MAO-A/B; COMT

In vitro, rosmarinic acid (RA) exhibits a variety of multifunctional characteristics, including antioxidant actions, inhibition of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO-A and MAO-B). In addition to inhibiting lipid peroxidation (IC50 of 19.6 μM), rosmarinic acid exerts antioxidant effects on hydroxyl (HO(•)) and nitric oxide (NO) free radicals (IC50 of 29.4 and 140 μM, respectively) [1]. Rosmarinic acid (RA) scavenges intracellular ROS produced by UVB rays, hence exhibiting notable cytoprotective properties. In H2O2-treated cells, N-acetyl-L-cysteine (NAC) has a 77% intracellular ROS scavenging activity, whereas 2.5 μM rosmarinic acid may scavenge 60% of intracellular ROS [2].

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Natural products are important sources of chemical diversity leading to unique scaffolds that can be exploited in the discovery of new drug candidates or chemical probes. In this context, chemical and biological investigation of ferns and lycophytes occurring in Brazil is an approach adopted by our research group aiming at discovering bioactive molecules acting on neurodegeneration targets. In the present study, Rosmarinic acid (RA) isolated from Blechnum brasiliense showed an in vitro multifunctional profile characterized by antioxidant effects, and monoamine oxidases (MAO-A and MAO-B) and catechol-O-methyl transferase (COMT) inhibition. RA showed antioxidant effects against hydroxyl (HO(•)) and nitric oxide (NO) radicals (IC50 of 29.4 and 140 μM, respectively), and inhibition of lipid peroxidation (IC50 of 19.6 μM). In addition, RA inhibited MAO-A, MAO-B and COMT enzymes with IC50 values of 50.1, 184.6 and 26.7 μM, respectively. The MAO-A modulation showed a non-time-dependent profile, suggesting a reversible mechanism of inhibition. Structural insights on RA interactions with MAO-A and COMT were investigated by molecular docking. Finally, RA (up to 5 mM) demonstrated no cytotoxicity on polymorphonuclear rat cells. Taken together, our results suggest that RA may be exploited as a template for the development of new antioxidant molecules possessing additional MAO and COMT inhibition effects to be further investigated on in vitro and in vivo models of neurodegenerative diseases. [1]

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This study was designed to investigate the cytoprotective effect of Rosmarinic acid (RA) on ultraviolet B (UVB)-induced oxidative stress in HaCaT keratinocytes. RA exerted a significant cytoprotective effect by scavenging intracellular ROS induced by UVB. RA also attenuated UVB-induced oxidative macromolecular damage, including protein carbonyl content, DNA strand breaks, and the level of 8-isoprostane. Furthermore, RA increased the expression and activity of superoxide dismutase, catalase, heme oxygenase-1, and their transcription factor Nrf2, which are decreased by UVB radiation. Collectively, these data indicate that RA can provide substantial cytoprotection against the adverse effects of UVB radiation by modulating cellular antioxidant systems, and has potential to be developed as a medical agent for ROS-induced skin diseases. [2] A common phenolic ester molecule found in many plants, particularly in Lamiaceae herbs including prunella vulgaris, salvia, and rosemary, is rosmarinic acid (RA). Through dual suppression of NF-κB and STAT3 activation, rosmarinic acid prevents colon inflammation in mice generated by dextran sulfate sodium (DSS). Treatment with rosmarinic acid (30, 60 mg/kg, oral) significantly reduced the generation of cytokines in a model of colitis produced by DSS [3].

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Ulcerative colitis (UC), a type of inflammatory bowel disease (IBD), is a chronic inflammatory disorder of the colon. Although UC is generally treated with anti-inflammatory drugs or immunosuppressants, most of these treatments often prove to be inadequate. Rosmarinic acid (RA) is a phenolic ester included in various medicinal herbs such as Salvia miltiorrhiz and Perilla frutescens. Although RA has many biological and pharmacological activities, the anti-inflammatory effect of RA in colonic tissue remains unclear. In this study, we investigated the anti-inflammatory effects and underlying molecular mechanism of RA in mice with dextran sulphate sodium (DSS)-induced colitis. In the DSS-induced colitis model, RA significantly reduced the severity of colitis, as assessed by disease activity index (DAI) scores, colonic damage, and colon length. In addition, RA resulted in the reduction of the inflammatory-related cytokines, such as IL-6, IL-1β, and IL-22, and protein levels of COX-2 and iNOS in mice with DSS-induced colitis. Furthermore, RA effectively and pleiotropically inhibited nuclear factor-kappa B and signal transducer and activator of transcription 3 activation, and subsequently reduced the activity of pro-survival genes that depend on these transcription factors. These results demonstrate that RA has an ameliorative effect on colonic inflammation and thus a potential therapeutic role in colitis [3].

A common phenolic ester molecule found in many plants, particularly in Lamiaceae herbs including prunella vulgaris, salvia, and rosemary, is rosmarinic acid (RA). Through dual suppression of NF-κB and STAT3 activation, rosmarinic acid prevents colon inflammation in mice generated by dextran sulfate sodium (DSS). Treatment with rosmarinic acid (30, 60 mg/kg, oral) significantly reduced the generation of cytokines in a model of colitis produced by DSS [3].

In the present study, rosmarinic acid (RA) isolated from Blechnum brasiliense showed an in vitro multifunctional profile characterized by antioxidant effects, and monoamine oxidases (MAO-A and MAO-B) and catechol-O-methyl transferase (COMT) inhibition. RA showed antioxidant effects against hydroxyl (HO(•)) and nitric oxide (NO) radicals (IC50 of 29.4 and 140 μM, respectively), and inhibition of lipid peroxidation (IC50 of 19.6 μM). In addition, RA inhibited MAO-A, MAO-B and COMT enzymes with IC50 values of 50.1, 184.6 and 26.7 μM, respectively. The MAO-A modulation showed a non-time-dependent profile, suggesting a reversible mechanism of inhibition. Structural insights on RA interactions with MAO-A and COMT were investigated by molecular docking. Finally, RA (up to 5 mM) demonstrated no cytotoxicity on polymorphonuclear rat cells[1].

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Decrease of hydroxyl radical generation [1]
Isolated RA/Rosmarinic acid was diluted in DMSO (final concentration 1%) and phosphate buffer (20 mM, pH 7.2), to obtain final concentrations ranging from 1.22 μM to 5 mM, which were added to the reaction system in 96-well plates, containing 2-deoxyribose, ferrous sulfate (FeSO4) and H2O2, following the method described by Lopes et al. Absorbance readings were performed at 532 nm, measuring the formation of malondialdehyde (product generated by degradation of 2-deoxyribose). Chlorogenic (CGA) and caffeic acids (CA) were used as positive controls. Experiments were performed in triplicate and results were expressed as IC50.

