4 Hery Winarsi_351.pmd A Supplement Based on Zn-Enriched Virgin Coconut Oil as an Antifungal agent for Vaginal Candidiasis Patients HERY WINARSI1*, HERNAYANTI1, AND AGUS PURWANTO2 1Biology Faculty, Universitas Jenderal Soedirman, Kampus Karangwangkal, Purwokerto 53122, Indonesia; 2 Margono Soekarjo Hospital, Purwokerto 53146, Indonesia This research was conducted to investigate the amount of Candida albicans in vaginal secretion of Vaginal Candidiasis patients administered with Zn-enriched virgin coconut oil. Thirty respondents were selected based on several criteria as follows: the number of C. albicans colonies in the vaginal secretion was more than 105 cfu.ml-1, voluntary, healthy, willing to sign the informed consent and resided in Purwokerto. In Group A, 10 women were administered 2 tablespoons per day of Zn-enriched virgin coconut oil. In Group B, 10 women were administered 1 tablespoon per day of Zn-enriched virgin coconut oil; and in Group C, 10 women served as control group. Vaginal secretions were taken 3 times, before intervention (at baseline time), at 1 month and 2 months after intervention. Samples were taken by collecting vaginal secretions from the vaginal proximal region using a sterile cotton bud, which was then put into a tube containing sterile carrier media. The vaginal secretions were tested for the number of total C. albicans using Pour Plate Method. Two months after treatment, the number of colonies decreased from 4.4x106 to 2.5x106 cfu ml-1 (p=0.03) in Group A. There was no significant difference in the number of colonies between Group A and Group B, the number of C. albicans colonies was still above the normal range. Therefore, the recommended dosage of intervention with Zn-enriched virgin coconut oil is one tablespoon a day. Key words: Candida albicans, vaginal candidiasis, virgin coconut oil, Zn _____________________________________________ ________________________ *Corresponding author, Phone: +62-281-638794, Fax: +62-281-631700, E-mail: winarsi@yahoo.com ISSN 1978-3477 Volume 2, Number 2, August 2008 p 69 - 72 Candida albicans is a normal microbiota that is commonly found in the vagina. However, excessive amount of this yeast can cause discomfort, pruritis, and pain. This condition is known as leukorrhea or Vaginal Candidiasis (VC). Many conditions can trigger the emergence of VC, such as trichomoniasis, diabetes mellitus, vaginitis senilis, inflammatory chronical inflammation of the pelvis, virus infection, disturbances in immune function, and Candidiasis. The last two conditions were commonly experienced by women (Sobel 2005; Winarsi et al. 2006). Transmorphism of C. albicans from yeast to mycelia is potentially pathogenic. The mycelium of C. albicans is capable of binding to the epithelium of the hosts cells mycelia and penetrating the surface with mycelium protein, which is then tightly cross- linked with the cells epithelium. Proteins contain amino acids that are able to act as a transaminase substrate of mammalian cell keratin. Binding of C. albicans enzymes to the epithelium of host cells leads to pathogenic process. Proteinase and phospholipase secreted by the mycelium are capable of digesting epithelium cells, which then facilitates the invasion by the mycelium. C. albicans itself may perform phagocytosis on the endothelium of the host cell. This ability enhances the virulence of C. albicans. Winarsi et al. (2006) reported that VC incidence in Purwokerto was 38%, which occured among women of 15-53 years of age that experienced Zn deficiency (Winarsi et al. 2005). Zn is a co-factor for enzyme that has antifungal, antiadenoma, antiprostatitis, and immunostimulator potency. It is important for maturation, activation, proliferation, and differentiation of T-cells. Therefore, the occurrence of VC is likely to be worse in women with Zn deficiency. The application of clinical medicine might cause immunosuppresor effect. Therefore, people prefer to use natural remedies such as Virgin Coconut Oil (VCO). VCO not only contains lauric acid, but also capric, caprilic, and myristic acids (Ingle et al. 1999). These fatty acids have antifungal, antibacterial, as well as antiviral potency, and they help maintain the immune system (Bergsson et al. 2001). Lauric acid can kill C. albicans and repair the metabolism energy (Portillo et al. 1998). The aim of this research was to investigate the growth of C. albicans in vaginal secretions of VC patients treated with Zn-enriched VCO. MATERIALS AND METHODS Zn-enriched VCO has been formulated by Winarsi et al. (2006). Thirty VC patients were selected based on the following criteria: number of C. albicans in the vaginal secret (more than 105 cfu ml-1), resident in Purwokerto, willing to volunteer for the research and sign an informed consent form. Subjects were divided into 3 groups with 10 patients in each group. Those in Group A were treated with 2 tablespoons per day, those in Group B were treated with 1 tablespoon per day, while those in the Group C were given a placebo and served as control group. Treatments were carried out for two months. Vaginal secretion were sampled at three periods: i.e at baseline and then continued by one and two months after