|Year : 2020 | Volume
| Issue : 3 | Page : 260-269
Effectiveness of red fruit (Pandanus conoideus Lam.) on Candida albicans (ATCC 10231) in the field of prosthodontics: An experimental study
Vita MP Novianti1, Lisda Damayanti1, Aprillia Adenan1, Yuti Malinda2
1 Department of Prosthodontics, Universitas Padjadjaran, West Java, Indonesia
2 Department of Biology Oral, Faculty of Dentistry, Universitas Padjadjaran, West Java, Indonesia
|Date of Submission||03-Sep-2019|
|Date of Decision||13-Dec-2019|
|Date of Acceptance||23-Dec-2019|
|Date of Web Publication||02-Jun-2020|
Dr. Vita MP Novianti
Sp. Pros Department of Prosthodontics, Faculty of Dentistry, Universitas Padjadjaran, Jl. Sekeloa Selatan I, Bandung 40132, West Java
Source of Support: None, Conflict of Interest: None
Aim: This research aimed at investigating the effectiveness of antifungal obtained from red fruit in the treatment of C. albicans. Materials and Methods: The disk diffusion method was used to examine the inhibition zone from red fruit in methanol, n-hexane, ethyl acetate, and water fractions with concentration of 25%, 50%, and 75%, and also chlorhexidine (2%) as control. The fraction that has a greatest antifungal effect toward C. albicans was further tested for minimum inhibitory concentration (MIC) with microdilution method and minimum fungicidal concentration (MFC) with potato dextrose agar media culture. Data analysis was carried out descriptively on inhibition zone test and MFC, whereas the experimental, analytical study was performed on MIC with Saphiro–Wilk test followed by one-sample t test. Results: The findings showed that the ethyl acetate fraction had the best antifungal effectivity against C. albicans as compared with methanol, n-hexane, and water fractions at all concentrations, although it was slightly less than the chlorhexidine control 2%. MIC of ethyl acetate fraction was 15.63mg/mL, whereas its MFC was 31.25mg/mL. MIC analysis with one-sample t test showed that ethyl acetate fraction of red fruit had a significant effect on C. albicans (P = 0.0053). Conclusion: This research showed that the ethyl acetate fraction from red fruit can be used as an antifungal agent for C. albicans.
Keywords: Antifungal, Candida Albicans, Red Fruit
|How to cite this article:|
Novianti VM, Damayanti L, Adenan A, Malinda Y. Effectiveness of red fruit (Pandanus conoideus Lam.) on Candida albicans (ATCC 10231) in the field of prosthodontics: An experimental study. J Int Oral Health 2020;12:260-9
|How to cite this URL:|
Novianti VM, Damayanti L, Adenan A, Malinda Y. Effectiveness of red fruit (Pandanus conoideus Lam.) on Candida albicans (ATCC 10231) in the field of prosthodontics: An experimental study. J Int Oral Health [serial online] 2020 [cited 2020 Aug 10];12:260-9. Available from: http://www.jioh.org/text.asp?2020/12/3/260/285571
| Introduction|| |
The condition of bad breath experienced by the community can be related to systemic conditions, but 85% of the causes come from oral cavity. One of the causes of bad breath on the denture wearers is poor denture hygiene, resulting from microbial degradation of oral organic substances. This colonization can be found in the acrylic resin-based denture surface. Fungus, such as Candida albicans sp., can be found and can cause the clinical infection problems, for example, candidiasis such as denture stomatitis. Epidemiological studies reported the prevalence of denture stomatitis in denture wearers, which varied from 15% to more than 70%.C. albicans in clinical conditions can be found in the healthy oral mucosa, which can be pathogenic, if the condition is decreased physiologically and immunologically.
C. albicans can increase the accumulation of colonization, both in denture and in the oral mucosal surface, and can become an opportunistic pathogenic fungus, caused by poor denture hygiene, the use of dentures continuously during the night, plaque accumulation in dentures, and bacterial and fungal contaminations on denture surface. In addition, dentures with poor adaptation can increase mucosal trauma, which causes mucosal lesions, and eventually inflammation.
