ANTI INFLAMATORYEFFECT OF STICHOPUS HERMANII EXTRACT AS ORAL CANDIDIASIS TREATMENT
Syamsulina Revianti1, Kristanti Parisihni1, Delianis Pringgenies2
1 Hang Tuah University, Surabaya, Indonesia
2 Diponegoro University, Semarang, Indonesia
Background: Sea cucumbers have been consumed by Asian community as food and traditional medicine. Recent research stated that sea cucumber extract have its biomedical properties. Golden sea cucumber (Sticophus hermanii) extract have been known to have antifungal agent and regarding to its contents is potential to be explored as treatment in oral candidiasis. Objective: The aim of this study was to examine the anti inflamatory effect of Stichopus hermanii extract againstCandida albicans in vivo. Material and Method: The study was an experimental laboratories research with post test only control group design. Sample were 30 male wistar rats, divided into 5 groups i.e negativecontrol, positivecontroland 3 treatment groups.Oral candidiasis condition were set up in all groups except negative control group, performed by spraying Candida albicans suspension to dorsal tongue of wistar rats, once in 2 days for 14 days.The treatment groups were treated by Stichopus hermanii extractusing feeding tube, consecutively on the dose of 4.25 ml/kgBW, 8.5 ml/kgBW, 17 ml/kgBW. On the 14th day rats were euthanazied and the tongue were biopzied.
Immunohistochemical examination was performed for anti Candida albicansantibody and TNFα, their expression were examine under light microscope. Data was analyzed by ANOVA and LSD test at 95% significance level.Result: Stichopus hermanii extract reduced the expression of Candida albicans antibody and enhanced the expression of TNFα (p<0.05). Conclusion: Stichopus hermanii extract had the anti inflamatory effect against Candida albicans in vivo.
Keywords : Stichopus hermanii, anti inflamatory, Candida albicans
Correspondence : Syamsulina Revianti, Department of Oral Biology Hang Tuah University, Jl. Arif Rahman Hakim 150 Surabaya 60111. Email : [email protected]
INTRODUCTION
Oropharyngealcandidiasis is the most common infection in oral mucosa caused Candida albicans. Candida albicans wasmicroflora commensal in themucocutaneous cavities of skin, vagina, andintestine of humans that cause infections underpathological conditions such as diabetes, prolongedbroad spectrum antibiotic administration, steroidalchemotherapy as well as AIDS(Kamai et al., 2001).The expression of Candida albicans virulence inthe oral cavity is strongly correlated withimpairment of the immune system(Coleman et al., 1997). Thepathogenicity lies with their adherence capacity,colonization, enzyme production and interactionswith host defences (Samaranayake, 1990).The opportunistic fungus Candida albicans is a major causeof oral and esophageal infections in immunocompromised patientssuch as human immunodeficiency virus (HIV)- infectedindividuals and the elderly. Other conditions predisposingindividuals to oral C. albicans infection include hyposalivation, diabetes mellitus, prolonged use of antibioticsor immunosuppressive drugs, use of dentures, and poororal hygiene. The clinical significance of the oral candidiasis,which is not life-threatening but causes significantmorbidity in patients, is increasing with time. Some drugssuch as azole antifungal agents are used for chemotherapy ofthis fungal infection. HIV-positive patients receiving thenew therapy (highly active antiretroviral treatment) demonstratesignificantly fewer episodes of oral candidiasis thanthose without highly active antiretroviral treatment.
However,long-term treatment with antifungal drugs still causes theappearance of drug-resistant Candida or side effects.
The antifungal drugs commonly used totreat candidiasis are nystain, ravuconazole,clotrimazole, fluconazole and ketoconazole. In recent years, polyenes andazole agents have been usedfor treating infections caused by C. Albicans(Kauffman & Carver 1997, Masci, 2000, Clemons& Stevans, 2001). Natural plant products have also been used since ancient times, but the recent recurrent infections have revived interest in the products. Some plant essential oils are known to have various health benefits, especially antifungal, antibacterial, anti-inflammatory and antioxidative properties(Chami et al., 2004). The lack of scientific validation of their use as preventive and therapeutic products restricts their application in human health.In oral candidiasis, herbal formulations and phytotherapies (Hammer et al., 1999; Manohar et al., 2001; Kalemba & Kunicka 2003) play a major role.
