Clinopodium Vulgare L. (Wild Basil) Extract and Its Active Constituents Modulate Cyclooxygenase-2 Expression in Neutrophils T
Total Page：16
File Type：pdf, Size：1020Kb
Food and Chemical Toxicology 124 (2019) 1–9 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Clinopodium vulgare L. (wild basil) extract and its active constituents modulate cyclooxygenase-2 expression in neutrophils T Kristiana M. Amirovaa, Petya Dimitrovab, Andrey S. Marcheva,c, Ina Y. Anevad, ∗ Milen I. Georgieva,c, a Center of Plant Systems Biology and Biotechnology, 4000, Plovdiv, Bulgaria b Department of Immunology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Georgi Bonchev Str., 1113, Sofia, Bulgaria c Group of Plant Cell Biotechnology and Metabolomics, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 139 Ruski Blvd., 4000, Plovdiv, Bulgaria d Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 1113, Sofia, Bulgaria ARTICLE INFO ABSTRACT Keywords: Clinopodium vulgare L. (wild basil) has a wide range of ethnopharmacological applications and accumulates a Wild basil broad spectrum of phenolic compounds, recognized for their anti-inflammatory and anticancer properties. The Clinopodium vulgare L. triggered cyclooxygenase-2 (COX-2) expression is creating an immunosuppressive microenvironment in the fi NMR pro ling inflamed tissue and considered to be the main cause of failure of even new anticancer-/immune-therapies. Phenolic compounds Nowadays, selective and novel plant-derived COX-2 inhibitors with safe profile are subject of profound research COX-2 interest. Neutrophils This study aimed to analyze the metabolic profile of C. vulgare and search for phenolic molecules with po- tential biological properties. By application of 1H and 2D-NMR (Nuclear Magnetic Resonance) profiling, caffeic, chlorogenic acids and catechin were identified along with a bunch of primary and secondary metabolites. Further, the biological effect of C. vulgare extract (CVE) and its constituents on zymosan-induced COX-2 ex- pression and apoptosis of murine neutrophils have been studied. The CVE, caffeic and chlorogenic acids in- hibited zymosan-induced COX-2 expression in bone marrow neutrophils, in vitro and in vivo activated. The ob- tained data indicate that CVE may have a good potential to manipulate neutrophil functions, however, its action may depend on the cellular state, the inflammatory milieu and the relative content of caffeic and chlorogenic acid in the extract. 1. Introduction concerning the molecular mechanisms and targets of the anti-in- flammatory and anticancer activity of CVEs referenced to specific mo- Clinopodium vulgare L. (wild basil; Lamiaceae) has diverse ethno- lecule or multiple constituents that formulate the activity of the ex- pharmacological applications and hence has been used for treatment of tracts. hemorrhagic disease, ulcer, diabetes, mastitis, prostatitis and skin in- Cyclooxygenases exist in two isoforms, and while COX-1 is con- flammation (Badisa et al., 2003). Contemporary studies revealed the stitutively expressed in the cells and has housekeeping functions, COX-2 multiple beneficial properties of C. vulgare aqueous or methanolic ex- is induced by inflammatory stimuli, which in turn accelerates the tracts (CVEs), i.e. anticancer, anti-inflammatory, DNA-protective, anti- synthesis of prostaglandins, and stimulates cancer cells proliferation oxidant and antibacterial properties (Burk et al., 2009). Comprehensive and their metastatic potential. Therefore, COX-2 is considered as a investigations accentuate on the selective and tissue specific anticancer molecular target for the development of novel and selective (natural) activity of the extracts against vast panel of human cancer cell lines anti-inflammatory drugs (Chen et al., 2019; Desai et al., 2018). Many (Badisa et al., 2003). However, to date, there is scarcity of information plant-derived molecules, such as caffeic and chlorogenic acid, as well Abbreviations: BM, Bone marrow; COX-1, Cyclooxygenase-1; COX-2, Cyclooxygenase-2; CVE, Clinopodium vulgare extract; HO-1, Heme oxygenase-1; iNOS, Inducible nitric oxide synthase; NF-kB, Nuclear factor-kappa B; NSAIDs, Non-steroidal anti-inflammatory drugs; Nrf2, Nuclear factor erythroid 2 p45-related factor 2; MAPK, Mitogen-activated protein kinase; MPO, Myeloperoxidase; Myd88, Myeloid differentiation primary response 88; NMR, Nuclear magnetic resonance; PGE2, Prostaglandin E2; TLR, Toll-like receptor ∗ Corresponding author. Center of Plant Systems Biology and Biotechnology, 4000, Plovdiv, Bulgaria. E-mail address: [email protected] (M.I. Georgiev). https://doi.org/10.1016/j.fct.2018.11.054 Received 22 September 2018; Received in revised form 21 November 2018; Accepted 24 November 2018 Available online 24 November 2018 0278-6915/ © 2018 Elsevier Ltd. All rights reserved. K.M. Amirova et al. Food and Chemical Toxicology 124 (2019) 1–9 as, catechin, were found to modulate COX-2 activity and immune re- 2.4. High performance liquid chromatography (HPLC) sponse in vitro and in vivo through suppression of phosphorylation of MAPKs, NF-kB p65 subunit