Antiallergic Activity of the Wild Mushrooms of Nepal and the Pure Compound Hispidin

Bioactive compounds in mushrooms, such as polysaccharides, terpenes, and phenolic compounds, are known to have anti-inflammatory ¹ and anti-allergic activities. Several mushroom species, including Ganoderma lucidum ² and Inonotus obliquus sclerotia ³ and mycelia, ⁴ have been found to suppress the release of proinflammatory mediators responsible for allergy. The increasing trend of allergic diseases, and the adverse effects caused by the long-term usage of some currently available antiallergic agents such as corticosteroids and decongestants, necessitates more effective and safer alternatives. ^5,6

Ethanol extracts of 90 mushroom samples were investigated in this study. The botanical origin, location, habitat, and extract preparation of the samples have been described in previously published reports. ^7,8 The suppression of β-hexosaminidase release (BHR) in calcium ionophore A23187-stimulated rat basophilic leukemia-2H3 (RBL-2H3) cells was used as the indicator of antiallergic activity. Antiallergic activity was tested according to a previously described method, with slight modifications. ⁹ Briefly, 40-h cultures of RBL-2H3 cells (seeded at 5 × 10 ⁴ cells per well in a 96-well plate) were treated with 0.5 μL of test samples or positive control (quercetin dihydrate at 12.5 μg/mL) or negative control (dimethyl sulfoxide [DMSO]) for 1 h. It was followed by stimulation of cell degranulation by the addition of 100 μL of 50 μg/mL of calcium ionophore A23187. The BHR was calculated as the percentage release compared with cells treated with DMSO. Also, cell viability (CV) was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, according to a previously described method. ¹⁰ The 50% inhibitory concentration (IC₅₀) values were investigated for 10 samples exhibiting the least BHR, while maintaining high CV. All assays were conducted at least three times, and the results are expressed as mean ± standard deviation.

The plot of the BHR percentage and CV percentage of the ethanol extracts of 90 samples dissolved in DMSO and tested at their maximum dissolved concentration (50–200 μg/mL) is provided in Figure 1; and the percentage values are provided as Supplementary Data in Supplementary Table S1. The samples were divided into four groups, based on their taxonomic order as Hymenochaetales, Polyporales, Agaricales, and Others. Since most of the samples fell into the first three groups, the few remaining samples were grouped together as “Others.” The positive control, quercetin dihydrate, showed a BHR of nearly 30%, with no cell cytotoxicity. Therefore, samples with BHR of <30% and CV of more than 90% were targeted for further investigation. As for the mushroom extracts, the Hymenochaetales group had the largest numbers of high antiallergic samples; with 6 of 20 samples showing a BHR of <30% while maintaining an almost 100% CV. Phellinus adamantinus showed the least BHR with only 19.4%, followed by Phellinus sp. 2 with 20.1% and Phellinus conchatus 1 with 21.6%. Also, I. clemensiae followed quite closely with a BHR of only 22.8%. The major bioactive compounds in Phellinus sp. are polysaccharides, flavones, pyranones, furans, terpenes, and steroids. ¹¹ Several researchers have demonstrated the antiallergic activity of Phellinus sp., using a variety of methods especially those involving RAW264.7 macrophages ¹² and animal models. ¹³

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FIG. 1.

The plot of BHR percentage and CV percentage of the ethanol extracts of 90 wild mushroom samples on RBL-2H3 cells. The extracts were tested at their maximum dissolved concentration (50–200 μg/mL). The top left corner of the plot region represents the target area with the highest antiallergic samples with BHR <30% and CV above 90%. 1. Inonotus clemensiae, 2. Inonotus sp. 2, 3. Phellinus conchatus 1, 4. Phellinus conchatus 2, 5. Phellinus adamantinus, 6. Phellinus sp. 2, 7. Ganoderma lingzhi 3, 8. Ganoderma endochroum, 9. Ganoderma sp. 1, 10. Mycorrhaphium sp. PC is positive control (quercetin dihydrate at 12.5 μg/mL). BHR, β-hexosaminidase release; CV, cell viability; RBL-2H3, rat basophilic leukemia-2H3.

Large variations were seen in the BHR percentage within the Polyporales group, with only 4 of 39 samples showing <30% BHR. Ganoderma lingzhi 3 showed the least BHR of 16.7%, which is also the least value among all the samples tested. It was followed by Mycorrhaphium sp. with 20.3%. Ganoderic acids C and D, isolated from the methanolic fraction of G. Lucidum, have been reported to inhibit the release of histamine from rat mast cells. ¹⁴ None of the 18 samples in the Agaricales group, and only one of the 13 samples in the Others group, Xylobolus princeps 4 fell within the target group, with a BHR of 27.0% and a near 90% CV.

The IC₅₀ values of all the tested samples are provided in Table 1. The samples included six samples from Hymenochaetales (I. clemensiae, Inonotus sp.2, P. conchatus 1 and 2, P. adamantinus, and Phellinus sp.2) and four samples from Polyporales (G. lingzhi 3, Mycorrhaphium sp., G. endochroum, and Ganoderma sp.1). Also, hispidin (Fig. 2), which is the major bioactive compound in I. clemensiae, ¹⁵ was analyzed for its IC₅₀ value, along with the positive control quercetin dihydrate. The anti-inflammatory activity of hispidin has been attributed to the suppression of reactive oxygen species-mediated nuclear factor kappa B pathway in RAW 264.7 macrophage cells. ¹⁶ However, this is the first report on the antiallergic activity of the compound on RBL-2H3 cells. Although the positive control quercetin dihydrate required a minimal concentration of 2.54 μg/mL, among the samples tested, I. clemensiae and P. adamantinus required the least concentrations of 51.24 and 50.65 μg/mL, respectively, for 50% BHR. Interestingly, the major active compound in I. clemensiae, hispidin, required a higher concentration of 82.47 μg/mL to achieve 50% BHR, which indicates that either the compound is more stable and bioactive within the sample matrix or there is the presence of some minor compounds in the extracts enhancing the antiallergic activity.

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FIG. 2.

Chemical structures of hispidin.

In conclusion, several Nepalese wild mushrooms, especially those belonging to Inonotus sp. and Phellinus sp, were identified as having strong antiallergic activity in the RBL-2H3 cells. Also, the bioactive compound in I. clemensiae, hispidin, was able to show a stronger antiallergic activity within the extract, than in its pure form. Further chemical characterization of other highly active samples shall reveal other bioactive compounds too. The promising findings of this study will be very important in establishing these largely inconspicuous mushrooms as a lucrative source of medicinal food.