Sea cucumbers such as Stichopus hermanii have high commercial value coupled with increasing global production and trade. Nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as Vitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), and minerals, especially calcium, magnesium, iron and zinc. Therapeutic properties and medicinal benefits of sea cucumbers can be linked to the presence of a wide array of bioactives especially triterpene glycosides (saponins), chondroitin sulfates, glycosaminoglycan (GAGs), sulfated polysaccharides, sterols (glycosides and sulfates), phenolics, cerberosides, lectins, peptides, glycoprotein, glycosphingolipids and essential fatty acids. Recent research stated that sea cucumber extract have its biomedical properties.The high-value components and bioactives as well as the multiple biological and therapeutic properties of sea cucumbers with regard to exploring their potential uses for functional foods and nutraceuticals.Golden sea cucumber (Sticophus hermanii) extract have been known to have antifungal agent and regarding to its contents is potential to be explored as treatment in oral candidiasis.
MATERIALS AND METHOD
The design of this research was post-test only control group design. The materials investigated in this study is the golden sea cucumber methanol extract (Sticopus hermanii) were tested for anti-fungal in vivo in Wistar rats induced Candida albicans for oral candidiasis condition.
Therapeutic efficacy studies were performed against Candida albicans. The culture was stored at -20º C in Sabouraud dextrose broth containing 15% glycerol until use. For inoculation, C. albicans was grown on Sabouraud dextrose agar plates at 30ºC for 24 h. Fungal colonies were then scraped off the agar, washed three times in Phosphate Buffered Saline (PBS; pH 7) and solution was adjusted to appropriate concentration using heamocytometer.
Stichopus hermanii was taken ± 100-250 grams weighs from Karimun Jawa. Sea cucumbers are cleaned, cut into pieces with a size of 3-10 cm, weighed wet weight after it is dried in the solar dryer rack for sample until it looks dry (3-4 days) to reduce the water content. Samples were dried sea cucumber, cut into pieces ± 1 cm, pulverized in a blender.The extraction process is done by the maceration process soak 250 grams of dried sample in 500 mL of solvent refined methanol until all samples submerged and allowed to stand at room temperature for 24 hours. After it was filtered with a filter paper to separate the filtrate and residue. The residue was then soaked again with 500 mL of methanol solvent for 24 hours. After it was filtered with a filter paper to separate the filtrate and residue. Filtrate thus obtained results maceration with 250 gram sample comparison / 1000 ml of solvent (1: 4 w / v). Filtrate methanol (polar) conducted homogenization with hexane solvent (non- polar) and 1,000 mL done with Separatory funel partition, then each layer of the filtrate solvent methanol and hexane solvent separated. Methanol filtrate done homogenization back with chloroform solvent (semi-polar) and 1,000 mL done with Separatory funel partition, then each layer of the filtrate solvent methanol and chloroform solvent separated. Each filtrate was then separated from the solvent using a rotary evaporator to obtain the extract (Pranoto et al, 2009).
The whole experimental plan was approved by Faculty of Dentistry Hang Tuah University ethical committee. 3 month-old, male Wistar strain rats weighing approximately 175 g each were used.
The rats were housed in 480x270x200 mm cages. Photoperiods were adjusted to 12/12 h light and darkness cycle daily, and the environmental temperature was constantly maintained at 21 ±1°C.
Access to food and water was allowed ad libitum to rats.
Prior to infection, all thetest animals were pre-checked for any pre-existingcandidiasis using sterile swabs rolled in the dorsal of lingue followed by a lawn on hi-chrome agarmedium.
Experimental infectionwas established by rolling of cotton swabimmersed in Candida albicans suspension containing 5 X 108 cells in the oralcavities of rat three times at 48-h intervals. Ratswere then randomly distributed into groups of 6animals each (n=6). Three days after the lastCandida inoculation, infected animals wereidentified by quantifying the number of CFU in theoral cavity as per the procedure summarizedbelow. The mean log10 CFU and standarddeviation per sample were calculated and used asthe basal level of infection before treatment.To assess the microbiological progression of infection the whole oral cavity, including buccal mucosa, tongue, soft palate, and other oral mucosal surfaces, was swabbed with a sterile cotton pad. The end of the cotton pad was then cut off and placed in a tube containing 5 ml of sterile physiological saline. After mixing with a vortex mixer to release Candida cells from the swab into the saline, 100 μl of undiluted cell suspension or its 100-fold dilutions were plated and incubated on Sabouraud dextrose agar (SDA) plates at 37°C for 20 h and the CFU of Candida cells were counted.
