Antifungal properties of hypericin, hypericin tetrasulphonic acid and fagopyrin on pathogenic fungi and spoilage yeasts

Abstract

Context: The role of hypericin-mediated photodynamic antimicrobial properties on pathogenic fungi and photodynamic therapy for human cancer cells is known. Antifungal properties of Hypericum perforatum L. (Hypericaceae) and Fagopyrum esculentum Moench. (Polygonaceae) extracts were also studied. The different polarities of solvents can cause complication in the identification of antifungal effects of separate biologically active compounds. In recent experimental work, we compared antifungal properties of purified hypericin, hypericin tetrasulphonic acid (hypericin + S) and fagopyrin, which is analogue of hypericin.

Objective: The antifungal properties of aromatic polyketide derivatives such as hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts have been studied.

Materials and methods: The antifungal properties of hypericin, hypericin + S and fagopyrin were determined using the broth microdilution method against a set of pathogenic fungi and spoilage yeasts including: Microsporum canis, Trichophyton rubrum, Fusarium oxysporum, Exophiala dermatitidis, Candida albicans, Kluyveromyces marxianus, Pichia fermentans and Saccharomyces cerevisiae. The tested concentrations of hypericin, hypericin + S and fagopyrin ranged from 750 to 0.011 μg/mL and MIC values were evaluated after 48 h incubation at 30 °C.

Results: The results confirm different antifungal properties of hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts. For pathogenic fungi, the minimum inhibitory concentrations of hypericin ranged 0.18–46.9 μg/mL, hypericin + S 0.18–750 μg/mL and fagopyrin 11.7–46.9 μg/mL. For spoilage yeasts, the MICs of hypericin and hypericin + S ranged 0.18–46.9 and 0.011–0.73 μg/mL, respectively.

Discussion and conclusion: The results obtained herein indicate that various chemical structures of hypericin, hypericin + S and fagopyrin can develop different antifungal properties.

Keywords:

• Naphthodianthrone derivatives
• pathogenic microorganisms
• hypericin tetrasulphonic acid

Introduction

Naphthodianthrone compounds present in Hypericum sp. (hypericin), including Hypericum perforatum L. and Hypericum erectum C.P. Thunberg ex A. Murray (Hypericaceae) and buckwheat plants (fagopyrin) have a photosensitizing effect (Frohne & Pfander 2005). Drug-induced photosensitivity refers to the development of cutaneous disease as a result of the combined effects of a chemical and light. This effect is currently used for photodynamic therapy of the treatment pathogenic microorganisms and cancer cells (Jori et al. 2006; Dai et al. 2009). Hypericin is also present in fungi Dermocybe sp. (Dewick 2002).

Aromatic polyketides derivative of hypericin, fagopyrin is found in much smaller quantities in the genus Fagopyrum (Polygonaceae). Brockmann and coworkers also extracted and purified hypericin via acid precipitation from H. perforatum (Brockmann & Sanne 1953). Fagopyrin is a naphthodianthrone with photosensitizing effect, which was isolated by Brockmann and Lackner for the first time in 1943 from the blossoms of the red flowering variety of Fagopyrum esculentum. The aromatic structure of fagopyrin is similar to that of hypericin, differing only in the presence of two symmetrically placed 2-piperidinyl groups in fagopyrin.

Recently, hypericin has been found to be highly active against tumour growth both in vivo and in vitro, only after photoactivation with either visible or UV light (Thomas & Pardini 1992; Vandenbogaerde et al. 1996). It is known that hypericin and fagopyrin are not well soluble in water (Wirz et al. 2002). Tetrasulphonation of hypericin provided the ideal means to enhance the water solubility of hypericin what should be sufficient for any biological purposes. The interactions of this material with certain biopolymers were shown to result in heteroassociation complexes (Falk et al. 1998). Roscetti et al. (2004) confirm the interesting role of H. perforatum L. in cancer therapy and strongly supports the hypothesis that agents, other than hypericin, present in the total extract can impair tumour cell growth acting separately or in a combined manner (Roscetti et al. 2004).

