International Journal of Microbiology

International Journal of Microbiology / 2017 / Article

Research Article | Open Access

Volume 2017 |Article ID 5273893 | 9 pages | https://doi.org/10.1155/2017/5273893

Effects of Medicinal Plant Extracts and Photosensitization on Aflatoxin Producing Aspergillus flavus (Raper and Fennell)

Academic Editor: Todd R. Callaway
Received12 Jan 2017
Revised29 Mar 2017
Accepted13 Apr 2017
Published02 May 2017

Abstract

This study was undertaken with an aim of exploring the effectiveness of medicinal plant extracts in the control of aflatoxin production. Antifungal properties, photosensitization, and phytochemical composition of aqueous and organic extracts of fruits from Solanum aculeastrum, bark from Syzygium cordatum, and leaves from Prunus africana, Ocimum lamiifolium, Lippia kituiensis, and Spinacia oleracea were tested. Spores from four-day-old cultures of previously identified toxigenic fungi, UONV017 and UONV003, were used. Disc diffusion and broth dilution methods were used to test the antifungal activity. The spores were suspended in 2 ml of each extract separately and treated with visible light (420 nm) for varying periods. Organic extracts displayed species and concentration dependent antifungal activity. Solanum aculeastrum had the highest zones of inhibition diameters in both strains: UONV017 (mean =  mm) and UONV003 (mean =  mm) at 600 mg/ml. Aqueous extracts had no antifungal activity because all diameters were below 8 mm. Solanum aculeastrum had the lowest minimum inhibitory concentration at 25 mg/ml against A. flavus UONV017. All the plant extracts in combination with light reduced the viability of fungal conidia compared with the controls without light, without extracts, and without both extracts and light. Six bioactive compounds were analyzed in the plant extracts. Medicinal plant extracts in this study can control conidia viability and hence with further development can control toxigenic fungal spread.

1. Introduction

Aspergillus flavus is ubiquitous, saprophytic, and a weak parasite [1]. The fungus contaminates a wide range of cereals and nuts like maize, wheat, sorghum, and groundnuts, which serve as staple foods in most parts of Africa. Toxigenic A. flavus have been reported to contaminate these products and produce aflatoxins which are carcinogenic, mutagenic, and lethal fungal metabolites [24]. Aflatoxins have been classified as class 1 poisons by the International Agency for Research on Cancer (IARC) [5]. Aflatoxins also contaminate feed; hence products like meat, milk, cheese, and eggs get contaminated when animals consume the aflatoxin contaminated feed [6, 7]. Aflatoxin B1 is the major type of aflatoxins produced by A. flavus [3]. Aspergillus flavus is the main fungi producing aflatoxin [1].

Aflatoxicosis was first reported in Kenya in 1982 [8]. More outbreaks have since been recorded in 2001, 2004, 2006, 2008, and 2010 [9, 10]. Records of aflatoxin contamination in food and feed are widespread in tropical and subtropical regions where climatic conditions and storage practices favor growth of fungi [11]. Aflatoxin production is influenced by aeration, moisture, temperature, and substrate and the control methods under trials like biological [12], cultural, and chemical ones [13] are all based on manipulation of these factors. Many African countries are now in the process of including regulation framework within their food policies to help control exposure to aflatoxins [1].

Traditionally, many plants have been used successfully for medicinal purposes [4]. Aromatic substances in plants, specifically secondary metabolites like alkaloids, flavonoids, saponins, glycosides, and tannins, are able to protect plants from invaders such as fungi, bacteria, and nematodes [14]. According to World Health Organization (2001), 80% of African and Asian communities rely on traditional herbal medicines for primary healthcare. This is because herbal medicines are safer and cheaper compared to synthetic medicines [15, 16].

