The in Vitro Effect of Berberine Sulfate and Berberine Chloride on the Growth and Aflatoxin Production by Aspergillus flavus and Aspergillus parasiticus

Introduction
In recent decades, the global trade of plant products such as grains, flours, and oilseeds has been raised significantly; however, the contamination of these products with various chemical compounds, especially mycotoxins, has become an essential global concern (Moretti et al., 2017; Santos Pereira et al., 2019). By growing on food products, fungi not only decrease the nutritional value of these products but also severely affect their quality by excreting mycotoxins (Vieira, 2003). 
Contamination of food and its essential components with mycotoxins may occur before harvest in the field due to the growth of pathogenic fungi on the plant or during the processing and storage of products due to the growth of saprophytic fungi (Gruber-Dorninger et al., 2019). Mycotoxins are secondary metabolites produced by many fungi. A group of mycotoxins is called aflatoxins. They are mainly produced by different species of the genus Aspergillus, in particular Aspergillus flavus and Aspergillus parasiticus, after harvest, during storage and processing (Nakavuma et al., 2020; Khorrami et al., 2022). So far, more than 20 metabolites of aflatoxins have been identified, but only 4 metabolites, B1, B2, G1, and G2, can poison humans and animals (Santos Pereira et al., 2019). The long list of harmful effects of these toxins on humans and animals include carcinogenesis, mutagenicity, weakening of the immune system, and liver and kidney poisoning (Nakavuma et al., 2020; Jard et al., 2011; Monson et al., 2015; Al-Mudallal, 2023; Mokhtari Hooyeh et al., 2022). The prevention of food contamination with aflatoxins is primarily based on confining these products from fungal spores and then controlling storage conditions such as temperature, humidity, and antifungal gas compounds. Another approach proposed the use of additives that prevent the mycelium growth of toxin-producing fungi and inhibit or reduce their production of aflatoxins (Gruber-Dorninger et al., 2019; Patil et al., 2014; Kadium et al., 2023). In recent years, the use of chemical fungicides has faced restrictions due to the health risks for humans and animals and the emergence of resistance to them. Therefore, using plant compounds with antifungal properties and preventing aflatoxin production has received much attention (Hu et al., 2017; Hasankhani et al., 2023). 
Plants have a wide range of herbal compounds with therapeutic and biological properties. These compounds are mainly classified as alkaloids, flavonoids, tannins, terpenoids, and steroids and have been widely used as medicine and additives by humans throughout history (Savoia, 2012). Berberine, a naturally occurring benzylisoquinoline alkaloid, is found in the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, and B. aristata (Ghavipanje et al., 2022). Berberine has been used for more than 3000 years in the traditional medicine of Iran and China as a herbal compound with many therapeutic properties against Alzheimer disease, Parkinson disease, cancer, obesity, and diabetes. Also, this composition has antiviral, bacterial, and fungal properties (Arayne et al., 2007). Berberine and its derivatives have inhibitory effects on the growth and production of toxins by fungi, and so far, this effect has been identified in Candida, Fusarium, Penicillium, and Aspergillus species (Da Silva et al., 2016; Ismail et al., 2020; El-Zahar et al., 2022). Recently, various studies have been conducted to evaluate this isoquinoline alkaloid as a natural preservative with significant antioxidant and antimicrobial properties (Geerlofs et al., 2019; Malekinezhad et al., 2021). So far, a few studies have investigated the effect of berberine on the growth and mycotoxin production by fungi. Therefore, this study aimed to evaluate the effect of berberine sulfate and berberine chloride on the growth and aflatoxin production by A. flavus and A. parasiticus.

Materials and Methods

Fungal strains
A frozen stock of A. flavus (ATCC 28539) and A. parasiticus (ATCC 15517) was obtained from the fungal collection of the Department of Mycology, Faculty of Veterinary Medicine, University of Tehran, Iran. 

Berberine salts
Berberine chloride and berberine sulfate were purchased from Sigma company (Sigma-Aldrich, St. Louis, MO, USA).

Preparation of Aspergillus suspensions
A. flavus and A. parasiticus were subcultured in potato dextrose agar (PDA) (Merck Co., Germany) at 28°C for 5 days. Then, 10 mL of PST solution (physiological salt solution containing 0.01% Tween 80) was poured on the surface of the colonies and gently scraped with a U-shaped glass rod. The resulting suspension was kept at room temperature without movement for 15 minutes to precipitate possible hyphae fragments. Then, the number of conidia present in 1 mL of the suspension was counted using a hemocytometer slide. The final concentration of the suspension was 2×106 conidia/mL.

