Inhibition and Eradication Effect of Javanese turmeric (Curcuma xanthorrhiza Roxb.) Extract Against Mature Phase Biofilm of Candida albicans

Abstract

Objective:

To analyze the potency of Javanese turmeric extract in inhibiting and eradicating the mature phase of C. albicans biofilm.

Material and Methods:

C. albicans ATCC 10231 was cultured in Sabouraud Dextrose Broth on a 96-well plate and was incubated at 37ºC. To analyze its inhibitory effect, C. albicans cultures were incubated for 1.5 hours before being exposed to various concentrations of Javanese turmeric extract, followed by a further 48-hour incubation to reach the maturation phase. To analyze the eradication effect, the 48-hour C. albicans cultures were exposed to the extract and incubated further for 24 hours. Nystatin (100,000 IU) was used as a positive control. The percentage of viable C. albicans cells on the 48-hour biofilm was determined by a methylthiazol tetrazolium assay. This value was converted into the percentage of the extract’s minimum inhibitory and eradication concentrations.

Results:

Against the mature phase C. albicans biofilm, the minimum biofilm inhibitory concentration of the Javanese turmeric extract was 40%, while the minimum biofilm eradication concentration was 45%. There were significant differences between the inhibition percentage of the positive control and that of the solutions exposed to Javanese turmeric with all the tested concentrations (p<0.05). There was a strong positive correlation between the increase in extract concentration and the eradication percentage of the mature C. albicans biofilm (r=0.981).

Conclusion:

Javanese turmeric extract is potential for inhibiting and eradicating mature phase C. albicans biofilm. The extract is more effective in inhibiting than in eradicating the biofilm.

Keywords:
Complementary Therapies; Phytotherapy; Dental Plaque; Candida albicans

Introduction

As an oral cavity commensal fungus, C. albicans can live in planktonic form in approximately 30-50% of population [¹]. The development of C. albicans from a commensal fungus to a pathogen is initiated by a change in the homeostasis of the oral cavity’s ecosystem, and by the yeast’s virulence factors, including morphological dimorphism of the yeast into hypha and the formation of biofilm [²]. The development of C. albicans biofilms consists of 3 phases: adhesion phase, intermediate phase, and maturation phase. The adhesion phase is the first stage and lasts for 11 hours. During this phase, planktonic C. albicans begins to adhere on a surface. The next phase, which lasts for 25 hours, is the intermediate phase, during which C. albicans changes form from yeast to hypha. The last phase is the maturation phase, which lasts for 34 hours. In this phase, the biofilm becomes more complex and the extracellular matrix thickens, thereby increase the resistence of C. albicans biofilm so that the penetration by antifungal agents becomes more difficult [³].

Previous studies have reported the potency of Javanese turmeric extract as an antifungal agent against C. albicans in its planktonic form and in the initial biofilm phase. It was previously reported that xanthorrhizol, which is isolated from Javanese turmeric root, had an antifungal effect in inhibiting C. albicans biofilm with a minimum inhibitory concentration (MIC) of 1-5 mg/L and a minimum fungicidal concentration (MFC) of 5-10 mg/L [⁴,⁵]. Javanese turmeric ethanol extract had been reported to eradicate the early phase of C. albicans biofilm [⁶]. An agent’s antifungal effect in penetrating and disrupting the development and morphology of the biofilm is known as its eradication effect [⁷].

It has been reported that Javanese turmeric ethanol extract has an antifungal effect against planktonic C. albicans and could eradicate C. albicans biofilm in the initial phase [⁴,⁶]. The aim of this study was to investigate the potency of Javanese turmeric ethanol extract in inhibiting and eradicating the mature phase of C. albicans biofilm.

Material and Methods

Candida albicans

For this study, we employed the laboratory strain of C. albicans (ATCC 10231). The negative control was the C. albicans biofilm unexposed to any antifungal agent, and the positive control was the C. albicans biofilm exposed to 100,000 IU of nystatin. The concentration of the C. albicans suspension employed in this study was determined by counting the yeast's colony-forming units (CFUs) after incubating the yeast in Sabouraud Dextrose Agar (SDA) for 24 hours at 37⁰C.