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Antioxidant capacity against nitric oxide radical [1]
To evaluate the scavenger potential of Rosmarinic acid/RA against nitric oxide (NOradical dot) radical, samples (1.22 μM–5 mM) were added to a sodium nitroprusside solution (10 mM) in 96-well plates. Incubations were performed for 60 min, at room temperature. The Griess reagent (1% sulphanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride) was added, followed by a 7 min incubation in the dark. Nitrite levels were measured at 546 nm, and the IC50 values were determined for triplicate experiments. The positive controls were CGA and CA.

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AChE and BChE inhibition [1]
The inhibitory properties of Rosmarinic acid/RA on cholinesterases were assessed using the Ellman’s method, modified by Di Giovanni et al. RA was tested in concentrations between 0.5 and 500 μM. Two independent experiments were performed in triplicate.

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MAO-A and MAO-B inhibition [1]
Monoamine oxidase inhibition assays were carried out with a fluorescence based method (end-point reading), using kynuramine as non-selective substrate for MAO-A and MAO-B. Briefly, reactions were performed in black, flat bottom 96-well plates containing potassium phosphate buffer (158 μL, 20 mM, pH 7.4), inhibitor solutions in DMSO (2 μL), 0.5 mM kynuramine solution (20 μL), using a final volume of 200 μL per well. Isolated Rosmarinic acid/RA and other structurally related compounds were tested at concentrations ranging from 0.5 to 500 μM. Solutions of clorgyline and pargyline (from 0.0001 to 1000 μM) were used as positive controls for MAO-A and MAO-B inhibition, respectively. Mixtures were incubated at 37 °C, and then 20 μL of diluted human recombinant MAO-A and MAO-B were added (0.009 and 0.015 mg/mL, respectively). Incubation was carried out at 37 °C, and reactions were stopped with 6 M sodium hydroxide (NaOH) solution (25 μL). The formation of the fluorescent product, 4-hydroxyquinoline (4-OH), was quantified at excitation/emission wavelengths of 310/400 nm. Experiments were performed in quadruplicate.

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Time-dependent studies on MAO-A [1]
To estimate the reversibility of MAO-A inhibition, a time-dependent assay using Rosmarinic acid/RA was employed, according to described by Passos et al. RA was preincubated with human MAO-A (final protein concentration of 0.03 mg/mL) for different periods (0, 15, 30, 60 min), at 37 °C, in potassium phosphate buffer (0.1 M, pH 7.4). For this purpose, RA concentration was equal to twofold the IC50 value determined for MAO-A inhibition. Subsequently, the reaction medium was diluted twofold, by addition of kynuramine to yield a final enzyme concentration of 0.015 mg/mL, and RA concentration corresponding to its IC50. This assay was also performed in 96-well microplates, and the final kynuramine concentration corresponded to 50 μM. Reactions were incubated at 37 °C, for a further 30 min period, and terminated with 6 M NaOH (25 μL). The rates of 4-OH formed in MAO-A reaction were measured and compared with negative control (1% DMSO) to estimate the % of inhibition. All measurements were carried out in triplicate.

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Inhibitory effects on COMT [1]
Modulation of COMT was assessed according to the method proposed by Passos et al., evaluating the conversion of esculetin to scopoletin catalyzed by the enzyme. Substrate conversion, in presence of COMT inhibitors, was performed by fluorescence measurements at λexcitation = 355 nm and λemission = 460 nm. Esculetin (final concentration 6 μM) was incubated with COMT (2.25 U.I./mL) and Rosmarinic acid/RA (15–500 μM) for 5 min, at 37 °C, followed by addition of S-adenosyl-l-methionine (SAM) (100 μM). Plates were incubated at 37 °C, and readings were performed in 4 min intervals, during 60 min, using 3,5-dinitrocathecol (DNC) (35 nM) as positive control. IC50 values were determined for two independent experiments, performed in triplicate.

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Cytotoxicity by LDH release assay (membrane damage) [1]
Cytotoxic effects of isolated Rosmarinic acid/RA on polymorphonuclear cells (PMN) were determined by release of cytosolic LDH (EC1.1.1.27), as described by Andrade et al. PMN (1.5 × 107 cells/mL) were obtained from male Wistar rats blood (180–220 g), and incubated with 0.5 and 5 mM RA, for 30 min, at 37 °C. The enzyme activity in supernatants was measured at 492 nm, using commercial LDH kit. Assays were performed in triplicate, and 1% Triton X-100 was used as positive control.

This study was designed to investigate the cytoprotective effect of rosmarinic acid (RA) on ultraviolet B (UVB)-induced oxidative stress in HaCaT keratinocytes. RA exerted a significant cytoprotective effect by scavenging intracellular ROS induced by UVB. RA also attenuated UVB-induced oxidative macromolecular damage, including protein carbonyl content, DNA strand breaks, and the level of 8-isoprostane. Furthermore, RA increased the expression and activity of superoxide dismutase, catalase, heme oxygenase-1, and their transcription factor Nrf2, which are decreased by UVB radiation. Collectively, these data indicate that RA can provide substantial cytoprotection against the adverse effects of UVB radiation by modulating cellular antioxidant systems, and has potential to be developed as a medical agent for ROS-induced skin diseases[2].

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Cell viability [2]
Cells were treated with Rosmarinic acid/RA (0.625, 1.25, 2.5, or 5 μM) and exposed to UVB radiation 1 h later. They were then incubated at 37°C for 48 h. At this time, MTT was added to each well to obtain a total reaction volume of 200 μl. After 4 h incubation, the supernatant was removed by aspiration. The formazan crystals in each well were dissolved in dimethyl sulfoxide (DMSO; 150 μl), and the absorbance at 540 nm was measured on a scanning multi-well spectrophotometer (Carmichael et al., 1987).

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DPPH radical detection [2]
RA/Rosmarinic acid (0.625, 1.25, 2.5, 5 μM) and 1 mM NAC was added to 0.1 mM DPPH and mixed well. The mixture was incubated for 30 min, after which the amount of residual DPPH was determined by measuring absorbance at 520 nm using a spectrophotometer.

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Intracellular ROS detection [2]
The DCF-DA method was used to detect intracellular ROS levels in HaCaT keratinocytes (Rosenkranz et al., 1992). Cells were seeded at a density of 1.5×105 cells/well in 24-well culture plates. Sixteen hours after plating, cells were treated with Rosmarinic acid/RA (0.625, 1.25, 2.5, 5 μM) or 1 mM NAC. After incubation for 30 min, cells were exposed to H2O2 (1 mM) and again incubated for 30 min. H2O2-treated cells were treated with DCF-DA (25 μM) solution and incubated for another 10 min to detect the fluorescence of DCF. Otherwise, cells were incubated with RA (2.5 μM) or 1 mM NAC for 1 h and exposed to UVB (30 mJ/cm2). Following 24 h, cells were further incubated with DCF-DA solution for 10 min. Fluorescence of DCF was detected using a PerkinElmer LS-5B spectrofluorometer.

