¹Department of Botany, Dr. Bhimrao Ambedkar Government Degree College, India
²Institute of Science, Banaras Hindu University, India

^*Corresponding author: Kumar A, Department of Botany, Dr. Bhimrao Ambedkar Government Degree College, Maharajganj-273303, Uttar Pradesh, India, Mobile: +91-8400945878; E-mail: ashokkumarbhu@gmail.com

Copyright: © Kumar A, et al. 2020. Th is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information: Submission: 15/09/2020; Accepted: 19/10/2020; Published: 22/10/2020

In the present study, stored wheat grains were found associated with spores of various storage moulds in which Aspergillus flavus exhibited the maximum relative density (33.48%). The chemical profi le of Ocimum Canum Essential oil (OcEO) showed 31 considerable peaks. E-citral (41.84%) was found as major component followed by Z-Citral (20.46%) and β-pinene (7.40%). OcEO and E-citral both exhibited broad spectrum fungitoxicity and their MIC against A. flavus LHPTA-07 was recorded at 0.6 and 0.4 mg/ml respectively while afl atoxin B₁ production was completely checked at 0.4 and 0.3 mg/ml respectively. The gradual reduction in ergosterol content with increasing OcEO and E-citral concentration indicates plasma membrane as the possible target site of antifungal action. OcEO and E-citral also exhibited signifi cant antioxidant potential with IC₅₀ value (16.72 and 15.44 μg/ml respectively) comparable to synthetic antioxidants (ascorbic acid, IC₅₀ 9.76 μg/ml) would be helpful to minimize lipid peroxidation. Fumigation of OcEO and E-citral signifi cantly reduced the population of A. flavus from wheat grains without adversely aff ecting seed germinability. Hence, OcEO and E-citral exhibited special virtues possessing antifungal, antiafl atoxigenic and antioxidant activity as well as no adverse eff ects on seed viability strengthening their safe exploitation as green preservative to enhance the shelf life of stored food commodities and other edible products.

Aspergillus flavus; Ocimum Canum; Essential oil; Antifungal; Aflatoxin; Antioxidant

Food commodities are susceptible to fungal and mycotoxin contamination during postharvest storage and illness due to consumption of these contaminated food products is a priority concern to public health. In developing countries, postharvest economic losses reach 25-40% or even more by fungal contamination and the mycotoxins produced by them [1]. Among storage moulds, diff erent species of Aspergillus potentially contaminate stored food commodities and adversely aff ect their nutritive values [2]. Toxigenic species of Aspergillus secrete afl atoxins in stored food commodities and majority of population in developing countries are exposed to afl atoxicoses [3]. Afl atoxin B₁ is potent hepatocarcinogenic and immunosuppressive, therefore, classifi ed as group-1 human carcinogen by International Agency for Research on Cancer [4].

Various synthetic fungicides (Carbendazim, Mancozeb, Benomyl, Ceresan, Ziram etc.) have been used for a long time and have greatly contributed in management of such losses but due to their harmful health eff ects, resistance development in pathogens and residual toxicity [5]. Synthetic preservatives are also produce partially reduced oxides such as superoxide (O₂⁻), hydrogen peroxide (H₂O₂) and also hydroxyl radicals (OH⁻) causing severe damage to biomolecules and also responsible for the stimulation of aflatoxin biosynthesis [6,7]. Hence, there is a need to develop some ecofriendly alternatives of these synthetic fungicides/preservatives.

Higher plants are great reservoir of various bioactive phytochemicals. Recently, plant based formulations are exploited as safe alternatives of synthetic preservatives and these green preservatives are Generally Recognized as Safe (GRAS) by U.S. FDA [8]. Various essential oils and their common bioactive components viz. terpenoids and phenolics have been used as antifungal [9], antimycotoxigenic [10], and pesticidal as well as potent free radical scavengers [11,12]. Some essential oil based formulations viz. TALENT, EcoSMART and EcoPCOR have been used on large scale in food and agriculture industries [13].

In the present study, Ocimum Canum Essential oil (OcEO) and its major component E-citral has been investigated for their fungitoxicity against toxigenic isolate of A. flavus, a potent storage fungus causing postharvest loses of food commodities. In addition, OcEO and E-citral was also studied for their antioxidant activity as well as eff ect on seed viability for recommendation of OcEO as plant based food preservative.

