Novel regulation of aflatoxin B1 biosynthesis in Aspergillus flavus by piperonal

Abstract The present study investigated its inhibitory role in aflatoxin (AF) biosynthesis. Treating only AFB1- and B2-producing Aspergillus flavus with piperonal completely inhibited AFB1 production with high sclerotial formation, resulting in 20-fold higher AFG2 production. On the other hand, benzodioxole and eugenol suppressed AFB1 production without AFG formation, while methyleugenol showed potent inhibition of AFB1 production with slight production of AFG1. These results indicate that natural products may change aflatoxin biosynthesis, and highlight a novel regulation of AFG2 production by piperonal. It is the first report for chemical regulation on AFG2 production in non-AFG producing-aspergilli.


Introduction
Aflatoxin B1 (AFB1) biosynthesis is a unique pathway to produce toxins by Aspergillus sp. that cause a severe disease, aflatoxicosis, in humans and other vertebrate animals (Monson et al. 2015). In recent report, oxidative stress was shown to induce sclerotial differentiation of Aspergillus flavus with increased AFB1 production (Grintzalis et al. 2014). In this regard, many natural antioxidants show antifungal and antiaflatoxigenic activities towards aspergilli (Chitarrini et al. 2014;Kazemi 2015).
Some direct inhibitors of AFB biosynthesis have been well studied, including some methylenedioxy-containing compounds such as piperine and pipernonaline that have been suggested to inhibit aspergilli growth and AFB production (Lee et al. 2002). Their inhibitory effects on AFB biosynthesis are based on the inhibition of cytochrome P450 activity, which is involved in many AFB biosynthesis steps.
Herein, we show that piperonal, a methylenedioxy-containing compound, changed the AFB1 biosynthesis to AFG2 biosynthesis by completely inhibiting AFB1 production and enhancing the sclerotial formation. Until now, chemical inhibition of AFB biosynthesis was only known to decrease aspergilli growth and inhibit AFB and AFG biosynthesis. Therefore, this is the first report to show a natural compound producing AFG2 from a typical insignificant AFG1 and G2-producing A. flavus.

Results and discussion
In this study, we show that piperonal ( Figure S1) completely inhibited AFB1 production, and stimulated 20-fold high AFG2 production than AFB1 production by the same aspergilli ( Figure 1). This was determined using HPLC with a C18 column (chromatogram shown in Figure S2). Piperonal at the tested concentration of 1 mg/mL decreased the growth of aspergilli than the control group as determined by dry weight measurement ( Figure S3). In addition, piperonal treatment enhanced sclerotial formation ( Figure S4).
AFB production by aspergilli is dependent on various parameters and the primary variables are the species and the degree of sclerotia differentiation induced by oxidative stress (Okoth et al. 2012;Grintzalis et al. 2014). Aspergillus species can be divided into two groups as S-and L-types according to the size of sclerotia. S-type aspergilli have fewer conidiospores and abundant sclerotia, which are usually smaller than 400 μm in diameter and produce Figure 1. aflatoxin production by Aspergillus flavus with or without treatment with different naturally occurring compounds at a concentration of 1 mg/ml: (1) control aspergilli; (2) benzodioxole-treated aspergilli; (3) piperonal-treated aspergilli; (4) eugenol-treated aspergilli; (5) methyleugenol-treated aspergilli; (6) piperine-treated aspergilli. relatively larger amounts of total aflatoxins, AFB toxins and lesser amounts of AFG toxins than the L-type strains (Horn & doner 1999;Okoth et al. 2012). L strains produce considerable amounts of AFB toxins and produce sclerotia that are larger than 400 μm in diameter (Cotty 1989;Okoth et al. 2012).
Our results are different from the previous studies because a complete inhibition of AFB1 production was seen with high amounts of sclerotial formation. A switch from AFB1 production to AFG2 was regulated by piperonal, which was partially isolated from Piper nigrum.
Two analogues of piperonal, benzodioxole and piperine were used to understand the structure-activity relationship ( Figure S1). Benzodioxole strongly inhibited fungal growth and AFB1 production without any production of AFG1 or AFG2 (Figure 1). Piperine did not show any inhibitory effect on A. flavus growth and AFB1 production. Two antioxidants eugenol and methyleugenol were also used in this study ( Figure S1). Methyleugenol showed potent inhibition of aspergilli growth and AFB1 production. The mode of action of methyleugenol may be inhibition of the enzyme aflatoxin B synthase (1.14.13.175) ( Figure S5). On the other hand, the inhibition by piperonal may be through two steps: (1) versicolorin B to versicolorin A transformation and (2) aflatoxin B synthase activity. Further studies should be undertaken to obtain spectrophotometric confirmation of AFB1 biosynthesis pathway intermediate accumulation when treated with piperonal ( Figure S5).

Conclusion
Piper, which is a typical methylenedioxy-containing molecule abundantly present in Piper fruits, shows a characteristic pattern of inhibitory effect on AFB1 production by A. flavus. A. flavus does not normally produce AFG type, but after treatment with piperonal, it produces 20 times higher AFG2 in comparison to the AFB1 production in the control. Taken together, the regulation of AFG2 formation by piperonal is highly unique and has never been reported before.

Supplementary material
Experimental details and Supplementary Figures S1-S5 relating to this paper are available online.