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Detection of the superoxide anion [2]
The superoxide anion was produced via the xanthine/xanthine oxidase system and reacted with a nitrone spin trap, DMPO. The DMPO/•OOH adducts were detected using a JES-FA electron spin resonance (ESR) spectrometer (Kohno et al., 1994). Briefly, ESR signaling was recorded 5 min after 20 μl of xanthine oxidase (0.25 unit/ml) was mixed with 20 μl each of xanthine (5 mM), DMPO (1.5 M), Rosmarinic acid/RA (2.5 μM). The ESR spectrometer parameters were set as follows: magnetic field, 336 mT; power, 1.00 mW; frequency, 9.438 GHz; modulation amplitude, 0.2 mT; gain, 500; scan time, 0.5 min; scan width, 10 mT; time constant, 0.03 sec; and temperature, 25°C.

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Detection of hydroxyl radical [2]
The hydroxyl radical was generated via the Fenton reaction (H2O2+FeSO4) and reacted with DMPO. The resultant DMPO/•OH adducts were detected using an ESR spectrometer (Li et al., 2004). The ESR spectrum was recorded 2.5 min after a phosphate buffer solution (pH 7.4) was mixed with 0.2 ml each of DMPO (0.3 M), FeSO4 (10 mM), H2O2 (10 mM), and Rosmarinic acid/RA (2.5 μM). The ESR spectrometer parameters were as follows: magnetic field, 336 mT; power, 1.00 mW; frequency, 9.438 GHz; modulation amplitude, 0.2 mT; gain, 200; scan time, 0.5 min; scan width, 10 mT; time constant, 0.03 sec; and temperature, 25°C.

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Protein carbonyl formation [2]
Cells were treated with Rosmarinic acid/RA at a concentration of 2.5 μM for 24 h. One hour later, cells were exposed to UVB and incubated at 37°C for another 24 h. The extent of protein carbonyl formation was determined using an Oxiselect™ protein carbonyl ELISA kit.

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Lipid peroxidation assay [2]
Lipid peroxidation was assayed by colorimetric determination of the levels of 8-isoprostane, a stable end product of lipid peroxidation, in medium from HaCaT cells (Beauchamp et al., 2002). A commercial enzyme immune assay was employed to detect 8-isoprostane. Lipid peroxidation was also assessed using DPPP as a probe (Okimoto et al., 2000). DPPP reacts with lipid hydroperoxides to generate a fluorescent product, DPPP oxide, thereby providing an indication of membrane damage. Cells were treated with 2.5 μM of Rosmarinic acid/RA for 1 h, followed by exposure to UVB (30 mJ/cm2). Twenty-four hours later, cells were incubated with 20 μM DPPP for 30 min in the dark. Images of DPPP fluorescence were captured on a Zeiss Axiovert 200 inverted microscope at an excitation wavelength of 351 nm and an emission wavelength of 380 nm.

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Nuclear staining with Hoechst 33342 [2]
Cells were treated with 2.5 μM Rosmarinic acid/RA and exposed to 30 mJ/cm2 UVB radiation 1 h later. After incubation for an additional 24 h at 37°C, 1 μl of the DNA-specific fluorescent dye Hoechst 33342 (stock, 15 mM) was added to each well of the 6-well plate. The plate was then incubated for 10 min at 37°C. The degree of nuclear condensation in the stained cells was determined by visualization with a fluorescence microscope equipped with a CoolSNAP-Pro color digital camera.

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DNA fragmentation [2]
Cells were treated with Rosmarinic acid/RA (2.5 μM) for 24 h. One hour later, the cells were exposed to UVB and incubated at 37°C for another 24 h. Cellular DNA fragmentation was assessed by analyzing cytoplasmic histone-associated DNA fragments using a kit from Roche Diagnostics.

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SOD activity assay [2]
Cells were seeded in a culture dish at a concentration of 1×105 cells/ml; 16 h after plating, the cells were treated with 2.5 μM Rosmarinic acid/RA. One hour later, cells were exposed to UVB and incubated at 37°C for an additional 24 h. The cells were then washed with cold PBS and harvested by scraping. The harvested cells were suspended in 10 mM phosphate buffer (pH 7.5) and then lysed on ice by sonicating twice for 15 sec. Triton X-100 (1%) was added to the lysates and incubated for 10 min on ice. The lysates were clarified by centrifugation at 5,000×g for 10 min at 4°C to remove cellular debris. The protein content of the supernatant was determined using the Bradford method. Total SOD activity was evaluated by measuring the level of inhibition of epinephrine auto-oxidation (Misra and Fridovich, 1972), as follows. Fifty micrograms of protein was added to 500 mM phosphate buffer (pH 10.2) and 1 mM epinephrine. Epinephrine rapidly undergoes auto-oxidation at pH 10 to produce adrenochrome, a pink-colored product, which was assayed at 480 nm using a UV/vis spectrophotometer in the kinetic mode. SOD inhibits the auto-oxidation of epinephrine. The rate of inhibition was monitored at 480 nm, and the amount of enzyme required to produce 50% inhibition was defined as 1 unit of enzyme activity. Total SOD activity was expressed as units/mg protein.

In this study, we investigated the anti-inflammatory effects and underlying molecular mechanism of RA in mice with dextran sulphate sodium (DSS)-induced colitis. In the DSS-induced colitis model, RA significantly reduced the severity of colitis, as assessed by disease activity index (DAI) scores, colonic damage, and colon length. In addition, RA resulted in the reduction of the inflammatory-related cytokines, such as IL-6, IL-1β, and IL-22, and protein levels of COX-2 and iNOS in mice with DSS-induced colitis. Furthermore, RA effectively and pleiotropically inhibited nuclear factor-kappa B and signal transducer and activator of transcription 3 activation, and subsequently reduced the activity of pro-survival genes that depend on these transcription factors. These results demonstrate that RA has an ameliorative effect on colonic inflammation and thus a potential therapeutic role in colitis.[3]\n