Sample were 30 male wistar rats, divided into 5 groups i.e negative control, positive control and 3 treatment groups. Oral candidiasis condition were set up in all groups except negative control group, performed by spraying Candida albicans suspension to dorsal tongue of wistar rats, once in 2 days for 14 days. The treatment groups were treated by Stichopus hermanii extract using feeding tube, consecutively on the dose of 4.25 ml/kgBW, 8.5 ml/kgBW, 17 ml/kgBW. On the 14th day rats were euthanazied and the tongue were biopzied. Neutral buffered formalin was used to preserve the tongue sections and then the tissues were embedded in paraffin. The embedded tissues were sectioned using microtome and stained with hematoxylin and eosin as described by Martinez et al. (2001).
Immunohistochemical examination was performed for anti Candida albicans antibody and TNFα, their expression were examine under light microscope. Data was analyzed by ANOVA and LSD test at 95% significance level.
RESULT
Figure 1. The expression of anti-candida albicans antibody in tongue ephitelial
Table 1. The expression of anti-candida albicans antibody and TNF-α in tongue ephitelial
Negative Control Positive Control
Stichopus hermanii 4,25 ml/kgBW
Stichopus hermanii 8,5 ml/kgBW
Stichopus hermanii 17 ml/kgBW ANTI
CANDIDA
TNF- ALPHA
ANTI CANDIDA
TNF- ALPHA
ANTI CANDIDA
TNF- ALPHA
ANTI CANDIDA
TNF- ALPHA
ANTI CANDIDA
TNF- ALPHA
0 3 26,00 3 10,00 17 7,00 16 2,00 18
0 4 22,00 5 9,00 12 2,00 15 5,00 19
0 3 21,00 3 11,00 18 5,00 18 3,00 15
0 3 23,00 4 10,00 12 8,00 12 2,00 12
0 4 19,00 2 12,00 13 4,00 17 3,00 17
0 4 24,00 5 9,00 11 5,00 17 2,00 12
0 3,50 22,50 3,67 10,17 13,83 5,17 15,83 2,83 15,50 Figure 2. The expression of TNFα in tongue ephitelial
cell on imunohistochemistry staining
Dependent Variable Sig.
ANTICANDIDA Negative control Positive control ,000
Stichopus hermanii 4,25 ml/kgBW ,000 Stichopus hermanii 8,5 ml/kgBW ,000 Stichopus hermanii 17 ml/kgBW ,006
Positive control Negative control ,000
Stichopus hermanii 4,25 ml/kgBW ,000 Stichopus hermanii 8,5 ml/kgBW ,000 Stichopus hermanii 17 ml/kgBW ,000 Stichopus hermanii 4,25
ml/kgBW
Negative control ,000
Positive control ,000
Stichopus hermanii 8,5 ml/kgBW ,000 Stichopus hermanii 17 ml/kgBW ,000 Stichopus hermanii 8,5
ml/kgBW
Negative control ,000
Positive control ,000
Stichopus hermanii 4,25 ml/kgBW ,000 Stichopus hermanii 17 ml/kgBW ,020 Stichopus hermanii 17
ml/kgBW
Negative control ,006
Positive control ,000
Stichopus hermanii 4,25 ml/kgBW ,000
0 5 10 15 20 25
Negative Control
Positive Control
Stichopus hermanii 4,25
ml/kgBW
Stichopus hermanii 8,5
ml/kgBW
Stichopus hermanii 17
ml/kgBW ANTI CANDIDA
ANTI CANDIDA
- 5.00 10.00 15.00 20.00
Negative Control
Positive Control
Stichopus hermanii
4,25 ml/kgBW
Stichopus hermanii
8,5 ml/kgBW
Stichopus hermanii
17 ml/kgBW
TNF-ALPHA
TNF-ALPHA
Stichopus hermanii 8,5 ml/kgBW ,020
TNFALPHA Negative control Positive control ,896
Stichopus hermanii 4,25 ml/kgBW ,000 Stichopus hermanii 8,5 ml/kgBW ,000 Stichopus hermanii 17 ml/kgBW ,000
Positive control Negative control ,896
Stichopus hermanii 4,25 ml/kgBW ,000 Stichopus hermanii 8,5 ml/kgBW ,000 Stichopus hermanii 17 ml/kgBW ,000 Stichopus hermanii 4,25
ml/kgBW
Negative control ,000
Positive control ,000
Stichopus hermanii 8,5 ml/kgBW ,126 Stichopus hermanii 17 ml/kgBW ,200 Stichopus hermanii 8,5
ml/kgBW
Negative control ,000
Positive control ,000
Stichopus hermanii 4,25 ml/kgBW ,126 Stichopus hermanii 17 ml/kgBW ,794 Stichopus hermanii 17