Denture cleaning is effective as an anti-activity against the formation of the C. albicans biofilm, both to remove adhesions and to become disinfectant. However, mechanical cleaning methods are still needed to increase the removal of the biofilm. In addition to being obtained from chemical substances, disinfectants can also be obtained from various plants in nature, such as sage (Salvia officinalis L.) from Mediterranean areas in which fungicidal effects on the surface of acrylic resins are tested. Red fruit (Pandanus conoideus Lam.) is a Papuan plant that is widely used by the community as a herbal plant to treat various health problems. This plant has substances for the treatment of degenerative diseases and has function as an antimicrobial. The research on the fractions of water, hexane, butanol, ethyl acetate, and ethanol from red fruit shows that red fruit has antibacterial sensitivity, even though the zone of inhibition is smaller than the antibiotics tested. However, no studies on the effectiveness of this plant on fungi, especially C. albicans, are available.
| Materials and Methods|| |
The proposal of our research has been approved by the ethical committee board in Faculty of Medicine, Universitas Padjadjaran, Indonesia. Informed consent was not applicable because we did not involve human as research subject. This research used a descriptive explorative research design on the test of the zone of inhibition of C. albicans (American Type Culture Collection [ATCC] 10231) with the disk diffusion method and on the minimum fungicidal concentration (MFC). Furthermore, analytic experimental design was conducted on the minimum inhibitory concentration (MIC). The sample was the fractions obtained from red fruit/fresh buah merah (P. conoideus Lam.) that was collected in 2017 from Papua, Indonesia. The specimen was determined and deposited at the Laboratory of Plants Taxonomy, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Bandung, Indonesia.
The process of making red fruit extract, methanol fraction, water fraction, ethyl acetate fraction, and n-hexane fraction is shown in [Figure 1] and [Figure 2].,
Red fruit was cut into small pieces, then immersed in organic methanol solvent for 3 × 24h, and filtered using a filter funnel and filter paper. Next, the solvent was evaporated using a rotary evaporator at 40°C to obtain a concentrated extract of methanol. The quantity of the red fruit sample was 2.3 kg, then we prepared 500 g extract methanol from the sample and 200 mL of water, which was sufficient in supporting the extraction process to get two phases (water and the solid extract). Afterwards, they were passed through a separating funnel, subsequently, partitioned with 400 mL of n-hexane for six repetitions to obtain the n-hexane and water fractions. Furthermore, the n-hexane fraction was evaporated again using a rotary evaporator [Figure 3] to obtain 48 g of the n-hexane fraction [Figure 4]. Meanwhile, the water fraction was partitioned using 200mL of ethyl acetate for six repetitions to obtain the water and ethyl acetate fractions. Hereinafter, each fraction was evaporated using a rotary evaporator to obtain 350g of water fraction and 2g of ethyl acetate fraction [Figure 5]., ,
Testing the zone of inhibition
This process was conducted in several fractions: methanol fraction, water fraction, ethyl acetate fraction, and n-hexane fraction.
- Rejuvenation of C. albicans (ATCC 10231): One inoculation loop measure of C. albicans (ATCC 10231) obtained from the stock was inoculated into a test tube containing 5mL of physiological NaCl suspension until a turbidity of McFarland was reached. Then, it was compared to the standard, and therefore the suspension contained fungus was obtained.
- Preparation of media and C. albicans (ATCC 10231): C. albicans (ATCC 10231) stock (1mL) was inoculated into a petri dish, then the nutrient agar (NA) media was also poured into it, and it was homogenized by shaking, and it was kept to rest at room temperature until the mixture solidifies, which was ready for use. Eight media were made for testing the zone of inhibition with two repetitions. Each media was given a label, such as A for the methanol fraction test, B for the n-hexane fraction, C for the ethyl acetate fraction, and D for the water fraction. Concentration of each fraction was made with concentrations of 25%, 50%, and 75%, respectively.