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\nInduction of colitis [3]
\nExperimental colitis was induced by giving mice drinking water ad libitum containing 5% (w/v) DSS for 7 days. Mice of each of the groups were monitored carefully every day to confirm that they had consumed an approximately equal volume of water containing DSS. For each experiment, the mice were divided into five experimental groups (n = 10/group). The first group was kept as the vehicle-treated control, and the second group was given drinking water with DSS only during the experimental period. The other three groups consisted of mice receiving 5% DSS who were administrated 5-ASA (75 mg/kg/day p.o.) or Rosmarinic acid (30 or 60 mg/kg/day p.o.) daily for 7 days, according to the experimental design. All materials were dissolved in a vehicle of 0.9% saline. Control groups were given the vehicle daily for 7 days as appropriate. Administration of each drug was initiated simultaneously with the DSS treatment.\n

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\nEvaluation of disease activity index (DAI) [3]
\nBody weight, stool consistency, and gross bleeding were recorded daily. Disease activity index (DAI) was determined by combining the scores for (i) body weight loss, (ii) stool consistency, and (iii) gross bleeding, divided by 3. Each score was determined as follows: change in body weight loss (0: none, 1: 1–5%, 2: 5–10%, 3: 10–20%, 4: > 20%), stool blood (0: negative, 1: +, 2: ++, 3: +++, 4: ++++), and stool consistency (0: normal, 1 and 2: loose stool, 3 and 4: diarrhoea). Body weight loss was calculated as the percentage of the difference between the original body weight (day 0) and the body weight on any particular day (Table 1). At the end of experiment, all mice were sacrificed and the large intestines were separated from the vermiform appendix to the anus. The colon length was measured between the caecum and proximal rectum. The spleens were dissected and their weights measured immediately.\n

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\nHistopathology [3]
\nThe resected mice colon tissues were fixed immediately in 10% formalin and embedded. For histopathological analysis, tissue samples were sectioned (5 μm) and stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). Both of the histologic processes were described previously.\n

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\nMeasurement of myeloperoxidase activity (MPO) and cytokine production [3]
\nColon tissues were washed with DMEM medium containing 0.2% FBS, streptomycin, and penicillin, and cut into smaller pieces. Afterwards, 0.5 cm of the tissue was placed in a 24-well plate filled with 1 ml DMEM medium containing 0.2% FBS, streptomycin, and penicillin, and incubated for 24 h at 37 °C in 5% CO2. The cell-free culture supernatants of the colon tissue were used to measure MPO activity and production of cytokines. Neutrophil sequestration in the colon was quantified by measuring tissue MPO activity. To estimate MPO activity, tissue samples were thawed and homogenized in 0.05 M phosphate buffer (pH 6) containing 0.5% (w/v) hexadecyltrimethylammonium bromide. The suspension was centrifuged (3,000 rpm, 20 min, 4 °C), and the supernatant was used for MPO assay. The reaction mixture consisted of the supernatant, 0.003% H2O2, O-dianiside in 0.05 M phosphate buffer (pH 6), and 0.5% HTAB. This mixture was incubated at 37 °C and terminated at 10 min. The absorbance was measured at 450 nm. The results were quantified as the amount from 10 min absorbance minus the amount from zero min absorbance, and expressed as unit per milligram of protein. In addition, the levels of IL-1β, IL-6, and IL-22 produced in the culture media were quantified using EIA kits, according to the manufacturer’s instructions.\n

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\nInflammation score [3]
\nThe inflammation score was evaluated based on Table 2. Inflammation was graded as follows: mucosal epithelial cell 1, prolonged epithelial cell or crypt; 2, destruction of barrier; 3, ulcer (30% < loss < 60%); 4, ulcer (loss > 60%), mucosal immune cell 1, infiltration (mild); 2, infiltration (moderate); 3, infiltration (severe) and submucosa’s immune cell 1, infiltration (mild); 2, infiltration (moderate); 3, infiltration (severe).\n\nImmunohistochemistry [3]
\nAll IHC was performed on formalin-fixed, paraffin-embedded samples. Paraffin blocks were sectioned to 5-μm thickness. Afterwards, poly-L-lysine-coated slides were used to promote adhesion of the paraffin-section to the slides, which were then dried. The dried slides were de-paraffinized, and antigen retrieval was performed by automated antigen retrieval machine for 20 minutes in cell condition solution (Ethylenediaminetetraacetic acid pH 9.0). Sections were blocked for 1 h with 15–20% normal goat serum, prior to incubation with primary antibody for 2 h at room temperature or overnight at 4 °C. Secondary rabbit antibodies were used to detect primary antibodies, followed by streptavidin-tagged horseradish peroxidase. Diaminobenzidine was used to induce signalling, and bluing reagent was used as a counterstain. Images of IHC slides were visualized by optical microscopy and rendered using Leica software. For IHC, p-STAT3 (Tyr705) and NF-κB p65 antibodies were used.\n

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\n\nDetection of thiobarbituric acid reactive substances (TBARS) [1]
\nExperiments to evaluate lipid peroxidation were performed in homogenates of cortex and hippocampus from male Wistar rats (180–220 g). Brain regions were washed with cold TRIS buffer (20 mM, pH 7.4) and homogenized. The pool of cells was centrifuged in a HT-MCD 2000 at 4592g for 5 min, and supernatant was removed.\nThe Rosmarinic acid/RA was diluted in phosphate buffer (20 mM, pH 7.2) containing 1% DMSO to obtain concentrations ranging from 0.001 to 5 mM, which were added to supernatant of brain homogenates. FeSO4 (2 mM) and ascorbic acid (0.02 mM) solutions were also added to the medium and incubated at 37 °C, for 60 min. Subsequently, trichloroacetic and thiobarbituric acids were added, followed by 20 min incubation, at 80 °C. Centrifugation at 7500 rpm for 10 min was taken, and supernatants were analyzed at 532 nm. Analyzes were carried out in triplicate and positive controls CGA and CA were employed. Percentage inhibition (%) were calculated comparing with the negative control (1% DMSO) and results were expressed as IC50 values.\n\n

