Research Article

Antifungal Effects of Rosmarinic Acid, α-Tocopherol and α-Tocopherol Acetate on Aspergillus parasiticus (NRRL 2999) and Candida albicans (MTCC 183) and Assessment of Chitin and Ergosterol Biomarkers

Vidya Chernapalli, Mir Zahoor Gul, Hanifa Mohammad Anwar, Naga Sai Susmitha Kuruganti and Karuna Rupula*

Department of Biochemistry, University College of Science, Osmania University, Telangana, India

Submission: August 23, 2024; Published: September 06, 2024

*Corresponding author: Karuna Rupula, Department of Biochemistry, UCS, Osmania University, Telangana, India Email: karunarupula@osmania.ac.in

How to cite this article: Vidya C, Mir Zahoor G, Hanifa Mohammad A, Naga Sai Susmitha K, Karuna R, et al. Antifungal Effects of Rosmarinic Acid, α-Tocopherol and Α-Tocopheryl Acetate on Aspergillus Parasiticus (NRRL 2999) and Candida Albicans (MTCC 183) and Assessment of Chitin and Ergosterol Biomarkers. Adv Biotech & Micro. 2024; 18(3):555987.DOI:10.19080/AIBM.2024.18.555987

Abstract

Fungal infections, primarily the ones associated with Candida and Aspergillus species are increasing every year and are challenging to eradicate despite the existence of antifungal drugs. For the past few decades, natural products have emerged as an essential source of antimicrobial agents particularly those derived from plants as promising alternatives to traditional drugs. They can selectively act on different targets with fewer side effects and moreover, phytotherapy is inexpensive. With this perspective, the present study reports the antifungal effect of selected natural compounds Rosmarinic acid, α-tocopherol and α-tocopherol acetate, on Aspergillus parasiticus (NRRL 2999) and Candida albicans (MTCC 183). The antioxidant capacity of these compounds was evaluated by DPPH assay that revealed Rosmarinic acid as a potent antioxidant than α-tocopherol and α-tocopherol acetate. Rosmarinic acid (RA), α-tocopherol (AT) and α-tocopherol acetate (ATA) showed significant antifungal activities against both A. parasiticus and C. albicans as studied by using well diffusion assay. RA exhibited significant inhibition against A. parasiticus and C. albicans with a minimum inhibitory concentration (MIC) of 15.62 μg mL-1 and 7.82 μg mL-1 respectively which is lower than the MICs of AT and ATA against the two fungi. RA, AT and ATA caused morphological changes in both A. parasiticus and C. albicans as studied by using scanning electron microscopy. The fungal growth biomarkers, chitin and ergosterol contents, were also lowered in all three antioxidants treated fungi with the potency of the compounds in inhibiting the chitin as well as ergosterol synthesis to be in the order of RA > ATA > AT. Thus, our experimental investigations suggest that RA, AT, an isoform of Vitamin E and ATA, a derivative of vitamin E are potent antifungal agents with RA being the most potent fungicidal compound.

Keywords: Antifungal; α-tocopherol; alpha tocopherol acetate; Aspergillus parasiticus; Candida albicans

Abbreviations: RA: Rosmarinic Acid; MIC: Minimum Inhibitory Concentration; ATA: Alphatocopherol Acetate; DMSO: Dimethyl Sulphoxide; DPPH: Diphenyl picrylhydrazyl; USDA: United States Department of Agriculture; IMTECH: Institute of Microbial Technology; MTCC: Microbial Type Culture Collection; YEPD: Yeast Extract Potato Dextrose; ZOI: Zone of Inhibition; CLSI: Clinical and Laboratory Standards Institute; NCCLS: National Committee for Clinical Laboratory Standards; GC: Growth Control; MC: Media Control; MIC: Minimum Inhibitory Concentration; DHE: Dehydro-Ergosterol; SD: Standard Deviation

Introduction

Fungi are eukaryotic, single cellular to multicellular varied clusters of living organisms distributed all over the biosphere. They are heterotrophic and differ in size, nature of life, and modes of reproduction. According to reports, more than 8000 species of fungi have been identified and classified with nomenclature [1]. Aspergillus parasiticus us is a saprophytic fungus that survives in soil and rotting plant material. It is one of the Aspergillus species, toxigenic that can produce aflatoxins B₁, _B2, _G1, & _G2 [2]. Other groups of fungi that produce aflatoxins include A. flavus and A. nomius [3]. Aflatoxin consumption causes major toxic effects including carcinogenic, hepatotoxic, immunosuppressive, teratogenic, mutagenic, genotoxic and cytotoxic effects [4,5]. Additionally, in humans, aflatoxins are known to be associated with Indian childhood cirrhosis, Reye’s syndrome, Kwashiorkor and respiratory diseases [6]. A. parasiticus breeds on several food and feed products, namely maize, rice, peanuts, cotton seeds, and milk, producing aflatoxins that cause food contamination [7].

