contains several polyphenolic components which exhibit antioxidant properties, but the predominant active components are the phenolic diterpenes, namely, carnosic acid and carnosol. Over 90% antioxidant activity of rosemary extract is attributed primarily to high content of lipophilic antioxidant molecules such as carnosic acid and carnosol. In addition to antioxidant components, rosemary extract also contains some volatile compounds. Rosemary extract bioactive compounds are also responsible for their distinctive aroma, color and flavor attributes. Rosemary extract can be applied in a wide range of oils and fats, and lipid-containing foods to delay lipid oxidation and to enhance the shelf-life of various food, feed and pet food products. This review delineates the chemistry, antioxidant mechanism, volatile flavor compounds, regulatory position, food applications, and stability of rosemary extract in foods.

Abstract: The phenolic diterpene carnosic acid (CA, C20H28O4) exerts antioxidant, anti-inflammatory, anti-apoptotic, and anti-cancer effects in mammalian cells. CA activates the nuclear factor erythroid 2-related factor 2 (Nrf2), among other signaling pathways, and restores cell viability in several in vitro and in vivo experimental models. We have previously reported that CA affords mitochondrial protection against various chemical challenges. However, it was not clear yet whether CA would prevent chemically induced impairment of the tricarboxylic acid cycle (TCA) function in mammalian cells. In the present work, we found that a pretreatment of human neuroblastoma SH-SY5Y cells with CA at 1 μM for 12 h prevented the hydrogen peroxide (H2O2)-induced impairment of the TCA enzymes (aconitase, α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH)) and abolished the inhibition of the complexes I and V and restored the levels of ATP by a mechanism associated with Nrf2. CA also exhibited antioxidant abilities by enhancing the levels of reduced glutathione (GSH) and decreasing the content oxidative stress markers (cellular 8-oxo-2'-deoxyguanosine (8-oxo-dG), and mitochondrial malondialdehyde (MDA), protein carbonyl, and 3-nitrotyrosine). Silencing of Nrf2 by small interfering RNA (siRNA) abrogated the protective effects elicited by CA in mitochondria of SH-SY5Y cells. Therefore, CA prevented the H2O2-triggered mitochondrial impairment by an Nrf2-dependent mechanism. The specific role of Nrf2 in ameliorating the function of TCA enzymes function needs further research.

Abstract: Weeds are rapidly developing resistance to synthetic herbicides, and this can pose a threat to the ecosystem. Exploring allelopathic species as an alternative weed control measure can help minimize the ecological threat posed by herbicide-resistant weeds. In this study, we aimed to evaluate the contribution of some polyphenols to the allelopathy of rosemary (Rosmarinus officinalis L.). The phytotoxic effects of rosemary (leaves, roots, inflorescences, and stems) crude extracts were tested on lettuce (Lactuca sativa L.). Soils incorporated with dried rosemary leaves were also tested on test plants. Reversed-phase high-performance liquid chromatography (HPLC) analysis was used to determine the content of some polyphenols (caffeic, ferulic, gallic, rosmarinic, carnosic, and chlorogenic acids) in rosemary. The specific activity and total activity of crude extracts and individual compounds were evaluated using lettuce. The crude extract of rosemary leaves showed the highest growth inhibitory effect among the rosemary organs tested. Soil amended with rosemary leaf debris reduced the dry matter and seed emergence of lettuce. Carnosic acid was the main compound detected in rosemary leaves and had a high specific activity when tested on lettuce. During the seed germination period, there was observed filter paper coloration among the test plants treated with carnosic acid (250 μg/mL). The high concentration and strong inhibitory effect of carnosic acid could explain the inhibitory activity of the rosemary leaf extract. Hence, we conclude based on the total activity estimation that carnosic acid among the other tested compounds is the major allelochemical in rosemary leaves.

