Carnosol Research Articles

Comparative Study of Green Sub- and Supercritical Processes to Obtain Carnosic Acid and Carnosol-Enriched Rosemary Extracts with in Vitro Anti-Proliferative Activity on Colon Cancer Cells

In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116). The processes, carried out under optimal conditions, were: (1) pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent) at lab-scale; (2) Single-step supercritical fluid extraction (SFE) at pilot scale; (3) Intensified two-step sequential SFE at pilot scale; (4) Integrated PLE plus supercritical antisolvent fractionation (SAF) at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight), this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA) and carnosol (CS) at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide), suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.

Determination of carnosic acid and carnosol in Rosmarinus officinalis L. by high-performance capillary electrophoresis

ABSTRACTCarnosol and carnosic acid in Rosmarinus officinalis L. were separated by high-performance capillary electrophoresis using a 50 cm capillary, a 12 mmol/L 80:20 pH 9.9 borax:methanol buffer using 25 kV of applied voltage at 30°C with detection at 280 nm. Rosmarinus officinalis L. was extracted with methanol with ultrasound at 170 W and 50 kHz for 20 min. The precision, repeatability, and recovery of the method were evaluated. The calibration relationships for carnosol and carnosic acid were y = 2.331x + 5.92 from 0.01 to 0.160 mg/mL and y = 7.980x + 4.32 from 0.005 to 0.080 mg/mL, respectively. The established method allows the separation and quantification of carnosic acid and carnosol in R. officinalis from various locations in China.

Anti-inflammatory and analgesic activity of carnosol and carnosic acid in vivo and in vitro and in silico analysis of their target interactions.

The diterpenoids carnosol (CS) and carnosic acid (CA) from Salvia spp. exert prominent anti-inflammatory activities but their molecular mechanisms remained unclear. Here we investigated the effectiveness of CS and CA in inflammatory pain and the cellular interference with their putative molecular targets. The effects of CS and CA in different models of inflammatory pain were investigated. The inhibition of key enzymes in eicosanoid biosynthesis, namely microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) was confirmed by CS and CA, and we determined the consequence on the eicosanoid network in activated human primary monocytes and neutrophils. Molecular interactions and binding modes of CS and CA to target enzymes were analyzed by docking studies. CS and CA displayed significant and dose-dependent anti-inflammatory and anti-nociceptive effects in carrageenan-induced mouse hyperalgesia 4 h post injection of the stimuli, and also inhibited the analgesic response in the late phase of the formalin test. Moreover, both compounds potently inhibited cell-free mPGES-1 and 5-LO activity and preferentially suppressed the formation of mPGES-1 and 5-LO-derived products in cellular studies. Our in silico analysis for mPGES-1 and 5-LO supports that CS and CA are dual 5-LO/mPGES-1 inhibitors. In summary, we propose that the combined inhibition of mPGES-1 and 5-LO by CS and CA essentially contributes to the bioactivity of these diterpenoids. Our findings pave the way for a rational use of Salvia spp., traditionally used as anti-inflammatory remedy, in the continuous expanding context of nutraceuticals. This article is part of a themed section on Principles of Pharmacological Research of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.11/issuetoc.

Carnosol and Related Substances Modulate Chemokine and Cytokine Production in Macrophages and Chondrocytes.