Absorption, Distribution and Excretion
This study aimed to determine the absorption, metabolism, and urinary excretion of rosmarinic acid (RA) in healthy individuals following a single administration of perilla extract (PE). A crossover design was used, enrolling six healthy men (mean age 37.2 ± 6.2 years, mean body mass index 22.0 ± 1.9 kg/m²). Each participant received a single administration of perilla extract containing 200 mg RA, while the other received a placebo, with a 10-day interval between administrations. Blood samples were collected before administration and at specified time intervals, and urine samples were collected at 0–6 hours, 6–24 hours, and 24–48 hours after administration. The concentrations of RA and its related metabolites in plasma and urine were determined using liquid chromatography-mass spectrometry (LC-MS). Following administration of rosmarinic extract (PE), rosmarinic acid (RA), methylrosmarinic acid (methyl-RA), caffeic acid (CAA), ferulic acid (FA), and trace amounts of mesocoumaric acid (COA) were detected in urine. RA, methyl-RA, and FA were also detected in plasma, reaching peak concentrations at 0.5, 2, and 0.5 hours after PE administration, respectively. Most components in plasma and urine were present in conjugated forms (glucuronide and/or sulfate). The proportion of RA and its related metabolites in urine was 6.3 ± 2.2% of the total dose, with approximately 75% excreted within 6 hours after PE administration. Rosmarinic acid is readily absorbed via the gastrointestinal tract and skin. This study aimed to investigate the percutaneous absorption, tissue distribution, and absolute bioavailability of rosmarinic acid (RA). In vitro experiments showed that the permeability of RA in isolated rat skin was approximately 8 times higher in alcoholic solution than in aqueous solution, suggesting that ethanol may act as an absorption enhancer. The osmotic fluxes of aqueous and alcoholic solutions were 4.4 and 10 μg/cm²/hr, respectively, with half-lives of 7.8 and 3.7 hours, respectively. Following intravenous injection, the distribution of RA best conformed to a two-compartment open model: t₁/₂ = 1.8 h, t₁/₂α = 0.07 h, V₁τ = 2.3 L/kg, V₁β = 15.3 L/kg. The absolute bioavailability of RA after topical application (in the form of an oil-in-water ointment, 25 mg/kg, 50 cm²) was 60%. RA was detected and measured in the brain, heart, liver, lungs, muscles, spleen, and bone tissue 0.5 hours after intravenous injection, showing the highest concentration in lung tissue (13 times the blood concentration), followed by the spleen, heart, and liver. Rosmarinic acid levels in blood, skin, muscle, and bone tissue were measured 4.5 hours after topical application of approximately 3 mg of rosmarinic acid over a 20 cm² area in the hind limb (peak time).

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Metabolism/Metabolites
Seven metabolites were found in the urine of rats orally administered rosmarinic acid (7), identified by spectroscopic and chemical data as trans-caffeic acid 4-O-sulfate (1), trans-mocoumaric acid 3-O-sulfate (2), trans-ferulic acid 4-O-sulfate (3), trans-caffeic acid (4), m-hydroxyphenylpropionic acid (5), trans-mocoumaric acid (6), and unmetabolized rosmarinic acid (7). Forty-eight hours after oral administration of rosmarinic acid, the total cumulative amount of 1-7 excreted in the urine was approximately 31.8% of the administered dose. On the other hand, no metabolites attributable to rosmarinic acid were detected in the bile. Therefore, it can be concluded that orally administered rosmarinic acid is primarily excreted via urine rather than bile, and its metabolic pathway includes ester bond cleavage, selective para-dehydroxylation, methylation, and sulfate conjugation. Metabolites 2, 3, 5, and 6 were also detected in plasma. Rosmarinic acid is a major hydroxycinnamic acid ester that accumulates in plants of the Boraginaceae and Lamiaceae families. Cytochrome P450 cDNA was isolated from Lithospermum erythrorhizon cultured cells using differential display technology, and its gene product was named CYP98A6 based on the deduced amino acid sequence. After expression in yeast, P450 was shown to catalyze the 3-hydroxylation of 4-coumaryl-4'-hydroxyphenyllactic acid, one of the final two steps in the formation of rosmarinic acid. Adding yeast extract or methyl jasmonate to Lactobacillus rubrum cells significantly increased the expression level of CYP98A6, and its expression pattern reflected changes in rosmarinic acid production induced by the inducer, indicating that CYP98A6 plays an important role in the regulation of rosmarinic acid biosynthesis. This study aimed to determine the absorption, metabolism, and urinary excretion of rosmarinic acid (RA) in healthy individuals after a single intake of perilla extract (PE). This study employed a crossover design, enrolling six healthy men (mean age 37.2 ± 6.2 years, mean body mass index 22.0 ± 1.9 kg/m²). Participants received either a single dose of PE containing 200 mg of RA or a placebo, with a 10-day interval between doses. Blood samples were collected before administration and at specified time intervals, and urine samples were collected at 0–6 hours, 6–24 hours, and 24–48 hours after administration. The levels of RA and its related metabolites in plasma and urine were determined using liquid chromatography-mass spectrometry (LC-MS). Results showed that after PE administration, RA, methylated RA (methyl-RA), caffeic acid (CAA), ferulic acid (FA), and trace amounts of mesocoumaric acid (COA) were detected in urine. RA, methyl-RA, and FA were detected in plasma, with peak concentrations reached at 0.5 hours, 2 hours, and 0.5 hours after PE administration, respectively. Most components in plasma and urine existed in conjugated forms (glucuronide and/or sulfate). The proportion of RA and its related metabolites excreted in urine is 6.3 ± 2.2% of the total dose, of which about 75% of the components are excreted within 6 hours after taking PE.