At the same time such naphthodianthrones like hypericin, hypericin + S and fagopyrin are natural photosensitizer considered for the new generation of photodynamic therapy drugs. It was found that hypericin had a high phototoxicity to Staphylococcus aureus, Enterococcus faecalis and Escherichia coli at extremely low drug concentrations (Kashef et al. 2013). It was found also that hypericin is inducible by pathogen/herbivore attack or if it could play a role in plant defence under plant pathogens Phytophthora capsici and Diploceras hypericinum (Çirak et al. 2005). The extracts from Hypericum sp. were monitored for antifungal activity (Fenner et al. 2005). The possibility of finding new clinically effective antifungal compounds for pathogenic fungal species is discussed nowadays.

Over the last 30 years more than one billion people around the world have suffered from different fungal infections (Novak Babič et al. 2016). Fungal infections range from superficial to deeply invasive and today they are among the most difficult diseases to manage, in humans. Superficial infections of the skin and nails are the most common fungal diseases and affect ∼25% (or ∼1.7 billion) of the general population worldwide. These infections are caused primarily by species Trichophyton, Microsporum and Candida (Brown et al. 2012).

Many fungi are commensal, forming part of our natural microbiota, and they have important roles in disease, being capable of switching to opportunistic pathogens. Fungal infections by opportunistic human pathogenic fungi are becoming an increasing health concern all over the world. The species of Exophiala, Fusarium are opportunistic pathogens on healthy or compromised humans (Novak Babič et al. 2016).

Spoilage yeasts can grow on raw and processed foods where the environmental conditions for most bacteria are unfavourable and cause deterioration of various products. Candida, Debaryomyces, Kluyveromyces, Pichia, Rhodotorula, Trichosporon and Yarrowia are considered as the most frequent food‐borne yeasts (Krisch et al. 2011; Kunicka‐Styczyńska 2011).

Nowadays many research topics in the area of phytochemistry were concentrated on the role of plant extracts, which could contain some biologically active compounds with antibacterial and antifungal properties (Milosevic et al. 2007; Maltas et al. 2013). It is also known that properties of pure biological active compounds can be different to the effect of plant extract, which affected by complex factors among which another antioxidants in the plant extract solution (Gadzovska-Simic et al. 2012). Therefore, the aim of this experimental work was to compare antifungal properties of photosensitizing compounds like hypericin, hypericin + S and fagopyrin on the selected pathogenic fungi and spoilage yeasts.

Materials and methods

Plant material

Plants were collected locally, while in flower and identified as Hypericum perforatum L. and buckwheat as F. esculentum Moench. It has been deposited in laboratory at the Department of Plant Physiology, Institute of Biology, Kiev National University of Taras Shevchenko, Kiev, Ukraine. After being dried in a cool, shaded, airy place, the flowers were minced and stored, protected from light and humidity.

Isolation of hypericin and fagopyrin

Hypericin and fagopyrin were prepared and purified with silica and Sephadex LH-20 column chromatography as described previously (Agostinis et al. 1996). Both compounds were characterized with HPLC analysis (UltiMate 3000 HPLC System, Dionex, Germany) and UV/vis spectrometry (UV-1800 ‘Shimadzu’, Japan), and the data were consistent with literature data (Falk & Schoppel 1992; Vandenbogaerde et al. 1998; Kentaro et al. 2009; Tavčar Benković et al. 2014). For the experiments with pathogenic fungi and spoilage yeasts, hypericin and fagopyrin (3 mg/mL) were dissolved in the sterile dimethyl sulphoxide (DMSO) and stored at −20 °C in dark conditions. Under these conditions, stock solutions were stable near 2 months. To test the susceptibility of pathogenic fungi and spoilage yeasts to hypericin and fagopyrin effects were used at 4% concentration of hypericin, hypericin + S and fagopyrin.

Sulphonation procedure for hypericin tetrasulphonic acid (hypericin + S)

Hypericin (41 mg) and 125 mg oleum were kept at the temperature and time given. The reaction mixture was gently diluted with ice water and saturated with NaCl. After centrifugation, the precipitated green crystals were washed with cold water and thoroughly dried in vacuum. The material in question was isolated by column chromatography on Sephadex-LH20 (GE Healthcare Bio-Sciences AB, Sweden) with methanol/water (4/1) as the eluent. Sulphonation of hypericin leads to its di-, tri- and tetrasulphonic acid derivatives (Falk et al. 1998).

