Elsevier

Fish & Shellfish Immunology

Volume 74, March 2018, Pages 76-83
Fish & Shellfish Immunology

Full length article
Alpinone exhibited immunomodulatory and antiviral activities in Atlantic salmon

https://doi.org/10.1016/j.fsi.2017.12.043Get rights and content

Highlights

  • Alpinone and Pinocembrine were isolated and purified from resinous exudates of Heliotropium.

  • Immune effects of Alpinone and Pinocembrine were studied in the kidney of Atlantic salmon.

  • Pinocembrine reduced in vivo the expression of TNF-α, IL-1, IL-12, IL4/13A and TGF-β1.

  • Alpinone increased in vivo the expression of TNF-α, IL-1, IFN-α, IFN-γ and TGF-β1.

  • Alpinone showed in vitro antiviral activity against Infectious Salmon Anaemia virus.

Abstract

In this study, we seek to identify flavonoids able to regulate the gene expression of a group of cytokines important for the control of infections in Atlantic salmon (Salmo salar). Particularly, we studied the potential immunomodulatory effects of two flavonoids, Alpinone and Pinocembrine, which were isolated and purified from resinous exudates of Heliotropium huascoense and Heliotropium sinuatum, respectively. The transcript levels of TNF-α and IL-1 (inflammatory cytokines), IFN-γ and IL-12 (T helper 1 type cytokines), IL4/13A (Th2-type cytokine), IL-17 (Th17 type cytokine) TGF-β1 (regulatory cytokine) and IFN-α (antiviral cytokine) were quantified by qRT-PCR in kidneys of flavonoid-treated and control fish. We demonstrated that the administration of a single intramuscular dose of purified Alpinone increased the transcriptional expression of five cytokines, named TNF-α, IL-1, IFN-α, IFN-γ and TGF-β1 in treated fish compared to untreated fish. Conversely, administration of purified Pinocembrine reduced the transcriptional expression of TNF-α, IL-1 and IL-12 in the kidney of treated fish. No other changes were observed. Interestingly, Alpinone also induced in vitro antiviral effects against Infectious Salmon Anaemia virus. Results showed that Alpinone but not Pinocembrine induces the expression of cytokines, which in vertebrates are essential to control viral infections while Pinocembrine reduces pro-inflammatory cytokines. Altogether results suggest that Alpinone is a good candidate to be further tested as immunostimulant and antiviral drug.

Introduction

Viral diseases result highly detrimental within intensive aquaculture because they cause significant mortalities with negative economical and social repercussions. Some of the most common viral diseases in the world farmed salmon production are Infectious Pancreatic Necrosis (IPN), Pancreatic Disease (PD), Infectious Haemorrhagic Necrosis (IHN), Viral Haemorrhagic Septicaemia (VHS), and Infectious Salmon Anaemia (ISA) (Crane) [1]. At present, vaccines for preventing fish viral diseases are limited and their efficacy is not satisfactory [2]. Furthermore, there are no effective drugs to treat fish virus disease in aquaculture therefore finding new anti-viral agents is an actual demand in this field [3].

Natural products have been and still are a great source of drugs. As they exhibit a wide array of compounds of unique chemical structures hard to produce in other ways, there is a greater chance that some of them fit the structural necessities to address the enormous diversity of biological targets [[4], [5], [6]]. Many screening efforts have been made to find anti-viral agents from natural sources not only acting on a particular viral target of one virus but also having broad-spectrum effects [4,6]. Some of this kind of anti-viral agents can be found among those molecules able to induce protective immune mechanisms in the host.

