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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 5  |  Issue : 2  |  Page : 229-234

Antifungal efficacy of lauric acid and caprylic acid – Derivatives of virgin coconut oil against Candida albicans


1 Department of Oral Pathology and Microbiology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India
2 Department of Oral Pathology and Microbiology, Seema Dental College and Hospital, Rishikesh, Uttarakhand, India
3 Department of Oral Pathology and Microbiology, GSL Dental College, Rajahmundry, Andhra Pradesh, India
4 Department of Oral Pathology and Microbiology, Andhra Pradesh, India

Date of Submission20-Apr-2021
Date of Acceptance01-Jun-2021
Date of Web Publication16-Jun-2021

Correspondence Address:
Satya Tejaswi Akula
Department of Oral Pathology and Microbiology, Vishnu Dental College, Vishnupur, Bhimavaram - 534 202, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_65_21

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  Abstract 


Background: Candida albicans is the primary causative agent of oral mycotic infections and can be fulminant, especially in immunocompromised individuals, which often necessitates a therapeutic intervention with antifungal drugs. However, with emergence of multidrug-resistant fungi and concomitant intolerance and adverse side effects of antifungal drugs, it is pivotal to find an alternative. One approach is to screen natural compounds which represent a rich source of novel antimicrobial agents. Aim: The present study focused on the pharmacological screening of active ingredients of virgin coconut oil, the medium-chain fatty acids – lauric acid and caprylic acid, for antifungal activity on C. albicans comparatively with those of standard antifungal drugs such as fluconazole and clotrimazole. Methods: The efficacy of lauric acid and caprylic acid against C. albicans was evaluated by using standard protocol of disc diffusion method. It was assessed by the presence or absence of inhibition zones, diameter of inhibition zones (in cm), and minimum inhibitory concentration (MIC) values. Results: Caprylic acid and lauric acid (MCFS) have potential anticandidal activity against C. albicans. Caprylic acid has the highest antifungal potential at MIC of 40μg/ml. Conclusion: Both the natural compounds have shown encouraging antifungal activity in the present study. However, further microbiological and clinical evaluation is essential to consider their utilization for therapeutic purposes.

Keywords: Candida albicans, caprylic acid, clotrimazole, fluconazole, lauric acid


How to cite this article:
Akula ST, Nagaraja A, Ravikanth M, Raj Kumar N G, Kalyan Y, Divya D. Antifungal efficacy of lauric acid and caprylic acid – Derivatives of virgin coconut oil against Candida albicans. Biomed Biotechnol Res J 2021;5:229-34

How to cite this URL:
Akula ST, Nagaraja A, Ravikanth M, Raj Kumar N G, Kalyan Y, Divya D. Antifungal efficacy of lauric acid and caprylic acid – Derivatives of virgin coconut oil against Candida albicans. Biomed Biotechnol Res J [serial online] 2021 [cited 2021 Jul 23];5:229-34. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/229/318440




  Introduction Top


Globally, infectious diseases remain the world's leading cause of death, accounting for 17 million deaths a year.[1] The oral cavity harbors an enormous number of different microbial species, including commensals, which become opportunistic pathogens during favorable environmental conditions within the host. Among these oral Candida infections accounted for 88% of all fungal infections and hence the epithet “the disease of the diseased” given to it.[2] Candida albicans, is the most common opportunistic fungal pathogen, predominant species found in denture wearers.[3] It is an asexual diploid fungus and thermally dimorphic. It exists in different morphological states (yeast, pseudohyphae, and hyphae).[4] Clinically, it presents in various forms such as oral thrush (pseudomembranous candidiasis), angular cheilitis, denture stomatitis, hyperplastic (Candida leukoplakia), chronic atrophic (erythematous), median rhomboid glossitis, endocrine candidiasis syndrome, and inflammatory papillary hyperplasia, which often necessitate a therapeutic intervention with antifungal drugs. However, with emergence of multidrug-resistant fungi and concomitant intolerance and adverse side effects of antifungal drugs, it is pivotal to find an alternative. One approach is to screen natural compounds that represent a rich source of novel antimicrobial agents. They are in great demand because of their wide biological and medicinal activities, low cost, ease of availability, and efficacy.[5] Emphasis is placed upon derived active components from natural compounds, which are generally bioactive secondary metabolites such as fatty acids, terpenoids, alkaloids, and phenols with better potential to treat different diseases.[6] Among these, it has been reported that medium-chain fatty acids (MCFAs), lauric acid and caprylic acid, derivatives of virgin coconut oil (VCO), show potential antimicrobial activity. Lauric acid (C12) accounts for 45%–53% of all fatty acid composition in coconut oil. It has the greatest antibacterial activity of all medium-chain aliphatic fatty acids. Caprylic acid (C8), octanoic acid, accounts for 25%–30% of all fatty acid composition in coconut oil. It is a clear, colorless or slightly yellowish, oily liquid, very slightly soluble in water.