Phenolic diterpenes present in Rosmarinus officinalis and Salvia officinalis have anti-inflammatory and chemoprotective effects. We investigated the in vitro effects of carnosol (CL), carnosic acid (CA), carnosic acid-12-methylether (CAME), 20-deoxocarnosol and abieta-8,11,13-triene-11,12,20-triol (ABTT) in murine macrophages (RAW264.7 cells) and human chondrocytes. The substances concentration-dependently reduced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-stimulated macrophages (i.e., acute inflammation). They significantly blunted gene expression levels of iNOS, cytokines/interleukins (IL-1α, IL-6) and chemokines including CCL5/RANTES, CXCL10/IP-10. The substances modulated the expression of catabolic and anabolic genes in chondrosarcoma cell line SW1353 and in primary human chondrocytes that were stimulated by IL-1β (i.e., chronic inflammation In SW1353, catabolic genes like MMP-13 and ADAMTS-4 that contribute to cartilage erosion were down-regulated, while expression of anabolic genes including Col2A1 and aggrecan were shifted towards pre-pathophysiological homeostasis. CL had the strongest overall effect on inflammatory mediators, as well as on macrophage and chondrocyte gene expression. Conversely, CAME mainly affected catabolic gene expression, whereas ABTT had a more selectively altered interleukin and chemokine gene exprssion. CL inhibited the IL-1β induced nuclear translocation of NF-κBp65, suggesting that it primarily regulated via the NF-κB signalling pathway. Collectively, CL had the strongest effects on inflammatory mediators and chondrocyte gene expression. The data show that the phenolic diterpenes altered activity pattern of genes that regulate acute and chronic inflammatory processes. Since the substances affected catabolic and anabolic gene expression in cartilage cells in vitro, they may beneficially act on the aetiology of osteoarthritis.

New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes’ transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells’ proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule.

Foodomics study on the effects of extracellular production of hydrogen peroxide by rosemary polyphenols on the anti-proliferative activity of rosemary polyphenols against HT-29 cells.

A number of studies have demonstrated a strong association between the antioxidant properties of rosemary polyphenols and their chemoprotective activity. However, the prooxidant effects of rosemary polyphenols have been rarely reported. In this work, a foodomics study is performed to investigate the in vitro autooxidation of carnosic acid (CA), carnosol (CS) and a polyphenol-enriched rosemary extract (SC-RE) in cell culture conditions. The results revealed that rosemary polyphenols autooxidation in culture medium generated H2 O2 at different rates. Generated H2 O2 levels by SC-RE and CA, but not CS, were correlated with intracellular reactive oxygen species (ROS) generation in HT-29 cells, and were partially involved in their anti-proliferative effect in this cell line. These compounds also induced different effects on glutathione metabolism. Results also indicated that high extracellular H2 O2 concentrations, resulting of using high (45 μg/mL) SC-RE concentration in culture media, exerted some artifactual effects related with cell cycle, but they did not influence the expression of relevant molecular biomarkers of stress.

Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence

Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

Pretreatment with carnosol in lung ischemia reperfusion-induced renal injury

Amac: Bu deneysel calismada, akciger iskemi/reperfuzyonu (I/R) ile induklenen bobrek hasari uzerine karnosolun koruyucu etkisi arastirildi. Ca­lis­ma­ pla­ni:­ Yirmi dort Yeni Zelanda beyaz tavsani uc gruba randomize edildi. Grup IR; akciger I/R grubu (60 dk. iskemi-60 dakika reperfuzyon), grup CIR; akciger I/R’si oncesi karnasol bolus enjeksiyonu ve grup S; plasebo grubu (pulmoner hilus klemplenmedi). Bobrek dokusu miyeloperoksidaz (MPO) ve interseluler adezyon molekulu -1 (ICAM-1) duzeyleri degerlendirildi. Bul gu lar: Bobrek dokusu MPO ve ICAM-1 duzeyleri grup IR’de, grup CIR ve grup S ile karsilastirildiginda, anlamli olarak daha yuksek idi (sirasiyla, p=0.021 ve p=0.0001). Grup CIR ve grup S arasinda ise olculen parametrelerde istatistiksel olarak anlamli fark tespit edilmedi. So­nuc:­ Calisma sonuclarimiz akciger I/R’sine bagli bobrek hasarinda aktif notrofil sekestrasyonu ile iliskili MPO ve ICAM-1 duzeylerinin arttigini ve karnosolun bu hasarda koruyucu etkisi olabilecegini gostermektedir. Anah tar soz cuk ler: Karnosol; iskemi reperfuzyon; akciger; bobrek hasari. Background:­This experimental study aims to investigate the protective effects of carnosol on kidney injury induced by lung ischemia/reperfusion (I/R). Methods: Twenty-four New Zealand white rabbits were randomized into three groups. Group IR; lung I/R group (60 min ischemia-60 min reperfusion), group CIR; bolus injection of carnosol before lung I/R, and group S; sham group (pulmonary hilum was not clamped). Renal tissue myeloperoxidase (MPO) and intercellular adhesion molecule-1 (ICAM-1) levels were evaluated. Results:­ Renal tissue MPO and ICAM-1 levels were significantly higher in the group IR compared to the group CIR and group S (p=0.021 and p=0.0001 respectively). No statistically significant difference in the parameters evaluated was detected between the group CIR and the group S. Conclusion:­Our study result suggests that lung I/R injury causes increased renal tissue MPO and ICAM-1 levels, which are related to activated neutrophil sequestration and carnosol may play a protective role against this kidney injury.