Stored wheat (Triticum aestivum L.) grains (variety-Malviya) were locally procured from retailers of Varanasi district of UttarPradesh, India. Th e grains were collected in sterilized low density poly ethylene bags to avoid further contamination. Th e grains were ground using a surface sterilized household blender. Th e powder was filtered through No. 50 mesh sieve and packed tightly in paper bags and stored at 5±2 ºC for further analysis [14].

For pH measurement, powdered wheat sample: distilled water suspension (1:10; w/v) was prepared and stirred for 24 h in 200 ml beaker. Th e pH of the suspension was measured using electronic pH meter. To determine moisture content, weighed amount of powered samples were dried at 100 ºC until their weights remained constant and per cent moisture content was calculated as follows [15] -

Storage moulds associated with wheat grains were assessed by serial dilution method [16]. Th e isolated fungal species were identifi ed on the basis of cultural and morphological characteristics [17,18]. Th e identifi ed fungal colonies were purifi ed and preserved on Potato Dextrose Agar (PDA) slant at 4±2 ºC. Th e per cent relative densities of diff erent fungi on raw herbal drug samples were calculated following [19].

Twenty isolates of A. flavus were randomly selected to determine their Afl atoxin B₁ producing potential by Th in Layer Chromatography (TLC) following [20]. Fifty μl conidial suspension (≈10⁶ conidia/ml) of selected A. flavus isolates were separately inoculated in 49.5 ml SMKY (Sucrose, 200 g; MgSO₄.7H₂O, 0.5 g; KNO₃, 0.3 g; Yeast extract, 7.0 g; Distilled water, 1000 ml) broth medium in 150 ml Erlenmeyer fl ask and mixed properly followed by incubation at 27±2 °C for 10 days. Content of each fl ask was filtered aft er incubation and filtrate was extracted with chloroform (40 ml) in a separating funnel. The separated chloroform extract was dried on water bath at 60-70 °C. Th e residue left aft er evaporation was re-dissolved in 1 ml chloroform and 50 μl of it was spotted on TLC plate (20×20 cm² of silica gel). The plate was developed in toluene: isoamyl alcohol: methanol (90:32:2; v/v/v) solvent system and intensity of AFB₁ was observed under ultra violate fluorescence analysis cabinet at an excitation wavelength of 360 nm [21]. Th e fluorescent blue spots on TLC plate containing AFB₁ were scraped in 5 ml cold methanol and centrifuged at 3000 rpm for 5 min. Absorbance of supernatant was recorded at 360 nm and AFB₁ content was quantified following [22].

Where, D = absorbance, M = molecular weight of AFB₁ (312), E = molar extinction coeffi cient of AFB₁ (21,800) and L = path length (1 cm cell was used)

For essential oil extraction, leaves of O. canum were collected from Banaras Hindu University campus, Varanasi. Th e plant was identifi ed with the help of Flora of BHU Campus and its voucher specimen (Lam/O-119/2019) was lodged in the herbarium of department of botany [23], Banaras Hindu University. Leaves of the plant were thoroughly washed with 1% Sodium hypochlorite followed by distilled water. Th e volatile fraction (EO) of leaves was extracted by Clevenger’s hydro-distillation apparatus. Th e extracted OcEO was stored in dark clean glass vial aft er removing water traces passing through anhydrous sodium sulphate and kept at 4-6 ºC [21].

To determine the chemical composition, OcEO was analyzed through gas chromatography (Perkin Elmer Auto XL GC) equipped with a fl ame ionization detector. Th e GC conditions were as follows: column, EQUITY-5 (60 m x 0.32 mm x 0.25 μm); H₂ was the carrier gas; column Head pressure 10 psi; oven temperature program isotherm 2 min. at 70 ºC, 3 ºC/min. gradient to 250 ºC, isotherm 10 min; injection temperature, 250 ºC; detector temperature 280 ºC. The GC-MS analysis was also performed using Perkin Elmer Turbomass GC-MS. Th e effl uent of the GC column was introduced directly into the source of MS. Spectra were obtained in the EI mode with 70ev ionization energy. Th e compounds were identifi ed by comparison of their relative retention times and the mass spectra with those of authentic reference compounds shown in the literature [24].

