**3. Discussion**

Due to their extreme environments, deep-sea fungi produce secondary metabolites with novel structures and potent bioactivities [21]. Recently, some secondary metabolites from deep-sea-derived fungi have been discovered to possess biological activities; however, they were poorly studied and seldom utilized. We have found that butenolides, polyketides, and penigrisacids have significant anti-allergic activities [27,29]. Unfortunately, few compounds have been evaluated in mouse models to further elucidate the associated mechanisms in vivo. In this study, viridicatol, a type of quinolone alkaloid, was reported as having anti-tumor activities [30]. However, the anti-allergic activity of viridicatol has not been reported. In this study, viridicatol was found to exhibit significant inhibitory effects on RBL-2H3 cell degranulation. In an OVA-induced mouse model, viridicatol significantly alleviated the allergic symptoms of diarrhea and hypothermia.

Food allergy is accompanied by immune disorders, which involves a variety of immune cells and cytokines [35]. In our study, treatments with viridicatol led to a moderate reduction in the population of splenic B cells, and may contribute to the reduction of serum antigen-specific IgE, which is involved in the process of MC and basophil activation [10]. To some extent, viridicatol was able to upregulate the population of Tregs, which is consistent with increased levels of IL-10. Tregs and the associated cytokine, IL-10, was reported to maintain immune balance and regulate MC activation [36]. MCs are the effector cells involved in inflammation and anaphylaxis, playing a crucial role in allergic reactions [13]. Notably, viridicatol decreased the population of MCs in a dose-dependent manner. Meanwhile, viridicatol could significantly decrease the concentration of serum mMCP-1, TNF-<sup>α</sup>, and histamine, which was related to a reduction of MC numbers and the inhibition of MC degranulation. On the basis of the results of the OVA-induced model of food allergy, viridicatol was

demonstrated to have a stronger effect on MCs than B and Treg cells. Thus, viridicatol may relieve food allergy in mice by decreasing the number and inhibiting the degranulation of MCs.

Previous studies have demonstrated that the occurrence of food allergy is related to damage of intestinal barrier function [37]. The intestinal barrier can be divided into mechanical and immune barriers. Moreover, food allergy can influence intestinal barrier function, which can also enhance the speed of food allergy developments [8]. In this study, viridicatol was found to significantly repair the intestinal barrier by reducing the damage of jejuna tissue villi and alleviating the degree of tissue inflammation. MCs residing in the mucosa of the gastrointestinal tract, as the center of the intestinal immune network, are not only limited to the antigen response, but are also involved in the regulation of the intestinal epithelial barrier and transport, changes in mucosal functionality, and production or amplification of signals to other cells [37]. Following treatments with viridicatol, the length of the cecum was significantly reduced. The activation of MCs resulted in diarrhea, which was likely a part of the mucosal defense response [38]. Bischoff et al. demonstrated that histamine plays a multifunctional role in the intestinal barrier [38]. In addition, Jacobs et al. found that MCs released proteases and TNF-<sup>α</sup>, which resulted in tight junction protein rearrangemen<sup>t</sup> and increased the colic epithelium permeability and intestinal inflammation [39]. The level of mMCP-1 in the blood reflected the extent of degranulation by mast cells in the local tissue [40]. Viridicatol reduced the number of MCs in the jejunum tissue, as well as inhibited MC degranulation and the release of mMCP-1, TNF-<sup>α</sup>, and histamine, which may be important to the repair of the intestinal barrier following exposure to a food allergen.

At the intersection of many classical signaling pathways, Ca2+ converts the received extracellular signals into intracellular signals and regulates lymphocyte activation, differentiation, and various transcriptional processes [41]. Ca2+ influx is considered to be a necessary condition for triggering immune function and a key factor for MC activation [42,43]. Ca2+ promotes MC degranulation, mediator release, and the activation of gene expression to accelerate a subsequent immune response [44]. Viridicatol was found to suppress Ca2+ influx, which was closely associated with MC degranulation. Jacobs et al. suggested that the destruction of the intestinal barrier was accompanied by MC degranulation and increased Ca2+ [42]. Therefore, the inhibitory effects of viridicatol on MC degranulation and Ca2+ influx may be beneficial for intestinal barrier repair and for relief of food allergy symptoms.