and mRNA expression (Fechtner et al., Prior to analysis the pure compounds and the C. vulgare extract were 2017; Kulabas et al., 2018; Lee et al., 2018). dissolved in 50% aqueous methanol and filtrated through 0.45 μm The NMR-based metabolite profiling appeared an effective and syringe filters. The standard solutions were prepared at concentrations unbiased approach used to extract useful analytical data for particular from 5 to 80 μg/ml, while the extract was 5 mg/ml. Caffeic acid (purity molecules from the spectra of complex plant extracts (Kim et al., 2010; 99.9%) and chlorogenic acid (99.8%) were purchased from Sigma Wolfender et al., 2013). Applied as a holistic approach in order to (Sigma Aldrich, St. Louis, Mo, USA), while catechin (96.0%) was sup- distinguish the possible therapeutic agents in herbal medicine, up to plied from Fluka (Fluka AG, Buchs, Switzerland). date, this technique is implemented in the assessment and generation of The analyses were performed on HPLC system, consisting of Waters standardized biomarkers of pharmacologically active molecules essen- binary pump, Waters dual λ absorbance detector (Waters, Milford, MA, tial to ensure the quality, safety and reproducibility of the natural USA) and controlled by Breeze 3.30 software. The molecules elution ® products (Cerulli et al., 2018; Deborde et al., 2017). In our research was performed on a reverse-phase Kinetex C18, 100 Å (150 × 4.6 mm, group, NMR-based profiling and metabolomics have been intensively 5 μm) core-shell column (Phenomenex, Torrance, CA, USA), operating applied towards identifying specific marker compounds in wide variety at 26 °C. of medicinal plant species (Georgiev et al., 2015; Marchev et al., 2017a, Chlorogenic and caffeic acids were determined following the pro- 2017b; Zahmanov et al., 2015). cedure reported by Elmastaș et al. (2017), with some slight modifica- In this study 1H and 2D-NMR profiling of C. vulgare has been per- tions. The mobile phase consisted of acetonitrile (phase A) and water, formed. In order to reveal the anti-inflammatory potential, the effect of acidified with 0.1% formic acid (phase B) at flow rate of 1 ml/min, CVE, caffeic, chlorogenic acid and catechin on the inducible COX-2 using the following gradient: 0–5 min, A 5/B 95; 5–20 min, A 10/B 90; expression in neutrophils from healthy or zymosan-injected mice has 20–40 min, A 15/B 85; 40–45 min, A 5/B 95. Catechin was analyzed been thoroughly studied. using isocratic mobile phase – water: methanol: phosphoric acid, at ratio 85: 15: 0.1 (v/v/v) and a flow rate of 1 ml/min (Nishitani and ff 2. Material and methods Sagesaka, 2004). Chlorogenic and ca eic acid were detected at λ = 360 nm, while catechin was at λ = 210 nm. 2.1. Plant material 2.5. Animals The Clinopodium vulgare L. samples were collected in 2014 from Pirin Mountains (Bulgaria) at 1144 m a.s.l. (latitude: 41° 82′65.2´´N, All experiments were approved by the Animal Care Committee at ff fi longitude: 23° 37′85.1´´E). The plant species was identified by Dr. Ina the Stephan Angelo Institute of Microbiology, So a in accordance Y. Aneva. The collected plants were further frozen, freeze-dried (VirTis with the National (directive 20/01.11.2012), European rules (directive BenchTop Pro with Omnitronics™, Genevac Ltd., UK) and stored at 2010/63/EU) and designed on the basis of ARRIVE guideline for animal −20 °C prior to analyses. research. ICR mice were purchased from the Slivnitza Experimental Animal Laboratory (Slivnitza, Bulgaria). Animals were housed in spe- cific-pathogen-free (SPF) Animal Facility (license No 352/30.01.2012; 2.2. Preparation of Clinopodium vulgare L. extract registration No 11130005 issued by National Food Agency) at tem- perature 25 ± 2 °C, humidity 50–60%, 12 h light/dark cycle and fed Aerial parts of the plant were grounded and extracted, in triplicate, with a standard chow diet and water ad libitum. with 50% aqueous methanol (1:30 w/v), in an ultrasonic bath at 35 kHz The ICR mice (female, 6 week-old, 25–26 g) were intraperitoneally ® frequency (UCI-50 Raypa R. Espinar S.L., Barcelona, Spain) for 20 min (i.p.) injected with 1 mg/g body weight of zymosan A (ZY) from each, at ambient temperature. The combined extracts were filtrated and ® Saccharomyces cerevisiae (Sigma-Aldrich, Munich, Germany) or with evaporated till dryness under vacuum at 40 °C (IKA -Werke GmbH & equal amount of endotoxin-free phosphate buffer saline (PBS) in the Co. KG, Germany) and further used for HPLC analysis and biological control group. After 24 h mice were sacrificed and femur and tibia were activity studies. collected as described before (Benigni et al., 2017). 2.3. Nuclear magnetic resonance (NMR) spectroscopy 2.6. Preparation of bone marrow (BM) suspension and cell purification The NMR analysis followed the protocol, described by Georgiev Bone marrow was collected by flushing of femur and tibia of mice et al. (2015). The C. vulgare freeze-dried leaf samples (6 biological re- with endotoxin-free PBS containing 2 mM ethylenediaminetetraacetic plicates, 50 mg each) were grounded and placed in Eppendorf tubes.