Comprehensive foodomics study on the mechanisms operating at various molecular levels in cancer cells in response to individual rosemary polyphenols.

In this work, the contribution of carnosic acid (CA) and carnosol (CS), two major compounds present in rosemary, against colon cancer HT-29 cells proliferation is investigated using a comprehensive Foodomics approach. The Foodomics study reveals that CA induces transcriptional activation of genes that encode detoxifying enzymes and altered the expression of genes linked to transport and biosynthesis of terpenoids in the colon cancer cell line. Functional analysis highlighted the activation of the ROS metabolism and alteration of several genes involved in pathways describing oxidative degradation of relevant endogenous metabolites, providing new evidence about the transcriptional change induced by CA in HT-29 cells. Metabolomics analysis showed that the treatment with CA affected the intracellular levels of glutathione. Elevated levels of GSH provided additional evidence to transcriptomic results regarding chemopreventive response of cells to CA treatment. Moreover, the Foodomics approach was useful to establish the links between decreased levels of N-acetylputrescine and its degradation pathway at the gene level. The findings from this work and the predictions based on microarray data will help explore novel metabolic processes and potential signaling pathways to further elucidate the effect of CA in colon cancer cells.

Carnosol, radiation and melanoma: a translational possibility

To compare the genoprotective and radioprotective effect of carnosol (COL) against damage induced by ionizing radiation with similar effects produced by different antioxidant compounds. The genoprotective effect was studied by means of the micronucleus test for antimutagenic activity in which the reduction in the frequency of micronuclei was evaluated in cytokinesis-blocked cells of human lymphocytes. The radioprotective effects were studied by cell viability test (MTT) in PNT2 (normal prostate) and B16F10 (melanoma) cell lines when they were administered before exposure to different X-ray doses (4, 6, 8, 10 and 0Gy). Carnosol shows a significant genoprotective capacity (p<0.001) against radiation with a protection factor of 50%, and a dose-reduction factor of 4.3. Cell survival obtained with COL administered before exposure to 10Gy of X-rays showed a protection factor of 55.1%, eliminating 39% of radiation-induced cell death in normal epithelial cells of prostate (PNT2) (p<0.001). However, in the melanoma cell lines (B16F10) assayed, COL acted not as a radioprotector, but as a sensitizing agent increasing the cellular death by 34% (p<0.01) and producing an enhancement ratio of 2.12. Carnosol may be developed as a radioprotective agent in the non-tumoral cells. However, in the B10F16 melanoma cells, melanogenesis is activated by COL leading to redistribution of the enzymatic balances of glutathione and cysteine-lyase production, which could compromise the intracellular redox defence system. This effect appears as an increase in the capacity of ionizing radiation-induced damage, and thus exhibits a paradoxical protective effect of COL on melanoma cells.