In considerations of these facts and a lack of current scientific approach regarding application of active compounds available from natural resources, the present study focused on the pharmacological screening of MCFAs – lauric acid and caprylic acid, predominant derivatives of VCO, for its antifungal activity on C. albicans. Numerous studies have proved the bactericidal ability of these MCFAs, but apparently, a very little or no work was done on these as antifungal agents and comparison with standard antifungal drugs.

With this background, the present study was conducted to assess anticandidal activity of the active compounds, MCFAs (lauric acid and caprylic acid), and compare their efficacy with the standard antifungal drugs, fluconazole and clotrimazole.


  Methods Top


Source of fatty acids

Caprylic acid and lauric acid were procured from Sigma Aldrich.

Preparation of fatty acid solutions and standard drug solutions

Standard solutions of fattyacids were prepared by dissolving the active compounds in 2.77M isopropyl alcohol as a solvent at different concentrations of 1μg/ml,10μg/ml, 50μg/ml, 100μg/ml,1mg/ml,10mg/ml.,70mg/ml,100mg/ml. These suspensions were warmed to approximately 70c for further solubilization of the fattyacids. Standard antifungal drugs, fluconazole and clotrimazole, were prepared as stock solutions by diluting in 0.1 N HCL plate in different concentrations, 0.25 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 2 μg/ml, 5 μg/ml, 7 μg/ml, and 10 μg/ml.

Micro organism

A loop full of freeze-dried Candida spores (7315) commercially procured from microbial type culture collection, Chandigarh, were mixed in 5 ml of distilled water to convert the inactive form into the active form [Figure 1].
Figure 1: Candida strain 7315 procured from microbial type culture collection

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Methodology

Active Candida strain of 0.1 μg was then inoculated with the aid of a preheated inoculating loop on the SDA plate. The sample-streaked agar plate was incubated at 37°C for 24–48 h [Figure 2]. The growth of C. albicans was visualized as convex, creamy white, smooth colonies with shiny surface. The obtained mycelium was confirmed as C. albicans mycelium by performing germ tube test. The test was performed by incubating inoculated serum for 3 h at 37°C, and on observation under microscope, germ tubes were evident on the wet smear. Cell densities (number) of the former colonies were measured according to McFarland standardization. It was performed by measuring optical density of 0.5 ml of barium chloride and 99.5 ml of sulfuric acid solution. This standard suspension was compared with the turbidity of the inoculum by using colorimeter. The resultant suspension was equivalent (approximately) to 1.5 × 108 CFU/ml.[7] Serial dilutions were prepared until the dilutions shows equal turbidity as that of standard suspension. Then, 500 μl of this standardized inoculum was spread on the SDA medium using pour plate method.
Figure 2: Commercially procured Candida albicans streaked in zig-zag manner on SDA

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Disc diffusion assay

Custom discs (sterile Whatman No. 1 filter paper was used to prepare discs with a diameter of 5.0 mm) were prepared to get inoculated with 5 μl of the specific drug concentrations. 5 μl/disc of active compounds and standard drugs were impregnated on the inoculated plate in different concentrations and were incubated for 24 h at 37°C. Clear inhibition zones around discs indicated the presence of anticandidal activity. Mean diameter of zones of inhibition was measured. The fungicidal activity was evaluated by measuring the mean diameter of zones of inhibition (with antibiotic zone scale) at each concentration and compared with the mean diameter of zones of inhibition of standard antifungal drugs.