The yeast Candida albicans is a dimorphic organism residing in healthy human gastrointestinal and urinogenital tracts. However, when the host defence mechanism of the host is compromised, it can cause severe infections [8]. The commensal or symbiotic C. albicans can transform into a pathogen in the presence of the virulence factors released by the organism such the hydrolases which allow it to penetrate the hosts, and in patients with comorbidities such as diabetes, alcohol consumption and smoking, antibiotic therapy, glucocorticoids, chemotherapy, radiotherapy, upper esophageal damage, and old age [9]. As C. albicans exhibits several morphological forms such as blastospores, pseudohyphae, and hyphae, it can spread to the skin and mucosal surface and may cause systemic infections in a variety of host niches.

Fungi possess a well-defined cell wall structure containing components that distinguish them from other eukaryotic cells and exhibit species-specific variations in their composition [10]. In fungal cell wall, is composed of different polysaccharides including chitin, cellulose, glucan, polyuronides and glycoproteins. The fungal cell wall. One of the chief constituents is chitin which is organized in the form of microfibrils and exhibits a significant contribution in maintaining the strength and integrity of the cell wall thus forming the major framework of the cell wall structure [11-13]. Chitin is a non-soluble polymer that contains β-1, 4-N-Acetylglucosamine (GlcNAc) monomers. Although it is found to be physically separated from the glucan within the cellular wall, it is chemically bonded to provide structural support [14,15]. In Candida albicans, chitin comprises 2-4% of its cell wall [15,16]. In filamentous fungi, chitin constitutes 10-30% of the dry weight of the cell walls. It is found in the spores and mycelia of the fungi. Chitin analysis was considered one of the significant methods for evaluating the fungal biomass. Polyoxins and Nikkomycins are promising antimycotics that target chitin biosynthesis.

The fungi cell membrane is composed of sterols and ergosterol is the most prevalent sterol. Sterols have a crucial role in maintaining fungal cell membranes by controlling their fluidity, heterogeneity, rigidity, resistance to water penetration, and overall integrity. The inhibition of the ergosterol biogenesis pathway results in reduced levels of cell membrane ergosterol levels thereby causing the growth inhibition of the fungi [17]. The azole based antifungal drugs mostly target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase. Among the azoles fluconazole is widely applied as antifungal agent but several fungal pathogens are emerging resistant to it [18]. As the ergosterol biosynthetic pathway influences the viability of the fungal cells and as it functions as an antifungal drug target, the ergosterol biosynthetic pathway additionally plays a part in the emergence of resistance of the fungus to the antifungal drugs [19].

Several studies projected ergosterol as a primary index for fungal contamination [20,21] and is widely recognized as a significant indicator of fungal presence in culinary and agricultural settings [22]. Studies conducted on mycorrhiza showed that a combination of chitin and ergosterol analysis proved to be a reliable indicator of fungal biomass [23]. It is reported that fungal infections, majorly the infections caused by Candida species and Aspergillus species are associated with the death of over one million individuals annually [24]. These underrated infections are difficult to eliminate, and the death rate associated with these diseases is increasing although antifungal treatments are available [25].

Fluconazole and Amphotericin are the widely used anti-fungal drugs, fluconazole being the primary choice of drug [26]. Other antifungal azole drugs are also in use viz ketoconazole, voriconazole and itraconazole. But most of these drugs show side effects such as itching, rashes, abdominal pain, diarrhoea and liver damage. This problem is further aggravated by the incidence of resistance of C. albicans to the available conventional antifungal drugs [27].

Therefore, to combat the resistant fungi, it has become vital for the discovery of new antifungal drugs with minimal side effects. Our previous studies also establish that neem seed kernel extracts exhibit antifungal and anti-aflatoxigenic potentials and may find applications in formulating antimicrobial preparations for crop protection in field and for human and animal healthcare [28]. In this context, natural products are found to be promising alternatives for the treatment of fungal infections, few of which are in clinical use [12,29,30]. Rosmarinic acid is a phenolic acid compound extracted from plant species belonging to Boraginaceae and subfamily Nepetoideae of the Lamiaceae plant species. The RA is found more in Liliaceae family plant species namely Rosmarinus officinalis, Perilla and Salvia species [31]. Rosmarinus officinalis essential oil was found to show antifungal and anti-aflatoxigenic activity against A. flavus. Salvia species extract was also found to exhibit antifungal activity against different Candida species [32- 34].

The role of vitamins in mitigating the detrimental effects of pathogenic fungi in chief crops and processed agricultural foods has been reported. In this regard, Vitamin E (α-tocopherol), a potential fat-soluble antioxidant compound sourced mainly from vegetable oils was found to exhibit a growth-inhibitory effect on A. flavus [35]. Vitamin E acetate, (an acetic ester of Vitamin E), α-tocopherol acetate (ATA) were reported to exhibit anti-inflammatory effects in an in vitro model of candidiasis [36]. Certain studies also reported the potency of Vitamin E in reducing microbial adhesion especially established for C. albicans. These microbes are known to adhere and produce biofilm on a variety of biomaterials and causing infections [37]. Similarly, ATA is also reported to display anti-biofilm effects against various bacterial species by hindering the bacterial colonization of medical devices thereby reducing healthcare-associated infections [38]. The present study focused on analysing the effect of three selected antioxidants, RA, AT and ATA on A. parasiticus and C. albicans microbial growth.