Abstract: Inflammation has an important role in the pathogenesis of many diseases. The aims of the present research were to assess the effectiveness of a combination of Astaxanthin, Lycopene rich Tomato extract (Lyc-O-Mato®) and Carnosic acid, at low concentrations, to prevent the release of the inflammatory mediators from LPS stimulated macrophages in vitro an in vivo using a mouse model of peritonitis. Addition of low concentrations of Astaxanthin Lyc-O-Mato® and Carnosic acid to peritoneal mouse macrophages 1 h before addition of LPS for 24 h caused a synergistic inhibition of NO, PGE2 and TNFα secretion. The supplementation of a nutrient combination in drinking water resulted in attenuation of basal, but not stimulated, superoxide production by recruited neutrophils to the peritoneal cavity and in inhibition of inflammatory mediators production by peritoneal macrophages. Our results indicate a potential use of these combinations in many inflammatory states.

Abstract: Purpose Carnosic acid (CA) is an important polyphenol mainly isolated from the famous spice and the medicinal plant Rosmarinus officinalis. CA has been shown to exhibit tremendous pharmacological properties which include, but are not limited to, anticancer, antioxidant and anti-inflammatory activities. The current study was designed to evaluate the anticancer effects of CA against chronic myeloid leukemia (CML) which is one of the rare but deadly malignancies both in men and women. Methods CML KBM-7 cell line was used in this study. Cell viability was assessed by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining, cell cycle analysis by flow cytometry and cell invasion by Boyden chamber assay. The microRNA-780 expression was determined by quantitative RT-PCR. Results Our results indicated that CA exhibits significant anticancer activity on CML KBM-7 cells with an IC50 of 25 μM. The anticancer activity was due to induction of apoptosis and cell cycle arrest. Moreover, it was observed that CA inhibits the proliferation and invasion of CML KBM-7 cells which could mainly be due to downregulation of microRNA- 780 expression as indicated by the quantitative RT-PCR analysis. Conclusion Taken together, we propose that carnosic acid could prove a potential lead compound in the treatment of CML and deserves further in vitro as well as in vivo study.

Abstract: Naturally occurring terpenes were combined by click reactions to generate sixteen hybrid molecules. The diterpene imbricatolic acid (IA) containing an azide group was used as starting compound for the synthesis of all the derivatives. The alkyne group in the terpenes cyperenoic acid, dehydroabietinol, carnosic acid γ-lactone, ferruginol, oleanolic acid and aleuritolic acid was obtained by esterification using appropriate alcohols or acids. The hybrid compounds were prepared by combining the IA azide function with the different terpene-alkynes under click chemistry conditions. The cytotoxic activity of the terpene hybrids 1–16 was assessed against Vero cells and tumour cell lines (HEP-2, C6 and Raw 264.7). Compounds 1, 2, 3 and 7 showed cytotoxic activity against the tested cell lines. The antiviral activity of the compounds was evaluated against HSV-1 KOS, Field and B2006 strain. For the pairs of hybrid compounds formed between IA-diterpene (compounds 3–8, except for compound 7), a moderate activity was observed against the three HSV-1 strains with an interesting selectivity index (SI ≥10, SI = CC50/CE50) for some compounds.

Abstract: PROBLEM TO BE SOLVED: To provide a method for exploiting the effects of an NRF2 (NF-E2-related factor 2) activator to treat and prevent neurodegeneration and inflammatory disease, as much as possible, while reducing the side effects as much as possible.SOLUTION: An NRF2 activator such as zonarol, carnosic acid, dimethyl fumaric acid, quercetin, catechin, and chlorogenic acids, is combined with an organic acid such as α-ketoglutaric acid, pyruvic acid, oxaloacetic acid.EFFECT: An amount of use of the NRF2 activator is restricted and the side effects are reduced as much as possible.SELECTED DRAWING: None

Abstract: Carnosic acid was used as starting material to synthesize royleanone derivatives featured C11-C14 para quinone. The importance of C-20 group of royleanone derivatives was verified by the cytotoxicity assay of royleanonic acid, miltionone I and deoxyneocrptotanshinone. Following our synthetic route, 15 amide derivatives were synthesized and 8 compounds exhibited moderate cytotoxic activities against three human cancer lines in vitro.

