Evaluation of the antifungal efficacy of different concentrations of Curcuma longa on Candida albicans: an in vitro study

Introduction

Candida species are human fungal pathogens capable of initiating various recurring superficial mycoses especially in the oral mucosa¹. Particularly Candida albicans is a major cause of oral and oropharyngeal infections in patients with various local and systemic causes like hyposalivation, denture wearers, diabetes mellitus, prolonged use of antibiotics, immunocompromised status etc. It is also known that up to 90% of patients with HIV infection or AIDS suffer from oropharyngeal candidiasis². The inadvertent use of antifungals in these patients has made them resistant to current antifungal therapy. Added to this the high cost and long duration of antifungal therapy have led to search for alternative antifungal agents with better efficacy and lesser side effects^3,4. Various studies have been carried out with the aim of examining the activity of natural products against fungi that cause infections.

Turmeric is a well-known indigenous herbal medicine belonging to the family Zingiberacaeae5. This tuberous rhizome has long held a place of honour in India’s traditional Ayurvedic medicine. The significance of turmeric in medicine has changed considerably since the discovery of the antioxidant and antimicrobial properties of its naturally occurring phenolic compounds. They also have antiinflammatory^6,7,8, anticancer^6,7,9, hepatoprotective¹⁰, antiallergic¹⁰, wound healing¹⁰, antispasmodic¹⁰ and anti-HIV properties^2,10.

The present study has been undertaken to evaluate the in vitro action of turmeric (C. longa species) against Candida albicans, the most common fungal species isolated from oral infection.

Objective of the study

The purpose of the present study was to evaluate the antifungal efficacy of various concentrations of turmeric extract against Candida albicans in vitro and to determine the minimum inhibitory concentration (MIC) of C. longa extract.

Materials and methodology    

This is a prospective lab trial study with no ethical concerns. Alcoholic extract of Curcuma longa and pure standard specimen of C. albicans (PTCC5027, Merck, Germany) were prepared in a way that is described below:

Alcoholic extract of C longa:

A slightly modified method from Singh RP et al was used for the alcoholic extract of C. longa. 5 g of dry powder of C. longa rhizome (turmeric) was soaked in 10 ml of absolute alcohol overnight in a refrigerator to avoid evaporation of alcohol. On the next day, it was centrifuged at a temperature of 22oC, speed of 3870 rotations per minute, timed for 12 minutes. At this point, a clear solution was visible at the top of the test tube and debris settled at the bottom. The clear solution at the top was carefully poured into a new test tube so that all debris remained in the original test tubes. This extract of turmeric in ethanol served as a stock for experimentation. This solution was stored at four degrees centigrade to avoid evaporation of the ethanol. The total mass of the debris was determined to be 3.37 grams. The net turmeric in the 10 ml of solution was determined to be 1.73grams¹¹. (Ref Table I for the amount of turmeric in different concentrations of extract)

Determination of in vitro antifungal activity:

Surface of vial containing fungi, was disinfected by alcohol and broken within cotton dipped in alcohol near the heat. Seaboard’s dextrose agar (SDA) slope was used to subculture from a stock culture of Candida albicans (PTCC5027, Merck, Germany) prepared using the necessary sterile precautions. This growth was used to prepare an inoculum in sterile saline and four different concentrations of the fungus was prepared using doubling dilution method comprising of 1:10, 1:20, 1:40 & 1:80 concentrations¹². An agar dilution assay was modified from the National Committee for Clinical Laboratory Standards (NCCLS, 2002) and used, for determination of the MIC¹³. SDA plates mixed with 7 different concentrations of 50, 100, 200, 400, 800, 1600 µl and 3200 µl) of alcoholic turmeric extract in 7 separate plates were prepared. Each plate was divided into four quadrants, different dilutions of fungal suspension was streaked onto the different quadrants of the culture plates and incubated for 48 hrs at 370C. Following incubation, the number of colonies was visually counted and the relative size of the colonies was visually inspected and data recorded.

MIC was the minimum concentration of extract of C longa that inhibited 100% of the growth of Candida.

Results

Antimicrobial activity of ethanolic turmeric extract was tested for Candida albicans by using agar dilution method. Alcoholic control was prepared to rule out the antifungal activity of ethanol (figure 1-A). Evaluation of plates showed that there was absolutely no antifungal effect in a concentration as low as 50 µl (Fig.  1-B). As the concentration of the alcoholic extract of turmeric increased the size and the number of the fungal colonies decreased. The size and number of colonies were inversely proportional to the concentration of turmeric. It was also noted that with increasing dilutions of candida albicans the number of colonies decreased, thereby the number of colonies were inversely proportional to the amount of dilution of Candida.

There was complete inhibition of the growth of all four dilutions of Candida at a concentration of 800 µl and greater (Fig.  1-E, F). 800 µl was considered as the Minimum Inhibitory Concentration (MIC) of alcoholic extract of turmeric on Candida albicans.