Materials and methods

Chemicals and Reagents

Rosamarinic acid [(RA) (R)-O-(3,4- Dihydroxycinnamoyl)-3-(3,4- dihydroxyphenyl) lactic acid, Alpha Tocopherol [(AT) 2,5,7,8-Tetramethyl-2-(4′,8′,12′- trimethyltridecyl)-6-chromanol, 5,7,8-Trimethyltocol, D-α- Tocopherol, Vitamin E] and Alphatocopherol acetate [(ATA) Vitamin E acetate, all-rac-α-Tocopherol acetate], Fluconazole, Dimethyl Sulphoxide (DMSO), 2,2- Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, St. Louis, USA. Tween-20 was obtained from Loba Chemie, Mumbai, India; All other chemicals and reagents used were of analytical grade

Fungal Strains and Media

Aspergillus parasiticus (NRRL 2999), a toxic fungal strain, was obtained from the United States Department of Agriculture (USDA), located in Peoria, Illinois, in the USA. The culture was inoculated and maintained on PDA slants at a temperature of 28°C for 8 days in a BOD incubator on PDA slants (Remi Laboratory Instruments, Mumbai, India), and was sub-cultured regularly.

Candida albicans (MTCC 183), a pathogenic strain, was purchased from the Institute of Microbial Technology (IMTECH), Chandigarh. The C. albicans were cultured and maintained on YEPD agar medium in the laboratory as specified in the Microbial Type Culture Collection and Gene Bank (MTCC) protocol.

Potato Dextrose Agar (PDA) medium

The PDA agar (3.9 %) media was prepared, and the pH was adjusted to 6.0. Fractions of 20 mL medium were distributed into 100 mL Erlenmeyer flasks, plugged with cotton and autoclaved at 103 kPa pressure, 121°C for 15 min [39].

Yeast Extract Potato Dextrose (YEPD) agar medium

Yeast agar medium with yeast extract 0.3 % (w/v), peptone 1% (w/v), dextrose 2 % (w/v) and agar 1.5 % (w/v) were dissolved in Milli-Q water and prepared as per the growth medium details specified by the MTCC and autoclaved at 103 kPa pressure, 121°C for 15 min (According to MTCC media composition).

YES, aqueous medium

YES, a medium containing 2% (w/v.) yeast extract, and 15% (w/v) sucrose was prepared, and the pH was adjusted to 6.0. Then 20 mL portions of the broth were transferred into conical flasks of 100 mL capacity, plugged with cotton, and sterilized for 15 min at 121°C, 1.05 kg cm-2 of pressure in an autoclave [40].

YEPD broth

YEPD broth with yeast extract 0.3 % (w/v), peptone 1% (w/v) and dextrose 2 % (w/v) was prepared as per the growth medium details specified by the MTCC. The 20 mL aliquots of the broth were transferred into Erlenmeyer flasks of 100 mL, plugged with cotton, and sterilized in an autoclave for 15 min at 121°C and 1.05 kg cm^-2 (103 kPa) of pressure.

Preparation of the RA, AT and ATA solutions

Dimethyl sulfoxide (DMSO) was used as a solvent for the preparation of antioxidant solutions. The RA stock was prepared at 1.0 mg mL^-1 while AT and ATA stock solutions were prepared at concentrations 100.0 mg mL^-1.

Estimation of antioxidant activity of the selected antioxidants: DPPH assay

Rosmarinic acid, AT and ATA, each of the concentrations, 0.5μg mL^-1, 1.0μg mL^-1, 2.5μg mL^-1, 5μg mL^-1, 10μg mL^-1, 25μg mL^-1, and 100μg mL^-1 were added individually to a solution of 0.002% of DPPH in methanol. The solutions were kept for incubation at room temperature for a duration of 30 min in the absence of light, and the absorbance measurement was conducted at a wavelength of 517 nm against a blank containing 0.002% of DPPH. Ascorbic acid at varied concentrations (0.5^-100μg mL^-1) was used as a reference standard. Triplicate assays at each of the concentrations for the test compounds were carried out and the absorbance was measured. The percentage (%) of inhibition of DPPH at each of the concentrations of the test samples were computed utilizing the subsequent equation:

% Inhibition = [(A_C-A_S)/ A_C] x 100

Where Ac denotes the absorbance of the control and As denotes the absorbance of the sample. The concentration of the sample that was required to scavenge 50% of DPPH (IC₅₀) was calculated [41].

Preparation of inoculum

The A. parasiticus cultures maintained on PDA slants were used to prepare fungal spore inoculum in 0.01% Tween 20 [40]. The spore suspensions containing 1×10⁶ spores were aseptically inoculated into the Erlenmeyer flasks comprising 20 mL of the YES medium prepared. The culture flasks were maintained in a cooling incubator at a temperature of 28±1°C under stationary conditions. The fungus was grown over a duration lasting 12 days. A loop of C. albicans culture was inoculated in the YEPD agar slants and incubated at 4°C. Subsequently, the cells were inoculated into 100 mL of freshly prepared YEPD media and incubated for a duration of 48 h at a temperature of 30°C. To obtain the cell suspension at 1.5 x 10⁶ CFU mL^-1, 30°C grown microbial cultures were diluted adequately in sterile YEPD broth to the 0.5 McFarland standard [42].