Fungitoxic effi cacy of OcEO and its major component E-citral was also recorded against 12 storage moulds viz. Alternaria alternata, Aspergillus candidus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, Cladosporium cladosporioides, Curvularia lunata, F. oxysporum, Penicillium italicum and Trichoderma viride recovered from powdered wheat samples. Requisite amount of OcEO and E-citral dissolved separately in 0.5 ml of 5% tween-20 mixed with 9.5 ml PDA medium in diff erent presterilized Petri dishes to attain final concentrations i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 and 2.0 mg/ml. Th e control sets were kept parallel to the treatment sets without OcEO. A positive control of bavistin (Carbendazim 50% WP), a prevalent synthetic fungicide was also kept parallel. A fungal disc (5 mm diameter) of each test fungus was inoculated separately and incubated at 27±2 °C for 7 days. Aft er incubation, Minimum Inhibitory Concentration (MIC) was recorded [25]. The concentrations on which OcEO and E-citral completely checked the visible fungal growth on PDA medium was considered as MIC.

Fungitoxic and aflatoxin inhibitory efficacy of isolated OcEO and its major component E-citral was tested against the toxigenic isolate of A. flavus LHPTA-07 using SMKY (Sucrose, 200 g; MgSO₂.7H₂O, 0.5 g; KNO₃, 0.3 g; Yeast extract, 7.0 g; Distilled water, 1000 ml) broth as nutrient medium following [20]. Requisite amount of the OcEO and E-citral dissolved separately in 0.5 ml of 5% tween-20 were pipetted aseptically to different presterilised Erlenmeyer fl asks (150 ml) containing 49.5 ml of SMKY broth to procure the final concentrations viz. 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/ml. The control sets were kept parallel to the treatment sets without OcEO and E-citral. Th en, fl asks were inoculated aseptically with 50 μl spore suspension (≈10⁶ spores ml^-1) of toxigenic isolate of A. flavus LHPTA-07 prepared in 0.1 % Tween-80 and incubated at 27±2 °C for 10 days [26]. Th e content of each fl ask was filtered (Whatman no. 1) and mycelium was oven dried at 100 °C till their weight remained constant for biomass determination. Mycelial biomass of treatment and control sets was measured and per cent mycelial inhibition was calculated [21]. Th e filtrates of control and treated sets were extracted separately with 50 ml chloroform in a separating funnel to quantify the aflatoxin B₁ production. Aflatoxin B₁ production in each set was estimated by aforementioned technique of [20].

Eff ect of OcEO on ergosterol content in plasma membrane of A. fl avus LHPTA-07 was assessed following [22]. Requisite amount of the OcEO and E-citral was dissolved separately in 0.5 ml of 5% tween-20 were pipetted aseptically to diff erent presterilised Erlenmeyer fl asks (150 ml) containing 49.5 ml of SMKY broth to procure the fi nal concentrations from 0.1 to 0.6 mg/ml. Th e fl ask without OcEO and E-citral was treated as control. Each fl ask was inoculated with 100 μl spore suspension of A. flavus LHPTA-07 followed by incubation at 27±2 °C for 5 days. Recovered mycelia from treated and control sets were subjected to extraction and quantifi cation of ergosterol.

Th e antioxidant activity of the OcEO and E-citral was determined by DPPH radical scavenging assay on TLC as well as its free radical scavenging activity was measured through spectrophotometery following [27]. Free radical scavenging activity of the OcEO and E-citral was measured by recording the extent of bleaching of the purple-coloured DPPH solution to yellow. Diff erent graded concentrations (1.0 to 20.00 μg/ml) of the samples were added separately to 4% DPPH solution in methanol (5 ml). Aft er a 30 min of incubation at room temperature, the absorbance was taken against a blank at 517 nm using spectrophotometer. Scavenging of DPPH free radical with reduction in absorbance of the sample was taken as a measure of their antioxidant activity following [28]. Ascorbic acid was used as positive control. Per cent Free Radical Scavenging Activity (FRSA) was calculated using the following formula –

Where, Ablank is the absorbance of the control (without test compound), and Asample is the absorbance of the test compound.

To determine the antifungal effi cacy of OcEO during storage, 500 g of surface sterilized wheat grains were kept separately in four sets in diff erent plastic containers having aerial volume 2.0 liters. Each set (three containers) was inoculated with 1 ml spore suspension (≈10⁶ spores/ml) of A. flavus LHPTA-07. Each set was fumigated with OcEO (0.6 mg/ml), E-citral (0.4 mg/ml) and bavistin (0.8 mg/ml) separately at their MIC against A. flavus one set run parallel as control without any fumigation. All the containers were kept air tight and stored for six months at room temperature i.e. 27±2 ºC [29,30]. Aft er storage, mycological analysis of fumigated wheat grains was performed to determine the effect of fumigation on number of isolates of A. flavus following [16].