Many anti-allergic drugs have some undesirable adverse effects such as dizziness and drowsiness. Thus, the anti-allergic activities of natural sources have attracted our attention due to their low side effects. As reported, terpenoids, flavenoids, and alkaloids have significant anti-allergic activities and different mechanisms of action [29,45,46]. Alkaloids are nitrogen-containing compounds that have many biological activities, including antiviral, anti-inflammation, and anti-allergy properties. Costa pointed that alkaloids may be associated with the inhibition of eosinophil and mast cell activities [46]. Viridicatol is a quinoline alkaloid that may be absorbed more readily due to the small molecular weight (253.26). Moreover, viridicatol was isolated from the deep-sea fungus *Penicillium griseofulvum*, which produces a variety of anti-allergic metabolites [29]. In this study, viridicatol inhibited the degranulation of RBL-2H3 cells and relieved the allergic reactions in mice, which may be related to its structure and thus it is worth further exploring. The bioactivity of these compounds is closely related to their structures. For example, the number and position of hydroxyl groups have been shown to be associated with anti-allergic activities [45]. Moreover, both in antigen-induced and A23187-induced RBL-2H3 cell degranulation models, the inhibitory effect of viridicatol on β-hexosaminidase were stronger than viridicatin, which may due to the presence of an extra phenolic hydroxyl. Interestingly, no significant effect of 3-*O*-methylviridicatin on the release of β-hexosaminidase were observed, which may have resulted from the different chemical structure of 3-*O*-methylviridicatin compared with viridicatol: one lost hydroxyl in ring C and one hydroxyl was replaced by a methoxy in ring B. Phenolic hydroxyl groups have been reported to contribute to the increased efficiency of these compounds [47].

Taken together, deep-sea-derived viridicatol could relieve OVA-induced allergic symptoms. Treatments with viridicatol had significant e ffects on decreasing the level of anti-OVA-IgE, histamine, mMCP-1, and TNFα. Moreover, viridicatol significantly upregulated the level of IL-10 in the serum. Viridicatol treatment was also found to downregulate the populations of B cells and MCs in the spleen, as well as upregulate Tregs in the spleen. Additionally, viridicatol repaired the intestinal barrier and alleviated the degree of tissue infiltration. Notably, viridicatol significantly suppressed MC degranulation in the jejunum of mice, which may be attributable to the decreased intracellular flow of Ca2+ in MCs. Compared to many alkaloids, viridicatol has a relatively small molecular weight, allowing it to be absorbed more readily, and it is expected to be stabilizing for MCs, which could possess a higher practical value. In conclusion, this research provided some insight into the prevention of food allergy and the application of marine fungi. Marine fungi have a large impact in terms of application in the pharmaceutical industry, since many of their metabolites have entered the clinical pipeline and have the potential of being exploited as novel drugs. The present findings demonstrated that viridicatol have the potential to be applied in the therapy of food allergies. Further systematic safety assessments and optimal dosage determination are necessary for the clinical application of viridicatol.

#### **4. Materials and Methods**

#### *4.1. Reagents an General Experimental Procedures*

Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). Roswell Park Memorial Institute (RPMI) 1640, Eagle's minimum essential medium (EMEM), penicillin–streptomycin solution, and trypsin 0.25% (1×) solution were purchased from HyClone Co. (Logan, UT, USA). Imject alum was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Evans Blue, anti-dinitrophenyl (DNP)-IgE, ovalbumin (OVA), calcium ionophore A23187, and 4-methyl-umbelliferyl-N-acetyl-β-D-glucosaminide were obtained from Sigma-Aldrich (St Louis, MO, USA). DNP-bovine serum albumin (BSA) was purchased from Biosearch (Petaluma, CA, USA). Goat anti-mouse IgG1 (ab98693) and IgG2a (ab98698) antibodies were purchased from Abcam (Cambridge, United Kingdom). Calcium kit-Fluo 3-AM was purchased from Dojindo Laboratories (Mashiki, Japan). ELISA (enzyme-linked immunosorbent assay) kits for histamine (EHP184), anti-OVA-IgE (500840), and IL-10 (BMS614INST) were obtained from IBL (Hamburg, Germany), Cayman (Ann Arbor, MI, USA), and eBioscience (San Diego, CA, USA), respectively. Antibodies for flow cytometry analysis were obtained from BioLegend (San Diego, CA, USA) and BD (New York, NY, USA). P-phycoerythrin (PE) anti-mouse CD117(c-Kit) (105807), Allophycocyanin (APC) anti-mouse FcεRI α (134316), PE anti-mouse CD19 (115508), Mouse Th/Treg Phenotyping Kit (560767) were also used. All other chemicals were of analytical grade. The HRESIMS spectrum was recorded on a Waters Xevo G2 Q-TOF mass spectrometer (Waters Corporation, Milford, MA, USA). The NMR spectra were recorded on a Bruker 400 MHz spectrometer (Bruker, Fällanden, Switzerland). The single X-ray crystal data were recorded with an XtaLAB AFC12 Kappa single di ffractometer using Cu K α radiation (Rigaku, Japan). Copies of the data can be obtained, free of charge, on application to Cambridge Crystallographic Data Center (CCDC), 12 Union Road, Cambridge CB21EZ, United Kingdom, (fax: +44(0)-1233-336033; email: deposit@ccdc.cam.ac.uk.
