*4.3. Animals*

Female BALB/c mice (aged 6–7 weeks) were purchased from Charles River Laboratories (Beijing, China). Six mice were randomly assigned to each experimental group and all mice were maintained in an automatic light/dark cycle (light periods of 12 h) environment and were acclimatized for 1 week before the experiment.

This experiment was performed in strict accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. All protocols were approved by the Animal Research Ethics Board of Jimei University (Xiamen, China, no. SCXK 2016-0006).

#### *4.4. Viridicatol Extraction, Isolation, and Purification*

On the basis of a previous method of fermentation, we defatted the crude extract with petroleum ether and CH2Cl2 by column chromatography over silica gel[29]. The MeOH layer was evaporated under reduced pressure to provide a defatted extract (55.4 g). The extract was then subjected to column chromatography on silica gel using a CH2Cl2-MeOH gradient (0→100%, 49 mm × 460 mm) to yield six fractions (Fr. 1−Fr.6). Fr. 5 (40.0 g) was separated by column chromatography over ODS (H2O-MeOH, 5→80%, 49 mm × 460 mm) to obtain 15 subfractions (sfrs. 5.1–sfrs. 5.15). Sfr. 5.15 was subjected to column chromatography on Sephadex LH-20 (MeOH) to obtain viridicatol (443.0 mg).

#### *4.5. Determination of Histamine and* β*-Hexosaminidase Release in RBL-2H3 Cells*

It has previously been reported that the level of β-hexosaminidase and histamine from RBL-2H3 cells activated by IgE–FcεRI complex were detected [45]. The cellular cytotoxicity of viridicatol was measured using MTT (3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide) assay.

#### *4.6. OVA-Induced Model of Food Allergy*

Groups of mice (each group of six) were sensitized with 2 mg alum and 100 μg OVA in 200 μL of phosphate-buffered saline (PBS) intraperitoneally (i.p.) on days 0 and 14. The mice were challenged with 50 mg OVA in 200 μL PBS by gavage every 3 days from days 28 to 40 for a total of 5 times. The mice in the treatment groups were treated with viridicatol by gavage every day starting on day 27. The mice in the PBS group were sensitized with 2 mg alum in 200 μL PBS and challenged with PBS, which served as the control group. The anti-allergic effect of viridicatol was determined on the basis of diarrhea, anaphylaxis, and rectal temperature [31]; mice were placed alone in cages, their feces were counted to calculate the proportion of loose stools in the total stools, and symptoms were scored from 0 to 5.

#### *4.7. Measurement of Immunoglobulins, Anaphylactic Mediators, and Cytokines in the Mouse Serum*

On day 41, the mouse eyeball sera were collected. OVA-specific IgE, IL-10, and TNF-α were measured by commercial ELISA kits. OVA-specific IgG1 and IgG2a were measured by an indirect ELISA, as previously reported [48]. Serum was obtained from the tail vein blood after 30 min of the final challenge on day 40 to detect the level of histamine and mMCP-1 using a commercial ELISA kit. All sera were stored at −80 ◦C.

#### *4.8. Morphology and Staining Analysis of the Intestinal Tissue*

On day 41, the mice were sacrificed, and the cecum tissue was collected for observation. The jejunum tissue was fixed, dehydrated, and coated with gold-palladium, and then the jejunum villi were observed by Phenom Pro Model desktop scanning electron microscopy (SEM, Phenom-world, Eindhoven, The Netherlands) as previously reported [48]. The jejunum tissue was fixed in 4% paraformaldehyde, and Servicebio was entrusted to conduct hematoxylin and eosin (H&E) staining and toluene blue staining for the jejunal tissues.

#### *4.9. Splenic Lymphocyte Population Analysis by Flow Cytometry*

As previously reported, single-cell suspensions were obtained on day 41 by density gradient centrifugation from the mouse spleen tissue suspension [45]. The cells were labeled with anti-CD3, anti-CD4, anti-CD19, anti-Foxp3, anti-FcεRI, and anti-c-KIT on the surface. Intracellular expression markers were stained with Foxp3-PerCP-Cy5.5 after fixing and permeabilizing as described in the protocol. The cells were gated and demonstrated by flow cytometry(Millipore, Billerica, MA, USA), and data were analyzed with a Guava easyCyte 6-2L system using Guava Soft 3.1.1 software.

#### *4.10. Release of* β*-Hexosaminidase and Ca2*+ *Influx of RBL-2H3 Cells Induced by A23187*

To measure the release of β-hexosaminidase induced by A23187, we inoculated RBL-2H3 cells in 48-well plates for 12 h and incubated them with or without viridicatol for 1 h. The cells were then stimulated with 2.5 μM A23187 for 15 min, after which β-hexosaminidase release was measured. The concentration of Ca2+ was measured using a Calcium kit-Fluo 3 according to the manufacturer's instructions. RBL-2H3 cells (5 × 10<sup>4</sup> in 100 μL) were seeded into a black 96-well plate and cultured for 16 h. The cells were then washed and incubated with Fluo-3 AM (10 μM) for 1 h. Next, the cells were washed and treated with viridicatol (2.5, 5, 10 μg/mL) in HBSS for 1 h. After we stimulated it with A23187 (2.5 μM), the fluorescent intensity was immediately monitored using the microplate reader at an excitation wavelength of 490 nm and an emission wavelength of 530 nm every 30 s. [Ca<sup>2</sup>+]i was calculated as follows: [Ca<sup>2</sup>+]i (nm) = Kd [(F − Fmin)/(Fmax − F)], Fmin: the background fluorescence of 5 mM Ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA), Fmax: the maximum fluorescence with of 0.1% Triton X-100, Kd: the dissociation constant of Ca2+ and Fluo-3 (450 nM). Moreover, the cells were photographed with a fluorescence microscope (Echo, San Diego, CA, USA) to observe the variation of intracellular Ca2+.
