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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

In vitro investigation of antifungal efficacy of diclofenac sodium salt against Aspergillus spp. strains causing food spoilage: a drug repurposing approach 

Ruturaj Kulkarni , Anurag R. Sawant , Atharva B. Jadhav , Nikhil Chavan , Mahadev A. Jadhav , Shivkrupa Devrao Halbandge *             

Department of Biotechnology, Deogiri college, Chh. Sambhaji Nagar, 431005, Maharashtra, India.

                                             

 

INTRODUCTION

Plant pathogens and pests are the major reasons for reduced yield and quality of crops^1,2.   Plant diseases are affecting economic development and food security³. More than 19,000 species of fungi are known to cause plant diseases. Some of these fungal species may live in a dormant state until conditions change to become proliferative⁴. The average annual loss due to phytopathogens is more than $200 billion⁵. Filamentous fungi have a special ability to grow on very simple substrates whose components are used as food. During their growth and development, they also synthesize different secondary metabolites⁶. 

Secondary metabolism of fungi is a complex process and different compounds are produced in response to different environmental signals. Some of them are pharmaceutical and industrial interests. Even though, fungal species can also produce a toxic metabolite known as mycotoxins^7,8,9. Filamentous fungi, especially Aspergillus flavus, are a major contaminant in different foods such as fruits, vegetables, and cereals. It is the most common species associated with food spoilage. It also secretes highly poisonous secondary metabolites like aflatoxin. Among all mycotoxins, aflatoxin is the most studied and strictly regulated because of its toxic effect on health and economic loss. It is a common natural carcinogen and is associated with the development of liver cancer. It is classified under group 1 of molecules that show carcinogenicity to humans and animals by the International Agency for Research on Cancer (IARC)¹⁰.  Aflatoxin is also well-known for its toxicity and teratogenic, hepatotoxic, mutagenic, and immunosuppressive properties. It can also target various aspects of the metabolic system. So, it is necessary to develop a safe and efficient way to control the spoilage of A.   flavus in food¹¹.   Therefore, it is necessary to develop an approach to prevent the contamination of this fungal species and their toxic mycotoxins to food and to restrict their harmful effect ¹².

Pesticide and fungicidal agents are commonly used to prevent the contamination of fungal agents. However, as they are associated with their own toxic effects, their use to fruits and vegetables has to be restricted¹³. Biological control and detoxification processes are other options for controlling the toxicity of contaminants to food. However, production of biopesticides is slow and difficult to be applied to food¹⁴.  So, chemical agents are still the preferable option¹⁵. Even though, large numbers of these weeds are not efficiently controlled by a number of agrochemicals. Additionally, antibiotic resistance was also observed in these species¹⁶.So, it is necessary to develop a novel approach to control these phytopathogens. Development of drugs is a complicated, long and costly process ¹⁷.  It takes nearly 20 years to develop a new drug and it can cost up to $2 billion for the whole process. Similarly, chemical agent discovery is also an expensive and time-consuming process¹⁸.  So, drug repurposing for already known drugs is one approach to the development and discovery of drugs as the pharmacokinetics and biological activities of these drugs are already known. Many of the drugs are already known for successfully repositioning¹⁹. The antifungal activity of diclofenac sodium has been demonstrated in various studies, showing its effectiveness against Candida albicans ²⁰ and Aspergillus fumigatus ²¹. Various studies are reported on antifungal potency of diclofenac sodium on human fungal pathogens its potency against phytopathogens such as Aspergillus flavus are not known. Additionally, Aspergillus flavus is an opportunistic human pathogen and can cause aspergillosis disease. On this basis, diclofenac sodium , a non-steroidal anti-inflammatory drug was tested against Aspergillus flavus and Aspergillus niger which can serve as the theoretical basis for the development of fungicides.

Objectives

The present study was carried out with two main objectives; i) to isolate Aspergillus spp. strains which cause the biodeterioration of peanuts (Arachis hypogea L.) ii) to determine the MIC of diclofenac sodium against Aspergillus spp. strains in liquid medium.

MATERIAL AND METHODS

Chemicals and culture media

All chemicals and culture media were purchased from Himedia Chemicals Ltd, Mumbai, India. 

Sampling

Contaminated peanut (Arachis hypogea L.) samples (with envelope, without envelope) were collected from different markets in the Chh. Sambhajinagar, India. Peanut samples were well mixed and kept in plastic bags. 