Determination of minimum inhibitory concentrations

Minimum inhibition concentrations (MICs) were determined using inhibitory concentrations in diffusion (ICD) method. The lowest concentration that inhibited the growth was noted and considered as the MIC value for that strain.[8],[9],[10]


  Results and Discussion Top


Virgin coconut oil (VCO)”, freshly prepared oil from coconut milk, referred as “drugstore in a bottle”, possesses a tremendous potential and deserves a special attention of the scientific fraternity. It emerge as a milestone for medical science of this millennium due to its various medicinal uses.[11]

Generally, lipids found in VCO are in the form of triglycerides that do not have bioactivity directly. In the human body, the synthetic fatty acids are absorbed as free fatty acids and could show bioactivity, whereas biologically metabolized fatty acids are re-esterified and formed into chylomicrons. Hence, direct usage of VCO to inhibit microorganisms may not be as effective as the use of synthetic-free fatty acids.[12] Marina et al. reported that MCFAs are one of the bioactive and beneficial components of VCO.[12] Among these MCFAs, lauric acid and caprylic acid were included in this present study.

A vast majority of microorganisms inhabiting the oral cavity are covered with a lipid (fatty) membrane and are unicellular. When these cells come in contact with other lipids, they adhere to each other and the high content of fatty acids in coconut oil may bring about a change in permeability of the cell wall and thereby alter cellular metabolism by allowing diffusion of essential metabolites extracellularly.[13] However, their bioactivity and mechanism of action have been emphasized against various bacterial species hitherto upon fungi.

A recent survey estimated that Candida species accounted for 88% of all fungal infections. These are the fourth most common cause of nosocomial bloodstream infections. Among them, C. albicans is the most common opportunistic fungal pathogen in humans and the predominant species found in denture wearers.[11]

Chemotherapeutic agents in vogue against these infections include nystatin, amphotericin B, and azoles. Currently, azoles are the mainstay in local treatment of candidal infections.[11] They show their fungicidal activity by altering the fungal cell membrane. Fungal cell wall is made up of specific compounds in contrast to bacterial and human cell wall that include chitin, β glucans, ergosterol. Mechanism of action is by inhibition of p450-cytochrome-dependent 14α-demethylase. Primary target of azoles is the heme protein, which co-catalyzes cytochrome p450-dependent 14α-demethylase of lanosterol. Inhibition of 14α-demethylase leads to depletion of ergosterol and accumulation of sterol precursors, including 14α-demethylated sterols (lanosterol, 4,14-dimethylzymosterol, and 24-methylene dihydrolanosterol), resulting in the formation of a plasma membrane with altered structure and function.[14]

Therefore, this study aimed to assess the efficacy of synthetically procured MCFAs (lauric acid and caprylic acid) against C. albicans comparatively with fungicidal activity of two azole derivatives (clotrimazole and fluconazole) rather than using direct VCO.

The MIC levels are considered as the gold standard for accurate measurement of cidal activity of antimicrobial agents. In the present study, MIC was determined using ICD method.[8],[9] The lowest concentration that inhibited the growth was noted and considered as the MIC value.[8],[9],[10] For tested compounds, if the zone diameter is <4 mm, it signifies that C. albicans was resistant to that particular concentration.[15]

A standard reference procedure, the paper disc diffusion method, which has been described by the National Committee for Clinical Laboratory Standards (NCCLS) was performed. The results obtained are similar to those obtained when the conventional agar dilution method is used.[16]

In the present study, we compared our results in accordance with NCCLS zone interpretative criteria for fluconazole (diameter between 15 and 18 mm) and clotrimazole (diameter between 15 and 18 mm)[15] [Table 1]. HCL (0.1 N) was employed for dissolution of fluconazole[17] clotrimazole to prepare stock solution and further serial dilutions. In this present study, fluconazole exhibited MIC at 16 μg/ml with a zone of diameter of 15 mm [Figure 3]. Clotrimazole exhibits MIC even at low concentration of 5 μg/ml with a zone of diameter of 15 mm. There was subsequent increase or decrease in zone of diameter according to the concentrations [Table 2], [Graph 1], and [Figure 4].
Table 1: Estimation of minimum inhibitory concentration of standard drugs by correlating with the interpretive criteria of National Committee for Clinical Laboratory Standards

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Table 2: Zone of inhibition values and respective inhibitory concentrations of fluconazole, clotrimazole, caprylic acid, and lauric acid

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Figure 3: Diameter of inhibitory zone of fluconazole

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Figure 4: Diameter of inhibitory zone of clotrimazole

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On consideration of zone interpretive break points as per NCCLS the present study found that MIC of Clotrimazole was 5 μg/ml that almost coincides with NCCLS interpretive values of standard drugs i.e.,(MIC>8 μg/ml). But Fluconazole had shown bioactivity at greater concentration 16 μg/ml.[18] [Table 1] Through disc diffusion method, they found that MIC of fluconazole was 20μg/ml which was almost equal to the MIC (16μg/ml) found in our study.[Table 2] and [Graph 1].