This study exploited the ability of known medicinal plant extracts to control the growth of A. flavus conidia. Photosensitization has also been reported to kill both metabolically active and dormant structures such as conidia, unlike conventional fungicides that kill only metabolically active cells [17]. It involves hitting of a photosensitizer by light of a specific wavelength, which makes the photosensitizer reactive thereby killing the toxigenic cells [18]. The technique has been reported as a safe and a potential control of mycotoxigenic fungi [19]. However, very few photosensitizers have been approved clinically for use against toxigenic microbes, hence needing identification of safe photosensitizers. Plant extracts, on the other hand, are biodegradable and hence environmentally friendly [20]. The aim of this study is to determine the antifungal activity and phytochemical composition of the plants extracts. The ability of visible light to stimulate the bioactive compounds in the plant extracts (photosensitization) and hence increase in the antifungal activity of the extracts against toxigenic A. flavus which causes aflatoxin production will also be tested.

2. Materials and Methods

2.1. Collection of Plant Material

Five plants known for their medicinal value were collected from Gakoe forest in Gatundu district, Central Region of Kenya. These were Ocimum lamiifolium leaves (LMM 2015/05), Prunus africana leaves (LMM 2015/03), Solanum aculeastrum fruits (LMM 2015/01), Lippia kituiensis leaves (LMM 2015/04), and Syzygium cordatum bark (LMM 2015/02). Fresh leaves of Spinacia oleracea (LMM 2015/06) were also collected from the local market. The identity of the plants was confirmed using reference material from the University of Nairobi herbarium where voucher specimens were deposited.

2.2. Crude Plant Extract Preparation

The selected plant parts were air-dried at room temperature, chopped, and ground into powder. Dichloromethane-methanol (1 : 1) mixture was used for organic extraction. Two hundred and fifty grams of each ground extract was soaked in 1 L of the organic solvents for 48 hours. A rotary evaporator was used to filter and concentrate the organic extract, hence obtaining a semisolid residue for use [4]. Distilled water was used for aqueous extraction. Two hundred and fifty grams of each ground extract was soaked in 500 ml of distilled water in a glass beaker sealed with aluminum foil for five days. The extract was then filtered using Whatman number 1 filter paper. The filtrate was evaporated and dried using a freeze-drier to get powder [21]. The resulting products were stored at 4°C.

2.3. Preparation of Fungal Spore Suspension

Toxigenic A. flavus strains used in this study were obtained from the School of Biological Sciences Mycology Culture Collection. The isolates used were UONV017 and UONV003 and they had been tested for toxigenicity through molecular characterization according to [3]. The isolates were transferred from the stock cultures into sterile PDA plates and incubated for 4 days at 29°C. Spores were aseptically harvested and suspended in sterile distilled water with three drops of Tween 80 solution and standardized to a turbidity of 1 McFarland solution (3 × 108 CFU/ml).

2.4. Determination of Inhibition Concentration

Antifungal activities of the plant extracts were evaluated using the disc diffusion as described by Sigei et al. [22] and according to National committee of clinical and laboratory standards NCCLS now CLSI [23]. The diameters of the inhibition zones produced around the test material were measured with a ruler and recorded in mm. Plant extracts that produced a zone of inhibition of 8–11 mm were said to be active. Those with zones above 11 mm were considered very active. Those with zone of inhibition below 8 mm were considered inactive [24]. The tests were replicated three times for each material.

2.5. Determination of Minimum Inhibitory Concentration

Minimum inhibitory concentration (MIC) was determined through the broth dilution technique. Different concentrations of the extracts were prepared and replicated three times. The extract concentrations were 100 mg/ml, 50 mg/ml, and 25 mg/ml. 5 ml of each concentration of the extract was poured aseptically into a sterile test tube. 1 ml of the toxigenic A. flavus (1 McFarland standard) was added. 1 ml of this mixture was poured aseptically into 5 ml of potato dextrose broth (serial dilution) [4]. All the tubes were incubated at 29°C for 72 hours. Observations were made for visible fungal growth. The lowest dilution without visible growth for each extract was regarded as the minimum inhibitory concentration

2.6. Treatment of Fungal Spores with Plant Extracts and Light

Concentrations of 450 mg/ml and 600 mg/ml of each plant extract were prepared as the working solutions. The 3 × 108 CFU/ml McFarland solution was serially diluted up to a concentration of 3 × 102 CFU/ml. 2 ml of this (3 × 102 CFU/ml) spore suspension was added to 2 ml of each extract separately. The mixture was well shaken and treated with visible light spectrum at a range of 420 nm provided by a special lamp from Multiplex Display Fixture [19]. The maximum absorption range of the plant extracts was tested using a spectrophotometer and found to be 420 nm. Irradiation of the plant extracts which were the photosensitizers was done for 10, 20, and 40 minutes. Effects of light irradiated on the photosensitizer (plant extract) for varying time periods (10 mins, 20 mins, and 40 mins) were tested on the viability of spores of the toxigenic A. flavus. Controls experiments involved adding plant extracts to the conidia suspension without light treatments, reacting conidia without plant extracts with light and conidia without light and plant extracts as described by [19]. Each treatment was replicated three times.

