Nigella damascena L. essential oil and its main constituents, damascenine and β-elemene modulate inflammatory response of human neutrophils ex vivo
Introduction
Inflammation is a complex, protective and non-specific immune response to different type of harmful factors. Pathogens, irritants or damaged tissues induce inflammatory process which under normal conditions is self-limiting (Ferrero-Miliani et al., 2007). A fever, hypotension and the release of adrenocorticotrophic hormone are the first symptoms of inflammation mediated by soluble proinflammatory cytokine interleukin 1 beta (IL-1β), which is released mainly by neutrophils and monocytes (Ferrero-Miliani et al., 2007). Neutrophils infiltrate infected or damaged tissue migrating through basement membranes via action of elastase and matrix metallopeptidase 9 (MMP-9) (Delclaux et al., 1996). MMP-9 also activates IL-1β and cleavage of several chemokines (Opdenakker et al., 2001). On site, neutrophils release chemokines and cytokines (interleukin 8, IL-8; IL-1β) enhancing inflammatory response of other white blood cells, which overstimulated initiate chronic inflammation (Czerwińska et al., 2018). To prevent development of prolonged inflammation and diseases related to inflammatory conditions the use of natural substances mitigating inflammation is of high importance.
Nigella damascena L belongs to Ranunculaceae family and is mentioned in Eastern traditional medicine for the treatment of high temperatures, regulation of menstruation, catarrhal affections, as a diuretic agent and against tapeworm (Alamgir, 2017; Boniand Patri, 1977; Fico et al., 2004; Fournier, 1948). The plant produces aromatic compounds which are stored especially in the seeds and can be obtained as essential oil. Damascenine and β-elemene are major constituents of N. damascena L. seeds essential oil (Fico et al., 2003; Sieniawska et al., 2018; Wajs et al., 2009). Current literature on activity of essential oil is limited to antimicrobial and molluscicidal assays (Fico et al., 2004; Sieniawska et al., 2018) however damascenine was studied for analgesic, antipyretic, anti-inflammatory and antiedematous effects (Bekemeier et al., 1967; Bekemeier and Schmollack, 1967). What is more, 3-hydroxyanthranilic acid, a precursor of damascenine in plants (Robinson, 1968), induced effectively the expression of hemeoxygenase-1, an antioxidant enzyme with anti-inflammatory and cytoprotective properties (Krause et al., 2011). Also β-elemene was recently proved to attenuate the lipopolysaccharide-induced murine macrophage activation and proinflammatory factors production (Fang et al., 2018). β-elemene treatment reduced inflammation (Patra et al., 2016) and suppressed the inflammation in experimental autoimmune encephalomyelitis of optic nerve in mice models (Zhang et al., 2010). Nonetheless, the anti-inflammatory activity of N. damascena essential oil and its main constituents damascenie and β-elemene was not studied in human neutrophils yet.
Damascenine is a methylated derivative of 3-hydroxyantranilic acid. So far, this compound was isolated using laborious procedure described in 1912 by Ewins. This included several hours of extraction of seeds, extract treatment with 5% solution of hydrochloric acid, shaking with kerosene multiplexer, basification of aqueous layer with ammonia and additional extraction into petroleum ether (Ewins, 1912). In 2004 another time consuming method was applied. Damascenine was obtained from imbibition water from seeds. The water was extracted repeatedly with butanol and butanolic extract was chromatographed on Sephadex LH-20 yielding numerous fractions which were then combined and rechromatographed by reversed phase high performance liquid chromatography (RP-HPLC) (Fico et al., 2004). In this study, for a separation of damascenine the modern high performance counter-current chromatography (HPCCC) technique was applied. HPCCC principle utilizes the continuous liquid-liquid extraction in a solvent system composed from two immiscible phases. Compounds partition between stationary and mobile phase pumped into the column in opposite directions. This can be obtained due to centrifugal force which ensure dynamic mixing between both phases and providing good retention of a high amount of the stationary phase. HPCCC is time-saving and lossless technique, where separation lasts from several minutes to 3–4 h and substances retained in a stationary phase can be fully recovered by evaporation of the solvent (Guzleket al., 2009; Skalicka-Woźniak and Garrard, 2015).
In order to evaluate possible mechanism of anti-inflammatory activity of N. damascena seed essential oil as well as damascenine and main compound β-elemene on ex-vivo LPS–stimulated human neutrophils, fast and efficient method for isolation by HPCCC technique was performed for the first time.
Section snippets
Chemicals
Analytical grade acetonitrile, acetone and petroleum ether were purchased from Polish Chemical Reagents (POCH, Poland), while acetonitrile, water and formic acid for LC-MS from J.T. Baker Chemicals (Witko, Poland). Helium 5.0 with 99.999% purity was provided by (PGNiG, Poland). F-MLP (N-formylmethionyl-leucyl-phenylalanine), Hanks’ balanced salt solution (HBSS), L-glutamine, fetal bovine serum (FBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), propidium iodide (PI), 3% dextran
Isolation and identification of damascenine
As the best separation of the compound can be obtained when partition coefficient value (Kval) is in a range between 0.5 and 2.0 (Skalicka-Wozniak and Walasek, 2014), series of solvent systems were tested. The satisfactory Kval was noticed when the mixture of petroleum ether/acetonitrile/acetone in the ratio 2:1.5:0.5 was applied (Table 1). Lower Kvals were determined for solvent systems 1 and 2, however solvent system 2 was not stable in the temperature above 23 °C and become a single phase.
Discussion
The fast, selective and effective separation of natural compounds is of high importance for their availability for biological testing. The information about biological activity of damascenine is very poor, most probably because of lack of method for its efficient separation. Hence, in this study, we elaborated the effective protocol for fast isolation of this compound. The HPCCC technique was proved to be the efficient and simple, one step separation of damascenine. 71% of damascenine present
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