Effect ofRosemarinus officinalisL. on MMP-9, MCP-1 Levels, and Cell Migration in RAW 264.7 and Smooth Muscle Cells

Atherosclerosis is a chronic and progressive inflammatory disease. Novel anti-inflammatory therapies may have promise as treatment strategies for cardiovascular risk reduction. Rosemary (Rosemarinus officinalis L.) has been used in folk medicine to treat headaches, epilepsy, poor circulation, and many other ailments. It was found that rosemary could act as a stimulant and mild analgesic and could reduce inflammation. However, the mechanisms underlying the anti-inflammatory and antiatherosclerotic effects of rosemary need more study. This study investigated effects of the rosemary components, carnosic acid (CA), and carnosol (CAR), on cell migration. Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) were determined by Western blot and gelatin zymography, respectively, in RAW 264.7 macrophages and vascular smooth muscle cells (VSMCs). VSMC migration was assessed by a Matrigel migration assay. Active compounds of rosemary extracts were also analyzed using a reversed-phase high-performance liquid chromatography. MMP-9 and MCP-1 activities were markedly diminished with methanol extract (RM), n-hexane fraction (RH), and CA in RAW 264.7 cells. RM, RH, CA, and CAR suppressed tumor necrosis factor-alpha-induced VSMC migration by inhibiting MMP-9 expression. Chromatograms of RM- and RH-containing CA and CAR revealed higher CA contents of RM (9.4%, 93.85 μg/mg dry wt.) and, especially, RH (18.4%, 184.00 μg/mg dry wt.), which were appreciably elevated compared with the similar CAR content in RM and RH (3.7%, 37.30 μg/mg dry wt.; and 2.5%, 25.05 μg/mg dry wt., respectively). Rosemary, especially its CA component, has potential antiatherosclerosis effects related to cell migration.

Antiproliferation effect of Rosemary (Rosmarinus officinalis) on human ovarian cancer cells in vitro

Rosemary (Rosmarinus officinalis L.) is a popular culinary/medicinal herb. Recent studies have shown it has pharmacologic activities for cancer chemoprevention and therapy. This study evaluated the antiproliferation activity of rosemary extract (RE) against human ovarian cancer cells, and whether the extract and its three main active ingredients carnosol (CS), carnosic acid (CA) and rosmarinic acid (RA) can enhance the antiproliferation activity of cisplatin (CDDP). Our study showed that RE has significant antiproliferation activity on human ovarian cancer A2780 and its CDDP resistant daughter cell line A2780CP70, with IC50 (50% inhibitory concentration) estimated at 1/1000 and 1/400 dilutions respectively. RE enhanced the antiproliferation effect with CDDP on both A2780 and A2780CP70 cells. A2780 cells were consistently more sensitive to CS, CA, and RA than A2780CP70 cells between 2.5 and 20μg/ml. CS and RA also showed synergistic antiproliferation effect with CDDP on A2780 cells at some concentrations. RE treated by ultrafiltration, dialysis, and removal of phenolics lost the antiproliferation activity suggested that the activity resides in phenolics with MW<1000Da. Apoptosis array study of A2780 cells treated with RE showed that the expression of a number of genes regulating apoptosis were modulated by the treatment. This study showed that RE inhibited the proliferation of ovarian cancer cell lines by affecting the cell cycle at multiple phases. It induced apoptosis by modifying the expression of multiple genes regulating apoptosis, and holds potential as an adjunct to cancer chemotherapy.

Carnosic Acid Induces the NAD(P)H: Quinone Oxidoreductase 1 Expression in Rat Clone 9 Cells through the p38/Nuclear Factor Erythroid-2 Related Factor 2 Pathway