Microdilution broth assay
The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) values of berberine salts were evaluated based on the Clinical and Laboratory Standards Institute (CLSI) document M38-A2 with some modifications (CLSI, 2008). The RPMI (Roswell Park Memorial Institute) 1640 medium containing 3-(N-morpholino) propane sulfonic acid (MOPS) buffer was prepared according to CLSI standard instructions, and its pH was set to 7. Finally, the medium was sterilized using a 0.22-µ syringe filter. At first, two-fold serial dilutions of berberine sulfate and berberine chloride were prepared in RPMI 1640 medium in rows of 96 cell culture plates. Each well in the row contained 100 µL of different dilutions of berberine salts ranging from 2000 to 15.6 µ/mL. Then, 100 µL of fungal suspension with a concentration of 0.4-5×104 conidia/mL was inoculated into each well, and the plates were incubated for 48 h at 28°C. For each experiment, a positive control without berberine containing fungi and a negative control without berberine and fungi were considered. All tests were performed in triplicate. The MIC was defined as the lowest concentration of completely inhibiting the growth of fungi. The MFC of berberine salts was determined by culturing from the MIC well and subsequent wells in PDA for 7 days at 28°C. MFC was estimated at concentrations in which either no fungi or less than three colonies were grown (CLSI, 2008).

Effect of berberine salts on the radial growth of A. flavus and A. parasiticus
The effect of berberine salts on the radial growth was measured through culture in a solid medium. Briefly, PDA plates containing 125, 250, 500, 1000, and 2000 µg/mL of berberine salts were prepared, and a sterile 5-mm blank disk was placed in the center of each plate. Ten microliters of Aspergillus suspensions containing 2×106 conidia/mL were inoculated into the disks. A plate without berberine was selected as a control for each species. The plates were incubated at 28°C, and the mean diameter of the colonies was measured after the incubation period. The antifungal effect was calculated as the percentage of radial growth inhibition according to the Equation 1:

, Where Dc represents the fungal colony diameter in the control plate, and DS represents the fungal colony diameter in the treated plates.

The effect of berberine salts on aflatoxin production by A. flavus and A. parasiticus
Berberine sulfate and berberine chloride at concentrations of MIC/2 and MIC/4 were added to 50 mL of flasks containing yeast extract broth (YEB) (Merck Co., Germany). Then, the flasks were inoculated with a 1.5×106 conidia/mL concentration. The flasks were kept for 10 days in an incubator with a temperature of 28°C and a rotation of 100 rpm. Also, flasks containing YEB without fungal inoculation were considered negative control, and those containing YEB without berberine were regarded as positive control.

Aflatoxin production assay
For evaluating aflatoxin formation, berberine sulfate and berberine chloride at concentrations of MIC/2 and MIC/4 were used. Spore suspension (1.5×106 conidia/mL) was added to 50 mL of flasks containing YEB containing different concentrations of berberine sulfate and berberine chloride. The flasks were kept for 10 days in an incubator with a temperature of 28°C and a rotation of 100 rpm. After incubation, the mycelia were dried to a constant weight at 80°C, and the weight of dried matter was estimated. Determination of aflatoxins B1, B2, G1, and G2 was performed by immunoaffinity column extraction using RP-HPLC (reversed-phase high-performance liquid chromatography) according to the Association of Official Agricultural Chemists (AOAC). Briefly, the filtrated content of each flask was mixed with 150 mL MeOH: H2O (80:20) and 2.5 g NaCl, followed by vortexing for 3 min. Sixty-five microliters of phosphate buffer solution (PBS) was added to 10 mL of this mixture, shaken vigorously, and passed through a glass fiber filter. Seventy milliliters of solution were transferred onto an immunoaffinity column (Puri-Fast AFLA IAC, Libios, France) at a 3 mL/min flow rate. The column was then washed with 15 mL PBS and dried by gently passing air through it. The aflatoxins were eluted with 500 and 750 µL methanol at 1 min intervals. The elution diluted with 1750 µL H2O, and the aliquot of 200 µL was injected into an HPLC system equipped with a separator module (2695, Waters, USA), a Nova-Pak LC-18 column, and a fluorescence detector (474, Waters, USA). Aflatoxins were derivatized by KB cell post-column derivatization system (Libios, Chemin de Plagne 69210 Bully, France) in a H2O–MeCN–MeOH mobile phase containing HNO3 and KBr at a flow rate of 1 mL/min and detected at an excitation wavelength of 365 nm and an emission wavelength of 435 nm. Aflatoxins were quantified using the peak height by Millenium 32 v 4.0 software (Waters, USA). Aflatoxin standards were purchased from Sigma (St. Louis, MO, USA). The Equation 2 calculated the inhibition percentage of aflatoxin production:

, where Ac is the amount of aflatoxin in the control sample, and As is the amount of aflatoxin in the treated sample (Hassan et al., 2015).