However, in this study, 2.7 M isopropyl alcohol was used as a solvent and microbiologically confirmed that it did not enhance the bioactivity of tested fatty acids (lauric acid and caprylic acid).

MIC of lauric acid was 10 mg/ml with an average inhibitory zone of diameter 4.0 mm [Graph 1] and [Figure 5] in this study.
Figure 5: After incubating for 24 h, appeared clear zones indicate inhibitory zones. Average diameter of inhibitory zone of lauric acid was calculated

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Caprylic acid was also tested similar to lauric acid. Caprylic acid had shown inhibition even at low concentrations. MIC of caprylic acid was 40 μg/ml at an inhibition zone of diameter 4.0 mm [Figure 6] and [Graph 2]. There was direct relationship between concentrations of the compound and zone of diameter found, considering MIC value as standard.
Figure 6: Diameter of inhibitory zone of caprylic acid

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In comparison among tested fatty acids, caprylic acid was more potent with high efficacy at low concentration, i.e., 40 μg/ml, than lauric acid concentration, i.e., 10 mg/ml [Graph 1], [Graph 3] and [Table 1].



Batovska et al. conducted a study to investigate MIC of MCFAs against various Gram-positive organisms, including Staphylococcus, Corynebacterium bacillus, Listeria, and Streptococcus. Among the MCFAs, lauric acid exhibits a potent MIC of 125 μg/ml against Gram-positive bacteria.[19] However, in our study, we found inhibition at higher concentrations, i.e., 10 mg/ml. This supports the variation in bactericidal and fungicidal activity of fatty acids.[13] It could be due to peptidoglycan polymers in the cell wall of bacteria and microfibrillar polymers of β glucans, chitin of the C. albicans cell wall.[20]

Lauric acid shows significant activity at 2.49 μmol/ml against Gram-positive and Gram-negative organisms and C. albicans within an incubation time of 18 h.[13] However, in the present study, MIC value was 10 mg/ml as it was estimated within longer incubation period of 24 h.

According to the Joint FAO/WHO Expert Committee on Food Additives, caprylic acid is considered safe (EFSA, 2009). This present study found that the MIC of caprylic acid against C. albicans was 40 μg/ml. In a study done by Sia CM et al., MIC of caprylic acid was assessed by broth microdilution method, and it was ranged between 3.67 and 4.33μg/ml for Salmonella strains. There was no significant anticandidal activity of caprylic acid, according to Bergsson et al.[20] However, MIC of caprylic acid against C. albicans according to the present study was 40 μg/ml. Antimicrobial effect of caprylic acid was probably by disrupting the electron transport chain and oxidative phosphorylation.[21]

In the present study, when compared among tested fatty acids, caprylic acid was more potent with high efficacy at low concentration, i.e., 40 μg/ml, than lauric acid (10 mg/ml) [Graph 1] and [Graph 3]. It is in contrast to previous studies.[21]

However, the present study reveals that octanoic acid (fatty acid with 8 carbon chains) showed greater bioactivity than lauric acid (fatty acid with 12 carbon chains). This is supported by the study done by Chandrasekaran M et al. in which the highest bioactivity was obtained with formic acid.[22] Thus, it indicates that the number of carbon chains in fatty acids did not play an important role in their bioactivity.


  Conclusion Top


Inhibitory concentrations of these tested drugs (caprylic acid and lauric acid) were almost equal, showing less variation when compared to standard drugs (fluconazole and clotrimazole). P values among these groups were insignificant, except between clotrimazole and lauric acid [Table 3]. Therefore, as they showed less variation with almost equal potency to the currently using azoles, these MCFAs (lauric acid and caprylic acid) can be used as an alternative therapeutic option. Although both were of almost equal efficacy, these fatty acids were preferred instead of azoles having advantages of less resistance, low cost, and minimal side effects.
Table 3: Mean, standard deviation, test of significance of zones of inhibition (in cm) among different groups

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Therefore, oral therapeutic antifungal preparations can be formulated by these concentrations to treat candidal infections. Future clinical trials are required to utilize these MCFAs against infections caused by C. albicans.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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