The anticarcinogenic effect of rosemary has been partly attributed to the modulation of the activity and expression of phase II detoxification enzymes. Here we compared the effects of phenolic diterpenes from rosemary on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Cells were treated with 1–20 μmol/L of carnosic acid (CA) or carnosol (CS) for 24 h. Both CA and CS dose dependently increased NQO1 enzyme activity and protein expression, and the induction potency of CA was stronger than that of CS. The increase in NQO1 enzyme activity in cells treated with 10 μmol/L CA and CS was 4.1- and 1.9-fold, respectively (P < 0.05). RT-PCR showed that CA and CS induced NQO1 mRNA in a dose-dependent manner. Furthermore, CA dose dependently induced transcription of nuclear factor erythroid-2 related factor 2 (Nrf2) and antioxidant response element (ARE)-luciferase reporter activity. Silencing of Nrf2 expression alleviated NQO1 protein expression and ARE-luciferase activity by CA. Moreover, the phosphorylation of p38 was mainly stimulated in the presence of CA. Pretreatment with SB203580 or silencing of p38 expression inhibited Nrf2 activation and NQO1 induction. These results suggest that the increased NQO1 expression by CA is likely related to the p38-Nrf2 pathway and help to clarify the possible molecular mechanism of action of rosemary phenolic compounds in drug metabolism and cancer prevention.

Effect of Some Biotic and Abiotic Elicitors on Phenolic Acids and Diterpenes Production from Rosemary (Rosmarinus officinalis L.) Leaf and Callus Analyzed by High Performance Liquid Chromatography (Hplc)

The yield of phenolic acids; rosmarinic acid (RA), caffeic acid (CAF) and phenolic diterpenes; carnosic acid (CA), carnosol (CAR), rosmanol (ROL) from rosemary (Rosmarinus officinalis L.) was investigated. Aqueous extraction was conducted for leaf and callus tissues, the chemical analysis for extracts was carried out using high performance liquid chromatography (HPLC). Data showed that RA and CAF production was 4.5, 2.7 µg/ml respectively. CA and CAR production reached 3.3, 2.8 µg/ml when the fungal elicitor; Fusarium oxysporum was added at the concentration 2.0 mg/l (2×10 4 CFU/ml), except ROL which was found at high levels (4.3, 4.2 and 4.6 µg/ml) only from leaf extracts, untreated callus and when callus treated with 0.4 mg/l of CaCl2 respectively.

Variation Regulation of Bioactive Components in Rosemary Leaves and Extraction Procedure

The variation regulation of five bioactive components including carnosic acid (CA), carnosol (CO), rosmarinic acid (RA), oleanolic acid (OA) and ursolic acid (UA) in rosemary leaves and extraction procedure was studied. The effects of rosemary sources, steam distillation time, activated carbon dosage and storage time of extraction solution on variation regulation of bioactive components were discussed in detail.

Suppression of COX-2, IL-1β and TNF-α expression and leukocyte infiltration in inflamed skin by bioactive compounds from Rosmarinus officinalis L.

In the present study, we evaluated the effects of extracts and purified compounds from fresh leaves of Rosmarinus officinalis L. Pretreatment with the major anti-inflammatory compounds, carnosic acid (CA) and carnosol (CS), inhibited phorbol 12-myristate 13-acetate (PMA)-induced ear inflammation in mice with an EC 50 of 10.20 μg/cm 2 and 10.70 μg/cm 2, respectively. To further understand the anti-inflammatory mechanism of these compounds, we analyzed the in vivo expression of several inflammation-associated genes in mouse skin by reverse transcriptase-polymerase chain reaction (RT-PCR). Our data showed that CA and CS reduced the expression of IL-1β and TNF-α but had less effect on fibronectin and ICAM-1 expression. Interestingly, both compounds selectively inhibited COX-2 but not COX-1. Histopathological analysis of hematoxylin and eosin (H&E)-stained tissue revealed a marked reduction in leukocyte infiltration and epidermal ulceration of PMA-treated ears when ears were pretreated with ethanolic extracts or pure CA. In vitro, we showed that ethanolic extract, carnosic acid and carnosol significantly inhibited the overproduction of nitric oxide (NO) in a dose-dependent manner in the RAW 264.7 murine macrophage cell line. For the first time in vivo, we showed that CA and CS differentially regulate the expression of inflammation-associated genes, thus demonstrating the pharmacological basis for the anti-inflammatory properties reported for CA and CS.