Antifungal Assay: Well Diffusion Method

The well diffusion assay method was used to measure the antifungal efficacy of RA, AT, and ATA against the two fungal strains, A. parasiticus and C. albicans. The spread plate technique was used to inoculate the PDA plates with 1 x 10⁶ fungal spores of A. parasiticus in a volume of 30 μl. In the case of C. albicans cell suspension at 1.5 x 10⁶ CFU ml^-1 was applied to the YEPD agar plates. The wells with a diameter of 8 mm were made in the plates. The wells were loaded with the test compounds in various concentrations namely RA in the concentrations of 1.5, 10, 25, and 50μg mL^-1; while AT and ATA were at higher concentrations of 0.5, 1.0, 5.0 and 10 mg mL^-1.s

The negative control wells were maintained with DMSO. As a positive control, the reference standard antibiotic Fluconazole (8.0μg mL^-1) was used. The treated PDA plates were maintained at a temperature of 28±1°C under stationary conditions and the A. parasiticus fungus was permitted to proliferate for 7 days. On the other hand, for C. albicans, the test plates were subjected to incubation for a duration of 48 h at 30°C in stationary conditions. The experiments were carried out in triplicates for each of the selected compounds and the zone of inhibition (ZOI) was quantified in mm by subtracting the well diameter from the overall diameter of the inhibition zone [43].

Determination of MIC by Micro broth dilution method

Using the broth micro-dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI, previously the National Committee for Clinical Laboratory Standards [NCCLS]), the fungicidal activity of RA, AT, and ATA against A. parasiticus and C. albicans was assessed [44,45].

The sterile polystyrene microtiter plate wells were loaded with various concentrations of the compounds and YES broth (100μL) containing 1 x 10⁶ spores of A. parasiticus. The plates were subjected to a 48h stationary incubation period at 28±1°C. In the case of C. albicans, the wells after being loaded with varying amounts of the test compounds, were inoculated with 100 μl of YEPD broth containing 1 x 10⁶ CFU mL^-1 of C. albicans and then the plates were kept for incubation for a duration of 48 h at 30±1°C. The fungal suspension without the test compounds was included in each dilution series as the growth control (GC) wells. Each dilution series also included media control (MC) wells that contained only the YES media free of A. parasiticus, YEPD media devoid of C. albicans and the test compounds that served as negative controls. An ELISA microplate reader (Micro Scan, MS5608A, ECIL, India) was used to measure the absorbance of each well. The minimum inhibitory concentration (MIC) values were monitored visually by measuring the turbidity that appeared in the wells and recording the absorbance at 546 nm. The MIC is characterized as the minimum concentration of the test substances that showed complete inhibition of the fungal growth in comparison to the controls. The experiment was carried out in triplicates for each of the test compounds.

Scanning Electron Microscopy Analysis

Using SEM with an accelerating voltage of 18 kV, the morphological alterations in A. parasiticus and C. albicans treated with the test drugs were observed.

parasiticus

The parasiticus fungal mycelial mat, formed in YES media after 7 days of incubation, was treated with RA, AT and ATA at their respective MIC concentrations and a negative control without the test compounds was also maintained. The treated and the control samples were incubated at 28±1°C for 24 h under stationary conditions. The samples were fixed overnight in a solution of glutaraldehyde [2.5% (v/v)] in 0.1M sodium phosphate buffer at a pH of 7.2. The mycelia were dehydrated using a sequence of water-acetone solutions with increasing acetone concentration (10% increments from 30-90%) for 60 min each. They were then exposed to 100% acetone for 180 min and left overnight in 100% acetone. Finally, the mycelia were immersed in hexamethyldisilazane and then preserved at 4°C. The carbon tape was covered with mycelium, dried in the air, and then coated with gold using a sputtering process. The sputtered sample was dried under vacuum and installed for imaging under the Scanning Electron Microscope (JEOL, JCM 6000 plus). The samples were observed in the magnification range of: x 1500 to 3000 [46].

albicans

C.albicans cell suspension (10 mL) bearing a concentration of 1x10⁶ CFU mL^-1 was subjected to incubation at 30°C, for 24 h on a YEPD broth containing RA, AT, and ATA at their MIC and the control group (i.e., the medium containing no test compounds). The test compound-treated yeast samples were cautiously washed with 0.1 M phosphate buffer (pH 7.2). Using 2% Osmium tetroxide, the post-fixation was carried out for 2 h at room temperature. Preliminary dehydration was carried out by placing specimens in a series of ethanol gradients. The first step of dehydration was done two times for 10 min using 50% and 70% ethanol, the second dehydration was carried out again two times but with 95% ethanol for 5 min and finally 100% ethanol two times for 1 min. Subsequently, the samples were dehydrated with acetone, two times for 30s each till they were dried by the critical point method in liquid CO₂. Later, the specimens were subjected to gold spluttering and the sputtered samples were examined under a scanning electron microscope [47].