treatment. Sample was taken by sweeping the vaginal proximal area using a sterile cotton bud, which was then put in a tube containing sterile carrier media. The sample was then tested for the total number of colonies of C. albicans using Pour Plate Method. RESULTS The amount of C. albicans at baseline time was above the normal range, being 8.38x106-1.24x 107 cfu ml-1 (Fig 1). One month after beginning treatment, the number of C. albicans 70 WINARSI ET AL. Microbiol Indones colonies decreased from 8.4x106 to 4.8x106 cfu ml-1 (p = 0.022) group receiving 2 tablespoons of VCO enriched with Zn per day (Group A). However, the number of colonies remained above normal. There was no significant difference on the amount of C. albicans between Group A and Group B (p = 0.32). Because the amount of C. albicans was still high, the time of treatment was lengthened. After 2 months of intervention, the number of C. albicans colonies in Group A decreased from 4.4x106 to 2.5x106 cfu ml-1 (p = 0.03). The growth of C. albicans is influenced by pH. Measurements of pH of vaginal secretions of VC patients are presented in Table 1. The pH at baseline time was relatively neutral approximately 6.0. One month after intervention, the pH decreased from 5.8 to 5.5, and continued to decrease 2 months after treatment, from 5.3 to 5.0 (Table 1). DISCUSSION At baseline time, the amount of C. albicans was similar among the 3 groups (p = 0.45), indicating that the groups were homogeneous before treatment. Therefore, a change in the number of colonies after treatment reflects the effect of Zn-enriched VCO. VC is a pathological infection condition. One factor causing VC is the excessive amount of C. albicans in vaginal secretions. In this case, the host immune system is disrupted by C. albicans. This causes rapid proliferation of yeast cells, which results in increasing amount of C. albicans. Fungal cells secrete enzymes that facilitate their invasion. Secreted Aspartyl Proteinase (SAP) produced by C. albicans increases the microorganism’s ability to colonize and penetrate into the host tissue. In the host’s body, the yeast cells are sometimes unrecognized by the host’s immune system (Zeppelin et al. 1998). SAP induces the release of mannan (a component of the fungal cell wall), which then inhibits and modulates the host’s immune system. This enzyme suppresses the host’s immunoglobulin and complement levels (Naglik et al. 2003). SAP hydrolyzes the mucous secreted by the host’s digestive tract, so that Candida cells may directly penetrate the mucous cells (Chaffin et al. 1998). Candida cells are still able to secrete SAP even after they have been subjected to phagocytosis by macrophages. Therefore, the activity of Candida cells is stronger than what the host’s body expected. This situation leads to suppression of the host’s immune system and thus causes infection. Mannoproteins and enolase (metabolites of C. albicans) are antigens that are able to stimulate the host’s humoral immune response. Thus, mannoprotein modulates the host’s immune response. It makes C. albicans unrecognized to the host’s immune system, so that the cells are not opsonicated or phagocytosized. The other mechanism by which C. abicans makes itself unrecognizable to the host immune system is through adhesion with components of the host’s cells, including thrombocyte, and complement of iC3b. This triggers phospholipase release by C. albicans, which assists the penetration to the host’s tissue and crushes the cell membrane. The activity of phospholipase is the primary factor affecting virulence in C. albicans. Candidiasis incidence is a consequence of C. albicans virulence, which is influenced by the metabolites of C. albicans and a predisposition factors of the host’s body. Regarding the immunocompetence between the organism and the host’s normal immune system, C. albicans acts as a normal microbiota on the skin, mucous surfaces, digestive tract, urethra, and genitalia. Accompanied by other normal microbiota, C. albicans balances the formation of colonies, so that the growth of the pathogenic microbes may be prevented and a balanced pH is maintained. In normal amounts, C. albicans is not pathogenic, because it can be controlled by the immune system and other normal microbiota. The existence of C. albicans and other normal microbiota have a competitive effect, especially on the adhesion and nutrition absorption ability of the host’s cells. Under certain conditions, the amount of C. albicans cells in the body increases when the activity of immune cells decreases. This condition disturbes the balance among other normal microbiota or other factors which triggers the growth of C. albicans. Candidiasis represents an opportunist i n f e c tion, so that infection usually occurs in immunocompromized individuals (Fridkin and Jarvis 1996). The decrease in the number of C. albicans colonies after 1 month of intervention with Zn-enriched VCO was related to the components of organisms cell walls. Chaffin et al. (1998) and Marcilla et al. (1998) stated that the cell walls of C. albicans were composed of glucan, chitin, mannoprotein (mannan binding to protein), protein, fat, and inorganic salts as