Discussion  

The interest in oral candidiasis has waxed and waned from the period of Hippocrates. The acquired immunodeficiency syndrome (AIDS) epidemic has certainly bolstered up these figures with diabetes and oral cancer being no exemption¹⁴. Over the years there has been a definite increase in yeast infections, the drugs available for their management, and the emergence of resistant isolates making antifungal drug susceptibility testing, an important component of current health care management. In 1995, in a landmark publication in the field of antifungal susceptibility testing, the National Committee for Clinical Laboratory Standards (NCCLS) published the Reference method for Agar Dilution Antifungal Susceptibility Testing of Yeasts, which proposed a standard method to determine yeast susceptibilities. This method measures and compares colony sizes of individual strains at different drug concentrations on solid agar medium. The colony size method is simple, fast, and inexpensive and requires no instrumentation. Even without using the microscope, simple comparisons of the colony sizes could usually determine the MICs for strains Multiple isolates could be streaked on a single petri dish. Most importantly, the colony size method could provide a statistical meaning to the MIC. In this study, the above method has been slightly modified and adapted¹³.

Oral candidiasis is frequently seen in both dental and general practice. It is mainly caused by Candida albicans. Several factors, including iatrogenic, pathophysiological, and behavioural, may promote oral candidiasis generating a disturbance in the oral microbial ecosystem and causing an increase in the ‘mycotic count’. Generally, treatment to control these episodes in small lesions is exclusively topical, employing nystatin in an oral suspension or other options such as clotrimazole tablets which dissolve in the mouth, and miconazole gel (Schechter & Rachid, 2004)⁶. The increase in resistance to antifungals and the long duration of antifungal therapy has led to various studies being carried out with the aim of examining the activity of natural products against fungi that cause infections⁶.

In our study we have used ethanol (absolute alcohol) as the solvent based on the methodology from Singh et al11 and a control plate was prepared to rule out its anti-fungal activity. The control plate showed maximum growth proving that there was no possible interference of alcohol in the antifungal activity11. In a study by Babaii et al15 methanol was used initially but it was revealed that Methanol has antifungal activity and it was replaced by 50% DMSO¹⁵.

Babaii et al¹⁵ conducted a study using three laboratory methods which included cell count technique, cup bioassay technique and disk diffusion method to evaluate antifungal effect of curcumin against C. albicans and this effect generally increases by increasing the dosage¹⁵.

A study by Jianping et al¹³ the MIC for fluconazole against candida albicans was determined by Agar dilution assay. It was observed that when different concentrations of fluconazole were tested against Candida, the diameter of the colonies decreased as the drug concentration increased. This concept has been adapted in our study to determine the MIC of turmeric¹³.

The ethanolic extract of turmeric was used because it contains many chemical components in their extracts including phenolic compounds and its derivatives, the esters of weak acid, fatty acid, terpenes and others. Since a large number of different chemical compounds are present in these crude extracts, they can affect multiple target sites against the fungus. The mode of action by which the turmeric extract inhibits the fungal growth is by alteration in the morphology of the hyphae which may appear severely collapsed, plasma membrane disruption, mitochondrial destruction, lack of cytoplasm, folding of the nuclear membrane, thickened cell wall caused by chemical components of spice extract¹⁶.

According to a study conducted by Martins et al (2008)17 Curcumin was a more potent antifungal than fluconazole. Curcumin intensely inhibited the adhesion of Candida species isolated from AIDS patients to buccal epithelial cells, indicating that curcumin is a promising lead compound that can be put to therapeutic use in immunocompromised patients¹⁷.

Alalwan et al¹⁸ investigated the biological impact of curcumin on C. license and showed that curcumin preferentially affected immature morphological forms (yeast and germlings), and actively promoted aggregation of the cells. Transcriptional analyses showed that key adhesins were downregulated (ALS1 and ALS3), whereas aggregation related genes (ALS5 and AAF1) were up-regulated. Collectively, the above data established that curcumin produces anti-adhesive effects and that induces transcription of genes integrally involved in the processes associated to biofilm formation. Curcumin and related polyphenols therefore have the capacity to be developed for usage in oral healthcare to amplify existing preventive strategies for candida biofilms on the denture surface¹⁸.

In a study by Sharma et al¹⁹, the use of curcumin alone or an antifungal drug (Nystatin or Amphotericin B) alone, did not create any halo after 48 hours. However, when curcumin was used simultaneously with each of two antifungal drugs, there was a 1-1.5mm halo because of synergic effect of drugs. This may be because they used 23μg/ ml concentration of curcumin and also 0.078g/ ml Amphotericin B, at such low concentrations these drugs may not have been effective alone¹⁹. In another study, curcumin was shown to enhance the activity of azole and polyene antifungals²⁰.

From the results of our study, it appears that, turmeric possesses definite antifungal properties. It shows static effects at lower concentration and fungicidal effects at higher concentrations. Since the extract used was crude; in other words, the active principles that endow it with an antifungal action were not isolated. It is possible that, after a purification step, the concentration required for an antifungal action would be lower than that obtained in the present work.

Conclusion  

Today, most pathogenic organisms are becoming resistant to drugs. To overcome this alarming problem, the discovery of novel active compounds against targets is a matter of urgency¹⁷. Most of the spices extract have biologically active compounds, which can be used in the synthesis of potent drugs. Thus spices, which are normal ingredients of our routine food preparations, can provide protection to a certain extent against infections. There should be encouragement for the assessment of phytomedicine products, represented in this study by the extract of C. longa, which can be considered a good candidate for a medicine for topical use in the form of mouthwashes, bearing in mind the antifungal performance demonstrated in vitro. Furthermore, the low and low cost are important points in favour of this drug in developing countries like India⁵. However further clinical studies are required to prove its efficacy in various infections.