In this study, planktonic C. albicans refers to the C. albicans cultured in Sabouraud Dextrose Broth (SDB) in a 96-well plate, exposed to various concentrations of Javanese turmeric or nystatin (positive control) and incubated for 48 hours at 37⁰C. The C. albicans biofilm refers to the biofilm already cultured in SDB in a 96 well-plate and incubated for 48 hours at 37⁰C.

Exposure of C. albicans to Javanese turmeric Extract

To test the inhibitory effect of the Javanese turmeric extract, the C. albicans biofilm was exposed to the extract after the culture had been incubated for 1.5 hours and further incubated for 48 hours to reach the maturation phase. To test the extract’s eradication effect, the 48-hour biofilm was exposed to the extract and incubated the biofilm for an additional 24 hours.

Javanese turmeric Extract

The Javanese turmeric ethanol extract was extracted by employing the maceration technique using 96% ethanol. We derived 4 kg of extract powder from 20 kg of Javanese turmeric root, which was further extracted to collect 672.5 mg of oil with a yield of 11.20%. To separate the oil into 3 layers, the extract oil was then centrifuged at 3700 rpm for 20 minutes. We used phytochemistry analysis to determine that the top supernatant layer contained 19.59% xanthorrhizol. We diluted the Javanese turmeric ethanol extract to various concentrations by adding SDB. For this study, the extract concentrations tested to achieve MICs were 0.25%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% and 45%. The extract concentrations tested to achieve minimum biofilm inhibitory concentrations (MBICs) were 15%, 20%, 25%, 30%, 35%, 40%, and 45%.

Minimum Inhibitory Concentration and Minimum Fungicidal Concentration of the Extract Against planktonic C. albicans

The extract’s MIC capable of inhibiting 90% or more of the growth of planktonic C. albicans was determined by calculating the optical density of the C. albicans culture exposed to Javanese turmeric extract [⁸]. A microplate reader with a wavelength of 595 nm was used to measure the optical density. The MFC was the minimum extract concentration capable of inhibiting the growth of planktonic C. albicans such that none of cultures presented even a single colony.

Minimum Biofilm Inhibitory Concentration and Minimum Biofilm Eradication Concentration

The extract’s minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were defined as the minimum extract concentration capable of inhibiting 50% or more or 90% or more, respectively, of the viable C. albicans cells in the 48-hour (mature) biofilm and were determined using a methylthiazol tetrazolium (MTT) assay. An equation was used to determine the inhibition and eradication percentages [⁸]. The application of this equation required the optical density of the experimental groups, positive controls, negative controls, blank group, negative blank and positive blank. The experimental groups were the C. albicans suspension with various concentrations of Javanese turmeric extract. The negative controls consisted of C. albicans suspensions in isolation. The positive controls consisted of C. albicans suspensions plus 100 µL nystatin. The blank group consisted of 100 µL SDB plus 100 µL of various concentrations of Javanese turmeric extract. The negative blank group consisted of 100 µL SDB in isolation, and the positive blank groups consisted of 100 µL SDB plus 100 µL nystatin.

MTT Assay

The concentration of the MTT solution employed in this study was 5 mg/mL. The MTT solution was kept in 15 mL tubes and was wrapped with aluminum foil until used. The C. albicans biofilm in the 96-well plate was washed with a 100 µL phosphate-buffered saline solution, then added to a 50 µL MTT solution and wrapped in aluminum foil before incubating for 3 hours at 37⁰C. Then, the solutions were removed from the incubator, and 100 µL acidified isopropanol was added to each well. Next, the well plate was shaken by an orbital shaker at 80 rpm for 1 hour. Finally, the solutions’ optical density was measured by a microplate reader at a wavelength of 570 nm.