Microorganisms

The fungi Microsporum canis NRCC/F35892, Trichophyton rubrum ATCC28188, Fusarium oxysporum NRCC/G-102 and the yeasts Exophiala dermatitidis ATCC28869, Candida albicans ATCC14053, Kluyveromyces marxianus NRCC/M-59, Pichia fermentans NRCC/M-86, Saccharomyces cerevisiae NRCC/M-99 were used in this study. All tested microorganisms strains were maintained on Sabouraud dextrose agar (Liofilchem, Italy) slants and stored at 4 °C.

Inoculum of each strain to be tested was prepared with fresh cultures by suspending the microorganisms in sterile water. The turbidity of the inoculum was adjusted by spectrophotometer according to the conidial size of the species (0.09–0.3 optical densities at 530 nm) (Espinel-Ingroff et al. 2009).

Antimicrobial properties of hypericin, hypericin + S and fagopyrin

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the hypericin, hypericin + S and fagopyrin were determined using a broth microdilution method. The 4% concentration of hypericin, hypericin + S and fagopyrin were prepared from stock solutions where these compounds were dissolved in DMSO. The 96-well plates were prepared by dispensing into each well 100 μL RPMI 1640 medium with l-glutamine without sodium bicarbonate (Sigma Aldrich, Germany). A 100-μL aliquot of the hypericin, hypericin + S and fagopyrin with concentrations (3 mg/ml) was added into the first wells. Then, 100 μL from their serial dilutions was transferred into eighth consecutive wells. Thereafter, each well was inoculated with 100 μL of suspension containing 10⁶ cfu/mL of the culture and incubated at 30 °C for 48 h. A suspension of microorganisms in the medium without hypericin, hypericin + S and fagopyrin was used as a positive control. A DMSO solution was used as a negative control for the influence of the solvents. After incubation, the growth of microorganisms was indicated by the presence of the turbidity and a pellet on the well bottom. MICs were determined presumptively as the first well, in ascending order, which did not produce a pellet. To confirm MIC and establish MFC, 10 μL of broth was removed from each well and inoculated on Sabouraud agar (Liofilchem, Italy). No visible colony growth after subsequent 24–48 h incubation was accepted as MFC. All tests were performed in duplicate (Wiegand et al. 2008).

Statistical analysis

The data are expressed as the mean ± SEM and were analysed using one-way ANOVA followed by the Bonferroni multiple comparison method. A p value <0.05 was defined as statistically significant.

Results

Experiments have been reported in regard to hypericin-mediated photodynamic antimicrobial properties on pathogenic fungi and spoilage yeasts (Yow et al. 2012). In our research, we compared antifungal properties of hypericin, hypericin tetrasulphonic acid (hypericin + S) and fagopyrin, which is analogue of hypericin, but we did not study photodynamic effects because the aim of this work was to investigate the effect of these compounds without it. All investigated compounds have differences in the chemical structure but present one class of naphthodianthrones compounds. It was supposed that tetrasulphonation of hypericin could enhance the water solubility of hypericin (Falk et al. 1998).

The hypericin was found to be more efficient than hypericin + S: the MIC and MFC varied in 0.18–46.9 μg/mL and 0.73–187.5 μg/mL, respectively, under the influence of hypericin, while MIC and MFC varied in 0.011–750 μg/mL and 0.046–750 μg/mL, respectively, under the influence of hypericin + S (Table 1).

Table 1. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of hypericin, hypericin + S and fagopyrin against pathogenic fungi.

