Elucidation of antifungal toxicity of Callistemon lanceolatus essential oil encapsulated in chitosan nanogel against Aspergillus flavus using biochemical and in-silico approaches
The antifungal and aflatoxin B1 (AFB1) inhibitory effect of chemically characterised Callistemon lanceolatus essential oil (CLEO), chitosan nanoparticles, and CLEO loaded chitosan nanoparticles (CLEO-ChNPs) were investigated. Scanning electron microscope observation exhibited the spherical shape of prepared CLEO-ChNPs with an average range of 20–70 nm. An in-vitro release study revealed the controlled volatilisation of CLEO from CLEO-ChNPs. The CLEO-ChNPs caused complete inhibition of growth (4.5 µl/ml) and AFB1 (4.0 µl/ml) production by A. flavus at a low dose compared to free CLEO (5.0 µl/ml). The antifungal and AFB1 inhibitory toxicity of CLEO-ChNPs were elucidated using biochemical (effect on ergosterol biosynthesis, membrane cations, mitochondrial membrane potential, C-sources utilisation and cellular methylglyoxal level) and in-silico (interaction with the gene product Erg 28, Cytochrome c oxidase subunit Va, Omt-A, Ver-1, and Nor-1) approaches.
Keywords: Aflatoxin B1; essential oil; mode of action; nanoparticles; in-silico
Graphical Abstract
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Introduction
Aflatoxin B1 (AFB1) is one of the most potent carcinogenic secondary metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. Consumption of AFB1 contaminated food commodities may cause acute and chronic toxicity to human and livestock. Among all known mycotoxins, AFB1 contamination in food commodities gain considerable attention throughout the world because of its hepatocarcinogenic, teratogenic, mutagenic, immunosuppressive effects and thermostable nature (IARC [12]). Therefore, the prescribed acceptable limit of AFB1 in the food product is regulated worldwide (Anukul et al. [3]).
Different chemical preservative agents such as sorbic, benzoic, propionic, nitrates, nitrites and their salts are commonly used to prevent the fungal and aflatoxin contamination in the food products. However, in view of green consumerism, the use of safe and natural food preservatives would have better prospects over synthetic chemicals. In this context, essential oils of aromatic plants possessing potent antimicrobial activity could be used as a safer alternative of synthetic antifungal agents (da Silva Bomfim et al. [7]). The current limitations of essential oils-based food preservatives such as strong aroma, and negative interaction with food components and surrounding environment could successfully be addressed by the use of nanotechnology approaches (Prakash et al. [23]). Nowadays, chitosan has gained considerable attention from food industries as a nanocarrier agent of plant volatiles due to its abundance, mucoadhesive, non-toxic and biological properties (Elgadir et al. [8]).
Callistemon lanceolatus (Sm.) Sweet is an aromatic plant of family Myrtaceae and widely distributed in subtropical and tropical regions. The essential oil of C. lanceolatus is known to have many biological properties such as antifungal (Shukla et al. [25]), antioxidant (Ahmad et al. [2]), and anti-inflammatory (Kumar et al. [17]). However, the low solubility, high volatility and strong aroma restrict its application in the food system. These disadvantages could be overcome by encapsulating C. lanceolatus essential oil in a nanocarrier. The antifungal efficacy of C. lanceolatus has been reported previously by Shukla et al. ([25]). However, there is no detailed mechanistic investigation in the literature on antifungal and aflatoxin B1 inhibitory potential of nano-encapsulated C. lanceolatus essential oil.
Therefore, in the present investigation, the nanoencapsulation of chemically characterised C. lanceolatus essential oil (CLEO) was carried out using chitosan nanogel to enhance its antifungal and aflatoxin B1 inhibitory activity. Further, the mechanism of toxicity of nanoencapsulated CLEO against the growth and AFB1 production by Aspergillus flavus has been investigated using biochemical and in silico approaches.