- Sample testing: The paper disk was inserted into 2% of chlorhexidine solution as a positive control, and then it was put into eight NA solid media that contained C. albicans (ATCC 10231). We used incremental concentrations comprising low (25%), medium (50%), and high (75%). We chose these concentrations in a simpler manner as a result of adoption from previous research. Each medium also included one inoculation loop measure concentrations of 25%, 50%, and 75% of the methanol fraction for media A; one inoculation loop measure of the concentrations of 25%, 50%, and 75% of the n-hexane fraction for media B; one inoculation loop measure of the concentrations of 25%, 50%, and 75% of the ethyl acetate fraction for media C; and one inoculation loop measure of the concentrations of 25%, 50%, and 75% of the water fraction for media D; then they were incubated for 24h at a temperature of 35°C–37°C. Furthermore, the diameter of the zone of inhibition was measured in millimeters (mm).
Minimum inhibitory concentration test
- Prepare a 96-well sterile microplate.
- Perform dilution with 25% of fraction concentration.
- Perform dilution 11 times.
- In Column A, add 100 μL potato dextrose broth (PDB) media and 100 μL buah merah fraction. In Column B, add 100 μL PDB media and 100 μL solvent. In Column C, add 90 μL PDB media, buah merah fraction, and C. albicans (ATCC 10231) with 5 μL turbidity of McFarland 0.5. In Column D, add 100 μL PDB media, 100 μL solvent, and C. albicans (ATCC 10231) with 5 μL turbidity of McFarland 0.5. Serial dilution was performed until the 12th tube within its column [Figure 6].
- The microplate subsequently was incubated at 37°C temperature for 24h, and turbidity was identified using microplate reader.
- MIC was shown by the tube that consisted of the lowest concentration that still can resist fungi growth in Column C (media, test compound, and fungi).
|Figure 6: Column on the microplate. Column A: media + test compound; Column B: media + solvent; Column C: media + test compound + fungus; Column D: media + solvent + fungus|
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Minimum fungicidal concentration test
For MFC, two solvents that were more concentrated and two that were of lower concentrations were used. These were subcultured on PDB media and incubated at 37°C temperature for 24h. To identify the degree of MFC, the amount of declination of colony was suggested. The media in which fungi colony was not found was identified as MFC.
In MIC test, data normality test (Saphiro–Wilk test) was performed, and then continued with one-sample t test to test the cell death of C. albicans as a result of red fruit fraction experiment. Analysis was performed using STATA Special Edition 15.1 (StataCorp, Texas, USA). One-sample t test was performed because the data were in normal distribution. Result was significant at P < 0.05 (confidence interval 95%), meaning that the red fruit fraction contributed significantly to the death of C. albicans.
| Results|| |
The inhibition zone tests on the red fruit sample (n-hexane, methanol, ethyl acetate, and water fractions) were conducted on C. albicans (ATCC 10231) using the disk diffusion method to investigate the inhibition of red fruit against the growth of the fungus C. albicans.
After incubating for approximately 24h at 37°C, it was found that the sensitivity of inhibition for each sample on the fungus C. albicans tends to increase along with the concentration of the extract being tested. The results of the red fruit test showed that sample C was more sensitive to the fungus C. albicans, followed by samples B, D, and finally sample A [Table 1]. This showed that ethyl acetate fraction was more sensitive to C. albicans as compared to n-hexane, methanol, and water fraction.
The inhibition zone test on the sample of n-hexane fraction showed that the average inhibition diameter with the concentrations of 25%, 50%, and 75% was 6 mm (each) with the average control diameter of 25.5 mm. The methanol fraction resulted in the average inhibition diameter of 9.5 mm with the concentration of 25%, 10.5 mm with the concentration of 50%, and 10 mm with the concentration of 75%, and 26 mm was the control diameter of chlorhexidine (2%).