100 ul of irradiated solution was transferred to PDA plates and incubated at 29°C. The control experiments were also treated in the same way. Colony forming units (CFUs) were counted after 72 hours of incubation to determine the viability of conidia.

2.7. Phytochemical Screening of Plant Extracts

The six organic and aqueous plant extracts obtained were subjected to phytochemical screening to determine the presence of bioactive agents like flavonoids, steroids, terpenoids, saponins, tannins, alkaloids, and glycosides. Plant extracts from the stock solution of 800 mg/ml were used for the phytochemical screening [4, 25].

2.8. Statistical Analysis

Data analysis was done using SPSS version 16. Data values were expressed as means ± standard error. Analysis of variance was used and when was significant (), comparison of means was done using Tukey’s test.

3. Results

3.1. Effect of Organic and Aqueous Plant Extracts on Growth of Toxigenic A. flavus

The crude organic extracts of five out of the six plants tested exhibited antifungal activity against the growth of toxigenic strains of A. flavus. The aqueous extracts did not show significant () antifungal activity because all zones of inhibition diameter were below 8 mm. Solanum aculeastrum and Syzygium cordatum plant extracts at 600 mg/ml against A. flavus UONV017 had inhibition diameters that had no significance difference ( and ) and hence compared favorably with the standard antifungal control Apron star (250 mg/ml) which is a class III Blue Active ingredient containing 20% thiamethoxam + 20% metalaxyl-M + 2% difenoconazole. Solanum aculeastrum organic extract had the highest antifungal activity followed by Syzygium cordatum against both strains of A. flavus (Tables 1 and 2). Apart from S. aculeastrum plant extracts against A. flavus UONV003 which had a higher inhibition diameter at 300 mg/ml (mean =  mm) than at 450 mg/ml (mean =  mm) and P. africana leaf extracts against A. flavus strain UONV017, which had a higher inhibition diameter at 450 mg/ml (mean =  mm) than at 600 mg/ml (mean =  mm), all the other extracts had the highest inhibitory activity at the highest concentration (600 mg/mL) and the lowest antifungal activity was at the lowest concentration (300 mg/ml) (Tables 1 and 2).


PlantsInhibition zones (mm) 600 mg/mlInhibition zones (mm) 450 mg/mlInhibition zones (mm) 300 mg/ml

S. aculeastrum
S. cordatum
L. kituiensis
P. africana
O. lamiifolium
S. oleracea
Positive control 250 mg/ml
Sig 0.000.000.00

Numbers are means of twelve replications. One-way Annova was used for analysis and means were separated by Tukey’s test. Numbers followed by the same letters in the same column are not significantly different ().

PlantsInhibition zones (mm) 600 mg/mlInhibition zones (mm) 450 mg/mlInhibition zones (mm) 300 mg/ml

S. aculeastrum
S. cordatum
L. kituiensis
P. africana
O. lamiifolium
S. oleracea
Positive control 250 mg/ml
(Sig )0.000.000.00

Numbers are means of twelve replications. One-way Annova was used for analysis and means were separated by Tukey’s test. Numbers followed by the same letters in the same column are not significantly different ().

Comparison of the activities of the organic plant extracts between both strains (UONV003 and UONV017) of A. flavus showed that A. flavus UONV003 had smaller inhibition diameters than A. flavus UONV017 at different concentrations (Figure 1).

Extracts of S. aculeastrum had the lowest minimum inhibitory concentration at 25 mg/ml against A. flavus (UONV017) and at 50 mg/ml against A. flavus UONV003. Syzygium cordatum had an MIC of 50 mg/ml on A. flavus UONV017 and 100 mg/ml on A. flavus UONV003.