Materials and methods
Test fungus,chemicals and equipment
The toxigenic strain of Aspergillus flavus (EC-03) previously isolated in our laboratory from the infested grain of Eleusine coracana was selected as test fungus (Kumar et al. [16]). All the chemicals used in the present investigation were procured from Sisco Research Laboratories (SRL) Pvt. Ltd., Hi-Media Laboratories Pvt. Ltd, Mumbai, India, and Sigma Chemical Co. (St. Louis, MO, USA). The low-molecular-weight chitosan (50–190 kDa) with CAS 9012–76-4 and 75–85 % deacetylation was purchased from Sigma. The major equipment used in the study were hydro-distillation unit (Merck Specialities Pvt. Ltd., Mumbai, India), Gas chromatograph-mass spectrometer (Perkin Elmer, Turbomass Gold, USA), Atomic absorption spectrophotometer (PerkinElmer, Analyst 800, USA), Spectrophotometer (Shimadzu UV1800, Japan), Cooling centrifuge (Remi-CPR-24 plus, India), Probe-type sonicator (Labman Scientific instrument, Pro-500, India), Lyophiliser (Christ, alpha D plus, Australia), Scanning electron microscope (Evo-18 researcher, Zeiss), Fourier transform infrared spectrometer (Perkin Elmer, USA) and X-ray diffractometer (Bruker D8 Advance).
Extraction of Callistemon lanceolatus essential oil and chemical characterisation
The aerial part of Callistemon lanceolatus was collected (March-April 2018) from the Varanasi district of Uttar Pradesh, India. Before extraction, the sample was washed with double distilled water to remove surface contamination and then subjected to Clevenger's hydro-distillation apparatus for 4 h (Prakash et al. [24]). The essential oil was stored in a clean glass. The chemical characterisation of CLEO was performed through GC-MS using PerkinElmer Elite-5 column (column length = 30 m, inner diameter = 0.25 mm, film thickness = 0.25), 70°C initial temp (2 min); 3°C/min to 250°C; hold 10 min; Inj = 250°C, Split = 20:1; Carrier Gas = He; Transfer Temp = 180°C; Source Temp = 160°C; Scan: 40 to 400 Da and solvent delay (4.00 min) following the previously described method of Prakash et al. ([24]). The chemical constituents of CLEO were identified by comparing the retention time (Rt) and mass spectral peaks available to the databases (NIST, Wiley, and NBS)(Adams [1]).
Preparation of CLEO-loaded nanoparticles (CLEO-ChNPs)
CLEO-loaded nanoparticles (CLEO-ChNPs) were prepared following the method described by Beyki et al. ([6]) and Kumar et al. ([16]) with slight modifications. In brief, 0.5% low molecular weight chitosan (LMC) solution was prepared in aqueous acetic acid solution. The prepared LMC solution was diluted with 85 ml methanol and mixed properly using magnetic stirring at 10 g for 30 min. The pH of the aqueous solution was adjusted to 3.5–4.0 followed by ultrasonication at 40 Hz for 5 min. Thereafter, a reaction mixture containing EDC (699 μl) and cinnamic acid powder (317.5 g) was prepared and added drop-wise to 75 ml of LMC solution under continuous stirring at 10 g for 24 h. Thereafter, the pH of solution was adjusted to 8.5–9.0 using NaOH for precipitation of the nanoparticles. The samples were centrifuged at 7790 g for 20 min (4°C) and the collected pellet was purified using sterile distilled water and kept for lyophilisation under vacuum (24 h).
Prior to incorporation of CLEO inside the hydrophobic core of the nanoparticles, the lyophilised sample was re-suspended in an aqueous acetic acid solution (pH 3.5–4.0). Subsequently, different doses of CLEO were added to prepared nanogel to obtain the desired ratio of LMC: CLEO (w/v) (1:0.25, 1:0.50, 1:0.75, 1:1). The samples were ultrasonicated at 40 Hz (20 min) for proper incorporation of the CLEO inside the cage of LMC nanoparticle.