Data Analysis

Data were analyzed using IBM SPSS Statistics for Windows Software, version 20 (IBM Corp., Armonk, NY, USA). One-way analysis of variance (ANOVA) and post-hoc tests were used. We used a Pearson correlation test to analyze the correlation between the increase in extract concentration and the increase in the inhibition and eradication percentage. For ANOVA and post-hoc tests the level of significance were p<0.05, while for the Correlation test the level of significance was p<0.01.

Results

Table 1 lists the results of the colony counts. The concentration of the C. albicans suspension that had an adequate number of colonies but could still be counted visually was 10^-4. Therefore, for this study, we employed the 10^-4 concentration of C. albicans suspension.

Table 1
Quantity of C. albicans colony at various concentrations.

Dilution Concentration of C. albicans	Number of C. albicans Colonies (CFUs/10 µL)
	I	II	Mean
10-2	260	257	258.5
10-4	2	30	16
10-6	0	25	12.5
10-8	0	3	1.5

The MIC of the Javanese turmeric extract against planktonic C. albicans was determined by measuring the cultures’ optical density using a microplate reader with a wavelength of 595 nm, which was then converted into the inhibition percentage value. As Table 2 shows, the minimum concentration of the extract capable of inhibiting 90% or more of planktonic C. albicans was 20%.

Table 2
Minimum inhibitory concentration of the Javanese turmeric ethanol extract against planktonic C. albicans

No.	Concentration of Javanese turmeric ethanol extract (%)	Inhibition (%)
1	0.25	55.30
2	0.5	63.65
3	1	60.92
4	5	74.41
5	10	68.72
6	15	74.57
7	20	97.50
8	25	82.68
9	30	77.77
10	35	89.31
11	40	94.85
12	45	83.23
13	Positive Control	100.0
14	Negative Control	0

Table 3 shows that the MFC of Javanese turmeric ethanol extract against planktonic C. albicans is 35%. At this concentration, exposure to the extract resulted in zero growth of the C. albicans colony.

Table 3
Minimum fungicidal concentration of Javanese turmeric ethanol extract against planktonic C. albicans.

No.	Concentration of Javanese turmeric ethanol extract (%)	Number of C. albicanscolonies, CFUs/10 µL
1	15	-
2	20	94
3	25	15
4	30	2
5	35	0
6	40	0
7	45	0
8	Positive Control	0
9	Negative Control	∞

As Figure 1 shows, exposure to 40% and 45% Javanese turmeric extract resulted in a >50% growth inhibition of the mature C. albicans biofilm. Therefore, the MBIC₅₀ of Javanese turmeric ethanol extract in this study was 40%. However, the extract’s MBIC₉₀ could not be determined in this study.

Figure 1
Minimum biofilm inhibition concentration of Javanese turmeric ethanol extract.

The post-hoc statistical analysis confirmed that there were significant differences between the inhibition percentage of the positive control and that of the solutions exposed to Javanese turmeric with all the tested concentrations (p<0.05). The relationship between the Javanese turmeric extract concentration and the inhibition percentage effect showed a strong positive correlation (r = 0.905), demonstrating that the higher the extract concentration, the higher its inhibition percentage.

As Figure 2 shows, the 45% Javanese turmeric ethanol extract was able to eradicate more than 50% of the mature C. albicans biofilm. Therefore, this study showed that the MBEC₅₀ of the Javanese turmeric ethanol extract against mature C. albicans biofilm was 45%. However, the extract’s MBEC₉₀ could not be determined.

Figure 2
Minimum biofilm eradication concentration of Javanese turmeric ethanol extract.

The post-hoc statistical analysis showed significant differences between the eradication percentage of the positive control and that of the experimental groups (p<0.05). There was a strong positive correlation between the increase in extract concentration and the eradication percentage of the mature C. albicans biofilm (r = 0.981), demonstrating that the extract’s eradication percentage increases as the extract’s concentration increases.

