The Measurement of Candida albicans Tolerance under The Influence of Moringa oleifera
Sri Rezeki1, Basri A. Gani2, Munifah Abdat3, Ridha Andayani2, Fitri Yunita Batubara4,
Nur Asmah5, Chusnul Chotimah6
1Oral Medicine Department, Faculty of Dentistry, Universitas Syiah Kuala,
Darussalam, Banda Aceh, Aceh, Indonesia.
2Oral Biology Department, Faculty of Dentistry, Universitas Syiah Kuala,
Darussalam, Banda Aceh, Aceh, Indonesia.
3Department of Dental Public Health, Faculty of Dentistry, Universitas Syiah Kuala,
Darussalam, Banda Aceh, Aceh, Indonesia.
4Department of Conservative Dentistry, Faculty of Dentistry,
Universitas Sumatera Utara, Medan, Sumatera Utara, Indonesia.
5Department of Conservative Dentistry, Faculty of Dentistry,
Universitas Muslim Indonesia, Makasar, Sulawesi Selatan, Indonesia
6Department of Protodonsia, Faculty of Dentistry,
Universitas Muslim Indonesia, Makasar, Sulawesi Selatan, Indonesia.
*Corresponding Author E-mail: iy_99fkg@usk.ac.id
ABSTRACT:
Natural ingredients such as M. oleifera have antibacterial and antifungal properties.The fungistatic and bacteriostatic properties are highly desirable for commensals.The tolerance properties of antifungals help create a balance among oral commensals such as Candida albicans (C. albicans). This study aimed to examine the tolerance properties of M. oleifera to C. albicans regarding biomass, pH stability, biostability, absorption, and opsonization properties. This study used Moringa leaves (M. oleifera) extracts and C. albicans as subjects. Tolerance properties were checked with analytical balance, pH meter, Sabouraud dextrose agar (SDA) medium, spectrophotometer, and 1% crystal violet. M. oleifera extracts at concentrations of 10% and 5% had excellent tolerance properties of index biomass properties, pH stability, biostability, absorption and residue, and opsonization. The M. oleifera have tolerance properties for C. albicans and significant differences between the concentrations tested. M. oleifera extract has good tolerance properties to C. albicans as an oral commensal.
KEYWORDS: Candida albicans, Moringa oleifera, tolerance.
INTRODUCTION:
We are researching the development of Moringa oleifera (M. oleifera) for application in oral candidiasis research, particularly concerningtolerance aspects of the C. albicanssuch as produce biomass, pH stability, biostability, absorption, and opsonization. The tolerance of these naturally occurring components found in extracts was evaluated before they were utilized as test subjects.
This test demonstrates that the use of natural substances is efficient in combating oral pathogenic pathogens and the hosts that they infect1. Recommendations for application to the oral cavity are needed to avoid infection of the oral cavity and toxicity to host cells, such as candidiasis. Its reference details pathogens' optimal concentration and capacity to restrict growth and maintain a healthy balance of pathogenicity2.
Candidiasis is an infection that is brought on by the yeast Candida. The fungus can typically be found on the skin and inside the body, particularly in the mouth. It is a benign fungus that colonizes human mucosae, but if it can translocate through the intestinal epithelium, it can induce fatal bloodstream infections3. Candidalysin is a cytolytic peptide toxin secreted by C. albicans in its invasive form, which is the human pathogenic form of the fungus. Candidalysin is an essential component for both mucosal and systemic infections and an effective activator of host cell activation, neutrophil recruitment, and type 17 immunity4.
A number of the infectious organisms responsible for oral infections are considered normal flora and must be present to maintain the biological equilibrium of the mouth cavity5. The maintenance of the growth of the pathogen is the purpose served by applying natural substances to sickness.Test materials' pharmacodynamic and pharmacokinetic features, such as medications, when delivered to the host to avoid infection of specific diseases3, are used to assess the tolerance concept.
Pharmacodynamic and pharmacokinetic qualities, related to immunotolerant and immunocompatibility properties, are the fundamental requirements for pharmaceuticals to meet to have an effect that lasts as long as possible inside the body6. A few examples of tolerance tests include the biomass index, shifts in temperature and pH, biostability, absorption, residual capacity, and the capability to opsonize against infections. The levels of tolerance that M. oleifera possessed were evaluated using C. albicans as the test organism. These findings can serve as a reference for ensuring that C. albicans and virulence factors remain in healthy equilibrium7.
MATERIAL AND METHODS:
Material:
This study used the test material for the ethanol extract of Moringa leaves (M. oleifera) and C. albicans ATCC 10231 as research subjects. M. oleifera tolerance test to C. albicans to measure the tolerance properties of M. oleifera in responding to C. albicans cellular activity. Tolerance tests carried out in this study included measurements of biomass index, pH stability, biostability, adsorption capacity, and opsonization of C. albicans cells. The M. oleifera tolerance assessment method was adopted from Gani's research (2022)8.