For the last 20 years, we have been isolating and characterizing several natural compounds obtained from the resin of species of the genus Heliotropium, section Cochranea [7]. The species of section Cochranea are restricted to the arid and hyperarid zones of the Atacama and Peruvian Deserts [7] therefore they are exposed to extreme environmental factors. These species produce a resinous exudate as a defense mechanism against environmental effects that covers their leaves and stems. The resin mostly contains flavonoids along with aromatic geranyl derivatives in minor quantities [[8], [9], [10], [11]]. Previous studies have shown that the components of the resin have chemical and biological properties, i.e., antioxidant, antimicrobial, antiviral and anti-fungal, which increase the resistance of the plants [[8], [9], [10], [12], [13], [14], [15], [16], [17], [18]] along with immunomodulatory effect [19] at non-cytotoxic concentrations. Flavonoids are natural compounds with a basic C6-C3-C6 structure containing two aromatic rings linked by an oxygenated heterocyclic ring [20] and have a wide range of beneficial health effects, i.e., they show antibacterial, antioxidant and anti-inflammatory activities [20]. Flavonoids isolated from the resin of H. sinuatum i.e., Naringenin, 7-O methyleriodictyol, Pinocembrin, Hesperidin and 3-O- methylgalangin inhibit the bacterial growth of E. coli and phytopathogenic bacteria [18,21], Naringenin and 3-O-methylgalangin showed bactericidal activity against P. salmonis, the bacteria that causes the highest mortality for infections in salmon farming [16], while 7-O-methyleriodictyol showed antiviral activity against ISAV, the virus responsible of the crash of Chilean salmon culture in 2007 [17]. Alpinone is also a flavonoid that we have obtained from sticky resinous exudate of Heliotropium huascoense. It is a flavanonol with hydroxyl substituent at positions 3 and 5 and methoxylated at position 7. There is little information about the chemical and biological properties of Alpinone. Some research shows that Alpinone has moderate antioxidant activity against ABTS radical cations [22] and induces maturation in dendritic cells, corresponding to the first stages of immune activation in higher vertebrates (Patent GB201410879A).

In this study, we seek to identify flavonoids able to regulate gene expression of a group of cytokines important for the control of viral infections. In mammals, cytokines are key secreted proteins regulating the immune system, they are able to activate inflammatory and regulatory signals, they regulate antigen-presenting cells (APC) and also play a role on the effector functions of T and B cells for anti-microbial responses [23]. In fish, many cytokines of the network have been described and so far functional studies indicate conserved roles in teleost immune responses [24]. For instance in salmonids, the type I interferons show antiviral activity and induce antiviral effectors such as Mx and ISG15 [25,26] and the type II interferon (IFN-γ) enhance antigen presentation and promote Th1 cytokine expression [27] just as in superior vertebrates. Thus we guess that flavonoids able to induce interferons and Th1 type cytokines will have anti-viral activity probably of broad-spectrum effects. Particularly, we here study the modulatory effects of Alpinone and Pinocembrine on the transcriptional expression of interferon, Th1 type cytokines and inflammatory cytokines of Atlantic salmon (Salmo salar). Potential antiviral effects were assayed in vitro against ISA virus.

Section snippets

Extraction and isolation of flavonoids

Heliotropium huascoense Johnston and Heliotropium sinuatum belong to the family Heliotropiaceae section Cochranea. The samples were collected in III region, Chile during the flowering season. A voucher specimen was deposited in the Herbarium of the Faculty of Biological Science of the Catholic University of Chile (ST-2580 and ST-2563 respectively). The resinous exudates were extracted by immersion of 900 g of fresh complete plant material (stems, leaves and flowers) in dichloromethane (CH2Cl2)

Immunomodulatory effects of flavonoids obtained from Heliotropium

Here we tested two flavonoids named Alpinone and Pinocembrine (Fig. 1) for the ability to induce the gene expression of cytokines in Atlantic salmon. Expression was examined in the kidney, the largest lymphoid organ of teleost fish. Fish were intramuscularly injected with 100 μg of the flavonoids and the transcript levels of cytokines were determined after 48 h post treatment, time point chosen based on previous results [19,32]. The cytokines tested were TNF-α and IL-1, which are

Discussion

In this study, we demonstrated that the administration of a single dose of Pinocembrine to Atlantic salmon reduced the transcriptional expression of pro-inflammatory cytokines TNF-α, IL-1 and IL-12 beta-like chain in the kidney. TNF-α and IL-1 fish paralogues exhibit similar bioactivity to that previously ascribed in superior vertebrates. In addition, TNF-α and IL-1 have overlapping functions and they are two of the early inflammatory cytokines expressed during infections [24]. The lack of the

Acknowledgement

FONDECYT1140261 and 1161015, Basal USA 1555, Basal USA 1298, Basal USA 1498 financed this work. We also thank Carolina Espinoza and Carolina Camus for outstanding technical assistance.

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