Physico-chemical characterisation of ChNPs and CLEO-ChNPs
The surface morphology and size of ChNPs and CLEO-ChNPS were observed with a field emission scanning electron microscope (EVO 18 SEM, Carl Zeiss, Germany). Air-dried samples coated with gold were used for the study.
Estimation of percent encapsulation efficiency (EE %), loading capacity (LC %), encapsulation...
The percent encapsulation efficiency (EE), loading capacity (LC) and encapsulation yield (EY) were determined following the previously described method of Keawchaoon and Yoksan ([14]) and Hosseini et al. ([11]) with slight modifications. In brief, five hundred microlitres of nano-emulsion was added to 4 ml of 2 M HCL solution followed by boiling at 95ᵒC (30 min). The samples were kept at room temperature for 1 h and subsequently 2 ml hexane were added. The mixture was centrifuged at 7790 g for 5 min at 25 ᵒC and optical density of the supernatant was recorded using a UV-vis spectrophotometer at 264 nm. The amount of CLEO was determined using a standard calibration curve (R2 = 0.999) of CLEO. A blank set without loaded CLEO was kept parallel to treatment. The percent EE, LC and EY were determined by using following equations.
(1)
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(2)
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(3)
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The maximum % EE, % LC and % EY were observed for the ratio of LMC/CLEO (1:0.75). Hence, further study was performed with this optimised dose.
For the in-vitro release studies, approximately five hundred microlitres CLEO-ChNPs were dispersed in 5 ml solution containing 60% phosphate buffer saline solution (pH 7.4) + 40% absolute ethanol. The mixture solution was kept at room temperature for 4 days under gentle agitation. The mixture solution was centrifuged at 7790 g (10 min) and 1.0 ml of the supernatant was removed and replaced with equal volume of fresh buffer at regular time duration. The absorbance of the supernatant was measured at 264 nm and percent cumulative release was determined following the equation.
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In-vitro antifungal and aflatoxin B1 inhibitory efficacy of free CLEO and CLEO-ChNPs
Effect of CLEO and CLEO-ChNPs on growth and aflatoxin B1 production by A. flavus (EC-03) was determined following the methods of Prakash et al. ([24]). In brief, the requisite amount of CLEO and CLEO-ChNPs were dissolved separately in 0.5 mL of 1% (v/v) Tween-20, followed by the addition of 24.5 ml SMKY broth medium to achieve the required concentrations (0.5–6.0 μl/ml). Subsequently, fifty microlitre spore suspension of a toxigenic isolate of A. flavus (EC-03) was inoculated in each flask and incubated for 10 days in the BOD incubator at 27 ± 2 °C. Two control sets (ChNPs without CLEO) and (0.5 ml Tween 20 without ChNPs and CLEO-ChNPs) were kept separately parallel to the treatment set. After incubation, the mycelia biomass was filtered (Whatman No.1), and dry weight was recorded. The percent inhibition of mycelial dry weight was calculated using equation 1. The filtrate was mixed with 20 ml chloroform in separating funnel. The AFB1 residue dissolved in chloroform was extracted after evaporation on a water bath (70°C). The residue was dissolved in 1 ml methanol, and 100 μl of this was loaded onto a thin-layer chromatographic plate (TLC) parallel to the standard of AFB1. The TLC was developed in mobile phase toluene: isoamyl alcohol: methanol (90:32:2 v/v/v). The blue fluorescent spots parallel to AFB1 standard were scraped off and mixed with 5 mL cold methanol followed by centrifugation at 870 g (10 min). The absorbance of supernatant was measured using UV-vis spectrophotometer at 360 nm and quantification of AFB1was determined following the equation 2. Two different control sets (one with ChNPs without oils while another with 0.5 ml 0.5 ml of 1% (v/v) Tween-20) was kept parallel to treatment set.
(4)
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A = Weight of mycelia in control, B = Weight of mycelia in treated samples
(5)
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Where, the molecular weight of AFB1 (312 kDa), the molar extinction coefficient of AFB1 (21 800), path length (1 cm).