Chitin analysis

Extraction of chitin from A. parasiticus and C. albicans

A 20 mL of YEPD broth taken in separate conical flasks, were inoculated with an individual Candida colony from a YEPD agar plate harboring culture and incubated overnight. To the culture the test compounds were added at their MIC; RA (7.82 μg mL^-1), AT (200 mg mL^-1) and ATA (100 mg mL^-1). Conical flasks with only the YEPD broth and C. albicans devoid of any test compounds were considered as controls. Similarly, A. parasiticus spore suspension in the volume of 100 μL containing 1x10⁶ spores was inoculated for seven days at 28±1°C in 20 mL of YES medium containing RA, AT, and ATA at their respective MIC values, i.e., RA at 15.62 μg mL- 1, AT at 200 mg mL^-1, and ATA at 200 mg mL^-1. Control samples were devoid of any test compounds. After the incubation period of 24 h and 7 days for C. albicans and A. parasiticus respectively, the samples were subjected to the process of centrifugation at 2,700 rpm for five minutes and subsequently, the cell pellet’s net wet weight was calculated and the pellet was suspended in one mL of 4M HCl and boiled for 4 h [48,49]. The hydrolysate obtained was further analyzed for the chitin content.

Estimation of chitin content in A. parasiticus and C. albicans

The number of amino sugar content within the hydrolysate obtained was estimated by the modified Elson-Morgan method [48], wherein N-acetyl-D-glucosamine (subjected to hydrolysis using the identical parameters mentioned earlier) served as a standard. The hydrolysates obtained were mixed with 19 mL of fresh distilled water to achieve a final concentration of 0.2 M HCl. Following this, 1 mL of acetyl acetone reagent, which consists of 2% (v/v) acetyl acetone and 1.25 M Na₂CO₃, was added to the 0.5 mL portion of the hydrolysates. The solutions were then subjected to incubation at a temperature of 90°C for 60 min and later were allowed to cool to the ambient temperature and thereafter treated with ten ml of C₂H₅OH and 1 mL of Ehrlich’s reagent. The resulting mixture was subjected to incubation at the ambient temperature for a duration of 60 min. The amount of hexosamine in the processed samples was determined spectrophotometrically by measuring OD at 530 nm with a spectrophotometer. All the sample analyses were carried out in triplicates. Glucosamine hydrochloride (0.020mg) was employed as a standard. A correction factor of 0.829 is required to represent values of the unknown/test sample in terms of free hexosamine.

Hexosamine (mg) in the sample =

(Optical density of the unknown)/ (optical density of the standard) X (0.020) (0.829).

M = m × 203/179

Where, m = mass of glucosamine as calculated by analysis

203 = molecular weight of acetylglucosamine anhydride (g mol^-1)

179 = molecular weight of glucosamine (g mol^-1).

Ergosterol analysis

Extraction of ergosterol from A. parasiticus and C. albicans

Extraction of ergosterol from fungal cultures was achieved successfully following the standard protocols [50,51]. A 100μL spore suspension of containing 1x10⁶ spores of the fungus A. parasiticus was inoculated in 20 mL of YES medium containing RA, AT and ATA at their respective MIC concentrations i.e., RA of 15.62 μg mL^-1, AT of 200 mg mL^-1 and ATA of 200 mg mL^-1. These cultures were kept for incubation for a period of seven days at a temperature of 28±2°C in a BOD incubator (Remi Laboratory Instruments, Mumbai, India). Samples without the compounds were considered as controls. In case of Candida, a single colony from an overnight YEPD agar plate culture was used to inoculate 20 ml of YEPD broth in flasks maintained separately each for control and RA (7.82 μg mL^-1), AT (200 mg mL^-1) and ATA (100 mg mL^-1) containing media. The cultures were incubated for 24 h at 30°C in a BOD incubator. After incubation, the cultures were harvested by centrifugation at 2,700 rpm for five minutes and cell pellets net weights were calculated after removing excess moisture. The samples obtained from Aspergillus and Candida were treated with 5 mL and 3 mL of 25% potassium hydroxide in alcohol solution respectively. The mixtures were then vigorously stirred for 2-3 min and kept for incubation at a temperature of 85°C for a duration of 4 h and 1 h for Aspergillus and Candida samples respectively. The sterols were extracted by combining a mixture of 2 mL of sterile distilled water and 5 mL of n-heptane for Aspergillus samples and in the case of Candida, 3 mL of n-heptane and one mL of sterile distilled water were added. The mixtures were then vigorously vortexed for 2-3 min following incubation, and the layer of heptane was transferred to a sterile borosilicate glass tube, screw-capped and preserved at a temperature of -20°C.

Estimation of ergosterol content in Aspergillus parasiticus and C. albicans

The n-heptane fractions of A. parasiticus and C. albicans cultures were analyzed using UV-vis spectrophotometry between 230 and 300 nm. Ergosterol displays absorbance at 282 nm and 24 [28] dehydro-ergosterol (DHE), the late sterol intermediate shows absorbance at 230 nm wavelength and 282 nm wavelength in the n-heptane layers [50]. Using the measurements of absorbance and moist weight of the initial pellet, the amount of ergosterol was determined as a percentage of the cell’s wet weight. The following formula is used to determine the amount of ergosterol:

%Ergosterol + %24(28) DHE = [(A_281.5/290) x F]/pellet weight,

%24(28) DHE = [(A₂₃₀/518) x F]/pellet weight,

%Ergosterol = [%Ergosterol + %24(28) DHE] -%24(28) DHE

Where F = factor for dilution in ethanol; 290 = percent extinction coefficient (E) values per centimeter determined for crystalline ergosterol in an absolute alcohol solvent; 518 = percent extinction coefficient (E) values per centimeter determined for 24 [28] DHE in an absolute alcohol solvent [52]. All the analyses of the samples were carried out in triplicates.