Th e viability of fumigated wheat grains was tested by seed germination test. Aft er six months of storage, 200 uninfested seeds were taken from each treated group and soaked in distilled water for 3 h. Th ereaft er, seeds were aseptically transferred to Petri dishes of 15 cm diameter (20 seeds per Petri plate) containing moist fi lter paper and incubated at 25±2 ⁰C. Two hundred healthy and uninfested seeds were taken from the market as control for comparison. Th e number of seeds germinated within a week was recorded as viable [31].

All the experiments were accomplished in triplicate and data were expressed as Mean±Standard error (SE) followed by one way ANOVA (P < 0.05) and Tukey’s multiple range tests. Th e soft ware SPSS (version 16.0) was used for statistical analysis of data.

Th e level of fungal contamination is mainly governed by pH and moisture content. Th e collected wheat grains having appropriate pH (6.90±0.047) whereas their relatively higher moisture content (17.64±0.347%) refl ects eff ect of conducive climatic conditions of Indian subcontinent which enhance fungal and mycotoxin contamination in food commodities during storage. During mycological analysis, total 1335 fungal isolates were recovered and among them A (Figure 1). fl avus was dominated with highest relative density (33.48%) followed by Cladosporium cladosporioides (20.92%) and A. niger (17.83%) while A. nidulans exhibited the lowest (1.05%) relative density (Figure 2). Th irty percent isolates of randomly selected A. flavus were found toxigenic and A. flavus LHPTA-07 was selected as test fungus due to its higher potential of afl toxin B₁ production (2392.654 μg/l) presented in Table 1. Th e Aspergilli usually exhibited relatively higher density due to their strong capability to produce some hydrolytic enzymes [32-34]. Fungal contamination also degraded the nutritive values of food commodities and rendering them unfi t for human consumption [2]. Some higher plant essential oils can be exploited as substitute of synthetic fungitoxicant against several moulds and their mycotoxin production [10,35]. An eff ort was made to evaluate the antifungal effi cacy of OcEO and its possible applicability in control of postharvest fungal deterioration of food commodities during storage and also enhancing their shelf life.

Th e OcEO was extracted through hydro-distillation is characterized with its pungent smell, yellow green colour and 0.96 % yield (w/w). Th e chemical profi le of EOs varies with age of the plant, season of collection, geographical area and soil characteristics [36,37]. Hence, OcEO was standardized to determine the chemical composition through GC-MS analysis before recommending for formulation. Th e GC-MS analysis of OcEO showed 31 considerable peaks in which E-Citral (41.84%) was found as major component followed by Z-Citral (20.46%), β-Pinene (7.40%), Caryophyllene oxide (5.04%), Epoxy ocimene (3.54%), Trans Carvone oxide (3.12%), β-Costol (2.67%) and 3-Heptadecen-5-yne (2.03%). Rest other identifi ed components were found in trace amount (Table 2). Th e major component of OcEO is diff erent from earlier fi ndings where, camphor [38], linalool [39], eucalyptol [40], thymol [41], etc. were reported as major components.

Th e OcEO and E-citral both exhibited remarkable broad fungitoxic spectrum at 1.0 mg/ml concentration against the 12 storage fungi isolated during mycological analysis and also showed superiority over synthetic fungicide bavistin (Figure 3). Th e broad spectrum fungitoxicity and superiority over bavistin would be suitable to provide complete protection from large number of fungal pathogens. Th e MIC of OcEO and E-citral against A. flavus LHPTA-07 was found to be 0.6 mg/ml and 0.4 mg/ml respectively whereas; OcEO and E-citral completely checked the AFB₁ production at 0.4 and 0.3 mg/ml concentration respectively (Table 3 and Figure 4). A direct relationship has been observed between fungal biomass and aflatoxin production. The AFB₁ production decreased with increasing concentrations of OcEO and E-citral. Fungal biomass and AFB₁ production exhibited a significant declining trend with increasing OcEO and E-citral concentrations. Therefore, to check AFB₁ production, mycelial growth must be below the threshold limit so that aflatoxin could not be produced [42]. Th e OcEO completely checked the AFB₁ production at lower concentration than earlier reported EOs viz. Cymbopogon flexuosus [31], Curcuma longa [43], Pimenta dioica [44], Coriandrum sativum [45], Artemisia nilagirica [46] etc. The antifungal and antiaflatoxigenic efficacy of OcEO may be due to bioactivity of different constituents or due to different metabolic pathways because; MIC and AFB₁ inhibition was recorded at different concentrations. E-citral, a well known antifungal agent [47,48] is major component (41.84%) of OcEO, may also played promising role in its fungitoxicity.