Interactions
Rosmarinic acid (RA) is a caffeic acid ester that exhibits insulin-sensitizing and antioxidant effects in a high-fructose-fed insulin resistance (IR) model. This study aimed to investigate whether RA supplementation could prevent cardiac abnormalities and hypertension in high-fructose-fed rats (FFR). Rats fed a high-fructose diet (60 g/100 g) for 60 days developed metabolic abnormalities, elevated plasma and cardiac lipid levels, and systemic insulin resistance. FFR rats showed significantly reduced cardiac antioxidant levels and plasma ferric reductive antioxidant capacity, while lipid peroxidation and protein oxidation product levels were elevated. Plasma levels of troponin T, creatine kinase-MB, aspartate aminotransferase, and lactate dehydrogenase were significantly elevated in FFR rats. Starting from day 16, RA supplementation (10 mg/kg) in FFR mice significantly improved insulin sensitivity, reduced blood lipid levels and oxidative damage, and decreased the expression of the p22phox subunit of nicotinamide adenine dinucleotide phosphate reductase, thereby preventing cardiac hypertrophy. RA also reduced fructose-induced hypertension by decreasing the activity of endothelin-1 and angiotensin-converting enzyme and increasing nitric oxide levels. Histological analysis showed reduced myocardial injury in FFR mice supplemented with RA. These results suggest that RA acts as a vasoactive substance and cardioprotective agent through its antioxidant properties. Therefore, RA may help reduce cardiovascular risk associated with insulin resistance.
Epidemiological and experimental studies have shown that diesel exhaust particulate matter (DEP) may be associated with the increase in lung diseases in recent years. DEP has been shown to generate reactive oxygen species. Intratracheal instillation of DEP can induce lung inflammation and edema in mice. Rosmarinic acid is a naturally occurring polyphenol with antioxidant and anti-inflammatory activities. This study investigated the effect of rosmarinic acid on lung injury induced by intratracheal injection of DEP (500 μg/mouse) in mice. Oral supplementation with rosmarinic acid (2 mg/mouse, for 3 consecutive days) inhibited DEP-induced lung injury characterized by neutrophil aggregation and interstitial edema. DEP enhanced the expression of keratinocyte chemokine (KC), interleukin-1β, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1α in lung tissue, while rosmarinic acid treatment inhibited these expressions. DEP also enhanced the expression of iNOS mRNA and the production of nitrotyrosine and 8-OHdG in lung tissue, which could also be inhibited by rosmarinic acid. These results suggest that rosmarinic acid inhibits diesel exhaust particulate (DEP)-induced lung injury by reducing the expression of pro-inflammatory molecules. The antioxidant activity of rosmarinic acid may also contribute to its protective effect. Rosmarinic acid (RA) is a polyphenolic phytochemical and a natural prolyl oligopeptidase inhibitor. This study found that RA has a significant anti-inflammatory effect in an in vivo model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Mice were pre-administered RA one hour before receiving stimulation with 0.5 mg/kg LPS. Twenty-four hours after LPS administration, bronchoalveolar lavage fluid (BALF) was collected to detect pro-inflammatory mediators and total cell counts. Compared with the LPS group, RA significantly reduced LPS-induced production of TNF-α, IL-6, and IL-1β. Pre-administration of rosmarinic acid (RA, 5, 10, or 20 mg/kg) significantly reduced the wet/dry weight ratio (W/D) of lung tissue and the total cell count, neutrophil count, and macrophage count in BALF. Furthermore, RA may enhance superoxide dismutase (SOD) activity during the inflammatory response of lipopolysaccharide (LPS)-induced acute lung injury (ALI). The authors further confirmed that RA exerts an anti-inflammatory effect in an in vivo ALI model by dose-dependently inhibiting the ERK/MAPK signaling pathway…
This study aimed to investigate the protective effect of rosmarinic acid against ethanol-induced DNA damage in mice. The antigenotoxicity of rosmarinic acid (100 mg/kg) was assessed using ethanol (5 g/kg) pretreatment, co-treatment, and post-treatment. Peripheral blood (1 h and 24 h) and brain cells (24 h) were evaluated using the comet assay, and bone marrow was analyzed using the micronucleus assay (24 h). Results were compared with data from the TBARS, antioxidant enzyme activity, and DCFH-DA assay. Peripheral blood and brain cell results showed that the mean damage index (DI) and damage frequency (DF) values in the ethanol group pretreated with rosmarinic acid were significantly lower than those in the ethanol group. In brain cells, the mean DI and DF values were significantly reduced in all different ethanol and rosmarinic acid treatment groups compared to the ethanol group and the negative control group. There were no significant differences in micronucleus frequency, antioxidant enzyme activity, and TBARS values among the groups. The DCFH-DA assay showed an 18% reduction in fluorescence intensity compared to the ethanol group. These results indicate that rosmarinic acid can reduce the level of DNA damage induced by ethanol, regardless of tissue type or treatment duration.
For more complete data on interactions of rosmarinic acid (13 in total), please visit the HSDB record page. Mouse LD50 (IV): 561 mg/kg. Future Drugs, 10(756), 1985.

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Antidotes and First Aid
/SRP:/ Immediate First Aid: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask, or simple breathing mask, and follow the training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, tilt the patient forward or to the left (head down if possible) to keep the airway open and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek medical attention. /Class A and Class B Poisons/
/SRP:/ Basic Treatment: Establish a clear airway (using an oropharyngeal or nasopharyngeal airway if necessary). Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via a non-invasive ventilation mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as necessary… If eyes become contaminated, flush immediately with water. During transport, continuously flush each eye with 0.9% normal saline (NS)… Do not use emetics. If swallowed, rinse mouth and dilute with 5 ml/kg body weight to 200 ml of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After disinfecting skin burns, cover with a dry, sterile dressing… /Class A and Class B Poisons/
/SRP:/ Advanced Treatment: For patients with altered consciousness, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation via bag-valve-mask may be effective. Consider medical treatment for pulmonary edema… Consider using a β-receptor agonist (such as salbutamol) to treat severe bronchospasm… Monitor heart rhythm and treat arrhythmias if necessary… Initiate intravenous infusion of 5% glucose solution (D5W)/SRP: “Keep it patent,” minimum flow rate/. If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution. For hypotension with signs of hypovolemia, administer fluids with caution. Watch for signs of fluid overload… Use diazepam or lorazepam to treat seizures… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/ Currance, PL Clements, B., Bronstein, AC (eds.); First Aid for Hazardous Substance Exposure. 3rd ed., Elsevier Mosby Publishers, St. Louis, Missouri, 2005, pp. 160-161