Culture of Candida albicans:
Refreshment of C.albicans was started by taking 30µL of supersized C. albicansand then cultured in Sabaround Dextron Agar (SDA)m (Sigma Aldrich, Germany) medium for 24hours. One C. albicans colony was cultured in a peptone medium for 48 hours. The suspension tested was then calibrated with MC. Farland 0.5 (0.5x10-8) or 300 CFU/mL.
Extraction of Moringa oleifera:
Moringa leaves (Moringa oleifera) that weigh up to one kilogram and have been dried are subjected to water washing. In order to obtain moringa leaf powder, the moringa leaves must first be ground up using a blender. The powdered leaves of moringa were put into a glass jar with a flat bottom and then steeped in 100mL of ethanol that was 96%. The solvent was changed after three days, and the residue and the filtrate were separated. At a temperature of 500C and a pressure of 75 mmHg, the filtrate was collected and then concentrated using a rotary vacuum evaporator.
Tolerance Assay:
Biomass index:
It was first necessary to weigh the empty tube (14mL), after which the tube was filled with 13mL of the test sample and resuspended for two minutes. Simultaneously, 1.3 milliliters of a C. albicans suspension diluted 10 to 1 was added, and the mixture was left to incubate at 370C for 48 h before being reweighed. The value of the difference in weight between the mass before and after treatment provides information on the activity level of activity of the active substance in response to C. albicans. The formula of Biomass Value = A B (A: before incubation) (B: after incubation). Index biomass value = C/D x 100%, (C: Biomass value); (D: Total Value of samples). Best (<20%); Good (21-50); 51-75 (Moderate); 76-100% (low effect).
pH Stability:
A suspension of the test material with C. albicans that had been cultured for 48 h at 370C, and then the pH was measured (three repetitions). The consistency of the reaction of the test material to variations in pH can be determined by comparing the value of the change in pH observed in the treatment group with that seen in the control group. pH response = A/B (pH of the treatment group); (B: Pathogen pH in medium or control).Meanwhile, pH stability = 100% - A (A: percentage of every sample of the number of pH changes) to assess the adaptation of pH of plant extracts using a formula. Best response (80-100%); Good responses (65-79%); moderate (40-64%); Low responses (<40%).
Biostability:
After incubating the extract suspension with C. albicans for 48 hat 370C, it was centrifuged at 3000 revolutions per minute for 15 minutes.The supernatant was collected at this point (product of bacterial reaction with the active test material). After that, spectrophotometry was used at a wavelength of 590nm to determine the reactivity value.The biostability formula is what's used to calculate the biostability value. Formula Index = A/B (A:OD Sample); (B:OD Control). The scale of the biostability index was optical density 1> (strong);OD 0.91-0.99 (moderate); <OD 0.9 (weak).
Table 1: Candida albicansTolerance under the influence of MoringaOleifera
|
M. oleifera |
N |
Biomass Index (%) |
pH Stability (%) |
Biostability (OD) |
Absorption (mm) |
||||
|
Value |
category |
Value |
category |
Value |
category |
Value |
category |
||
|
30% |
3 |
25 |
Moderate |
77 |
Good |
0.97 |
Moderate |
0.67 |
Moderate |
|
20% |
3 |
22 |
Moderate |
81 |
Best |
0.93 |
Moderate |
0.51 |
Moderate |
|
10% |
3 |
17 |
Good |
92 |
Best |
1,23 |
Strong |
0.47 |
Strong |
|
5% |
3 |
18 |
Good |
90 |
Best |
1.11 |
Strong |
0.42 |
Strong |
|
2.5% |
3 |
21 |
Moderate |
65 |
Good |
0.91 |
Moderate |
0.56 |
Moderate |
|
1.25% |
3 |
26 |
Moderate |
72 |
Good |
0.87 |
Weak |
0.74 |
Weak |
|
Nystatin |
3 |
18 |
Good |
93 |
Best |
1.78 |
Strong |
0.21 |
Strong |
|
p-Value |
0.047 |
0.027 |
0.0111 |
0.037 |
|||||
Absorption and Residual Value:
The SDA medium was made with a 6 mm well, then 100 µL of test material and 30µL of C. albicans suspension were added, then incubated for 24 hours. The residual area and absorption zone were measured with a caliper (mm). The absorption value refers to the absorption formula.
Absorption Level formula:
Ratio Diameter Residual
-------------------------------------------------------------
Total Diameter Residual + Diameter absorbs zone
Strong (<0.50mm), Moderate (0.51-0.75mm), Weak (0.76-1mm), and Negative (1mm>).
Opsonization of Candida albicans cells:
A grid of 2000 was used to create a rough surface on the glass.After that, it was submerged in a saline solution for an hour. After that, the abject glass was allowed to dry while suspended above the shaker at an acceleration of 500 xg, and the surface was obstructed by a square centimeter.After that, a layer of assay material extracts totaling 200mL was applied all over the exterior.After 15min of incubation, 20µL of C. albicans diluted at a ratio of 1:10 was added to the glass, and it continued to be incubated for another 24h. The C. albicans test material was removed, washed with buffer saline, and allowed to remain at room temperature for 15min before the visualization of the cell opsonization mass was modified. After administering the crystal violet 1% in 200µL for five min, the area was cleansed with buffered saline.After that, it was left to incubate at 370C for an additional twenty minutes, after which an optilab viewer-assisted microscope was used to examine the glass (Miconos).