Statistical analysis
The quantitative data of fungal growth and HPLC analyses were subjected to variance (one-way ANOVA) in the Tukey range (SPSS software, version 16). The differences with P<0.05 were considered significant. 

Results

MIC and MFC
Based on the broth microdilution method, berberine sulfate revealed MIC values of 250 and 500 µg/mL for A. flavus and A. parasiticus, respectively (Table 1). Berberine chloride exhibited stronger activity than berberine sulfate, with MIC values of 125 and 250 µg/mL against A. flavus and A. parasiticus, respectively. Subcultures of these treated inoculums were negative, confirming MFC against A. flavus and A. parasiticus at 500 to 2000 µg/mL (Table 1).

The effect of berberine sulfate and chloride on the growth of A. parasiticus and A. flavus
As demonstrated in Table 2 and Figure 1, all concentrations of berberine sulfate and berberine chloride exhibited significant growth inhibition of A. flavus and A. parasiticus compared to the control group, suggesting a dose-dependent pattern (P˂0.05).

Berberine sulfate (2000 µg/mL) and berberine chloride (1000 µg/mL) exhibited a 100% growth inhibition of mycelia production by A. flavus. In addition, berberine sulfate at a concentration of 2000 µg/mL and berberine chloride at a concentration of 1000 µg/mL inhibited the growth of mycelia production by A. parasiticus by 96.7% and 100%, respectively (Table 2).

The effect of berberine chloride and berberine sulfate on aflatoxin production
In our study, when the certain concentrations (MIC/2 and MIC/4) of berberine sulfate and berberine chloride were added to the cultures, significant reductions in aflatoxins production were observed by A. flavus and A. parasiticus compared to control (P˂0.05) (Table 3, Table 4, and Figure 2). As shown in Table 3, berberine chloride exhibited a higher inhibitory effect on aflatoxin production than berberine sulfate by A. flavus (P˂0.05). Berberine chloride caused significant reductions down to 100% for aflatoxin G1 and aflatoxin G2 by A. flavus.

According to Table 4, aflatoxin production by A. parasiticus treated with berberine sulfate and berberine chloride at MIC/2 concentration was significantly lower than MIC/4 concentration (P˂0.05). Also, berberine chloride exhibited a higher inhibitory effect on aflatoxin production than berberine sulfate by A. parasiticus (Figure 2). Berberine chloride caused a significant 100% reduction for aflatoxin G1 by A. parasiticus (Table 4).

At MIC/2 concentration, berberine chloride decreased aflatoxin production by A. flavus and A. parasiticus by 96.81% and 98.12%, respectively, 100% for aflatoxin B2, 98.9% for aflatoxin G1, 100% for aflatoxin G2 and 97.5% for total aflatoxin (P<0.05) (Figure 2).