Mycobiota Analysis 

The fungi present in the samples were extracted using the suspension-dilution method. A mixture of 10 grams of contaminated crushed peanuts was combined with 90 ml of distilled water and subsequently mixed with tween 80. This mixture was homogenized by stirring for 15 minutes. Serial dilutions were performed starting from the stock solution. From each dilution, 100 microliters were subcultured onto Potato Dextrose Agar (PDA). The plates were incubated at 28 °C for a period of 5 to 7 days. Representative isolates from each sample were purified and preserved on Potato Dextrose Agar (PDA). The identification of the main fungal species was carried out on specific medium and on the basis of morphological characteristics. Purified fungal strains of Aspergillus flavus (RAN5) and Aspergillus niger (ARN7) were isolated and stored on PDA at 4 ° C.

Effect Of Diclofenac On Mycelial Growth of Aspergillus spp. strains 

The antifungal properties of diclofenac were examined on the mycelial growth of A. flavus strain (RAN5) and A. niger (ARN7). To assess the impact of diclofenac on mycelium growth, aliquots of the drug were separately dissolved in distilled water and aseptically added to PDA medium at a temperature ranging from 45 to 50°C. Solid potato dextrose agar plates were prepared with different concentrations of diclofenac, varying from 75 to 2400 μg/mL. Control plates were maintained without test substances (diclofenac sodium). Agar discs (9 mm in diameter) containing the edge of a 5-day-old culture of A. flavus (RAN 5) and A. niger (ARN7) were placed at the center of each Petri dish. Subsequently, the plates were sealed with parafilm. The incubation of the plates was carried out at 28 ± 2 °C. The growth rate of the colony was assessed by measuring the diameter of the expanding colony. The effect of diclofenac was evaluated after 5 days by calculating the average of two perpendicular diameters of each colony. The antifungal impact of diclofenac on the mycelial growth, in comparison with the control, was determined on the 5th day using the following formula:

Percentage of mycelial inhibition = [𝑑𝑐 – 𝑑𝑡/𝑑𝑐] × 100

 where, 𝑑𝑐 (cm) is the mean radius of the colony in the control plates and 𝑑𝑡 (cm) is the mean colony radius value of colonies grown in PDA media containing diclofenac ²².

Determination of Minimum Inhibitory Concentration (MIC) in liquid medium

The minimum inhibitory concentration (MIC) of diclofenac was assessed using the CLSI broth microdilution technique described by Borman et al. (2017) with minor adjustments. A spore suspension of A. flavus (RAN5) and A. niger (ARN7) was introduced into potato dextrose liquid medium to achieve a final concentration of 1×10⁴ CFU/ml, and this medium was distributed into the wells of a 96-well plate. Diclofenac sodium was subsequently added to the wells at final concentrations ranging from 75 to 2400 μg/mL. Control tubes containing potato dextrose medium were inoculated solely with the fungal suspension and were incubated at 35⁰C for 48 hours. The plate was incubated at 35°C for 48 hours, after which the growth of A. flavus (RAN5) and A. niger (ARN7) in each well was evaluated, and the MIC was established. The IC₅₀ represents the concentration of the drug necessary to eliminate or inhibit 50 % of the growth of the isolates²³.

Statistical analysis

Values presented are the means with standard deviations, obtained from three different observations. Values in the control and treatment groups for various concentrations were compared using the student’s t-test. A value of p < 0.05 was considered statistically significant.

RESULTS

Mycological analysis

In the examined peanut samples, the pH levels were noted to be between 6 and 7. Two critical factors influencing the growth and spread of mold in stored food samples are moisture content and pH. Our investigation revealed that pH in the peanut samples was suitable for fungal development. The mycological assessment of the peanut samples indicated a prevalence of Aspergillus species. A. flavus (RAN-5), isolated from peanut samples, was identified and was chosen as the test fungus and A. niger (ARN7) was also evaluated.