Antifungal and aflatoxin B1 inhibitory mechanism of action of CLEO-ChNPs
The antifungal and aflatoxin B1 inhibitory toxicity of CLEO-ChNPs were elucidated using biochemical (effect on ergosterol biosynthesis, membrane cation, mitochondrial membrane potential, C-sources utilisation, and cellular methylglyoxal), and in-silico (targeting the gene products such as Erg 28, Cytochrome c oxidase subunit Va, Omt-A, Ver-1, and Nor-1) approaches.
Biochemical approaches
Effect on ergosterol biosynthesis
The effect of different doses (1.0, 2.0, 3.0, 4.0 µl/ml) of CLEO-ChNPs on ergosterol content in the plasma membrane of A. flavus was investigated following the method of Tian et al. ([27]). The ergosterol % was calculated based on the absorbance and initial pellet weight using the following formula.
Graph
Graph
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Where, 290 and 518 are the E values (in percentages per cm) determined for crystalline ergosterol and 24(28) dehydroergosterol, respectively and pellet weight is the net wet weight (g).
Effect on membrane cation
The effect of different doses (2.25, 4.5, and 9.0 µl/ml) of CLEO-ChNPs on leakage of cellular cations (Ca+2, K+ and Mg+2) from the membrane of A. flavus was investigated following the method of Helal et al. ([9]). The content of cations Ca+2, K+ and Mg+2 were estimated in the supernatant using an atomic absorption spectrometer (Perkin Elmer, Analyst 800, USA).
Effect on mitochondrial membrane potential (MMP)
Effects of different doses of CLEO-ChNPs (2.25, 4.50 and 9.0 μl/ml) on MMP of A. flavus were determined using rhodamine 123 dye following the method of Kumar et al. ([15]).
Effect on C-sources utilisation
The PDA Petri-plate contain A. flavus was fumigated with CLEO-ChNPs (4.50 µl/ml) for four days in the BOD incubator at 27 ± 2ºC. Thereafter, the spores were collected into FF-inoculating fluid, and the transmittance was adjusted to 75%. Subsequently, the spore containing FF-inoculating fluid was poured into 96 well BiologFF Microplates (catalogue #1006) and incubated for 24 h at (27 ± 2°C). After incubation, the samples were analysed using the Biolog instrument at 490 nm and the percent decrease and increase in the utilisation of different carbon sources compared to the control set were determined (Singh [26]).
Effect on cellular methylglyoxal (MG) level
The effect of CLEO-ChNPs on cellular methylglyoxal (MG) level of A. flavus was determined following the method of Yadav et al. ([29]). Three-days-old mycelia of A. flavus grown in 10 ml SMKY medium were exposed to different doses of CLEO-ChNPs to obtained the desired concentration (1.0–6.0 μl/ml) for 12 h. After incubation, 0.3 g mycelia were extracted in 3 ml of 0.5 M perchloric acid and incubated on ice for 15 min. Thereafter, the mycelial extract was centrifuged at 11,000 g at 4 °C for 10 min. The supernatant was neutralised by a saturated solution of potassium carbonate and recentrifuged at the same g value. The supernatant was analysed for the quantification of MG using the standard curve for MG.
In silico analysis of receptor-ligand complex
The SWISS-MODEL was used to develop 3D models of the respective protein sequences (Erg 28, Cytochrome c oxidase subunit Va, Omt-A, Ver-1, and Nor-1) procured from the NCBI Protein database. Ligand structures were developed via coordinates generated from their SMILES string using UCSF Chimera 1.14 (Armandoet al. [4]; Kumar et al. [16]).
Thereafter, molecular docking of CLEO major compound eucalyptol (ligand) with targeted gene products (receptors) was performed by AutoDock 4.2 (AutoDock tools-1.5.6) to make predictions based on Receptor-ligand interactions (Morris et al. [21]). Furthermore, the visual screening and result prediction (highest affinity-bound-ligand) were performed using the PyMOL Molecular Graphics System v2.3.1 (Hemachandran et al. [10]).