Discussion
In the present study, we showed a new biological activity for berberine as an inhibitor of aflatoxins B1, B2, G1, and G2 by A. flavus and A. parasiticus, besides its ability for strong fungal growth inhibition. MIC and MFC techniques assessed the fungistatic and fungicidal properties of berberine sulfate and berberine chloride. Several studies have been carried out on the chemical composition of B. vulgaris and have shown that the most important constituents of this plant are isoquinoline alkaloids such as berberine (Tabeshpour et al., 2017). In the case of the antifungal effects of berberine, few studies approved the high potential of berberine against some pathogenic fungal strains (Da Silva et al., 2016; Mahmoudvand et al., 2014). According to Ghareeb et al. (2013) study, a 62% berberine ethanolic extract from dried B. vulgaris roots displayed antifungal activity against five fungal infections at dosages ranging from 1:1 to 1:8 (Penicillium verrucosum, Fusarium proliferatum, A. parasiticus, A. niger, and A. flavus) (Ghareeb et al., 2013). In a study by El-Zaher (2022), the MIC values of B. vulgaris leaf and root extracts for A. flavus were 70 and 90 µg/mL, respectively, while these values for A. parasiticus were found to be 85 and 100 µg/mL (El-Zahar et al., 2022). Lei et al. (2011) showed that the MIC range of berberine over the 42 strains of Aspergillus spp. was 4–256 μg/mL. The growing rate of Trichophyton mentagrophytes treated with berberine hydrochloride was significantly lower than those obtained in untreated control, demonstrating that berberine hydrochloride was fungicidal (Xiao et al., 2019). Additionally, a few studies have found that B. vulgaris and its major component, berberine, have antifungal action against Candida spp. In a study conducted by Da Silva et al. (2016), fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 µg/mL and 16 µg/mL, respectively (Da Silva et al., 2016). Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis (Da Silva et al., 2016). Li et al. (2013) demonstrated that berberine has a strong antifungal effect on Candida albicans, causing cell cycle arrest and DNA damage. Other studies have also suggested that berberine can bind to DNA, affecting DNA replication, transcription, and cell cycle (Bhadra & Kumal, 2011).
In this study, berberine salts inhibited the radial growth of A. flavus and A. parasiticus mycelium (Table 2). El-Zahar et al. (2022) showed that B. vulgaris root extract inhibited the mycelial growth of P. verrucosum, F. proliferatum, A. ochraceous, A. niger, and A. flavus. For P. verrucosum and A. ochraceous, the maximum inhibition zones ranged from 1.7 to 2.35 cm at the 100 µL concentration. In a study by Lei et al. (2011), Aspergillus treated with berberine exhibited smaller colony size, slower mycelial growth, and reduced conidia. These cultures also lost conidial pigment such that the conidial surface observed was white rather than green-gray (Lei et al., 2011). These results demonstrated that berberine can restrain Aspergillus growth, development, and conidial pigmentation. Some studies demonstrated that berberine significantly inhibits gene expression in the Aspergillus ergosterol biosynthesis pathway and that berberine is significantly more effective than azoles at inhibiting expression of the Erg5, Cyp51A, Cyp51B, and IMP genes, which are related to pigment production in Aspergillus conidia. The IMP gene is closely associated with cell wall biosynthesis, and by inhibiting its expression, berberine may thus inhibit the biosynthesis of fungal cell walls and cause growth and developmental aberrations in Aspergillus (Ouyang et al., 2010). Da Silva et al., (2016) demonstrated that the berberine concentration necessary to inhibit both planktonic cells and preformed biofilm cells is similar. This finding indicated that berberine may reduce planktonic cell growth and inhibit cell viability in preformed biofilms at concentrations of 8 µg/mL and 37.5 µg/mL, respectively. 
Up to now, there has been no research on aflatoxin inhibition by berberine, but a few investigations reported the effect of B. vulgaris on aflatoxin production. For example, Ghareeb et al. (2013) reported that ethanolic extract of B. vulgaris could inhibit the production of 44% and 98.3% of aflatoxin B1 and 67.2 and 89% of aflatoxin B2 at concentrations of 0.01% to 0.1%, respectively. Safari et al. (2020) exhibited that the inhibition of aflatoxin B1 production by A. flavus in B. vulgaris extract (6 mg/mL) was significant. Their findings demonstrated a highly significant correlation between the gene expression and the aflatoxin B1 biosynthesis, such that certain doses of the extract reduced or blocked the expression of the aflR, aflM, and aflP and consequently reduced the synthesis of aflatoxin B1. Interestingly, compared to the regulatory gene (aflR), the down-regulation of expression in the structural genes (aflM and aflP) was more consistent and correlated with the inhibition of aflatoxin B1 production. In another study by Tintu et al. (2012), α-amylase inhibitors, such as berberine, can control the growth of A. flavus and the production of aflatoxins. Malekivezhad et al. (2021) showed that the addition of different levels of berberine to chickens challenged with aflatoxin reduced the negative effect of this toxin on broiler feed intake. Also, supplementation of aflatoxin B1-contaminated diets with berberine improved growth performance and reduced vascular congestion, inflammatory cell infiltration into the liver portal space, and hepatocyte apoptosis. Furthermore, it protects against toxin-induced damage to the ileal epithelium. These findings suggested that berberine could be a useful dietary strategy to prevent the effects of aflatoxicosis in animals and humans.

Conclusion
In summary, our findings indicated the potential of berberine as a natural inhibitor of the growth and aflatoxin production by A. flavus and A. parasiticus, the well-known agents of food-borne aflatoxicosis.

Ethical Considerations

Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This work was supported by the Research Council of the University of Tehran.

Authors' contributions
Project administration: Mohammad Sadegh Moradi and Samin Kamkar; Formal analysis: Jalal Hassan; Funding, supervision, and writing: Aghil Sharifzadeh and Javad Abbasi; Final approval: All authors.

Conflict of interest
The authors declared no conflict of interest.

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