Antifungal Effect of Diclofenac sodium on mycelial growth and determination of MIC 

The inhibitory effects of various concentrations of Dic on the colony growth of A. flavus and A. niger in vitro are illustrated in Figures 01 and 02 . We observed that different concentration of Dic led to a decrease in fungal growth, and the reduction in mycelial growth was correlated with the higher concentrations of Dic. The Dic resulted in a notable decrease in mycelium growth for A. flavus (RAN 5) at concentrations of 300, 600, 1200, and 2400 𝜇g/mL, with reduction percentages of 82, 87, 90, and 97 respectively. Likewise, for A. niger (ARN7), Dic demonstrated a significant decline in mycelial growth at 300, 600, 1200, and 2400 𝜇g/mL concentrations, with inhibition percentages of 89,90,92, and 94 respectively.

The minimum inhibitory concentration (MIC) of Dic was evaluated against foodborne fungi using the dilution method in a liquid environment. Conducting the MIC study is essential for identifying the lowest concentration needed to inhibit fungal growth. At a concentration of 1200 𝜇g/mL, Dic effectively reduced growth of A. flavus (RAN-5) by 72%. Additionally, Dic was found to inhibit A. niger (ARN-7) at 300 𝜇g/mL, resulting in a 64% reduction. The IC₅₀ values were determined to be 300 𝜇g/mL for A. flavus (RAN-5) and 1200 𝜇g/mL for A. niger (ARN-7) respectively (Fig 03). Here, we found MIC value of Dic is higher in A. niger than A. flavus.

 

 

      

Figure 1: Effect of different concentrations of diclofenac sodium on mycelial colony growth of (a) A. flavus (b) A. niger. The plates were incubated at a temperature of 28 ± 2⁰C for 5 days. Values are means (𝑛 = 3) ± standard deviations.

                                

                      

(2 A)                         (2B)        (2a)                              (2b)

Figure 2: Effect of diclofenac sodium on mycelial colony growth of A. flavus; control(2A), 300 µg/ml (2B) and A. niger; control (2a), 300 µg/ml. The plates were incubated at a temperature of 28 ± 2⁰C for 5 days.

Figure 3: Effect of different concentrations of diclofenac sodium on growth of (a) A. flavus (b) A. niger in liquid medium. The plates were incubated at a temperature of   35 ⁰C for 48 hrs. Values are means (𝑛 = 3) ± standard deviations.

 

 

DISCUSSION

Due to the emergence of drug resistance, repurposing of drugs may be a promising strategy for drug development. It is a promising approach to finding effective drugs and overcoming the problem of the emergence of drug resistance. There are various studies done with a drug repurposing approach to screen a library of drugs with anti-inflammatory, anti-tumour, and antimicrobial efficiency²⁴. In the same way, in the agricultural field, the efficacy of halofuginone, kaempferol, honokiol and tavaborole against phyto-pathogens has been reported ^24,25. An et al in 2023, screened 600 drugs against pathogenic fungi and 82 drugs were reported effective against Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Phytophthora capsici, Fusarium graminearum and Fusarium oxysporum, respectively²⁴. In this present work, we have investigated the potential of Dic against aflatoxin producing Aspergillus flavus and Aspergillus niger isolated from contaminated peanuts. In vitro antifungal assay, Aspergillus spp. was found sensitive to the different concentrations of diclofenac sodium salt. Our data clearly show a significant reduction in mycelium growth of both fungal strains tested. The diclofenac sodium salt at 300 µg/ml showed significant mycelium inhibition for both Aspergillus species. There are a number of findings available suggesting antifungal effects of Dic against human pathogens. Dic at 500 µg/ml showed significant activity in the human fungal pathogen Aspergillus fumigatus ²¹.  Rusu et al in 2014 reported the antifungal efficacy of Dic in combination with antifungal agents against C. albicans and C. krusei²⁶. However, there are no studies evaluating the effects of Dic on A. flavus and A niger isolated from contaminated food. Dic significantly inhibited the growth of A. flavus and A niger in a concentration-dependent manner. Our results are in agreement with the above findings. These findings suggest the efficiency of Dic as an antifungal in food protection. However, further study is required to find out the toxicity and its effect on the quality of food.

 

CONCLUSION

Our study reports antifungal efficacy of diclofenac sodium salt against A. flavus and A. niger. It shows significant inhibitory effects on the growth of Aspergillus spp. in a concentration-dependent manner. This observed efficacy of Dic against mycelial growth of Aspergillus spp. suggests the repurposing diclofenac as an antifungal agent, particularly for food protection applications. Our findings align with previous findings on the antifungal efficacy of diclofenac and expand its scope to use against foodborne fungal pathogens. However, further investigations are needed, including in vivo studies, toxicity, and its effect on food quality. We suggest diclofenac would be a cost-effective and efficient solution for managing fungal contamination and spoilage in food.