Statistical analysis

The experimental investigations were carried out in triplicates. The values were reported in mean±standard deviation (SD). The GraphPad prism software (version 9.3) was used for the statistical analysis and plotting of graphs. A ‘p’ value less than 0.05 was deemed to be statistically significant.

Results

DPPH Assay

The change in absorbance observed by the reduced DPPH was employed to gauge the antioxidant capacity of the test samples. The results depicted a decrease in absorbance caused by varying concentrations of RA, AT, ATA and ascorbic acid. Ascorbic acid was used as a reference standard for the analysis. The analysis revealed that among the three compounds selected for the study, RA is more potent than AT and ATA as graphically represented in (Figure 1). The IC₅₀ values for RA, AT and ATA were found to be 4.32±0.04 μg, 13.31±0.23μg and 16.49±0.17μg respectively when compared with that of ascorbic acid which was,, found to be 3.32±0.06 μg and is presented in (Table 1).

Each value is expressed as a mean ± standard deviation (n = 3). Significant p value (*p<0.05) were obtained by Student’s t test analysis.

Well Diffusion Assay

All three samples RA, AT and ATA exhibited antifungal effects on A. parasiticus and C. albicans as depicted in (Figure 2) by the agar well diffusion method. The ZOI in mm at different concentrations of RA, AT and ATA were measured and the results are depicted in (Table 2). The highest concentrations of RA, AT and ATA used for the assay are 50 μg mL^-1, 10mg mL^-1 and 10mg mL^-1 respectively. At the highest concentrations chosen, RA, AT and ATA exhibited ZOI of 20.00, 15.67 and 16.33 mm against A. parasiticus respectively. In the same way, RA, AT and ATA exhibited ZOI of 15.67, 12.00 and 11.67 mm against C. albicans respectively (Table 2). Dimethyl sulphoxide which was used as a solvent for preparing various concentrations of the test compounds was considered as a negative control and did not show any inhibition zones in both the microbial species. The antibiotic fluconazole used as positive control displayed 21.80 and 16.67 mm ZOI against A. parasiticus and C. albicans respectively. The zone of inhibition visualized is depicted in (Figure 2.1 & 2.2).

RA= Rosmarinic acid, AT= α-tocopherol, ATA= α- tocopherol acetate, ND= Not detected

MIC Determination

The minimum inhibitory concentration (MIC) of RA, AT and ATA against A. parasiticus and C. albicans were determined based on the absorbance recorded in the fungal cultures and are depicted in (Figure 3). The positive control chosen for the analysis was the standard drug fluconazole. The MICs of RA, AT and ATA against A. parasiticus were 15.62 μg mL^-1, 200 μg mL^-1 and 100 μg mL^-1 respectively and against C. albicans were 7.82 μg mL^-1, 200 μg mL^-1 and 100 μg mL^-1 respectively (Table 3). The MIC was lowest for RA against both A. parasiticus and C. albicans. The MIC values obtained indicated that RA is more potent against C. albicans and the most effective compared to the other two antioxidants used in the study. Interestingly, the MIC of AT (200 mg mL^-1) was observed to be the same for both A. parasiticus and C. albicans. Likewise, the MIC of ATA (100 mg mL^-1) was found to be identical for both fungal species. However, ATA displayed lower MIC than AT indicating that the acetate form of AT holds more ability in showing fungal inhibitory activity.

Scanning Electron Microscope studies

The effect of RA, AT and ATA at their MIC on the morphology of A. parasiticus was examined using SEM investigation. The SEM photographs demonstrated that in untreated control, the fungi mycelium was normal while in the treated samples, the mycelium was irregular, shrunken and damaged as depicted in (Figure 4). Similarly, the SEM analysis of the C. albicans at MIC of RA, AT and ATA-treated cells showed irregularities on their surface and had scars indicating morphological abnormalities compared to the control which appeared normal as depicted in (Figure 5).

Chitin analysis

The fungal samples were analyzed for chitin as per Yabe et al., 1996. The results observed are presented in (Figure 6). The chitin content in the untreated control sample was 1.454±0.039 mg g^-1. In RA, AT and ATA exposed fungal cells the chitin content was estimated to be 0.868±0.087 mg g^-1, 1.340±0.077 mg g^-1 and 1.179±0.067 mg g^-1 respectively. Thus, the fungal samples treated with the compounds under the study exhibited a decrease in the chitin levels in comparison to the control with a p value of statistical significance i.e. < 0.05. Further, the potency of the test compounds in influencing the chitin synthesis was found to be in the order, RA>ATA>AT. Similarly, the chitin levels were also evaluated in C. albicans samples which were treated with RA, AT and ATA at the specified MIC. The chitin levels in the untreated control yeast samples were estimated to be 2.414±0.463 mg g^-1. Rosmarinic acid, AT and ATA downregulated the chitin levels in C. albicans, and the chitin levels recorded were 1.320±0.175 mg g^-1, 1.844±0.103 mg g^-1 and 1.778±0.234 mg g^-1 respectively. RA was found to be the most potent followed by ATA and AT.