Statistical analysis
The experiments were performed in triplicate and data (mean ± standard error) were compared by one-way analysis of variance (ANOVA). The significant differences were determined using Tukey's multiple range test (p <.05).
Results and discussion
CLEO isolation and chemical characterisation: GC-MS analysis
CLEO was isolated by the hydro-distillation method using the Clevenger apparatus and yield was found to be 0.5% w/v. The chemical profile of CLEO has been explored by GC-MS analysis. The details of the major and minor components of CLEO are summarised in Table 1. Eucalyptol has been identified as the major compound, followed by α-terpineol and α-thujene. A similar supportive observation on the chemical profile of CLEO was previously reported by Misra et al. ([20]).
Table 1. Chemical profile of Callistemon lanceolatus essential oil (GC-MS analysis).
| S. N. | Compounds | RT | % |
| 1. | α-Thujene | 4.08 | 3.28 |
| 2. | Eucalyptol | 4.46 | 66.61 |
| 3. | Phenol, 4-ethyl-3-methyl- | 5.23 | 0.88 |
| 4. | Benzene, (1,1-dimethyldecyl)- | 6.08 | 0.14 |
| 5. | α-Terpineol | 6.74 | 5.46 |
| 6. | Methanone | 7.90 | 2.29 |
| 7. | Limonin | 8.82 | 0.82 |
| 8. | Cinnamyl cinnamate | 10.22 | 0.03 |
| 9. | (n)-Valachine | 12.22 | 0.42 |
| 10. | Cytochalasin e | 12.80 | 0.26 |
| 11. | Delsoline | 20.23 | 0.21 |
| Total percentage | | 80.4 |
Synthesis of CLEO-ChNPs and characterisation
The CLEO-loaded nanoparticles (CLEO-ChNPs) were prepared using low molecular weight chitosan, cinnamic acid and EDC. All three chemicals (chitosan, cinnamic acid and EDC) used for the synthesis of nanogel have a negligible toxic effect and therefore would have better prospects as a carrier agent of food preservative (Beyki et al. [6]; Prakash et al. [23]). The interaction between EDC, chitosan and cinnamic acid leads to the formation of micellar due to the hydrophobic/hydrophilic effects which entrapped the CLEO.
The structural morphology of prepared ChNPs and of CLEO- ChNPs were examined by SEM analysis. The shapes of both ChNPs and CLEO- ChNPs were found to be nearly spherical with an average particle size ranged between 20–30 nm and 20–70 nm, respectively (Figure 1(a)) (Beyki et al. [6]; Kumar et al. [15]). The spherical shape of the nanoparticles provides a large surface area resulting in the enhancement of antifungal efficacy and control release of entrapped CLEO (Kumar et al. [15]).
PHOTO (COLOR): Figure 1. Characterisation and in vitro release of CLEO-ChNPs (a) SEM analysis of ChNPs and CLEO-ChNPs (b) Encapsulation efficacy and loading capacity (c) In-vitro release of CLEO-ChNPs.
Encapsulation efficiency (EE), loading capacity (LC) and encapsulation yield (EY)
The 1:0.75 w/v ratio of CS/CLEO exhibited better encapsulation efficiency and loading capacity, therefore, it has been selected for further detail investigation (Figure 1(c)). The encapsulation efficiency (EE) and loading capacity (LC) of CLEO-ChNPs ranged between 20–55% and 1.5–6.0% respectively while yield of the encapsulated CLEO (EY) at the ratio of 1:0.75 (LMC:CLEO) was found to be 34.56 %. A similar supportive observation was reported for oregano EO (Hosseini et al. [11]) and lemongrass EO (Natrajan et al. [22]).
In vitro release study
The in vitro release of CLEO-ChNPs is shown in Figure 1(d). The amount of CLEO released by CLEO-ChNPs was determined at a different time intervals (12 to 96 h) using the λ max of CLEO. The release of CLEO from CLEO-ChNPs occurs in a biphasic way with an initial burst (12 h) followed by slow release (48 h). The release of CLEO might be due to diffusion, surface erosion, disintegration, and desorption (Hosseini et al. [11]).