The inhibition zone test on the ethyl acetate fraction [Figure 7] and [Figure 8] showed that the average inhibition diameters were 22.5, 24, and 23.5 mm with the concentrations of 25%, 50%, and 75%, respectively, with the average control diameter of 27 mm. The inhibition zone test on the water fraction showed that the average inhibition diameter was 25 mm with the concentrations of 25% and 50%, and 7 mm with the concentration of 75%, with the average control diameter of 26.5 mm [Graph 1].
|Figure 7: Result of the inhibition zone from ethyl acetate (repetition 1)|
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|Figure 8: Result of the inhibition zone from ethyl acetate (repetition 2)|
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Overall, the ethyl acetate tested had a strong antifungal sensitivity on the concentrations of 50% and 75%, as it was more than 20 mm, whereas the fractions of hexane, methanol, and water had a weak antifungal sensitivity, which was less than 20 mm.
Next step was MIC test from the fraction that has the highest inhibitory zone based on the growth resistance zone test (red fruit ethyl acetate fraction). The MIC test was initiated with 25% concentration.
The percentage of C. albicans death in 25%, 12.5%, 6.25%, and 3.125% concentrations inclined by 99.926, 130.612, 171.988, and 225.707, respectively. Concentration of 1.56% declined by 194.633, whereas concentrations of 0.781%, 0.391%, 0.195%, and 0.0098% showed negative value, which were –537.131, –81.092, –371.922, and –222.967. Concentration of 0.049% increased by 24.714, but concentrations of 0.024% and 0.012% showed negative value (–51.062 and –97.859, respectively).
[Table 2] and [Table 3] and [Graph 2] show that ethyl acetate fraction had fungi static activity against C. albicans by minimum concentration of 1.563% or 15.63mg/mL, and its solvent (methanol redistillation) also had fungi static activity against C. albicans by minimum concentration of 12.5% or 125 µL/mL.
|Table 2: Minimum inhibitory concentration test result on Candida albicans|
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MFC test of ethyl acetate fraction showed that based on the observation on red fruit ethyl acetate fraction in 25%, 12.5%, and 6.25% concentration, negative growth of C. albicans was found. In 3.125% concentration test also, negative growth was found [Figure 9]. However, in 1.563% and 0.781% concentrations, fungi growth was found [Figure 10]. MFC in 50% concentration of methanol solvent showed no growth in both repetitions. But in 25% and 12.5% concentrations, the MFC showed positive growth of C. albicans.
|Figure 9: Minimum fungicidal concentration ethyl acetate fraction 3.125% concentration|
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|Figure 10: Minimum fungicidal concentration ethyl acetate fraction 1.653% concentration|
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Ethyl acetate fraction of red fruit had fungicidal activity on C. albicans by minimum concentration of 3.125% or 31.25mg/mL. Its solvent (methanol redistillation) also had fungicidal activity on C. albicans by minimum concentration of 50% or 50 µL/mL [Table 4]. [Table 5] shows the values of inhibition zone, MIC, and MFC.
|Table 4: Minimum fungicidal concentration test result on Candida albicans|
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|Table 5: Inhibition zone, minimum inhibitory concentration, and minimum fungicidal concentration description of red fruit toward Candida albicans|
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[Table 6] shows that ethyl acetate fraction has minimum concentration that can inhibit the growth of C. albicans (ATCC 10231). Result was significant (P = 0.0053 and degrees of freedom = 11), meaning that the red fruit fraction contributed significantly to the death of C. albicans.
| Discussion|| |
Previous researches have been conducted to test the antimicrobial effect of various plants existed in nature. For example, C. Albicans, the test was conducted by Assob et al. to five species of medicinal plants. On the basis of the research on 60 plant extracts (66 species) in Tanzania, it was found that 48% of plant extracts had anticandidal activity. Red fruit was also a medicinal plant used in the Papua area, and it had been investigated to have antibacterial effect.