	Hypericin		Hypericin + S		Fagopyrin
Microorganisms	MIC (μg/mL)	MFC (μg/mL)	MIC (μg/mL)	MFC (μg/mL)	MIC (μg/mL)	MFC (μg/mL)
Candida albicans	11.7	11.7	750	750	11.7	11.7
Exophiala dermatitidis	0.18	0.73	0.18	46.9	11.7	11.7
Microsporum canis	11.7	187.5	2.93	11.7	11.7	46.9
Fusarium oxysporum	46.9	187.5	750	750	–	–
Trichophyton rubrum	2.93	46.9	750	750	46.9	46.9

Hypericin had the strongest inhibitory effect on S. cerevisiae and E. dermatitidis, the concentration of hypericin lower than 1 μg/mL already had negative effect on these yeasts. All investigated fungi – M. canis, F. oxysporum and T. rubrum – and only one yeast K. marxianus were more resistant to hypericin. It is interesting that the same concentrations of hypericin are required that both inhibitory and fungicidal activities on cells of yeast C. albicans, P. fermentans and K. marxianus. However, the concentration of hypericin needed was 16 times higher to obtain the MFC on fungi M. canis and T. rubrum and yeast S. cerevisiae in comparison with the concentration of hypericin for the inhibitory effect on these microorganisms.

Hypericin + S was distinguished by the high activity on S. cerevisiae (Table 1): the MIC and MFC of hypericin + S was 16 and 64 times lower, respectively, in comparison with the MIC and MFC of hypericin. Hypericin + S was also more biologically active on K. marxianus and especially on M. canis than hypericin. The inhibitory and antifungal activities of hypericin + S against C. albicans and fungi F. oxysporum and T. rubrum were similar and found to be well moderate against these microorganisms (MIC and MFC values 750 μg/mL) in comparison with hypericin.

The antifungal properties of fagopyrin was analysed only against two yeasts and two fungi (Table 2). The yeasts were the more sensitive to fagopyrin than fungi. Also, the MIC of fagopyrin against microorganisms coincides with MFC of fagopyrin in all cases (excluding fungi M. canis where the MFC was four times higher than MIC). C. albicans exhibited same sensitivity to both fagopyrin and hypericin, however, the antifungal properties of fagopyrin on C. albicans was 64 times stronger in comparison with the effect of hypericin + S, whereas the sensibility of E. dermatitidis to fagopyrin was lower: the concentration of fagopyrin was necessary 16 times and even 65 times higher than hypericin than MFC or, respectively, cells of E. dermatitidis. The MIC of fagopyrin on fungi M. canis was the same as hypericin; however, a four time lower concentration of fagopyrin was necessary to kill this fungus. The fagopyrin also more weakly influenced on M. canis in comparison with hypericin + S. The antifungal properties of fagopyrin on fungi T. rubrum conversely was lower and higher in comparison with hypericin and hypericin + S, respectively.

Table 2. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of hypericin and hypericin + S against food spoilage yeasts.

	Hypericin		Hypericin + S
Microorganisms	MIC (μg/mL)	MFC (μg/mL)	MIC (μg/mL)	MFC (μg/mL)
Pichia fermentans	2.93	2.93	11.7	46.9
Kluyveromyces marxianus	46.9	46.9	0.73	11.7
Saccharomyces cerevisiae	0.18	2.93	0.011	0.046

Discussion

The effectiveness of hypericin-mediated photodynamic killing can be strongly affected by cellular structure and photosensitizer uptake. Photodynamic therapy is based on the use of a photosensitizing compounds (like hypericin, hypericin + S, fagopyrin), which in the presence of oxygen produces reactive oxygen species upon light activation at a wavelength matching its absorption spectrum (Dougherty et al. 1998). Hypericin and porphyrin derivatives showed strong preference for lipid membranes (Maisch et al. 2005) with ability to reach the interior of a cell (Maisch et al. 2005).

In this experimental work, hypericin had the strongest inhibitory effect on S. cerevisiae and E. dermatitidis. The inhibitory effect of hypericin was also confirmed for Gram-positive methicillin-sensitive and -resistant S. aureus cells (Yow et al. 2012). In this research, combination of hypericin and light irradiation could induce significant killing of Gram-positive methicillin-sensitive and -resistant S. aureus cells, but was not effective on Gram-negative E. coli cells. The difference was caused by different cell wall/membrane structures that directly affected cellular uptake of hypericin (Yow et al. 2012).

Results of our experiment can confirm that different chemical structure of representatives of class naphthodianthrones can have different effects on pathogenic fungi and spoilage yeasts. It was found that in the main the antifungal properties of hypericin were stronger than hypericin + S, except K. marxianus, S. cerevisiae and M. canis. The hypericin + S affected these three microorganisms stronger can be connected with higher water solubility of hypericin + S, which ensures reproducible photodynamic effect on these microorganisms (Kubin et al. 2008).

