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Honey bees, Apis mellifera L., are the most economically valuable pollinators of many crops, fruits, and wild plants worldwide. Bee populations have been declining dramatically worldwide, and a major contributing factor is infection with the pathogenic fungus Ascosphaera apis which cause chalkbrood disease (Aronstein and Murray, 2010; Hedtke et al., 2011). Though Asc. apis does not destroy entire honey bee colonies, the chalkbrood disease can weaken the colony, reduce honey yields, and increase susceptibility to other bee pests and diseases. In addition, this pathogen causes a significant reduction in bee numbers and honey-bee related commercial productivity (Hedtke et al., 2011).

Even though the occurrence of chalkbrood disease has been increasing, there are no registered control systems or chemicals available. Burning all potentially contaminated hive material is the only effective means to destroy Asc. apis (Shin and Kim, 2016) because chemical treatments should be avoided due to the contamination of honey products and honey bees. In the recent years, secondary metabolites from plants have been screened as new drugs because of their potentially lower toxicity to human and the environments than synthesized chemical compounds (Mourad et al., 2005; Kloucek et al., 2012; Shin and Kim, 2016).

This study suggested the enhanced antifungal effects of new compound candidates, 4-hydroxyderricin and acetylshikonin, aginst Asc. apis. Therefore 4-hydroxyderricin and acetylshikonin can be helped to prevent Asc. apis infection to the honey bees.

We screened 640 compounds from NPBANK (http://www.npbank.kr, Korea) using a modified broth microdilution assay and found 32 compounds that inhibited the growth of Asc. apis (Table 1). 4-hydroxyderricin (MIC of 3.125 and 6.25 mg/L after 24 h and 48 h treatment, respectively) showed the strongest anti-Asc. apis activity, followed by acetylshikonin (12.5 mg/L after 24 h and 48 h treatment) (Fig. 1, Table 1).

Fig. 1. 
Chemical molecular structure of (A) 4-hydroxyderricin and (B) acetylshikonin.

To determine if 4-hydroxyderricin inhibited the growth of fungi other than Asc. apis, we investigated the antifungal activity against several fungal species. 4-Hydroxyderricin specifically inhibited the growth of the fungi Asc. apis and R. oryzae (MIC of 0.78 mg/L for both 24 h and 48 h treatment) (Table 2). Acetylshikonin had low antifungal activity against R. oryzae and S. cerevisiae, but its effect was too weak to classify acetylshikonin as a growth inhibitor of these fungal species (Table 2). Although previously identified anti-microbial compounds were effective when first described, abuse of broad-spectrum anti-microbial compounds has increased the occurrence of antibiotic-resistant infections. Accordingly, there is demand for novel anti-microbial agents with a narrow spectrum of anti-microbial activity (Bax and Green, 2015).

4-Hydroxyderricin is a chalcone and one of the phytochemical components of Angelica keiskei. It has various biological activities including anti-inflammatory, anti-diabetic, anti-bacteria, and anti-tumor activities (Ohnogi, et al., 2012). 4-hydroxyderricin suppressed the growth of Staphylococcus aureus by inhibiting seryl-tRNA synthetase (Battenberg et al., 2013). The antifungal activity of this compound might be due to similar mechanisms, but this needs to be confirmed in future studies.

Acetylshikonin is a natural naphthoquinone derivative that is abundant in the roots of traditional medical herbs such as Lithospermum erythrorhizon and Onosma species. Previous studies have demonstrated that acetylshikonin is significantly cytotoxic toward various types of cancer cells (Cho and Choi, 2015). In addition, acetylshikonin has antifungal activity, but not as high as that of fluconazole or amphotericin B (Sasaki et al., 2002). The detailed antifungal mechanism of acetylshikonin has not been studied and requires further elucidation.

We next investigated if 4-hydroxyderricin and acetylshikonin can be cytotoxic to humans. 4-Hydroxyderricin showed very low cytotoxicity toward HepG2 and HaCaT cells, but acetylshikonin showed some cytotoxicity toward HepG2 cells, but not HaCaT cells (Fig. 2).

Fig. 2. 
Cytotoxic effects of (A) 4-hydroxyderricin and (B) acetylshikonin against HepG2 or HaCaT cells for 24 h. Data represent mean±SD of three independent experiments.

One of the disadvantages of using natural compounds is the cost of preparation. Combined treatment with natural compounds and miconazole, a well-known synthetic antifungal compound, would increase the cost-effectiveness of 4-hydroxyderricin and reduce the cytotoxicity associated with miconazole usage. We therefore explored that synergy effect of 4-hydroxyderricin or acetylshikonin with micinazole. Acetylshikonin had a synergy effect with miconazole weakly (FICI=0.83±0.12 and 0.88±0.15 after 24 h and 48 h treatment, respectively), but 4-hydroxyderricin had a more effect with miconazole (FICI=0.63±0.10 and 0.65±0.13 after 24 h and 48 h treatment, respectively) (Table 3). 4-Hydroxyderricin at 0.78 μg/mL inhibited Asc. apis growth when combined with 0.78 μg/mL of miconazole (Table 2).

4-Hydroxyderricin has antifungal activity against Asc. apis with less cytotoxicity against human cell lines. The narrow anti-fungal spectrum indicates that it could potentially be used to control honeybee infectious fungal diseases without affecting beneficial fungal species.