Ergosterol analysis

The ergosterol levels were evaluated spectrophotometrically in A. parasiticus cultures treated with RA, AT and ATA at their respective MICs and incubated for seven days. The results revealed that the selected compounds had a negative impact on the ergosterol synthesis thereby decreasing the ergosterol level in comparison to the control sample. Rosmarinic acid significantly (p<0.05) decreased the ergosterol levels by 72% in comparison with the control and showed a better potency than AT and ATA. The α-tocopherol (AT) and ATA were significantly able to reduce the ergosterol content by 62% and 65% respectively compared to the control fungal cells as depicted in (Figure 7). Likewise, RA, AT and ATA were tested at their MICs against C. albicans incubated for 48h, to analyze their potency in altering the amount of ergosterol in the C. albicans cellular membrane. All the selected compounds were able to lower the ergosterol levels in C. albicans. The percentage decline in the amount of total ergosterol was 63%, 53 % and 60% in the presence of MICs of RA, AT and ATA respectively.

Discussion

The Candida genus is the major pathogen in the spread of yeast-based infections in humans among which C. albicans is the predominant (90%) virulent species [15,53]. Aspergillus species are the common fungal organisms for the incidence of disorders in various plants and contaminate the plant products directly or mediated through mycotoxins [54]. Antimycotics, the antifungal agents employed in the management of fungal infections are limited and are comparatively less than their antibacterial counterparts. The mechanisms through which antifungal drugs target are also associated with side effects and the fungi have developed alternative ways to adjust. The resistance to antifungal drugs is one of the chief reasons for advancing the research studies of antifungal medication development. Hence, it is challenging to generate drugs with maximum selectivity and fewer side effects on humans. All these create a need for the emergence of designing novel antifungal drugs of natural origin [19,55]. Our earlier study demonstrated the binding affinity of Rosmarinic acid, α-tocopherol and their derivatives with CYP51 of Candida spp. and their drug-likeliness potencies in in silico [56].

Rosmarinic acid (RA) and plant extracts containing RA such as extracts of rosemary, are the most widely utilized natural antioxidants that are included in food products [57]. Alpha-tocopherol is a biologically active compound and is one of the most potent lipophilic antioxidants that has a significant role in protecting cells from reactive oxygen species [58]. Similarly, an investigation revealed that α-tocopherol and α-tocopherol acetate have comparable effects as free radical traps in rat liver microsomes that are supplemented with tert-butyl hydroperoxide [59]. Based on the earlier reports, in the present work, DPPH analysis was carried out to understand the antioxidant abilities of purified RA, AT and ATA. The DPPH analysis revealed that RA possesses a significantly higher antioxidant capacity than AT and ATA with IC₅₀ of 4.322±0.043μg. This result is supported by an earlier study that reported, RA possesses higher antioxidant potency in comparison to α-tocopherol, BHT, TBHQ and BHA and can be a better natural substitute for the antioxidants used in the food industry [60].

Rosemary extracts contain Rosmarinic acid as one of the primary bioactive materials showing antibacterial properties [61]. The essential oil of rosemary was reported to show fungicidal effect against C. albicans and A. niger [62]. Rosmarinic acid is one of the major phenolics found in mints namely Mentha piperata and Mentha spicata [63]. Mentha species essential oil showed inhibitory efficacy against C. albicans and Aspergillus spp. α-tocopherol is an important constituent of Ficus carica leaves accounting for an amount of 57 mg 100 g^-1 of dried leaves [64] and leaf extracts of Ficus carica exhibited antifungal activity against the Aspergillus species [65]. Similarly, other studies indicated that methanolic fractions of Ficus carica displayed a 100% inhibitory effect on C. albicans [66]. In this aspect, the well diffusion assays carried out in our study demonstrated that all three selected antioxidants namely RA, AT and ATA had significant inhibitory activity (p<0.05) on the growth of A. parasiticus and C. albicans.

There were significant variations in the MIC values exhibited by RA, AT and ATA. The MIC of RA was less in both the fungal species in comparison with AT and ATA and the MIC of RA in C. albicans (7.82 μg mL^-1) was less in comparison to A. parasiticus (15.62 μg mL^-1). The S. montana and S. subspicata extracts, containing Rosmarinic acid as the most prevalent phenolic compound with a concentration ranging from 1.11% to 3.31% (w/w), were found to be effective against clinical isolates of Candida spp. with MIC values ≤ 32.9 μg mL^-1 [67]. Similarly, Cynara cardunculus extracts were found to contain phenolic compounds and flavonoids as the major bioactive compounds, Rosmarinic acid being one of the phenolic compounds. The ethanolic extracts of the artichoke showed antifungal activity against eight species of mycotoxigenic fungi with MIC values of 0.87-4.16 mg mL^-1 [68]. There are no studies to date about the effect of purified RA on A. parasiticus, however, there are limited studies that reported the antimicrobial potency of purified RA on C. albicans. Further studies revealed that RA possesses antimicrobial potential against 11 Candida strains with MIC ranging from 100-200 μg mL^-1 [69].