Abbreviations

Dic-Diclofenac sodium salt, NSAID-Non steroidal anti-inflammatory drug

Conflict of interest: Authors declare no conflict of interest.

Acknowledgment: Authors are thankful to Dr. Ashok Tejankar, Principal of Deogiri college for his kind assistance.

Author Contributions: All authors have equal contribution in the preparation of manuscript and compilation.

Source of Support: Nil

Funding: The authors declared that this study has received no financial support.

Informed Consent Statement: Not applicable. 

Data Availability Statement: The data presented in this study are available on request from the corresponding author. 

Ethical approval: Not applicable

 

REFERENCES

1. Bebber DP, Holmes T, Gurr SJ. The global spread of crop pests and pathogens. Global Ecology and Biogeography. 2014;23(12):1398-1407. https://doi.org/10.1111/geb.12214

2. Zhang Q, Men X, Hui C, Ge F, Ouyang F. Wheat yield losses from pests and pathogens in China. Agriculture, Ecosystems & Environment. 2022 ;326:107821. https://doi.org/10.1016/j.agee.2021.107821

3. Horbach R, Navarro-Quesada AR, Knogge W, Deising HB. When and how to kill a plant cell: infection strategies of plant pathogenic fungi. Journal of plant physiology. 2011 ;168(1):51-62. https://doi.org/10.1016/j.jplph.2010.06.014 PMid:20674079

4. Jain A, Sarsaiya S, Wu Q, Lu Y, Shi J. A review of plant leaf fungal diseases and its environment speciation. Bioengineered. 2019;10(1):409-424. https://doi.org/10.1080/21655979.2019.1649520 PMid:31502497 PMCid:PMC6779379

5. Li Y, Aioub AA, Lv B, Hu Z, Wu W. Antifungal activity of pregnane glycosides isolated from Periploca sepium root barks against various phytopathogenic fungi. Industrial crops and products. 2019;132:150-155. https://doi.org/10.1016/j.indcrop.2019.02.009

6. Nguefack J, Dongmo JL, Dakole CD, Leth V, Vismer HF, Torp J, Guemdjom EF, Mbeffo M, Tamgue O, Fotio D, Zollo PA. Food preservative potential of essential oils and fractions from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against mycotoxigenic fungi. International journal of food microbiology. 2009 ;131(2-3):151-156. https://doi.org/10.1016/j.ijfoodmicro.2009.02.009 PMid:19268382

7. Frisvad JC, Skouboe P, Samson RA. Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov. Systematic and applied microbiology. 2005 ;28(5):442-453. https://doi.org/10.1016/j.syapm.2005.02.012 PMid:16094871

8. Prakash B, Shukla R, Singh P, Kumar A, Mishra PK, Dubey NK. Efficacy of chemically characterized Piper betle L. essential oil against fungal and aflatoxin contamination of some edible commodities and its antioxidant activity. International journal of food microbiology. 2010 ;142(1-2):114-119. https://doi.org/10.1016/j.ijfoodmicro.2010.06.011 PMid:20621374

9. Reddy KR, Raghavender CR, Salleh B, Reddy CS, Reddy BN. Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains. International journal of food science & technology. 2011 ;46(1):161-165. https://doi.org/10.1111/j.1365-2621.2010.02468.x

10. Boudjaber K, Miri YB, Benabdallah A, Bennia N, Hamadi C, Soumati B, Djenane D, Simal-Gandara J. Evaluation of Antifungal and anti-aflatoxin B1 efficacy of some crude extracts of Chamaerops humilis L. against Aspergillus flavus isolated from peanuts (Arachis hypogea L.). Food control. 2023 ;152:109858. https://doi.org/10.1016/j.foodcont.2023.109858

11. Joseph GS, Jayaprakasha GK, Selvi AT, Jena BS, Sakariah KK. Antiaflatoxigenic and antioxidant activities of Garcinia extracts. International journal of food microbiology. 2005 ;101(2):153-160. https://doi.org/10.1016/j.ijfoodmicro.2004.11.001 PMid:15862877

12. Giray B, Girgin G, Engin AB, Aydın S, Sahin G. Aflatoxin levels in wheat samples consumed in some regions of Turkey. Food control. 2007;18(1):23-29. https://doi.org/10.1016/j.foodcont.2005.08.002