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Human Toxicity Excerpt
/Human Exposure Studies/ Rosmarinic acid is known to have anti-inflammatory and immunomodulatory activities. This study aimed to evaluate the effects of rosmarinic acid on atopic dermatitis (AD, an inflammatory skin disease). Twenty-one patients with mild AD (14 women and 7 men, aged 5–28 years) participated in this study. A 0.3% rosmarinic acid emulsion was applied topically to the flexor surface of the elbow joint in AD patients twice daily (morning and evening). All subjects underwent pre-treatment skin condition assessment at their first visit, followed by reassessment at 4 and 8 weeks post-treatment. According to the local atopic dermatitis severity score, erythema in the cubital fossa significantly decreased at both 4 and 8 weeks post-treatment (P < 0.05). Transepidermal water loss in the cubital fossa significantly decreased at 8 weeks post-treatment compared to pre-treatment levels (P < 0.05). …PMID: 19239556
/Alternatives and In Vitro Experiments/ This study investigated the protective effect of rosmarinic acid (RA) against H₂O₂-induced neurotoxicity in the human dopaminergic cell line SH-SY5Y. The results showed that RA significantly attenuated H₂O₂-induced reactive oxygen species (ROS) production and apoptosis. Rosmarinic acid effectively inhibited the upregulation of Bax and the downregulation of Bcl-2. Furthermore, rosmarinic acid stimulated the expression of the antioxidant enzyme heme oxygenase-1 (HO-1). …Rosmarinic acid-induced HO-1 expression is associated with the protein kinase A (PKA) and phosphatidylinositol-3-kinase (PI3K) signaling pathways. These results suggest that rosmarinic acid can protect SH-SY5Y cells from oxidative stress damage by regulating the apoptosis process…PMID:18644421
/Alternative and In Vitro Assays/…This study…evaluated the cytoprotective effects of two Sage extracts (aqueous and methanolic extracts) against tert-butyl hydroperoxide (t-BHP)-induced HepG2 cytotoxicity. The most abundant phenolic compounds in the extracts were rosmarinic acid and luteolin-7-glucoside. Both extracts significantly protected HepG2 cells from death when co-incubated with the toxin. The methanol extract had a higher content of phenolic compounds than the water extract, thus providing better protection… Both extracts were tested at concentrations that protected 80% of cells from death (IC(80)). The results showed that they significantly inhibited tert-butyl hydroperoxide (t-BHP)-induced lipid peroxidation and glutathione (GSH) depletion, but had no significant effect on DNA damage assessed by the comet assay… PMID:17349617
/Alternative and In Vitro Assays/ UVA radiation induces the production of reactive oxygen species (ROS), leading to oxidative stress in exposed cells, causing widespread cell damage and cell death, including apoptosis or necrosis. One way to protect human skin from the harmful effects of UV radiation is to use herbal compounds as photoprotectants. This study evaluated the protective effects of Prunella vulgaris L. (Lamiaceae) and its main phenolic acid component, rosmarinic acid (RA), against UVA-induced damage in human keratinocytes (HaCaT). Human keratinocytes exposed to UVA (10–30 J/cm²) were treated with Prunella vulgaris extract (1–75 mg/L) or rosmarinic acid (0.9–18 mg/L) for 4 hours. Results showed that both Prunella vulgaris and rosmarinic acid reduced UVA-induced cell viability loss, which was monitored by neutral red retention and lactate dehydrogenase (LDH) release. Prunella vulgaris extract and rosmarinic acid significantly inhibited UVA-induced reactive oxygen species (ROS) production, manifested as decreased intracellular lipid peroxidation and increased ATP and reduced glutathione levels. Furthermore, post-treatment with Prunella vulgaris extract and rosmarinic acid significantly reduced DNA damage. Furthermore, treatment with common purslane (P. vulgaris) and retinoic acid (RA) inhibited UVA-induced caspase-3 activation. Both common purslane extract and retinoic acid exhibited concentration-dependent photoprotective effects (reaching maximum at 25–50 mg/L and 9 mg/L, respectively). These results suggest that common purslane and retinoic acid used in skincare cosmetics may help combat UVA-induced oxidative stress and may serve as supplements to photoprotective skin formulations. PMID:16631374

[1]. Combining in vitro and in silico approaches to evaluate the multifunctional profile of rosmarinic acid from Blechnum brasiliense on targets related to neurodegeneration. Chem Biol Interact. 2016 Jul 25;254:135-45.

[2]. Rosmarinic Acid Attenuates Cell Damage against UVB Radiation-Induced Oxidative Stress via Enhancing Antioxidant Effects in Human HaCaT Cells. Biomol Ther (Seoul). 2016 Jan;24(1):75-84.

[3]. Rosmarinic acid suppresses colonic inflammation in dextran sulphate sodium (DSS)-induced mice via dual inhibition of NF-κB and STAT3 activation. Sci Rep. 2017 Apr 6;7:46252.

Therapeutic Uses
Lemon balm (Melissa officinalis L., Lamiaceae family) has been used in traditional medicine to treat nervous system disorders and lower abdominal discomfort, and in recent years has also been used to treat herpes simplex virus infection. This study evaluated the antiviral activity of the hydroalcoholic extract of lemon balm leaves against herpes simplex virus type 2 (HSV-2) using a Vero cell (ATCC CCL-81) cytopathic effect inhibition assay, and compared it with acyclovir. Previously, the cytotoxicity of the extract against Vero cells was tested by assessing cell death and validated by trypan blue staining. The results showed that within the non-toxic concentration range of 0.025–1 mg/mL (based on the starting crude herb), lemon balm could reduce the cytopathic effect of HSV-2 on Vero cells. The inhibition rate reached its maximum (60%) at a concentration of 0.5 mg/mL. Virus binding assays showed that the extract could not prevent HSV-2 from entering cells, suggesting that its mechanism of action may occur after the virus has entered the cell. The extract was also chemically characterized by nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC); the results showed that it contained cinnamic acid compounds, with rosmarinic acid (4.1% w/w) as the main component. These experimental results support the use of lemon balm for the treatment of herpes simplex lesions and encourage clinical trials of this medicinal plant. Mechanism of Action… To determine the effect of rosmarinic acid on melanin production and to elucidate the molecular mechanism by which rosmarinic acid induces melanin production, we conducted several experiments in B16 melanoma cells. In this study,… rosmarinic acid increased melanin content and tyrosinase expression in a concentration-dependent manner. Furthermore, after rosmarinic acid increased melanin content, protein kinase A (PKA) inhibitors H-89 and KT 5720 decreased melanin content, while p38MAPK inhibitor SB203580 or PKC inhibitor Ro-32-0432 had no such effect, indicating that PKA is involved in rosmarinic acid-induced melanin production. Consistent with this, rosmarinic acid induced phosphorylation of CRE-binding protein (CREB) but had no effect on the inhibition of p38MAPK phosphorylation or Akt phosphorylation. Furthermore, rosmarinic acid induced activation of the cAMP response element (CRE) but had no effect on cAMP production, indicating that rosmarinic acid-induced melanin production is mediated by PKA, which is downstream of cAMP production. This result is further supported by the fact that rosmarinic acid-induced CREB phosphorylation can be inhibited by H-89 but not by the MEK1 inhibitor PD98059 or the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. H-89 attenuates rosmarinic acid-induced tyrosinase protein expression. Based on these results, ... rosmarinic acid induces melanin production through the PKA-activated signaling pathway. PMID:17651699
Rosemary acid (RA)…inhibits multiple complement-dependent inflammatory processes and may have the potential as a therapeutic agent to control complement activation in diseases. Rosemary acid has been reported to affect both classical pathway C3 convertases and cobra venom factor-induced alternative pathway convertases. To clarify its inhibitory mechanism, this study examined the effects of RA on classical and alternative pathway cleavage, C1q binding, classical pathway convertases, alternative pathway convertases, membrane attack pathway cleavage, and the generation of C3 and C5 fragments during activation in vitro. The results showed that RA had a stronger inhibitory effect on classical pathway cleavage than on the alternative pathway. This inhibition was dose-dependent, with the maximum inhibition of classical pathway cleavage observed at a concentration of 2.6 mmol/L RA. RA had minimal effect on C1q binding and classical and alternative pathway convertases. However, in the presence of RA (1 mM), dilution of human or rabbit serum significantly inhibited the cleavage of pre-formed EA43b cells, accompanied by inhibition of C5a generation. Therefore, it was concluded that the inhibitory effect of RA is related to C5 convertase. This inhibitory effect may be beneficial to the host, especially in diseases where terminal attack sequences are involved in pathogenesis. PMID:1761351
Rosemary acid (RA) is a naturally occurring polyphenolic flavonoid that has been reported to inhibit TNF-α-induced NF-κB activation in human dermal fibroblasts. However, the exact mechanism of RA in TNF-α-mediated anticancer therapy remains poorly elucidated. In this study, the authors found that RA treatment significantly enhanced TNF-α-induced apoptosis in human leukemia U937 cells by inhibiting nuclear transcription factor-κB (NF-κB) and reactive oxygen species (ROS). RA treatment significantly enhanced TNF-α-induced caspase activation. However, pretreatment with the caspase-3 inhibitor z-DEVD-fmk significantly restored cell viability after combination therapy. RA also inhibited NF-κB activation by inhibiting IκBα phosphorylation and degradation, as well as nuclear translocation of p50 and p65. This inhibitory effect is associated with the inhibition of NF-κB-dependent anti-apoptotic proteins (IAP-1, IAP-2, and XIAP). RA treatment also restored TNF-α-induced ROS production to normal. Furthermore, U937 cells ectopically expressing Bcl-2 reversed combination therapy-induced cell death, cytochrome c release into the cytosol, and mitochondrial membrane potential collapse. These results indicate that rosmarinic acid (RA) inhibits TNF-α-induced reactive oxygen species (ROS) production and NF-κB activation, and enhances TNF-α-induced apoptosis. PMID:19619938
Rosmarinic acid (RosA) is a hydroxylated compound commonly found in herbal medicines, primarily possessing anti-inflammatory and antioxidant activities. Previous studies have shown that RosA inhibits T-cell antigen receptor (TCR)-induced interleukin-2 (IL-2) expression and subsequent T-cell proliferation in vitro. This study investigated the inhibitory mechanism of RosA on the TCR signaling pathway, which ultimately activates the IL-2 promoter by activating transcription factors such as nuclear factor-activated T cells (NF-AT) and activated protein-1 (AP-1). Interestingly, RosA inhibited NF-AT activation but not AP-1 activation, indicating that RosA only inhibits the Ca²⁺-dependent signaling pathway. RosA strongly inhibited upstream signaling events of NF-AT activation, such as the production of inositol 1,4,5-triphosphate, Ca²⁺ mobilization, and tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1). PLC-γ1 tyrosine phosphorylation mainly depends on three protein tyrosine kinases (PTKs): Lck, ZAP-70, and Itk. The researchers found that RosA effectively inhibited TCR-induced Itk tyrosine phosphorylation and its subsequent activation, but had no inhibitory effect on Lck or ZAP-70. In the presence of RosA, ZAP-70-dependent signaling pathways (such as tyrosine phosphorylation of LAT and SLP-76 and serine/threonine phosphorylation of mitogen-activated protein kinase (MAPK)) remain intact, confirming that RosA inhibits TCR signaling in a ZAP-70-independent manner. …/Conclusion is/RosA inhibits TCR signaling leading to Ca2+ mobilization and NF-AT activation by blocking proximal membrane events, particularly tyrosine phosphorylation of inducible T cell kinase (ITk) and PLC-γ1. PMID:12511421