Discussion

The MIC and MFC of Javanese turmeric ethanol extract against planktonic C. albicans in this study were 20% and 35%, respectively. This study had higher MIC and MFC values than a previous study that reported extract MIC and MFC of 10% and 20%, respectively [⁶]. This difference could be due to the different xanthorrhizol content in the Javanese turmeric extracts used in the studies. However, the two studies showed similar results in that the MFC was higher than the MIC, and the extract concentration required to inhibit planktonic C. albicans is lower than the concentration required to inhibit the C. albicans biofilm.

Similar results were also reported by several previous studies on the antifungal agent [⁹-¹¹]. In this study, the inhibitory effect of Javanese turmeric extract was examined by exposing the C. albicans culture to the extract after the culture had been incubated for 1.5 hours. By doing so, the yeast culture was given the chance to adhere to the well surface and start the biofilm formation, given that planktonic C. albicans requires 1 to 2 hours to adhere to a surface [³,¹²].

The results of this study show that the extract concentration needed to inhibit (MBIC) and to eradicate (MBEC) more than 50% of the growth of the mature C. albicans biofilm were 40% and 45% respectively. However, the MBIC₉₀ and MBEC₉₀ could not be determined in this study. These results confirm that eradication of C. albicans biofilm is more difficult than its inhibition and, therefore, the extract concentration required to eradicate the biofilm was higher than the concentration needed to inhibit it. The results also showed that the extract could only inhibit or eradicate approximately 60% of the growth of the mature C. albicans biofilm, even at a 45% concentration. Conversely, the positive control (nystatin) was capable of inhibiting and eradicating 100% of the growth of the mature C. albicans biofilm. These results indicate that although it is not as strong as nystatin, Javanese turmeric ethanol extract has sufficient potency to inhibit or eradicate the C. albicans biofilm even during the maturation phase.

From a previous study, we know that at a 35% concentration, the Javanese turmeric extract can eradicate 90% of early-phase C. albicans biofilms, both during the adhesion phase (1.5 hours) and during the proliferation phase (3 hours) [⁶]. From these two studies, we can see that the more mature the C. albicans biofilm, the higher the extract concentration required to eradicate the biofilm and the weaker the extract’s eradication effect. At a 35% concentration, Javanese turmeric extract can eradicate 90% of early-phase C. albicans biofilm, while at a 45% concentration, the extract can only eradicate approximately 60% of the mature C. albicans biofilm.

The activity of xanthorrhizol in inhibiting C. albicans biofilm is influenced by the extract concentration and the phase of the C. albicans biofilm. It has been reported that xanthorrhizol can eradicate 100% of early-phase C. albicans biofilm at 8 µg/mL but can only eradicate 67.48% of the mature biofilm at 32 µg/mL [⁴]. These results indicate that the efficacy of Javanese turmeric extract is in line with the efficacy of xanthorrhizol, which is its active antifungal component. During the maturation phase, C. albicans biofilm is more complex and its extracellular matrix is denser, which makes penetration more difficult [³,¹³]. Both nystatin and xanthorrhizol contain OH chains capable of disrupting the C. albicans cell membrane [¹⁴]. This antifungal mechanism could explain the antifungal effect of Javanese turmeric extract.

Conclusion

The antifungal effect of Javanese turmeric extract against mature C. albicans biofilm is not as strong as its effect on early-phase C. albicans biofilm. However, Javanese turmeric extract is potent in inhibiting and eradicating mature C. albicans biofilm. The inhibitory effect of Javanese turmeric ethanol extract is stronger than its eradication effect against mature C. albicans biofilm.

• Financial Support: This study was supported by a grant from the Directorate of Research, Universitas Indonesia (Hibah PITTA 2017).

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Edited by

Academic Editors: Alessandro Leite Cavalcanti and Wilton Wilney Nascimento Padilha

Publication Dates

• Publication in this collection
  31 Oct 2019
• Date of issue
  2019

History

• Received
  05 Feb 2018
• Accepted
  26 Apr 2019
• Published
  09 May 2019