13. García-Cela E, Ramos AJ, Sanchis V, Marin S. Emerging risk management metrics in food safety: FSO, PO. How do they apply to the mycotoxin hazard? Food Control. 2012;25(2):797-808. https://doi.org/10.1016/j.foodcont.2011.12.009

14. Jochum CC, Osborne LE, Yuen GY. Fusarium head blight biological control with Lysobacterenzymogenes strain C3. Biological Control. 2006;39(3):336-344. https://doi.org/10.1016/j.biocontrol.2006.05.004

15. Wang L, Li C, Zhang Y, Qiao C, Ye Y. Synthesis and biological evaluation of benzofuroxan derivatives as fungicides against phytopathogenic fungi. Journal of agricultural and food chemistry. 2013;61(36):8632-8640. https://doi.org/10.1021/jf402388x PMid:23937418

16. Sparks TC, Lorsbach BA. Perspectives on the agrochemical industry and agrochemical discovery. Pest management science. 2017 ;73(4):672-677. https://doi.org/10.1002/ps.4457 PMid:27753242

17. Zheng W, Sun W, Simeonov A. Drug repurposing screens and synergistic drug‐combinations for infectious diseases. British journal of pharmacology. 2018;175(2):181-191. https://doi.org/10.1111/bph.13895 PMid:28685814 PMCid:PMC5758396

18. Wall G, Lopez-Ribot JL. Screening repurposing libraries for identification of drugs with novel antifungal activity. Antimicrobial Agents and Chemotherapy. 2020;64(9):110-128. https://doi.org/10.1128/AAC.00924-20 PMid:32660991 PMCid:PMC7449171

19. An Q, Li C, Chen Y, Deng Y, Yang T, Luo Y. Repurposed drug candidates for antituberculosis therapy. European journal of medicinal chemistry. 2020;192:112175. https://doi.org/10.1016/j.ejmech.2020.112175 PMid:32126450

20. Barbarossa A, Rosato A, Carrieri A, Tardugno R, Corbo F, Clodoveo ML, Fracchiolla G, Carocci A. Antifungal Biofilm Inhibitory Effects of Combinations of Diclofenac and Essential Oils. Antibiotics. 2023;12(12):1673. https://doi.org/10.3390/antibiotics12121673 PMid:38136707 PMCid:PMC10740460

21. Nargesi S, Rezaie S. Investigation an antifungal activity of diclofenac sodium against hyphae formation in Aspergillus fumigatus with attention to the expression of Ef-1 gene. Iranian Journal of Public Health. 2018;47(5):770-2.

22. Tian J, Zeng X, Zeng H, Feng Z, Miao X, Peng X. Investigations on the antifungal effect of nerol against Aspergillus flavus causing food spoilage. The Scientific World Journal. 2013;2013(1):230795. https://doi.org/10.1155/2013/230795 PMid:24453813 PMCid:PMC3884799

23. Borman AM, Fraser M, Palmer MD, Szekely A, Houldsworth M, Patterson Z, Johnson EM. MIC distributions and evaluation of fungicidal activity for amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin and 20 species of pathogenic filamentous fungi determined using the CLSI broth microdilution method. Journal of Fungi. 2017;3(2):27. https://doi.org/10.3390/jof3020027 PMid:29371545 PMCid:PMC5715917

24. An Q, Li C, Chen Y, Deng Y, Yang T, Luo Y. Repurposed drug candidates for antituberculosis therapy. European journal of medicinal chemistry. 2020;192:112175. https://doi.org/10.1016/j.ejmech.2020.112175 PMid:32126450

25. Zhang S, Cai J, Xie Y, Zhang X, Yang X, Lin S, Xiang W, Zhang J. Anti-phytophthora activity of halofuginone and the corresponding mode of action. Journal of Agricultural and Food Chemistry. 2022 ;70(39):12364-12371. https://doi.org/10.1021/acs.jafc.2c04266 PMid:36126316

26. Rusu E, Sarbu I, Pelinescu D, Nedelcu I, Vassu T, Cristescu C, Musat S, Cojocaru M. Influence of associating nonsteroidal anti-inflammatory drugs with antifungal compounds on viability of some Candida strains. Romanian J. Infec. Dis. 2014 ;17(2).