Food commodities are also deteriorated by free radical mediated oxidation of unsaturated lipids during storage [49]. Oxidative stress stimulates A. flavus to produce more AFB₁ during storage [7] which results quantitative as well as qualitative losses to stored commodities and reduces their shelf life. Th e OcEO and E-citral exhibited signifi cant radical scavenging activity as their IC₅₀ values (16.72 and 15.44 μg/ml respectively) in concentration dependent manner (Figure 5), which were found comparatively higher than ascorbic acid (9.76 μg/ml). Th e IC₅₀ values of OcEO and E-citral were found greater than EO of O. sanctum, O. gratissimum, O. basilicum etc. but quite lower than some earlier reported EOs and also comparable to synthetic antioxidants [39,50,51]. Th e presence of various phenolic compounds and/or synergistic eff ect among compounds also play major role in antioxidant activity of EOs [52,53]. Owing to free radical scavenging activity, the OcEO and E-citral may serve as plant based antioxidants in shelf life enhancement as well as protection from oxidative stress by decelerating oxidative rancidity of lipids.

Ergosterol is specifi c sterol in fungal cell membrane providing membrane integrity and fl exibility as well as stability of membrane associated enzymes and signifi cant alteration of its biosynthesis adversely aff ect fungal growth [54,55]. Ergosterol content of cell membrane was found decreasing with increasing concentration of OcEO and E-citral. Th e per cent inhibition of ergosterol contents by OcEO treatment at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/ml concentration was 33.86%, 55.02%, 77.14%, 86.43%, 92.86% and 100% respectively. Whereas per cent ergosterol content inhibition by E-citral at 0.1, 0.2, 0.3 and 0.4 mg/ml concentration was found 43.57%, 65.71%, 85.71% and 100% respectively (Figure 6). Th e decrease in ergosterol level with increasing OcEO and E-citral concentration clearly denotes that the bioactive components of OcEO targeted the cell membrane and rendering them more permeable to leakage of ion and other cell content [56].

Th e OcEO and E-citral drastically reduces the A. flavus population in fumigated wheat samples i.e. 75.19 and 80.73% at 0.6 mg/ml and 0.4 mg/ml concentrations respectively (Table 4). Th e fumigant activity of OcEO and E-citral against A. flavus was high and also comparable to vabistine where 73.95% reduction was recorded (Table 4). Reduction of A. flavus isolates in fumigated wheat samples of OcEO and its major constituent also showed their preservative nature. OcEO and E-citral fumigated wheat grains showed 81.5% and 78.0% germination, which was also noteworthy (86.5%) to prevalent synthetic fungicide vabistine strengthens their non-phytotoxic nature even six months aft er application (Table 5).

Th e plant O. canum is also used as phytomedicine in diff erent diseases [57,58] exhibited its non-mammalian toxic nature. Th us, non-phytotoxic and non-mammalian toxic nature of the OcEO strengthens its possible exploitation as a safer plant based preservative of food commodities during storage. Th e fi ndings may draw the attention of food industries to conduct further experiments regarding large scale exploitation of OcEO as botanical preservative for food commodities during storage. Th e attraction of modern society in ‘green consumarism’ [59] desiring fewer synthetic ingredients in foods and recommendation of herbal products as ‘Generally Recognized as Safe’ (GRAS) in the developed countries may lead scientifi c interest in OcEO as food preservative.

Th e fi ndings of present study reveals that, OcEO exhibited antifungal, antiafl atoxigenicity, broad fungitoxic spectrum, free radical scavenging activity and non-phytotoxicity which strengthening its exploitation as a substitute of synthetic preservatives for enhancing the shelf life of stored food commodities and other edible products during storage.

Th e authors are thankful to Head, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, India, for providing laboratory facilities.