3.2. Effect of Aqueous Plant Extracts and Photosensitization on A. flavus UONV017 at Different Concentrations and Different Timings

Interaction between aqueous plants extracts and visible light at 600 mg/ml was statistically significant (; DF = 18; ). Different plant extracts fungi suspensions had different counts of CFU after light treatment at varying time durations. Solanum aculeastrum (mean = 2 CFUs) had the lowest CFU reading at 40 minutes and hence was the most effective. Other than the controls, S. oleracea (mean = 13 CFUs) had the highest CFU reading at 10 minutes and hence was the least effective at 600 mg/ml. At 450 mg/ml, interaction between the aqueous extracts and light caused significant (; df = 18; ) reduction of CFUs. Solanum aculeastrum (mean = 3 CFUs) was the most effective with the lowest number of CFUs while O. lamiifolium (mean = 14 CFUs) was the least effective with the highest number of CFUs at 10 minutes.

Comparison of photosensitization activities within different time durations proved that treatments kept under light for the highest duration of time (40 minutes) had the lowest CFU counts, hence proving the highest inactivation of fungal spores. Treatments that were under light for the shortest time duration (10 minutes) exhibited a higher number of CFUs; samples with no light and no extract treatment had the highest CFU count (Table 3).


PlantsColony forming units at 600 mg/mlColony forming units at 450 mg/ml
10 (min)20 (min)40 (min)0 minutes (ctrl)10 (min)20 (min)40 (min)0 minutes (ctrl)

S. aculeastrum
P. africana
S. cordatum
L. kituiensis
O. lamiifolium
S. oleracea
0 extracts (ctrl)

Numbers are means of three replications. Two-way Annova was used for analysis and means were separated by Tukey’s test. Numbers followed by the same letters in the same row within each concentration are not significantly different ().
3.3. Effect of Aqueous Plant Extracts and Photosensitization on A. flavus (UONV003) at Different Concentrations and Different Timings

Statistically significant (; DF = 18; ) interaction existed between aqueous plants extracts and light in their activity against A. flavus strain UONV003 at 600 mg/ml. Syzygium cordatum (mean = 3 CFUs) had the lowest CFU reading at 40 minutes and hence was the most effective. Lippia kituiensis (mean = 16 CFUs) had the highest CFU reading at 10 minutes and hence was the least effective. At 450 mg/ml, interaction between aqueous extracts and light was also significant (; DF = 18; ). Solanum aculeastrum (mean = 3 CFUs) was the most effective while L. kituiensis (mean = 17 CFUs) was the least effective at 10 minutes (Table 4).


PlantsColony forming units at 600 mg/mlColony forming units at 450 mg/ml
10 (min)20 (min)40 (min)0 minutes (ctrl)10 (min)20 (min)40 (min)0 minutes (ctrl)

S. aculeastrum
P. africana
S. cordatum
L. kituiensis
O. lamiifolium
S. oleracea
0 extracts (ctrl)

Numbers are means of three replications. Two-way Annova was used for analysis and means were separated by Tukey’s test. Numbers followed by the same letters in the same row within each concentration are not significantly different ().
3.4. Effect of Organic Plant Extracts and Photosensitization on A. flavus (UONV017) at Different Concentrations and Different Timings

Organic extracts at both concentrations of 600 mg/ml displayed significant (; DF = 18; ) photosensitization activity. Syzygium cordatum (mean = 4 CFUs) had the lowest CFU reading at 40 minutes at a concentration of 600 mg/ml. Ocimum lamiifolium (mean = 19 CFUs) had the highest CFU reading at 10 minutes at 600 mg/ml. At 450 mg/ml, there was also significant (; DF = 18; ) photosensitization activity. Syzygium cordatum (mean = 7 CFUs) was the most effective at 40 minutes (Table 5).


PlantsColony forming units at 600 mg/mlColony forming units at 450 mg/ml
10 (min)20 (min)40 (min)0 minutes (ctrl)10 (min)20 (min)40 (min)0 minutes (ctrl)

S. aculeastrum
P. africana
S. cordatum
L. kituiensis
O. lamiifolium
S. oleracea
0 extracts (ctrl)