ml/kgBW
Negative control ,000
Positive control ,000
Stichopus hermanii 4,25 ml/kgBW ,200 Stichopus hermanii 8,5 ml/kgBW ,794
The result of this research showed that the expression of anti-Candida albicans antibodies in all groups induced Candida albicans, whereas the negative control group were not induced Candida albicans not found any expression of anti-Candida albicans antibodies. Results of ANOVA and LSD test showed a significant difference between the control and treatment groups (p <0.05) The extract Sticophus hermanii in all treatment groups decreases the amount of expression of anti-Candida albicans antibodies significantly. The larger the dose of the extract Sticophus hermanii the smaller number of expressions of anti-Candida albicans antibodies.
The result of this research showed that the expression of TNF-α antibodies in all groups induced Candida albicans were increased, whereas the negative control group were not induced Candida albicans, the expression of TNF-α antibodies were decreased. Results of ANOVA and LSD test showed a significant difference between the control and treatment groups (p <0.05) The extract Sticophus hermanii in all treatment groups increased the amount of expression of TNF-α antibodies significantly. The larger the dose of the extract Sticophus hermanii the smaller number of expressions of anti-Candida albicans antibodies but the larger the dose of the extract Sticophus hermanii the larger number of expressions of TNF-α antibodies.
DISCUSSION
In vivo studies used Wistar rats that had been commonly used in experimental candidiasis because it has several advantages that are relatively easy to manage and have adequate oral cavity size for inoculation and sampling. Inoculation of C. albicans and sampling conducted on the tongue with a tongue consideration that the area is an area of predilection for candida infection that most often, both for erythematous candidiasis, atrophic, median rhomboid glossitis, and pseudomembranous (Samaranayake, 2001). Negative control group is a group of normal mice were not inoculated C.albicans, whereas the positive control group and the group treated for 14 days induced candidiasis.
Sea cucumber extract gold done systemically per sonde with three concentrations, compared with positive and negative control group were given only 1% CMC solution.
After inoculation of C. albicans does not appear to be any significant differences in the clinical manifestations between the normal group and the induced candidiasis. Administration during the time span of 14 days is not expected to provide significant clinical manifestations appear but the immunohistochemical examination is expected to give an idea about the condition of oral candidiasis by examining the expression of anti-Candida albicans antibodies. Based on the presence of the expression of anti-Candida albicans antibodies is assumed that there is the presence of Candida albicans in a certain amount of tongue that indicates a condition of oral candidiasis.
In the negative control group not found the fungus C. albicans, which is in line with the assumption that in the normal animals there is no process candidiasis, Candida albicans which is not the normal flora of the oral cavity of mice. In the positive control group and the treatment of acquired antibodies anti C. albicans, whereas in the positive control group who were not given the amount of sea cucumbers is higher than the group treated given the golden sea cucumber extract (p <0.05).