Both AT and ATA showed the same MIC values against A. parasiticus and C. albicans. Earlier, a research group reported that whole ground black cumin followed by black cumin oil had higher α-tocopherol content which inhibited A. flavus and its aflatoxin production [70]. Additionally, Curtisia dentata acetone extract (30%) showed antifungal activity against A. flavus & A. ochraceous with MIC of 0.63 mg mL^-1 and 0.08 mg mL^-1 respectively. Further, the phytochemical analysis of the extract revealed vitamin E as one of the major constituents [71]. When compared, our studies suggest that since A. parasiticus is a highly toxigenic strain the concentration of compounds needed to inactivate it might be higher than on other fungal species.

All three antioxidants, RA, AT and ATA damaged the morphology of A. parasiticus and C. albicans with irregularities and scars on the surface as examined by SEM. Similar observations were reported wherein it was shown that Salvia officinalis, a good source of many biologically active compounds particularly Rosmarinic acid as one of the phytochemicals that was used to synthesize zinc oxide nanoparticles cause morphological changes in the C. albicans clinical isolates [72]. The SEM analysis revealed the development of lesions and furrows on the cell wall and cell membrane leading to cell death. The nanoparticles stabilized by the phytochemicals were also able to disrupt the cell membrane by inhibiting the production of ergosterol.

It is well established that chitin analysis can be used as a suitable marker for the assessment of fungal growth in various samples. The hydrolysis of chitin polymer leads to the formation of N-acetyl-D-glucosamine and its colorimetric estimation was formerly used as a biomarker for fungal contamination [22]. In the fungal cell membrane, the ergosterol biosynthetic pathway is the major antifungal drug target. Ergosterol is a major component of the fungal cell membrane and has been established as a good indicator of fungal growth owing to its correlation with the metabolically active biomass [73]. Thus, not only the determination of ergosterol as biomarker for fungal contamination allow for monitoring changes during fungal growth in food samples but also its biosynthesis pathway is a target for the major antifungal drugs [22]. In the present study, the chitin and ergosterol were extracted from A. parasiticus and C. albicans cultures and quantified spectrophotometrically.

Rosmarinic acid at MIC of 15.62μg mL^-1 lowered the chitin levels by 40% and ergosterol levels by 72% in A. parasiticus. In C. albicans, RA reduced the ergosterol by 63% and the chitin content was lowered by 45%. Studies by earlier researchers revealed that phenolic compounds extracted from plants exhibit antifungal activity by blocking ergosterol synthesis [74,75]. These compounds were found to diffuse through the cell membrane and enter the cell where they alter the synthesis of chitin and ergosterol by interfering in the biosynthetic pathways. Essential oil from Coriandrum sativum was reported to be effective against moulds and lead to the decrement of the ergosterol amount in the fungal cell membranes [76]. A decrease in the cell wall chitin levels in C. albicans when treated with Cleome viscosa essential oil was also reported [77].

So far there have been no studies that evaluated the direct effect of AT and ATA on fungal chitin and ergosterol synthesis. In this perspective, our study demonstrated that AT and ATA lowered the chitin levels in A. parasiticus and C. albicans with the reduction of chitin content being significantly more in A. parasiticus treated compounds than the Candida albicans. Between α-tocopherol and α-tocopherol acetate, the ATA was more potent in reducing the chitin content in both A. parasiticus and C. albicans. Similarly, AT and ATA also affected the ergosterol synthesis in both the fungi under the study with ATA reducing the ergosterol content significantly than AT. Similar observations were reported about the essential oils of Mentha piperata that contained α-tocopherol and Rosmarinic acid as two of the major constituents and the leaf oils showed antifungal activity by lowering the ergosterol levels [73].

Conclusion

Our present study revealed that Rosmarinic acid (RA), α- tocopherol (AT) and α-tocopherol acetate (ATA) exhibited antifungal activities against A. parasiticus and C. albicans. Among the three antioxidants, RA was found to be more potent than AT and ATA and C. albicans was more susceptible. The morphological damage and the inhibitory effects on chitin and ergosterol in both the fungal organisms due to the compounds were significant. These findings suggest that all the selected antioxidants are potent antifungal agents and among the three antioxidants, RA could serve as a valuable natural alternative to the commercial chemical antifungal drugs in usage. Further, in vivo investigations are needed to explore the antifungal potential of RA, AT and ATA and to decipher the mechanisms by which the selected test compounds could target the chitin and ergosterol biosynthetic pathways. Additionally, the synergistic effect of RA, AT and ATA can be studied and may hold the potential for formulating topical treatments alongside the existing medications or on par with the existing antifungal drugs. Such a formulation could provide clinicians with additional tools for managing the diseases caused by A. parasiticus and C. albicans. Moreover, the topical sprays of the concoctions of the selected antioxidants may protect the crops affected by the Aspergillus spp. thus evading the deleterious effects of the fungi on the crops and their yield.

Author’s Contribution

VC carried out all the experimentation, acquisition and analysis of data and drafting of the manuscript. MZG assisted with the acquisition and analysis of data and drafting of the manuscript. HMA and KNS assisted in the drafting of the manuscript. KR conceived, designed, and supervised the study and revised the manuscript. All authors have read and approved the final manuscript.

Declaration of competing interest

The authors declare that there are no conflicts of interest.

Acknowledgements

All the authors are thankful to the Department of Biochemistry, Osmania University, Hyderabad. VC is also thankful to the Department of Biochemistry, Government City College, Hyderabad for the support during the Ph.D. studies.

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