Abstract: The invention relates to a method for extracting and separating rosemary essential oil, rosemary acid, ursolic acid and carnosic acid from rosemary leaves. The method mainly comprises the following steps that rosemary dry leaves are crushed and are then put into a water solution; temperature rise distillation is performed for 0.5h; distillates are collected to obtain the rosemary essential oil; after the distillation is completed, eight times of volume of 75-percent ethanol solution is added; extraction is performed for 2h at 60 DEG C; filtering is performed to obtain filtering liquid A; six times of volume of 75-percent ethanol solution is added into filtering slag; extraction is performed for 1h at 60 DEG C; filtering is performed to obtain filtering liquid B; the filtering liquid A andthe filtering liquid B are merged; concentration and centrifugation are performed to obtain a solid part and a water phase part; the water phase part is subjected to concentration spray treatment to obtain the rosemary acid; 60-percent ethanol solution is added into the solid part; after the dissolution, centrifugation is continuously performed and is repeated for three times to obtain the ursolicacid; the liquid part is subjected to concentration drying treatment to obtain the carnosic acid. The extracting method has the advantages that the process is simple; green and environment-friendly effects are achieved; the operation is convenient; the cost is low; the yield is high; the method is suitable for industrial production.

Abstract: The following study demonstrated the influence of 1,25D/CA and 1,25D/CUR combinations on Nrf2 expression in primary leukemic cells derived from newly diagnosed CML and AML patients of hematological department of Karaganda Regional Clinical Hospital (Karaganda, Kazakhstan). Methods. The research was conducted by using quantitative real-time PCR. Mononuclear cells were isolated by centrifugation in a Ficoll-PaqueTMPREMIUM density gradient. Cell cultures were incubated in RPMI 1640 medium with test agents. RNA extracted from mononuclear cells was used for cDNA molecules synthesis by reverse transcription. Quantitative real-time PCR was carried out in a DT-322 PCR machine. The relative expression level was quantified by using the 2-DDCt method. The statistical analysis was carried out by GraphPadPrism 6.0 program. Results. It was found 1.25D, CUR, CA alone and their combinations do not lead to an increase in the level of expression of the Nrf2 gene in peripheral blood mononuclear cells of AML and CML patients. The expression of NQO1 gene did not change much in the all cell groups of CML patients, but is pronounced in cells of AML patients grown in a medium supplemented with combinations of 1.25D/CUR, 1.25D/CA and CUR, CA alone. 1.25D/CUR and 1.25D/CA combinations do not enhance the expression level of the Nrf2 gene, but increase the amount of its protein, which in turn augments the expression level of the NQO1 gene.

Abstract: A method for improving the carnosic acid content in a rosemary fat-soluble antioxidant belongs to the technical field of plant extraction. The method uses rosemary leaves as a raw material, and comprises the following steps of: preparing a rosemary extract, preparing a carnosic acid crude extract, preparing a carnosic acid refined solution, preparing a carnosic acid eluent, preparing a rosemary extract refined product and preparing a regenerated D370R or D315 anion exchange resin column, thereby preparing a rosemary extract refined product with the carnosic acid content exceeding 90%. In the method provided by the invention, a ceramic membrane is used for separating macromolecular impurities in the rosemary extract, and D370R or D315 weakly basic anion exchange resin is used for separatingand purifying carnosic acid in the rosemary extract, so that the separation specificity is strong, the separation efficiency is high, and the yield is high; the rosemary waste leaves and other byproducts generated in an implementation process are subjected to deep processing treatment, thus realizing comprehensive utilization and environmental protection; and the operation condition is mild, theproduction cost is low, and the prepared product can be widely applied to the fields of food, health products, medicines and the like.