Statistical Analysis:
All experiments were carried out in technical and biological triplicates. One-Way ANOVA analyzed the tolerance data of C. albicans in the ethanol extract of M. oleifera. p<0.05 was considered statistically significant
Results and discuSsion:
Each plant has antibacterial and antifungal compounds. The antifungal properties of commensal must be considered for survival or biological balance. Determination of this property is studied for several concepts, including testing the tolerance properties of plant extract test materials in maintaining and eliminating the development of pathogens9. The Assessment of the biomass index and the stability of pH changes to measure the effectiveness of active plant compounds when interacting with C. albicans (Table 1). This test tries to quantify the total amount of dissolved solids, also known as biomass, created directly within 48 hours by the reaction between plant extracts and pathogens. Analytical scales were used to determine the biomass index value, and the results were based on the difference in sample weight before and after incubation. The difference in value justifies whether plant extracts can eradicate oral infections or preserve growth equilibrium. It is determined by the fact that a greater index biomass suggests more significantfungal activity, indicating that the material is less susceptible to infections. On the other hand, a lower index biomass shows that the material can better maintain the equilibrium of fungal development.
The measured pH stability relates to the pH's balancing response (6-7). Increases in pH levels over 7 indicate the dominance of C. albicans activity.In contrast, if the pH stability is between 6-7, the test substance can counteract the growth of C. albicans10. This test measures fluctuations in the pH of the solution as a result of the oral activity of the pathogen to maintain a stable environment favorable to the pathogen by lysing various active plant components to prevent death. An examination of the solution's temperature and pH can be utilized as a possible indicator of the existence of pathogenic activity (Table 1). Gani (2012) revealed through his research that each oral pathogen independently influences changes in saliva pH11.
Assessing the biostability of active plant compounds in responding to pathogens is necessary to determine the effectiveness of the active compounds' working lives when interacting with pathogens and their environment. This treatment showed that M. oleifera had good biostability properties suppressing the development of C. albicans for up to 48 hours12. This potential can illustrate that the active compounds contained in nature,M. oleifera have a relatively stable working period.Fig1 shows that the interaction of M. oleifera with C. albicans for 24 hours showed good absorption properties with little residual.Besides reducing the activity of C. albicans cells, M. oleifera was also appropriately absorbed for 24 hours.M. oleifera may have good pharmacodynamic and pharmacokinetic properties when interacting with pathogens and the fate of active compounds in host tissues.Gupta (2021) reported that the absorption intensity of this dynamic material when it is in the tissue could be a reference to the power of the host's immune response13.
Fig 1: The absorption profile of M. oleifera on SDA medium.(a) SDA medium, (b) Residual area, (c) absorption zone, and (d) wells filled with M. oleifera and C. albicans.1000x magnification
This test aims to assess whether the test material, an extract from a plant, can be absorbed by the target cells so that it can execute its antibacterial and antioxidant effects14. It is done to prevent an overwhelming response from target cells, as evidenced by the residual value, a signal of unabsorbed metabolism by cells (fungal cells and host cells)8. During this test, the reference concentration for subsequent testing on hosts and bacteria is determined if the absorption value is greater than the residual value. However, if the residual is substantial, it is necessary to filter the molecular size of the test sample (plant extract) to lower the residual value.In order to use the culture medium for testing the absorption and residual power of plant extracts, a well was constructed. Then the medium's surface was coated with antibiotics to prevent fungal contamination15.
Figure 2 shows that the ethanol extract of M. oleifera could opsonize C. albicans cells.Its ability indicates that several active compounds possessed by M. oleifera can penetrate and be toxic to C. albicans cells. Soraya (2022) reported that several antibacterial compounds from M. oleifera can damage cell membranes through active diffusion and passive diffusion pathways, which cause changes in hydrostatic pressure so that cells lose balance and communicate with other pathogenic cells.16
We are researching the opsonization of pathogenic cells to understand better the biological reaction that plant extracts have when they come into touch with pathogenic cells and assess whether they successfully remove pathogenic cells. This investigation aims to determine the standard or number of opsonization cells as an indicator of cytotoxicity and lysis, which can diminish the pathogen's virulence in oral infections. This examination seeks to ascertain the standard or quantity of opsonization cells. This study aims to set a standard or determine the number of opsonization cells.In general, oral pathogenic cells that have been opsonized by a plant extract (antifungal) can disrupt communication amongst other oral pathogenic cells during the phases of biofilm creation, colonization, and adherence on the tooth surface and oral mucosa17.
M. oleifera extract has good tolerance (fungistatic) properties to C. albicans as an oral commensal. Concentrations of 5% and 10% provide excellent tolerance properties related to biomass index, pH stability, biostability, absorption, and opsonization of C. albicans cells.
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Received on 07.05.2021 Modified on 17.04.2022
Accepted on 20.02.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(6):2579-2583.
DOI: 10.52711/0974-360X.2023.00423