Efficacy of CLEO and CLEO-ChNPs against the fungal growth and aflatoxin B1 production
Antifungal and aflatoxin B1 (AFB1) inhibitory efficacies of CLEO and CLEO-ChNPs are summarised in Figure 2(a,b). The CLEO-ChNPs caused complete inhibition of A. flavus (EC-03) growth and AFB1 production at low doses (4.5 µl/ml and 4.0 µl/ml respectively) compared to free CLEO (5.0 µl/ml). However, ChNPs had no significant effect on growth and AFB1 production up to 5.0 µl/ml. Thus, it could be concluded that essential oil incorporated ChNPs exhibited strong antifungal activity that might be due to increase in particle size, surface area, and targeted delivery. The positive charge of ChNPs and negative charge of cell membrane fungus cause electrostatic interactions that enhance the specific site of action of essential oil loaded nanoparticle (Lu et al. [18]; Bedoya-Serna et al. [5]; Prakash et al. [23]).
PHOTO (COLOR): Figure 2. Antifungal and aflatoxin B1inhibitory efficacy of (a) CLEO (b) CLEO-ChNPs.
Mechanism of action of CLEO-ChNPs against A. flavus growth and aflatoxin B1production
Antifungal and AFB1 inhibitory modes of action of CLEO-ChNPs were elucidated targeting the ergosterol content, ion leakage, mitochondrial membrane potential (MMP), C-source utilisation, and methylglyoxal synthesis. The percent inhibition of ergosterol content in A.flavus exposed to different doses (1–4 µl/ml) of CLEO-ChNPs is given in Figure 3(a). The effects of different doses of CLEO-ChNPs (2.25, 4.5, 9.0 µl/ml) on membrane cation (Ca++, K+, Mg++) content compared to the control are summarised in Figure 3(b). A significant decrease in ergosterol content and an increase in ions leakage were observed in a dose-dependent manner. Ergosterol is the prime sterol that maintains the integrity, fluidity and homoeostasis of the cell membranes. However, the Ca2+, K+ and Mg2+ play an important role in osmotic balance. The disruptions in ergosterol biosynthesis machinery with the elevation of membrane cations by essential oil distort the membrane fluidity, integrity and internal cellular pH, and homoeostasis leads to cell death (Tian et al. [27]; Kumar et al. [15]).
PHOTO (COLOR): Figure 3. Biochemical mechanism of action of CLEO-ChNPs against A. flavus (a) % reduction of ergosterol content, (b) Ion leakage, (c) MMP, (d) % inhibition of monosaccharide, (e) disaccharide carbon source utilisation, (f) Methylglyoxal content.
The decrease in fluorescence intensity of RH123 dye in A. flavus spores exposed to different doses of CLEO-ChNPs (2.25, 4.5, and 9.0 µl/ml) are summarised in Figure 3(c). In general, RH123 dye is used for the study of the dysfunction of MMP. The decrease in fluorescence intensity is a positive sign of impairment in MMP function leading to a decrease in ATP production require for normal cellular functioning (Kumar et al. [15]).
The utilisation pattern of different carbon sources (mono and disaccharide) by A. flavus exposed to MIC dose (4.5 µl/ml) of CLEO-ChNPs is presented in Figure 3(d,e). A significant decline in the utilisation of various mono and disaccharide carbon sources was observed (Figure 3(d,e)). However, considerable perturbation was observed for arbutin, D-fructose, α-D-glucose, D-raffinose, D-ribose and L-sorbose (monosaccharide), and α-cyclodextrin, maltose, maltotriose, D-melibiose, palatinose, stachyose and sucrose (disaccharide). These carbon sources play an important role in the production of secondary metabolites and various metabolic pathways of glycolysis, pentose phosphate pathway and tricarboxylic acid (Wang et al. [28]; Kumar et al. [16]). Thus, it can be concluded that CLEO-ChNPs may negatively interfere with metabolic pathways related to growth and aflatoxin B1 production in A. flavus.