Activated Glutathione Metabolism Participates in Protective Effects of Carnosic Acid against Oxidative Stress in Neuronal HT22 Cells

In our previous studies, we have reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protects cortical neurons by inducing phase 2 enzymes, the induction of which was initiated by activation of the Keap1/Nrf2 pathway , , . In the present study we address the nature of the effector of these neuroprotective effects downstream of the phase 2 enzyme induction. From our results we conclude that activated glutathione (GSH) metabolism may participate in these protective effects. First, we performed cDNA microarray analysis in order to identify the gene(s) responsible for the actions and found that various enzymes involved in the metabolism of GSH (glutathione S-transferase, alpha 4; glutathione S-transferase, alpha 2; and formylglutathione hydrolase) constituted 3 of the top 5 CA-induced genes. The other 2 genes encoded phase 2 enzymes [NAD(P)H-quinone oxidoreductase1and aldehyde dehydrogenase family 3, subfamily A1]. Next, we compared the physiologically-active compounds originating from rosemary (CA, CS, luteolin, genkwanin, rosmarinic acid, caffeic acid, and verbenone) by 3 criteria (enhancement of total glutathione levels, transcriptional activation, neuroprotective effects). By all of these criteria, CA and CS were the most active. In contrast, the other compounds were only weakly active or totally inactive. These results suggest that pro-electrophilic compounds such as CA and CS may protect cortical neurons by causing the following sequential events: S-alkylation --> activation of the Keap1/Nrf2 pathway --> transcriptional activation --> induction of phase 2 enzymes --> activation of GSH metabolism --> neuroprotection.

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.

Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes

Carnosic acid (CA) and carnosol (CS) are phenolic diterpenes present in several labiate herbs like Rosmarinus officinalis (Rosemary) and Salvia officinalis (Sage). Extracts of these plants exhibit anti-inflammatory properties, but the underlying mechanisms are largely undefined. Recently, we found that CA and CS activate the peroxisome proliferator-activated receptor gamma, implying an anti-inflammatory potential on the level of gene regulation. Here we address short-term effects of CA and CS on typical functions of human polymorphonuclear leukocytes (PMNL). We found that (I), CA and CS inhibit the formation of pro-inflammatory leukotrienes in intact PMNL (IC 50 = 15–20 μM [CA] and 7 μM [CS], respectively) as well as purified recombinant 5-lipoxygenase (EC number 1.13.11.34, IC 50 = 1 μM [CA] and 0.1 μM [CS], respectively), (II) both CA and CS potently antagonise intracellular Ca 2+ mobilisation induced by a chemotactic stimulus, and (III) CA and CS attenuate formation of reactive oxygen species and the secretion of human leukocyte elastase (EC number 3.4.21.37). Together, our findings provide a pharmacological basis for the anti-inflammatory properties reported for CS- and CA-containing extracts.

Relevance of phenolic diterpene constituents to antioxidant activity of supercritical CO2 extract from the leaves of rosemary

Isolation of phenolic diterpene constituents from the freeze-dried leaves of Rosmarinus officinalis has been obtained by supercritical extraction with carbon dioxide. To determine the ideal conditions for the maximum yield of extract, nine different conditions using three levels of pressures (3000, 4000 and 5000 psi) in combination with three temperatures at 40, 60 and 80°C, respectively, in combination with the analyses of the corresponding antioxidant activities and constituents which existed in extracts has been investigated. The antioxidant activity of each obtained extract was determined by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals test. GC/MS method was used as an alternative to conventional HPLC method for the determination of the principal antioxidant constituents in extract, including phenolic diterpenes carnosic acid (CA) and carnosol (CAL). The confirmation of CA and CAL in extract was forward performed by subjecting HPLC isolates from extract into an ion trap mass spectrometer through an electrospray ionization (ESI) interface for MS/MS analysis. These results indicate that an ideal extraction process was obtained at 5000 psi and 80°C with an extraction yield of 4.27% (w/w) and rich in phenolic antioxidants CA and CAL as contents of 35.23 and 0.46 mg g−1 in extract, respectively.