The research on the inhibition zone from red fruit was in line with the research about traditional medicine in Cameroon regarding five species of medicinal plants, such as Piptadeniastrum africana, Cissus aralioides, Hilleria latifolia, Phyllanthus muellerianus, and Gladiolus gregasius, which had antimicrobial activity against all microorganisms tested, including C. albicans with the various zones of inhibition between 10 and 28 mm, whereas in red fruit, the value varied between 6.5 and 24 mm.
Likewise, in the research on red fruit, the ethyl acetate fraction had the significant value of the zone of inhibition compared to other fractions which is also found in the research on Euclea crispa against Aeromonas hydrophilla with the most inhibition zone had the value of 25.5 ± 0.50 mm indicating this plant extract had a source of bioactive components against membrane as one mechanism of biocidal action.
The ethyl acetate fraction from C. araliodes had the greatest inhibition zone, which was 20 mm, compared to the n-hexane and methanol fraction of the plant extract with the value of 12 and 10 mm. In line with this research, the results indicated that the ethyl acetate extract had the most sensitivity against C. albicans as compared to other extracts with the value of 22.5 and 24 mm, whereas in P. muellerianus, the methanol extract had the most sensitivity against C. albicans as compared to the hexane and ethyl acetate of the plant extract with the value of 28 mm, and it was considered as the most sensitive fraction of all plant extracts tested, whereas nystatin was used as control with the inhibition zone of 20 mm. If referring to the aforementioned research with nystatin used as control with the zone of inhibition of 20 mm, the ethyl acetate fraction from red fruit was found to have the most sensitivity against C. albicans with the value of 22.5–24 mm, although this value was lower than the control of chlorhexidine (2%) of 25.5–27 mm.
The research on the antioxidant and antimicrobial activity of Vernonia cinerea (a medicinal plant in India, Bangladesh, Sri Lanka, and the Malay islands) showed that the anti-C. albicans activity was lower than the ethyl acetate fraction from red fruit, which was 10.2 mm on the fractions of n-hexane, chloroform, and ethyl acetate.
The research on the extract of Himatanthus articulatus (Sucuba), which is a plant in the northern Amazon region used as a medicinal plant in the local Amerindian area, showed an antifungal activity that inhibited C. albicans from ATCC and also the one obtained clinically with an inhibition zone value between 13 and 19 mm in all methanol extracts of tree bark, leaves, and stems. This value was higher than the methanol extract from red fruit, but it was lower than the ethyl acetate extract from red fruit. The activity was considered due to the principle of polarity of the solution from the responsible extract against the inhibitory action.
Antimicrobial activity against mangroves (Lumnitzera littorea) showed that the n-hexane extract was the most effective extract, especially against gram-positive bacteria (Bacillus cereus), but interestingly, there was no inhibition zone against C. albicans, which indicated that this plant extract had resistance to the test concentrate, so it cannot be used as an antifungal agent. This was not in line with the findings of research studies with red fruit that actually had an antifungal effect on C. albicans.
Research on the substance of other Pandanus plants, namely P. tectorius, as an antioxidant, antibacterial, that has cytotoxicity effects, stated that the results of qualitative phytochemical analysis of this fruit contain phenol, flavonoid, terpenoid, steroid, and glycoside. The ethyl acetate extract from cores had the highest antioxidant capacity (inhibitory concentration 50% [IC50] = 0.8 ± 0.20mg/mL), whereas the ethyl extract from keys had the highest antibacterial activity with the zone of inhibition of 10–15 mm, even though this value was lower than that of the antibiotics found in the market, and it was proven that all extracts of this plant did not have cellular cytotoxic activity. If referring to this research, with this inhibition zone value, it was found that the antifungal activity of Pandanus genus was greater than the antibacterial activity.
The extract from red fruit had a sensitivity effect on various bacteria, which was found using Kirby–Bauer method, although the inhibition zone had a smaller value compared to other antibiotics. In this test, the water fraction could inhibit Salmonella typhi, B. cereus, and Streptococcus pyogenes. The hexane fraction could inhibit S. typhi, Escherichia coli, B. cereus, and S. pyogenes. The butanol fraction could inhibit E. coli, and the ethyl acetate fraction and ethanol fraction could inhibit Klebsiella pneumoniae and Staphylococcus aureus.