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Rosmarinic acid exhibits interesting antioxidant properties, characterized by reducing the levels of oxygen and nitrogen free radicals and inhibiting lipid peroxidation. This compound shows activity against targets associated with neurodegenerative diseases, namely MAO-A and COMT enzymes. Time-dependent inhibition studies of MAO-A strongly suggest that RA is a reversible inhibitor of this target. Cytotoxicity studies on PMN cells showed that RA did not cause extensive damage to the cell membrane at concentrations higher than its antioxidant and enzyme inhibitory activity levels. Molecular docking helped to understand the binding patterns of RA, CGA, and CA with MAO-A and COMT enzymes and their main interactions. In addition, the reasons for the differences in the inhibitory efficacy of these compounds against MAO-A and COMT were revealed. In summary, these data help to rationally design novel multifunctional derivatives with a 3,4-dihydroxycinnamic acid backbone. [1] In conclusion, the cytoprotective activity of rosmarinic acid/RA against UVB radiation may be related to the scavenging of reactive oxygen species (ROS), which can alleviate oxidative damage to cellular components and induce apoptosis. Therefore, RA can be used as a therapeutic agent to protect the skin from the harmful effects of UVB radiation. These findings may provide an experimental platform for further research on the bioavailability and photoprotective activity of rosmarinic acid (RA), including in vitro and in vivo studies, and its potential mechanisms. [2] In summary, our study showed that rosmarinic acid/RA significantly improved systemic symptoms in a mouse DSS-induced colitis model and inhibited the expression of pro-inflammatory cytokines and inflammatory mediators by regulating the activation of NF-κB and STAT3. Therefore, we believe that RA deserves further investigation and is expected to become a potential therapy for inflammatory diseases such as colitis. [3]

C18H16O8

360.3148

360.084

C, 60.00; H, 4.48; O, 35.52

537-15-5

Rosmarinic acid;20283-92-5

5315615

Crystalline solid

1.5±0.1 g/cm3

694.7±55.0 °C at 760 mmHg

171-175ºC(lit.)

254.5±25.0 °C

0.0±2.3 mmHg at 25°C

1.714

1.7

5

8

7

26

519

0

O(C(/C(/[H])=C(\[H])/C1C([H])=C([H])C(=C(C=1[H])O[H])O[H])=O)[C@@]([H])(C(=O)O[H])C([H])([H])C1C([H])=C([H])C(=C(C=1[H])O[H])O[H]

DOUMFZQKYFQNTF-ZZXKWVIFSA-N

InChI=1S/C18H16O8/c19-12-4-1-10(7-14(12)21)3-6-17(23)26-16(18(24)25)9-11-2-5-13(20)15(22)8-11/h1-8,16,19-22H,9H2,(H,24,25)/b6-3+

3-(3,4-dihydroxyphenyl)-2-[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxypropanoic acid

Rosmarinate; 537-15-5; Rosmarinic acid (racemate); CHEMBL66966; CHEBI:17226; NSC687846; Benzenepropanoic acid,a-[[3-(3,4-dihydroxyphenyl)-1-oxo-2-propenyl]oxy]-3,4-dihydroxy-; Rosemarinic Acid; DTXSID70896992; 537-15-5; alpha-[[3-(3,4-Dihydroxyphenyl)-1-oxo-2-propen-1-yl]oxy]-3,4-dihydroxybenzenepropanoic acid; alpha-((3-(3,4-Dihydroxyphenyl)-1-oxo-2-propen-1-yl)oxy)-3,4-dihydroxybenzenepropanoic acid; DTXCID30810952; Rosmarinate; rosmarinate acid; Labiatenic acid pound>>(R)-rosmarinic acid pound>>Rosemary acid;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~75 mg/mL (~208.15 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.94 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)