The evaluation of the in vitro fungicidal effect of hypericin photodynamic therapy on various Candida spp. has been observed 3-log fungicidal effect for two C. Albicans strains, C. Parapsilosis and C. krusei in combination with light irradiation (Rezusta et al. 2012). It was observed that C. albicans exhibited the same sensitivity to both fagopyrin and hypericin, however, the antifungal properties of fagopyrin on C. albicans was 64 times stronger in comparison with hypericin + S. At the same time the yeasts were the most sensitive to fagopyrin than fungi.

Increased fungicidal effects of hypericin is usually induced by exposure of cell cultures to a light-emitting diode lamp. With optimal incubation time, 60 min, a 3-log fungicidal effect was achieved with hypericin concentration ranges of 10–20 μM for T. rubrum (Paz-Cristobal et al. 2014). In our experimental work after incubation at 30 °C for 48 h with normal light condition was achieved results that T. rubrum is more resistant to the hypericin. At the same time hypericin + S found to be more effective compared to hypericin. It is possible that effectiveness of hypericin + S can be different under additional exposure to light-emitting diode lamp.

The potential genotoxicity and antigenotoxicity of non-photoactivated hypericin was investigated in a yeast (S. cerevisiae) assay. It was found that hypericin did not increase the frequency of mitotic crossovers or total aberrants at the ade₂ locus, the number of convertants at the trp5 locus or the number of revertants at the ilv1 locus (Miadokova et al. 2010). At the same time, none of the crude methanolic extracts of Hypericum sp. showed activity against E. coli or S. cerevisiae (Dall'Agnol et al. 2003). In the presented experimental work was estimated not high hypericin antifungal properties on S. cerevisiae but hypericin + S showed appreciable by the high activity.

The present study showed that the antifungal properties of investigated biologically active compounds varied in a wide range (0.011–750 μg/mL). Süntar et al. (2016) investigated the antibacterial effects of different polarity sub-extracts (with polar solvents water, butanol, ethyl acetate and non-polar solvents hexane, chloroform) of Hypericum perforatum against oral pathogenic bacteria and established that Streptococcus mutans, S. sobrinus, Lactobacillus plantarum and E. faecalis were sensitive against at MIC values from 8 to 32 μg/mL only. The different polarities of solvents influenced the different solubility of biologically active compounds of H. perforatum. For example, the main constituents of ethyl acetate were found flavonoids, hypericins and hyperforins (Avato et al. 2004); the hyperforin was also isolated from the nonpolar extracts of H. perforatum (Schempp et al. 1999). The use of extracts complicate identification of effects of antimicrobial properties of separate biologically active compounds, the effects of activities of compounds solute in extracts can be additive (the synergistic effect) or an antogonistic effect can come into play.

Most present information about the antibacterial and antifungal capacities of Hypericum perforatum extracts includes extracts which contain phenolic compounds and other antioxidant (Saddiqe et al. 2010; Mašković et al. 2011). The highest inhibitory effect on the growth of F. oxysporum, Aspergillus glaucus and Phialophora fastigiata was exhibited by the ethanol extract of Hypericum perforatum L. (Mašković et al. 2011). In our experimental work it was estimated that all investigated fungi – M. canis, F. oxysporum and T. rubrum were more resistant to pure hypericin. The fungicidal properties of hypericin + S against fungi F. oxysporum and T. rubrum was found to be moderate against these microorganisms in comparison with hypericin.

Conclusion

The results confirm different antifungal properties of hypericin, hypericin-S and fagopyrin on the selected pathogenic fungi and spoilage yeasts because of structural differences of the investigated photosensitizing compounds. It is known that using plant extracts complicates identification of antimicrobial activity of separate biologically active compounds. The comparison of antifungal properties of pure naphthodianthrones compounds on pathogenic microorganisms provides new information that can help us understand the antifungal properties of plant extracts from Hypericum sp. or Fagopyrum sp. The results of this research can be useful for preliminary screening of the antifungal properties of hypericin, hypericin + S, fagopyrin for developing photodynamic methodology with light irradiation.

Disclosure statement

The authors declare no conflicts of interest.