Elsevier

Food and Chemical Toxicology

Volume 125, March 2019, Pages 161-169
Food and Chemical Toxicology

Nigella damascena L. essential oil and its main constituents, damascenine and β-elemene modulate inflammatory response of human neutrophils ex vivo

https://doi.org/10.1016/j.fct.2018.12.057Get rights and content

Highlights

  • Nigella damscena essential oil, damascenine and β-elemene modulate the response of LPS-stimulates human neutrophils ex vivo.

  • Damascenine was isolated using fast and efficient high performance countercurrent chromatography technique.

  • Detailed spectroscopic characteristic of damascenine was provided for the first time.

Abstract

Nigella damascena L belongs to Ranunculaceae family and is mentioned in Eastern traditional medicine for the treatment of high temperatures, regulation of menstruation or catarrhal affections. The anti-inflammatory activity of compounds present in the essential oil obtained from seeds of this plant can be found in literature, however no studies on immunomodulatory activity are provided. Hence, in this work anti-inflammatory activity of N. damascena seed essential oil as well as damascenine and main compound β-elemene was evaluated on ex-vivo lipopolysaccharide (LPS)–stimulated human neutrophils. For isolation of damascenine fast and efficient protocol was elaborated using high performance countercurrent chromatography technique for the first time. Also detailed spectroscopic characteristic of damascenine was provided for the first time. Damascenine was separated from the essential oil in a mixture of petroleum ether/acetonitrile/acetone (2:1.5:0.5 v/v/v) in reversed phase mode in 12 min with 99.47% purity. Essential oil, damascenine and β-elemene presented immunomodulatory activity evaluated in LPS–stimulated neutrophils ex vivo. All studied samples significantly inhibited release of interleukin 1 beta (IL-1β) and interleukin 8 (IL-8). What is more, damascenine and β-elemene decreased matrix metallopeptidase 9 (MMP-9) production similar to dexamethasone. The release of tumor necrosis factor (TNF-α) was also inhibited in all range of concentrations, however the activity was weaker then activity of dexametasone. The previously reported anti-inflammatory activity of damascenine and β-elemene investigated in murine models was confirmed in our study on human neuthrophils suggesting their possible strong inhibitory effect on inflammatory response progression.

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

References (37)

  • S. Patra et al.

    Anti-inflammatory effects of royal poinciana through inhibition of toll-like receptor 4 signaling pathway

    Int. Immunopharm.

    (2016)
  • E. Sieniawska et al.

    Carrot seedless entail oil—source of carotol and cytotoxicity study

    Ind. Crop. Prod.

    (2016)
  • K. Skalicka-Woźniak et al.

    Comprehensive classification of solvent systems used for natural product purifications in countercurrent and centrifugal partition chromatography

    Nat. Prod. Rep.

    (2015)
  • R. Zhang et al.

    Downregulation of IL-17 and IFN-gamma in the optic nerve by beta-elemene in experimental autoimmune encephalomyelitis

    Int. Immunopharm.

    (2010)
  • A.N.M. Alamgir

    Therapeutic use of medicinal plants and their extracts: volume 1: pharmacognosy

  • H. Bekemeier et al.

    Antipyretic, antiedematous and analgetic effects of damascenine in comparison with acetylsalicylic acid and phenylbutazone

    Arch. Int. Pharmacodyn. Ther.

    (1967)
  • H. Bekemeier et al.

    Ethoxose edema of the rat paw and its influencing by acetylsalicylic acid, damascenine, phenylbutazone and salicylamide

    Acta Biol. Med. Ger.

    (1967)
  • U. Boni et al.

    Scoprire, riconoscere, usare le erbe

    (1977)
  • Cited by (21)

    • The ethnobotany, phytochemistry, and biological properties of Nigella damascena – A review

      2022, Phytochemistry
      Citation Excerpt :

      Traditionally, in Tunisia and Italy, N. damascena is used to treat trachoma, hypertension, and cardiac diseases (Leporatti and Ghedira, 2009). Furthermore, other authors have reported its use as an antihelmintic for children and as a treatment for haematuria, skin diseases such as itchiness and eczema (Geraci et al., 2018), and tapeworm (Sieniawska et al., 2019). Our research on the chemical compounds present in N. damascena has led to the formation of two main classes, namely alkaloids and terpenes, which are divided into diterpenoids, triterpenoids, flavonoids, and aromatic compounds.

    • The essential oil of the leaves of Verbesina macrophylla (Cass.) S.F.Blake has antimicrobial, anti-inflammatory and antipyretic activities and is toxicologically safe

      2021, Journal of Ethnopharmacology
      Citation Excerpt :

      The essential oils or their constituents can act as immune-modulating of the inflammatory process under macrophages, natural killer cells (NK) and T and B lymphocytes, production of cytokines, chemokines and interleukins, among other mechanisms, becoming potential sources of new compounds treatments for regulation of persistent stimuli that can lead to autoimmune diseases, allergies, arthritis, cancers and atherosclerosis (Gandhi et al., 2019; Anastasiou and Buchbauer, 2017; Han et al., 2017). Effects of anti-inflammatory potential with immune-modulation of the process can be observed in investigations such as a study with essential oil of Nigella damascena which presented anti-inflammatory activity by modulating the release of pro-inflammatory levels of TNF-α, IL-1β, IL-8 (Sieniawska et al., 2019). The essential oil of Artemisia princeps and its main constituents eucalyptol and α-terpineol inhibit the expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and the nuclear transcription factor κappa B (NF-kB), in addition to increasing the expression of the anti-inflammatory cytokine IL-10 (Trinh et al., 2011).

    • Inhibition of the pro-inflammatory mediators in rat neutrophils by shanzhiside methyl ester and its acetyl derivative isolated from Barleria prionitis

      2020, Journal of Ethnopharmacology
      Citation Excerpt :

      LPS (100 ng/mL)-stimulated neutrophils were considered as stimulated control (st. control) cells. Indomethacin (50 μg/mL) was used as a standard in the MMP-9 assay while dexamethasone (10 μg/mL), an immunosuppressant drug, according to Sieniawska et al. (2019), was used as a standard control in the IL-8 and TNF-α assays. The results are expressed as the percent release of enzyme in rat neutrophils.

    View all citing articles on Scopus
    View full text