The effective concentration from red fruit in inhibiting the growth of these bacteria was found to be 80% in S. typhi (32 mm), E. coli (20.87 mm), K. penumoniae (20.27 mm), S. aureus (21.13 mm), and S. pyogenes (19.47 mm). The research showed that the red fruit test on various fractions had an antibacterial effect. In line with the antibacterial test, the current antifungal test conducted showed that various red fruit fractions (methanol, n-hexane, ethyl acetate, and water) with the concentrations of 25%, 50%, and 75% had an antifungal effect. In addition, the ethyl acetate fraction with the concentration of 50% had the average value of 24 mm, which is considered to be the most sensitive, although it is still lower than the test on S. typhi.
Effective concentration of red fruit in inhibiting bacterial growth was 80%, and it inhibited the growth of S. typhi (32 mm), E. coli (20.87 mm), K. penumoniae (20.27 mm), S. aureus (21.13 mm), and S. pyogenes (19.47 mm). The study showed that red fruit test in various fractions had antibacterial power. Corroborating with antibacterial test, the antifungal test that we studied showed various antifungal power in red fruit (methanol, n-hexane, ethyl acetate, and water) fractions at 25%, 50%, and 75% concentrations. Ethyl acetate fraction at 50% concentration was the most sensitive (mean: 24 mm), but it was lower than that of the test against S. typhi.
MIC of ethyl acetate fraction in our study showed fungi static value of 1.563% or 15.63mg/mL and its solvent (methanol redistillation) had minimum concentration of fungi static activity of 12.5% or 125 µL/mL. Fungicidal activity of ethyl acetate fraction had MFC value of 3.125% or 31.25mg/mL, and its solvent (methanol redistillation) had minimum concentration of fungicidal activity on C. albicans of 50% or 50 µL/mL.
MIC variation in five herbal plants was tested against C. albicans, and the results showed values between 0.31 and 20mg/mL in hexane, methanol, and ethanol extracts. These values showed that MIC of ethyl acetate red fruit extract fell within that range (15.63mg/mL). Ethyl acetate V. cinerea (L.) fraction had MIC value of 1.56mg/mL and MFC value of 3.13mg/mL against C. albicans. These values were in fact much lower than the MIC and MFC values of ethyl acetate red fruit fraction. In Combretum erythrophyllum test against C. albicans, ethyl acetate and acetone fraction was found to have antifungal activity against C. albicans (1.25mg/mL), using control (amphotericin B) with an MIC value of 0.020mg/mL.
However, this was different with that of raspberry, which has the potential to prevent oral biofilm of C. albicans. In the study, hexane extract and ethyl acetate extract from raspberry were found to have anti-adhesive activity against C. albicans in low concentration (IC50 = 15.6–62.5 µg/mL).
On the basis of the aforementioned result and discussion of this research, it was found that the ethyl acetate fractions from red fruit (P. conoideus Lam.) were effective as an antifungal agent against C. albicans (ATCC 10231), with the MIC value of 15.63mg/mL and MFC value of 31.25mg/mL.
We would like to acknowledge Laboratory of Plants Taxonomy, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia for determining and depositing the specimen, Chemistry Laboratory and Dikdik Kurnia, MSc., PhD from Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia for permission of using the lab as well as direction to conduct the extracting process, and Central Laboratory of Universitas Padjadjaran for permission of conducting MIC and MFC experiments. We would also like to thank Fedri Ruluwedrata Rinawan, MD, PhD, from Department of Public Health and Health System Research Centre, Faculty of Medicine, Universitas Padjadjaran, Indonesia, for permission and direction of using the STATA Special Edition 15.1 license.
Financial support and sponsorship
This study was self-funded by the authors.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Graph 1], [Graph 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]