The effect of different doses of (1.0 to 6.0 µl/ml) CLEO-ChNPs on cellular methylglyoxal (MG) level has been summarised in Figure 3(f). The result shows a dose-dependent decrease in the MG level. Methylglyoxal is a well-known inducer of aflatoxin production and has a toxic effect on the functioning of the cellular organelles (Martins et al. [19]; Kaur et al. [13]). Hence, the decrease in the MG level could be one of the possible reasons for inhibition of aflatoxin B1 by the CLEO-ChNPs.
In silico mode of action
All the receptor proteins of targeted genes were modelled and found suitable (≥ 90 % of their amino acid residues are found in the favourable regions) for in-silico studies using the SWISS-MODEL. The targeted genes such as nor1, ver1 and omt1 are the structural genes that play key roles in the aflatoxin biosynthesis pathway. The nor1 gene product (an NADP-dependent oxidoreductase enzyme), converts norsolorinic acid (NOR) into averantin (AVN), while ver1 gene product (the versicolorin a dehydrogenase enzyme) transforms versicolorin A (VERA) to dimethyl sterigmatocystin (DMST) and omt1 gene product (O-methyl transferase A) converts di-hydrosterigmatocystin (DHST) to dihydro-o-methylsterigmatocystin (DHOMST) (Yu et al. [30]). The Cytochrome c oxidase subunit Va serves as one of the constituent elements in the 'Cytochrome c oxidase' or 'complex IV,' which plays a key role in ATP synthesis and Erg 28 is responsible for ergosterol biosynthesis in the fungal cell membrane.
The interactive behaviour of eucalyptol (major compound of CLEO) with all the receptor proteins was analysed based on the binding energy and inhibition constant obtained for the ligand-receptor protein complex. The result shows that eucalyptol inhibits the aflatoxin biosynthesis pathways via interfering with the functioning of biosynthetic gene products viz. Ver1, Omt1 and Nor1 (Figure 4). It also affects the ergosterol biosynthesis (Erg28) and mitochondrial membrane potential (Cytochrome c oxidase subunit Va) in a limited way. Hence, it can be concluded that eucalyptol may cause the inhibition of fungal growth and aflatoxin B1 biosynthetic pathway via inhibiting several gene products required for the cell growth and toxin production.
PHOTO (COLOR): Figure 4. In-silico analysis of interactive behaviour of eucalyptol (ligand) with all the receptor proteins.
Conclusion
The study demonstrates that the incorporation of CLEO inside the chitosan biopolymer enhances the antifungal effectiveness of CLEO. The deciphered antifungal and toxin-inhibitory actions of CLEO-ChNPs consist of cell membrane damage, with perturbation in the mitochondrial membrane, C-source utilisation and toxin-biosynthetic gene. The results highlight the possible use of the chitosan nanoparticles as a carrier agent for the CLEO-based green preservative against the fungal and aflatoxin B1 contamination. Furthermore, the diverse inherent chemical profiles of CLEO may target different cellular sites of Aspergillus flavus, therefore, further study is warranted to explore the role of minor components to improve the antifungal effect of CLEO.
Acknowledgments
The author gratefully acknowledges the Science and Engineering Research Board (Scheme No. ECR/2016/000299) New Delhi, India for the financial support. Akshay Kumar is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial support as a junior research fellowship (09/013(0747)/2018-EMR-1). We are thankful to the Head, CAS in Botany, Banaras Hindu University, Varanasi for Instrumental facilities. We are also thankful to Indian Institute of Technology, Banaras Hindu 545 University, Varanasi for SEM.
Disclosure statement
The authors declare that there are no conflicts of interest.
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By Prem Pratap Singh; Akshay Kumar and Bhanu Prakash
Reported by Author; Author; Author