minor components. These components build up the yeast cell walls and mycelium in the relatively same amount. Glucan builds up cell structure, while chitin maintains integrity of the cell wall structure (Marcilla et al. 1998). Mannoprotein and other proteins are predominant in the external layer of the cell wall with only small amounts being found in the 1.40E+07 1.20E+07 1.00E+07 8.00E+06 6.00E+06 4.00E+06 2.00E+06 0.00E+00 0 1 2 A m o u n t o f C . a lb ic a n s ( c fu m l- 1 ) Fig 1 The amount of Candida albicans colonies. A, group treated with Zn-enriched VCO, 2 tablespoons/day; B, group treated with Zn- enriched VCO, 1 tablespoon per day; C, control group. A B C A A B B C C Table 1 The average pH of vaginal secretion pH in the period of intervention (month) Group 0 1 2 A B C 6 . 1 6 . 1 6 . 0 5 . 8 5 . 6 5 . 5 5 . 3 5 . 1 5 . 0 A, group treated with Zn-enriched VCO, 2 tablespoons per day; B, group treated with Zn-enriched VCO, 1 tablespoon per day; C, control group. Period of intervention (month) Volume 2, 2008 Microbiol Indones 71 internal layer. Mannoprotein is covalently bound to β-glucans and protein chains (Chaffin et al. 1998). Mannoprotein is reported to trigger host immune responses to Candidiasis, because this compound is thought to be involved in the changing of cell morphology. Mannoprotein has immunomodulator potency to the host’s body. It generally controls the host’s immune system, including natural killer, phagocytes (macrophage), as well as cellular and humoral immune cells (Chaffin et al. 1998; Marcilla et al. 1998). Zn- enriched VCO may attract mannoproteins, the primary component of C. albicans exterior cell walls, causing destruction of the cell walls. This will cause glucan and chitin, other components of C. albicans cell walls, unable to main- tain the integrity of the entire cell walls, thus making the cell walls very brittle. A brittle cell wall can be easily lysed, and unable to maintain C. albicans structure. Brittleness of C. albicans cell walls may be related to glycoprotein because the ability of the external layer of cell walls to act as an adhesion mediator on host epithelial cell surface is interrupted (Chaffin et al. 1998). These conditions could therefore suppress the growth of C. albicans. Goyal and Khuller (1992) reported that the cell membrane of C. albicans consisted of a phospholipid layer, which was one of its energy sources. When C. albicans is in contact with lauric acid in the host body, the lipid compounds of cell wall will be lysed. As the lipid content of the membrane is destroyed, the cell content leaks out. Therefore, the growth of C. albicans is inhibited and the C. albicans can even be killed. Caprilic acid derivatives of VCO could also kill VC causing C.albicans. The potency of VCO as an antifungal agent is shown by the activity of lauric and caprilic acids. Lauric acid in the body is converted into monolaurine, while capric acid is converted into monocaprine compounds. These compounds are monoglycerides which have antimicrobial activities capable of regulating the growth of C. albicans (Bergsson et al. 2001). Zn also has an antimicrobial potency, especially towards pathogenic microbes. In the form of free ions, they can directly attack microbes. Zn is also a component of human membrane cell structure and it is an essential antioxidant for cells with short half-life, such as immune cells (Filipe et al. 1995). Therefore, Zn fortified by VCO will improve the capacity of the host immune and defense systems. Specific protein compilers of the cell wall are responsible for C. albicans dimorphism (Marcilla et al. 1998), but this dimorphism also depends on temperature, pH, and CO 2 concentration (Ernst 2000). C. albicans optimally grows at 37°C and neutral pH. At neutral pH (at baseline time), the organism in a mycelial state, but at lower pH (after 1 and 2 months of treatment) it is in yeast state (Molero et al. 1998). In the yeast form, adhesion of fungal cells to the host epithelial cells is stronger. Protein and glycoprotein components of the C. albicans cell wall surface are also important in adhesion (Senet 1998), which is the first step of colonization and infection (Chaffin et al. 1998). Adhesion occurs at a minimum pH of 3-4, and is optimal at pH 6 (Sundstrom 2000). At relatively neutral pH (at baseline time), cell colonization could occur, so that the number of C. albicans colonies were higher than normal. At decreased pH, although not significant, the number of C. albicans colonies declined. Kanbe and Cutler (1994) argued that C. albicans was capable of producing a lactic acid compound in vaginal secretion. Lactic acid causes the cell wall of C. albicans, which consists of mannan, mannoprotein and chitin were denaturated, and reduce the level energy of this micro- organism. This finding supports the statement of Klotz and Smith (1995), that some microorganisms are more sensitive to acidic environment. As proteins, enzymes will be denatured by acid. This causes physiological disturbance and a decrease of the microbe’s life-time. A decrease of pH will also improve the inhibition of C. albicans growth by VCO. 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