Extracts of some sea cucumber extract is known to have anti-fungal properties which contained compounds and is thought to act as antifungal saponins, alkaloids, and triterpenes (Mayer et al, 2005; Hua et al, 2009; Pranoto et al, 2012). Saponins are natural glycosides and triterpenes consists of sugar, has a broad spectrum of biological activities including hemolytic, antifungal, cytotoxic and membranotropik reaction. Saponins have the ability to lysis of erythrocytes and forms a complex with the cell membrane resulting in the formation of holes and affect cell permeability, are like 'detergent' that contain both hydrophilic and hydrophobic parts (Podolak, et.al, 2010). Saponins may also result in apoptosis because it can damage the mitochondrial membrane cell, lowering the transmembrane potential, increase cytosolic calcium and activates apoptosis pathways through calcium (Wojtkielewicz, 2007).
Bordbar et al. (2011) reported, identified saponins from the sea cucumber. Its chemical structure is quite comparable to the bioactive ganoderma, ginseng and other herbal remedies.
Saponins produced as a form of chemical defense mechanism for sea cucumbers in nature. In addition to alleged as a defense from predators, also believed to have biological effects, including anti fungal, cytotoxic against tumor cells, hemolysis, immune activity, and anti-cancer (Zhang et al, 2006).
Saponin is a class of compounds that inhibit or kill microbes by interacting with membrane sterols, contribute as antifungal with membrane sterols mekanismemenurunkan surface tension of the cell wall of C. albicans, sehinggapermeabilitasnya increased (Podolak et al, 2010; Pranoto et al, 2012), similar to the mechanism nystatin action.
In this study used whole cucumber extract contains saponins which have not been exposed to the maximum likelihood. Efforts to maximize the active anti-fungal substances carried by sea cucumber extract gold with methanol according to research Pranoto et. al (2012). Antifungal compounds in polar sea cucumbers suspected because soluble in methanol, as in the study of Abraham (2002) using a polar solvent to test the antifungal and antibacterial activity of Holothuria, as well as Thanh (2006) who isolated compounds of sea cucumber triterpene sand in Vietnam using polar solvent and shown to function as an antifungal, antibacterial, and cytotoxic agents.
This study is a continuation of previous studies which have known of the power of anti fungal extracts against C. albicans Sticophus hermanii in vitro (Parisihni et al, 2013). It is estimated that there are other possible mechanisms of saponins in the potential anti-fungal immunomodulatory capabilities that triggers macrophages in response to an infection in which this mechanism is not seen with the in vitro antifungal test that is done in vivo in the present study. The results showed that administration of sea cucumber extract gold in all treatment groups decreases the amount of expression of anti-Candida albicans antibodies were significantly (p <0.05). Hermanii Sticophus extract has anti-fungal power against Candida albicans in vivo. The results of this research can be the basis for further research exploration Sticophus hermanii extracts as a source for the biological treatment of oral candidiasis.
Sea cucumbers are one of the potential marine animals with multiple biological activities and medicinal value. Therefore, marine echinoderms can be explored as a sustainable natural source for the discovery of novel antifungal agent. Bioactive and antiagent substances in sea cucumbers, such as triterpene glycosides, enzymes, amyloses, fatty acids, cytotoxins, etc. with potential capabilities to antifungal activities and increase immunity as well as contribute to immunopotentiation.In this study,
methanol extract of Stichopus hermanii showed promising antifungal and immunostimulantor activity in vivo. It was shown that Stichopus hermanii extracts were effective against candida albicans at a concentration of 4,25 mg/kg BW until 17 mg/kg BW.They inhibited fungal growth start the lowest until highest tested extract concentration. This shows that the amount of extract present related with its antifungal activity.
A wide variation of bioactivities among the sea cucumbers extracts suggests that a variety of substances are capable of antifungal activities. Prior research showed thath sea cucumber protein, especially produced from body wall, is rich in glycine, glutamic acid and arginine. Glycine can stimulate production and release of IL-2 and B cell antibody and thus contributes to enhancing phagocytosis. Arginine can enhance cell immunity by promoting activation and proliferation of T-cell.
Due to these amino acid components, sea cucumbers have remarkable function in immune regulation.In this study showed that Stichopus hermanii extract reduced the expression of candida albicans antibody and enhanced the expression of TNFα (p<0.05). Stichopus hermanii extract had the antiinflammatory effect against Candida albicans in vivo
ACKNOWLEDGEMENT
This research was supported by a grant from Fundamental Research Program, funded by The Directorate of Higher Education Indonesia 2012-2013.
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